The Role of Per1 and EDN1-As in Mineralocorticoid and Salt-Sensitive Hypertension

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The Role of Per1 and EDN1-As in Mineralocorticoid and Salt-Sensitive Hypertension
Solocinski, Kristen
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University of Florida
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Doctorate ( Ph.D.)
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University of Florida
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Medical Sciences
Biochemistry and Molecular Biology (IDP)
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circadian -- endothelin -- per1 -- pressure
Biochemistry and Molecular Biology (IDP) -- Dissertations, Academic -- UF
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Cardiovascular disease is the cause of death for 1/3 of Americans every year. Hypertension is one of the main risk factors for developing cardiovascular disease and thus is a large target for treatment to prevent cardiovascular disease as well as other associated morbidities such as chronic kidney disease and renal injury. While many treatments can be extremely effective for some patients, about half of the hypertensive population has uncontrolled hypertension with about 10% of the hypertensive population being resistant to treatment with multiple drugs including diuretics. Even with the best medications we have, around 8 million Americans still do not have their blood pressure under control. Clearly, better medications are needed and to accomplish this, we need a better understanding of the progression of hypertension. The Gumz lab and others have clearly demonstrated the importance of the circadian clock to blood pressure as every clock gene knockout animal exhibits a blood pressure phenotype. Period 1 (Per1) is one of four core clock proteins and coordinately regulates many aspects of renal sodium reabsorption. Per1 has been implicated in the pathogenesis of hypertension, including salt-sensitive hypertension. We have shown that male mice lacking the Per1 gene display a harmful non-dipping phenotype while on a high salt and mineralocorticoid diet. However, female Per1 knockout mice on the same diet do not show any significant phenotype compared to wild type mice, in line with reported sex disparities with respect to cardiovascular and kidney disease. Per1 regulates Endothelin-1 (ET-1) which is itself an important contributor to hypertension as it is one of the most potent endogenous vasoconstrictors. Our lab has discovered a novel long non-coding RNA (lncRNA) antisense to the Endothelin-1 gene (EDN1), named EDN1-AS. It appears to be regulated by the circadian clock, which suggests the possibility that both Per1 and EDN1-AS are novel, potential targets for the treatment of hypertension. ( en )
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Thesis (Ph.D.)--University of Florida, 2017.
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by Kristen Solocinski.

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2017 Kristen Solocinski


To My Family and Friends


4 ACKNOWLEDGMENTS First and foremost, I have to thank my mentor, Dr. Michelle Gum z, for all of her support and help. When I first came to UF, I was so nervous about only having three skip the others. Over the years, Michelle has pushed me to write a rticles, present posters and submit abstracts. Indubitably, I would not have accomplished half of what is on my CV without her guidance and the least aggressive push to write imaginable. I also thank Michelle for her wonderfully bubbly personality which ma kes it very hard for people to have anything bad to say about her at all. It is not only great to work for someone like that, but it is great for me in terms of networking because everyone is happy to talk to her It is also a big bonus to have a boss who likes the same great TV shows as you. I thank Michelle for being not only a mentor but a mentor/friend/mom hybrid over the years. In a similar vein, I have to thank Dr. Brian Cain for his mentorship and role as my lab grandpa as well. I have lost count o f the times he has explained the antisense of the Dr. Cain has always been such a welcoming, generous person going so far as to invite me to his home for Thanksgiving and C hristmas. He has also given up so much time in order to offer thoughtful criticisms of countless talks, posters and papers. I thank both Dr. Gumz and Dr. Cain for always having an open door whenever I needed to talk or troubleshoot. I am also extremely gra teful for the glowing letters of recommendation you have written for me. I also need to express my gratitude to my other committee members. I thank Dr. Peter Sayeski for always responding to my emails and writing me great letters of


5 recommendation. He has always listened thoughtfully to my presentations and given me helpful critiques and recommendations. I also thank Dr. Debbie Scheuer for her assistance on my committee. I apologize for never doing as much brain related work as I had hoped (and possibly pro mised). Nevertheless, Dr. Scheuer was always helpful in her thoughtful critiques and suggestions for future experiments. I thank Dr. Jorg Bungert for his role on my committee. He has always kept his thoughts and critiques short and sweet which I appreciate He has also offered some very insightful experiments to confirm that what I have seen with my antisense is real This is greatly valuable to me because I always want to be sure of my results. Finally, I thank Dr. Kevin Brown as an honorary committee memb er. In addition to an incredible amount of help with my CRISPR/Cas9 project, Dr. Brown has given me countless hours of time discussing anything from job opportunities to science to politics. To all of my committee members, I cannot thank you enough for you r valuable time and input over the past four years. In addition to my committee mentors, I have had a number of non official mentors I would like to thank as well. I thank Dr. Charles Wingo for his helpful input at lab meetings as well as the thoughtful co nversations we have shared about science. I owe him many thanks for his assistance with mice and his allowance to use his facilities at the VA. I also have to thank him for his wonderful fashion sense so I have a new bow tie to look forward to at our meeti ngs. Finally, I have to thank Dr. Wingo for his excellent taste in food, especially Cubans (sandwiches, not cigars), which I have been treated to numerous times. I also have to give great thanks to I. Jeanette Lynch for the countless hours she has spent te aching and explaining an y aspect of physiology which I was curious about. Any time I would drop in to her lab, she always had time to talk or help


6 me with whatever I needed. She has also been invaluable with respect to our mouse studies, as all of the tele metry surgeries on which this dissertation relies so heavily were done by her. Lastly, I thank Jeanette for being a good friend and person to talk to. Although she was not with the Wingo lab for a very long time, I also thank Dr. Luciana Morla for her time and contributions to all of our labs. She always had a funny story to brighten the day and made some good macaroons. For her guidance with immunohistochemistry and perfusion fixation, I thank Dr. Jill Verlander. Her protocols have been the most reliable o nes I have used in this lab and her attention to detail is no doubt what gives us such great results. Her tough yet accurate criticisms have given me extreme faith in the immunohistochemistry work I have done here. I also thank Dr. Rajesh Mohandas for his thoughtfulness about my work and his great personality. He literally always has something funny to say to brighten my day and I could not have picked a better person to have in the lab I also have to thank the entire Nephrology Department for having such great people to talk to about science or ot her topics. I cannot thank Shiy u Li enough, whose sweet smile and simple wave brightens the day with more force than our big centrifuge generates. I could not have been half as successful without the guidance o f Dr. Jacob Richards and Dr. Sarah Barilovits. Jacob started me off in the lab and showed me the tenure in a lab. We also shared many insightful conversations as well as laughs, for which I am most indebted. While I did not spend much time with Sarah, she started off the EDN1 AS project for me and really helped me to get on my way with it. She also produced the first evidence of the antisense in multiple cell lines, which I use as opening


7 data in all of my presentations. Dr. Lauren Douma has also been a great source of knowledge and laughter, for which I thank her. She has been invaluable when it comes to RNA isolation and has golden hands which produce the most beautiful r esults. I also cannot overstate how much I appreciate her help with the undergraduate students. Additionally, I thank Thomas Cheng for his help in the lab with mice, comedy and foreign goodies from his many travels. When Thomas left for his many trips, he always made sure that all the orders were in and things were in place before he left. He also made sure to send post cards which decorate the lab. Undergraduate students all deserve my gratitude as well. I thank Sean All and Alex Diaz who were always willi ng to help with anything at all and made great contributions to the lab in the form of data and laughter. I thank Kareme Alder for his professional attitude and dedication to work at all times. I also thank him for immersing me in the Jamaican culture, bro adening my knowledge of duppies, Tevin and rolling calf. I thank Ravi Patel for his countless efforts to produce the perfect Western blot and aid in troubleshooting the ECL reagents. I thank Amber Miller for her continuation of and her willingness to help with anything I needed. I thank Sarah Masten for continuing to return to lab after being given hundreds of tubes of urine compensation. Finally although technically not an undergraduate in the lab anymore, I thank Maggie Holzworth for her obsessive efforts to clone an EDN1 AS plasmid and continual effort where most others would have given up. I thank her for her enthusiasm to continue my work on the EDN1 AS project and give it just as much love as I did. I


8 also thank her for being a great friend outside of lab as well and watching my dogs which ultimately brought even more urine into her life. Finally, I have to thank all of the people outside o f lab who have helped me. I thank all of my ex boyfriends for showing me just how much I can put up with and still be successful. I thank Natalie Ross for always being there for me even though we are over a thousand miles apart. I thank my cousins, Mike L awrence, Laura Bush and Billy Bush for being there when I need it and making time to see me during the two times that I came home during my studies. I am so glad to be part of the CCC. I thank Jason never having a single mean thing to say to me. He is the best baby brother a girl could ask for. I thank my mom for raising me as a parent, not a friend, so I became independent and able to handle stressful situations, which is graduate school in a nut she ll. I also thank her for her continual support, be it monetary, emotional or otherwise. has done justice to just how grateful I am to all of you who have helped me along the wa y.


9 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ .......... 12 LIST OF FIGURES ................................ ................................ ................................ ........ 13 LIST OF ABBREVIATIONS ................................ ................................ ........................... 15 ABSTRACT ................................ ................................ ................................ ................... 18 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 20 Background ................................ ................................ ................................ ............. 20 The Kidney ................................ ................................ ................................ .............. 23 Hormones and the Kidney Clock ................................ ................................ ............ 25 The Kidney Clock ................................ ................................ ................................ .... 2 9 PER1 Action in the Kidney ................................ ................................ ...................... 34 Development ................................ ................................ ................................ ........... 38 Regulation of the Kidney Clock ................................ ................................ ............... 41 Potassium ................................ ................................ ................................ ............... 43 Clinical Implications ................................ ................................ ................................ 45 2 TRANSCRIPTIONAL REGULATI ON OF NHE3 AND SGLT1 BY THE CIRCADIAN CLOCK PROTEIN PER1 IN PT CELLS ................................ ............. 51 Background ................................ ................................ ................................ ............. 51 Methods ................................ ................................ ................................ .................. 53 Animals ................................ ................................ ................................ ............. 53 Cell Culture ................................ ................................ ................................ ....... 54 RNA Isolation and quantitative real time PCR ................................ .................. 54 Membrane Protein Analysis, Nuclear Protein Isolation, and Western Blot Analysis. ................................ ................................ ................................ ........ 54 hnRNA Analysis. ................................ ................................ .............................. 55 Chromatin Immunoprecipitation (ChIP). ................................ ........................... 56 Ouabain sensitive 86 Rb uptake ................................ ................................ ......... 56 Statistical Analysis. ................................ ................................ ........................... 57 R esults ................................ ................................ ................................ .................... 57 D iscussion ................................ ................................ ................................ .............. 60 3 DOCP SALT TREATMENT LEADS TO NONDIPPING HYPERTE NSION IN PER1 KNOCKOUT MICE ................................ ................................ ....................... 73


10 Background ................................ ................................ ................................ ............. 73 Methods ................................ ................................ ................................ .................. 75 Mice ................................ ................................ ................................ .................. 75 Tissue dissection and quantitative real time RT PCR (qPCR) .......................... 76 Flame Photometry ................................ ................................ ............................ 76 Aldosterone ELISA ................................ ................................ ........................... 76 Statistics ................................ ................................ ................................ ........... 76 Results ................................ ................................ ................................ .................... 76 Discussion ................................ ................................ ................................ .............. 80 4 LOSS OF THE CIRCADIAN CLOCK PROTEIN PER1 DOES NOT LEAD TO NONDIPPING IN FEMALE C57BL/6 MICE ................................ ............................ 91 Background ................................ ................................ ................................ ............. 91 Methods ................................ ................................ ................................ .................. 93 Mice ................................ ................................ ................................ .................. 93 Tissue dissection and Real Time PCR ................................ ............................. 93 Flame Photometry ................................ ................................ ............................ 93 Aldosterone ELISA ................................ ................................ ........................... 93 Statistics ................................ ................................ ................................ ........... 94 Results ................................ ................................ ................................ .................... 94 Discussion ................................ ................................ ................................ .............. 96 5 IDENTIFICATION AND INITIAL CHARACTERIZATION OF A lncRN A ANTISENSE TO THE EDN1 GENE: EDN1 AS ................................ .................... 105 Background ................................ ................................ ................................ ........... 105 Methods ................................ ................................ ................................ ................ 108 Exploration of EDN1 Chromatin State and Regulation with the UCSC Genome Browser ................................ ................................ ........................ 108 Strand Specific Primer Design ................................ ................................ ....... 109 RNA I solation and DNase Treatment ................................ ............................. 109 cDNA Conversion and PCR ................................ ................................ ........... 109 Cell Culture ................................ ................................ ................................ ..... 110 Aldosterone Treatment ................................ ................................ ............ 110 Dexamethasone Treatment ................................ ................................ ...... 110 Chromatin Immunoprecipitation ................................ ................................ ...... 111 Mice ................................ ................................ ................................ ................ 111 CRISPR/Cas9 ................................ ................................ ................................ 111 gRNA Design ................................ ................................ ........................... 111 Creation of the gRNA Plasmid ................................ ................................ 112 Puromycin Kill Curve ................................ ................................ ................ 112 Creation of Virus and Infection of Cells ................................ .................... 112 96 well cloning ................................ ................................ ......................... 113 10 cm dish cloning ................................ ................................ ................... 113 Nomenclature ................................ ................................ ........................... 114 CRISPR Genotyping Primer Design ................................ ............................... 114


11 Sequencing ................................ ................................ .............................. 114 Mycoplasma Decontamination and Detection ................................ ................ 114 ET 1 ELISA ................................ ................................ .............................. 115 Results ................................ ................................ ................................ .................. 115 Discussion ................................ ................................ ................................ ............ 118 6 CONCLUSIONS AND FUTURE DIRECTIONS ................................ .................... 136 Conclusions ................................ ................................ ................................ .......... 136 Future Direc tions ................................ ................................ ................................ .. 138 LIST OF REFERENCES ................................ ................................ ............................. 141 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 168


12 LIST OF TABLES Table page 2 1 Sequences and exon numbers. ................................ ................................ .......... 64 2 2 Primer sets for each gene. ................................ ................................ ................. 64 3 1 Baseline characteristics of WT and Per1 KO mice. ................................ ............ 89 3 2 Cosinor analysis of HS/DOCP effects on MAP in WT and Per1 KO mice. ......... 90 3 3 Analysis of MAP by JTK Cycle. ................................ ................................ .......... 90 3 4 Analysis of MAP by Cosinor Analysis. ................................ ................................ 90 4 1 Cosinor analysis of mean arterial BP on different diets. ................................ ... 104 4 2 Baseline Characteristics of WT and KO Mice. ................................ .................. 104 5 1 Cut size and expected amplicon size for all gRN A combinations. .................... 135


13 LIST OF FIGURES Figure page 1 1 Diagram of Two Nephrons and Representative Cell Types.. .............................. 48 1 2 Circadian Expression of Core Clock Genes in the Kidney ................................ .. 50 2 1 Pharmacological blockade of Per1 nuclear entry in vivo results in decreased NHE3 and SGLT1 ex pression, but not SGLT2 expression in the renal cortex ... 65 2 2 Per1 knockdown results in decreased NHE3 and SGLT1 expression, but not SGLT2 expression in HK 2 cells in vitro ................................ ............................. 65 2 3 Pharmacological blockade of Per1 nuclear entry results in decreased NHE3 and SGLT1 expression, but not SGLT2 expression in vitro ............................... 66 2 4 Pharmacological blockade of Per1 nuclear entry results in decreased nuclear Per1 in vitro ................................ ................................ ................................ ....... 67 2 5 Pharmacological blockade of Per1 nuclear entry results in decreased transcription of NHE3 and SGLT1 ................................ ................................ ...... 68 2 6 Pharmacological blockade of Per1 nuclear entry leads to decreased occupancy of CLOCK a nd Per1 on the NHE3 promoter in HK 2 cells ................ 69 2 7 Pharmacological blockade of Per1 nuclear entry leads to decreased occupancy of CLOCK and Per1 on the SGLT1 promoter in HK 2 cells .............. 70 2 8 Pharmacological blockade of Per1 nuclear entry results in decreased membra ne and intracellular protein expression of NHE3 and SGLT1 in HK 2 cells ................................ ................................ ................................ .................... 71 2 9 Na + /K + ATPase activity is reduced in HK 2 and HKC11 cells following pharmac ................................ ................................ .. 72 3 1 Differences in renal gene expression between WT and Per1 KO mi ce at noon and midnight on control diet. ................................ ................................ ............... 85 3 2 There are no significant differences in expression of Pcsk9 or ETA in WT or Per1 KO mice at noon and midnight ................................ ................................ .. 86 3 3 Inactive/active averages and 72 hour telemetry recordings of MAP in WT and Per1 KO mice on control diet ................................ ................................ .............. 86 3 4 Inactive/active averages and 72 hour telemetry recordings of MAP in WT and Per1 KO mice on HS/DOCP diet ................................ ................................ ........ 87 3 5 Per1 KO mice lose the inactive/active difference in MAP on HS/DOCP diet. \ ... 87


14 3 6 Paral lel changes in DBP, SBP and MAP ................................ ............................ 88 3 7 WT and Per1 KO mice maintain inactive/active activity patte rns on control and HS/DOCP diet ................................ ................................ ............................. 89 4 1 Circadian Rhythm of BP on Control Diet. ................................ ......................... 100 4 2 Mean Arterial Pressure Averaged Over Multiple Days of Diets. ....................... 100 4 3 Avera ge MAP for All Diets ................................ ................................ ................ 101 4 4 Changes in DBP and SBP on Control and HS/DOCP Diets ............................. 101 4 5 Real Time PCR Expression of Selected Genes in Kidney Cortex and Medulla 102 4 6 Additional Real Time PCR Expression of Selected Genes in Kidney Cortex and Medulla ................................ ................................ ................................ ...... 103 5 1 The UCSC genome browser predicted chromatin state of EDN1 in HUVEC (human umb ilical vein endothelial cells) ................................ ........................... 123 5 2 Transcription Factor Binding Sites at the EDN1 AS Predicted Promoter Region ................................ ................................ ................................ .............. 123 5 3 Diagram of strand specific primer placement ................................ ................... 124 5 4 Dexamethasone Treatment of HK 2 Cells and RNA Isolation .......................... 12 4 5 5 Rationalization for gRNA Desig n in Human EDN1 ................................ ........... 125 5 6 96 well Cloning Method for CRISPR/Cas9 Knockout C ells .............................. 126 5 7 EDN1 AS is expressed in several human cell lines and in human kidney. ....... 127 5 8 EDN1 AS expression is increased in the murine renal outer medulla .............. 128 5 9 EDN1 AS Expression in Mouse Aorta ................................ .............................. 129 5 10 EDN1 AS expression m ay exhibit sex dif ferences in murine renal cortex ........ 130 5 11 EDN1 AS expression is regulated by aldosterone in mmIMCD3 cells .............. 131 5 12 EDN1 AS is regulated by the circadian clock ................................ ................... 132 5 13 RNA polymerase II, GR and Per1 are present at the EDN1 AS promoter ........ 133 5 14 Representative Genotyping Gel for CRISPR/Cas9 Knockout ........................... 134 5 15 ET 1 ELISA of Media from CRISPR Cell Lines ................................ ................. 134


15 L IST OF ABBREVIATIONS 2 0 HETE 20 hydroxyeicosatetraenoic acid HSD 3 beta dehydrogenase isomerase ACE angiotensin converting enzyme ACTH adrenocorticotropic hormone ADH antidiuretic hormone Ang II Angiotensin II ANP atrial natriuretic peptide ARB Ang II receptor blocker BP BP Cav 1 Caveolin 1 ChIP chromatin immu noprecipitation casein kinase 1 isoform delta/epsilon CKD chronic kidney disease CLOCK circadian locomotor output cycles kaput Cry cryptochrome CVD cardiovascular disease DBP diastolic BP DCT DCT DNA deoxyribonucleic acid DOCP desoxycorticosterone p ivalate ECF extracelluar fluid ENaC ENaC eRNA enhancer derived RNA ET 1 Endothelin 1


16 GAPDH glyceraldehyde 3 phosphate dehydrogenase GFR glomerular filtration rate HK 2 huma n kidney PT cells HLF hepatic leukemia factor hnRNA heterogeneous nuclear RNA HPA h ypothalamic pituitary adrenal HR heart rate HS/DOCP high salt + DOCP m IMCD3 mouse inner medullary CD cells K potassium KO knockout lncRNA lon g non coding RNA MAP mean arterial pressure mpkCCDc14 mouse principal cortical CD cells Na sodium NCC sodium chlo ride cotransporter NHE3 sodium hydrogen exchanger isoform 3 NKCC2 sodium potassium 2 chloride co transporter Per period PT PT RAAS Renin Angiotensin Aldosterone System RNA ribonucleic acid ROR retinoid related orphan receptor SBP systolic BP SCN suprach iasmatic nucleus


17 SGLT1 sodium glucose transporter isoform 1 SHR spontaneously hypertensive rats TAL thick ascending Loop of Henle TEF thyrotroph embryonic factor Ube2e3 Ubiquitin Conjugating Enzyme E2E3 WNK with no lysine kinase WT WT ZT Zeitgeber t ime


18 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE ROLE OF PER1 AND EDN1 AS IN MINERALOCORTICOID AND SALT SENSITIV E HYPERTENSION By Kristen Solocinski December 2017 Chair: Michelle L. Gumz Major: Medical Sciences Biochemistry and Molecular Biology Cardiovascular disease is responsible for 1/3 of the total American deaths every year. Hypertension is one of the mai n risk factors for developing cardiovascular disease and thus is a large target for treatment to prevent cardiovascular disease as well as other associated morbidities such as chronic kidney disease and renal injury. While many treatments can be extremely effective for some patients, about half of the hypertensive population has uncontrolled hypertension with about 10% of the hypertensive population being resistant to treatment with multiple drugs including diuretics Even with the best medications we have, around 8 million Americans still do not have their blood pressure (BP) under control. Clearly, better medications are needed and to accomplish this, we need a better understanding of the progression of hypertension. The Gumz lab and others have clearly d emonstrated the importance of the circadian clock to BP as every clock gene knockout animal exhibits a BP phenotype. Period 1 (Per1) is one of four core clock proteins and coordinately regulates many aspects of renal sodium reabsorption. Per1 has been impl icated in the pathogenesis of hypertension, including salt sensitive hypertension. We have shown that male mice


19 lacking the Per1 gene display a harmful non dipping phenotype while on a high salt and mineralocorticoid diet. However, female Per1 knockout mic e on the same diet do not show any significant phenotype compared to WT (WT) mice, in line with reported sex disparities with respect to cardiovascular and kidney disease. Per1 regulates Endothelin 1 (ET 1) which is itself an important contributor to hyper tension as it is one of the most potent endogenous vasoconstrictors. Our lab has discovered a novel long non coding RNA (lncRNA) antisense to the Endothelin 1 gene (EDN1), named EDN1 AS. It appears to be regulated by the circadian clock, which suggests the possibility that both Per1 and EDN1 AS are novel, potential targets for the treatment of hypertension.


20 CHAPTER 1 INTRODUCTION Background Many aspects of behavior and physiology cycle over the course of ~24 hours, being sync hronized to the day/night cycle, and are accordingly called circadian (latin for Circadian rhythms in physiological function are critical to maintaining health and the biochemical pathways mediating thes e effects are highly conser ved and are present in species from archaebacteria to humans (reviewed in 2 ). The pacemaker of the circadian clock is located in the suprachiasmatic nucleus (SCN) of the brain and is entrained by light 3, 4 For this central clock, light is a dominant nal for entrainment. The central clock synchronizes the peripheral clocks, located in other areas of the brain and in tissues throughout the body, via neuronal and humoral signaling. While light is the dominant Zeitgeber for central clock entrainment, me tabolic cues likely act as an additional Zeitgeber for peripheral clocks such as those l ocated in the liver and kidneys. While the circadian clock is composed of many genes, the core molecular clock mechanism is comprised of four key circadian genes: Clo ck Bmal1 Period ( Per homologs 1, 2 and 3) and Cryptochrome ( Cry homologs 1 and 2). These genes encode proteins that function in a feedback loop to regulate transcription of clock controlled genes (for an excellent review on this mechanism, see 4 ). Briefly, circadian proteins CLOCK and BMAL1 form a heterodimer which binds to E box elements of clock _________________________ *Reprinted w ith Permission from: 1 Solocinski K, Gumz ML. The circadian clock in the regulation of renal rhythms. J Biol Rhythms 2015;30:470 486


21 controlled genes. This BMAL1/CLOCK heterodimer also binds to E box elements in the promoters of the Cry and Per genes t o activate transcrip tion, thus forming the positive arm of the feedback loop. In the negative arm of the loop, PER and CRY then act on BMAL1/CLOCK to inhibit their own transcription. Two nuclear receptors involved in the molecular clock regulatory loops ar e the retinoid related orphan receptor (ROR) and REV Bmal1 whereas REV action on Bmal1 Kinase and phosphatase mediated post translational modifications provide an additional layer of regulation for clock proteins. For example, nuclear entry of PER and CRY is regulated via phosphorylation by Casein 5, 6 The molecular circadian clock mediates the regulation of rhythmic physiological fu nction via transcriptional control and post transcriptional control of downstream clock target genes and this regulation occurs in a tissue specific manner. In an elegant genome wide study in the liver, Koike et al. demonstrated that the core circadian clo ck proteins interact with regulatory sequence elements in several thousand genes 7 Studies such as these are increasing our understanding of circadian clock function and shedding light on the tissue specific effect s of the clock. A recent landmark genomics study characterized circadian gene expression over a 48 hour period in wild type mice in a variety of tissues, including the kidney 8 This work demonstrated that more than 40% of all expressed genes in the tissues tested exhibited circadian variations in expression. In terms of the absolute numbers of cycling genes, the kidney was second only to the liver with several thousand genes exhibiting circadian expression.


22 In his 1985 Bowditch Lecture, Moore Ede discussed the concept of physiological homeostasis, or equilibrium 9 In the 19 th century, Claude Bernard popularize d the notion physiologists developed the concept of homeostasis: that the body makes constant changes to maintain a steady state. It was thought that the body reacted t o daily activities in order to maintain homeostasis. For instance, in response to sodium intake, the body would then react to excrete excess sodium from the body. In his lecture, Moore Ede talked about predictive homeostasis in which the body starts to ena ct changes to counter activities it can anticipate because they occur in a predictable, cyclical manner. Thus, if meals are frequently eaten at the same time each day, the body can start to prepare for this predicted meal before it occurs by inducing expre ssion of necessary solute transporters and metabolic enzymes in the liver, gut and kidney, for example. Discovery of the molecular clock components provided evidence that this predictive homeostasis occurs due to the activity of the circadian clock; the cl ock allows the body to keep track of the time using multiple cues such as light and food intake. While laying down at night suppresses urinary sodium, potassium and water excretion, excretion is not suppressed by laying down during the day 10 The body predicts we are going to bed at night and prevents loss of precious nutrients. The kidney provides a textbook illustration of predictive homeostasis. Here, we review the evidence for circadian control of rhythms in renal function and present the current state of knowledge regarding the regulation and function of the clock in the kidney.


23 The Kidney The kidneys function to filter the blood, maintaining fluid and ion homeostasis 11 This is crucial and tightly regulated because even slight changes in filtrate reabsorption can lead to large changes i n blood chemistry and blood pressure (BP) Sustained hypertension (high BP ) can lead to a number of diseases and is a main risk factor for chronic kidney disease (CKD) and cardiovasc ular disease, the main cause of about 1/3 of American deaths ever year 12 The kidney achieves this fine level of regulation via the many specialized cell types located along the segmented nephron, the functional unit of the kidney (Figure 1 1). An adult kidney contains about 1 million ne phrons on average and each one is tightly controlled to avoid large fluctuations in BP Blood enters every nephron and is filtered through the glomerulus. The filtrate flows through the nephron and the many specialized cell types in each segment of the nep hron reabsorb or secrete solutes according to the needs of the body. The final filtrate flows into the ureter to eventually become the urine. The kidney carefully maintains homeostasis for sodium, potassium and several other key solutes in addition to mai ntaining acid/base balance. Each segment of the nephron has specific channels and transporters dedicated to the transport of sodium, potassium, hydrogen and bicarbonate, in addition to other important ions and solutes. Each is regulated differently, allowi ng for fine tuned control of each segment in order to maintain homeostasis. Because much of the work on the circadian clock in the kidney has focused on sodium balance and effects on BP this review will focus on regulation of renal sodium handling by the molecular clock. After passing through the glomerulus, the filtrate from the blood enters the proximal tubule (PT) where about 60% of sodium reabsorption takes place (reviewed


24 in 11 ). Cells in this segment of the nephron express an apical (facing the lumen of the tubule) sodium hydrogen exchanger (NHE3) and sodium glucose transporter (SGLT1); expression of these transporters appears to be regulated by the circadian clock 13 (CircaDB 14 Filtrate then moves to the Loop of Henle where around 25% of sodium reabsorption occurs in the thick ascending portion (TAL). In this segment, the clock appears to regulate expression of the sodium potassium 2 chloride co transporter (NKCC2) and the estrogen related receptor be (CircaDB 14 ) 15 The next segment, the distal convoluted tubule (DCT), accounts for about 10% of sodium reabsorption and this occurs mainly through the sodium chloride co transporter (NCC). NCC is regulated by a kinase cascade involving members of the with no lysine (WNK) family. Increasing evidence suggests that NCC and the WNK pathway are subject to regulation by the circadian clock 16, 17 The final segment the filt rate enters is the collecting duct (CD) where only about 5% of sodium reabsorption occurs. However, the distal nephron is the most highly regulated segment of the nephron. In addition to transporters and exchangers, ion channels are also integral to sodium reabsorption. In principal cells of the CD, sodium entry occurs through the apical epithelial sodium channel regulators of sodium transport in this segment have all been linked to the clock 18 20 five human subjects in which activity, light, temperature, diet and other variables which may affect circadian rhythms were carefully con trolled and monitored. Rhythms in urine flow and pH were observed in addition to sodium (Na + ) and potassium output (K + ), bo th peaking shortly after waking 21 In 1975, the circadian rhythm of potassium excretion


25 was observed in three ma les who maintained a constant supine position and subsisted on a liquid diet administered every three hours 22 These studies provided the first evidence for a kidney specific clock. Subsequent work continued to demonstrate that renal function appears to vary with a circadian rhythm. For example, ma ny aspects of kidney function exhibit circadian fluctuations, such as glomerular filtration rate (GFR) 23 sodium excretion and renal blood flow 24 At a tra nscriptional level, expression of clock genes including Clock Bmal1 Cry1 Cry2 Per1 and Per2 oscillate with a ~24 hour rhythm 25 Urinar y sodium excretion exhibits a circadian pattern in rodents, primates and humans (reviewed in 26 ) and this may be explained by the clock mediated regulation of a number of renal sodium transport genes 13, 18, 20 As illustrated in Figure 1 1 circadian clock mediated regulation of a several key transport genes in the kidney has been established. In the following sections, the evidence for this regulation is considered in detail. Hormones and the Kidney Clock The kidney is both an important ta rget and a source of hormones critical for maintaining ion homeostasis and BP control. The circadian rhythm of plasma sodium is in phase with many hormones involved in BP homeostasis including angiotensin II, aldosterone 27, 28 and vasopressin 29 as well as angiotensin converting enzyme (ACE) 30 and plasma renin activity 27, 28 detailed below. Thus, proper regulation of these hormones is critical for BP homeostasis. All forms of Mendelian (inherited) hypertension are due to a defect in the Renin Angiotensin Aldosterone System (RAAS) 31 The RAAS acts to maintain BP homeostasis. When low BP is sensed, juxtaglomerular cells in the nephron release the enzyme renin. Renin then converts angiotens inogen to angiotensin I. Angiotensin I then


26 is further converted to Angiotensin II (Ang II) by ACE. Ang II acts on the vessels as a vasoconstrictor but can also stimulate the adrenal glands to secrete aldosterone. Aldosterone further stimulates sodium tran sporters in the aldosterone sensitive distal nephron, leading to an increase in sodium reabsorption and BP An intrarenal RAAS exists in addition to the well characterized systemic RAAS 32 34 further solidifying a role for the in the kidneys in the development of high BP In a human study, Ang II levels were examined from 15 patients around 14 years of age who had monosymptomatic nocturnal enuresis (MNE) and 10 normal patients of a similar age 35 They found that within the MNE group, Ang II was expressed in a time dependent manner with nighttime levels being more than twice as high as daytime levels in patients without polyuria (exces s urination). The MNE group with polyuria did not show any significant variation in Ang II levels between day and night, suggesting that disrupted circadian rhythms of Ang II may play a part in the pathogenesis of MNE. The peptide hormone atrial natriureti c peptide (ANP) is made in the heart and is a vasodilator. Its actions on the kidney can lead to water and electrolyte excretion which, combined with vasodilatory properties, lead to an overall decrease in BP Reports of daily, rhythmic changes in levels o f this peptide in humans are conflicting and often have small subject numbers. In one study, ANP levels in humans (10 normotensive patients and 10 hypertensive patients), display a rhythm that appears to be antiphase to BP and HR rhythms 36 Furthermore, in normotensive subjects, there was a correlation between ANP, renin and aldosterone levels while this was not observed in the hypertensive group. In another study of 12 males, the authors state no rhythm of ANP but a 15% increase at night 37 In 21 healthy, middle aged men, ANP has


27 a significant rhythmic expression, with the peak of expression occurring at night 38 These results are similar to those obtained for 20 year old men and women 39 While it is definitely possible that daily variation of ANP can vary between humans and other animals, more studies would be helpful to con firm these findings. Urodilatin is encoded by the same gene as ANP but has four extra amino acids on its N terminus 40 Unlike ANP, urodilatin is only secreted in the kidney and is not secreted into the sys temic circulation. It causes inhibition of sodium and water reabsorption when high BP is sensed. In 1991, ten years after ANP was discovered, Drummer et al. described the time dependent rhythm of urodilatin 41 In six healthy men, a rhythm of urodilatin was shown with a maximal expression during the day. However, in this study, a sign ificant rhythm for ANP immunoreactivity was not observed. Dopamine is released from nerves in the kidney and it is synthesized by cells in the PT (reviewed in 42 ). In the kidney, dopamine inhibits sodium and water reabsorption when extracelluar fluid (ECF) volume is increased. Under constant light and under nor mal 12:12 LD, there were time dependent changes in dopamine expression in the striatum and nucleus accumbens of rats 43 There was no circadian rhythm of dopamine expression under constant darkness, however, demonst ration that variations in dopamine levels are likely dependent on time cues. While the circadian expression of dopamine in the kidney has not yet been investigated, it does appear to have a circadian rhythm in mouse adrenal glands and skeletal muscle (Circ aDB 14 ). It would be interesting to investigate the expression of dopa mine in the kidney as well, given that it regulates water and sodium handling and in turn, BP


28 Vasopressin, also called antidiuretic hormone (ADH), is released when there is a decrease in extracellular fluid volume. It causes an increase in water reabsorpt ion in the CD thus decreasing diuresis as the name implies. Challet et al. looked at the rhythm of vasopressin in mice lacking folate, vitamin B 9 44 It has previously been shown that folate may be a cofactor for cryptochromes 45 It has also been shown that rats lacking folate have decreased amplitude of melatonin secretion 46 which is important for maintaining the sleep wake cycle. This group found that their folate deficient mice had decreased rhythms of vasopressin expression compared to normal mice 44 The authors further point out that folate reduction is com monly seen with aging. Decrease or loss of circadian rhythms has also been associated with increasing age 47 although whether it is a cause or result of the aging process is unclear. It also leads to the possibility that these disruptions in circadian rhythms may be implicated in patho genesis of many diseases associated with aging. The kidney is mainly responsible for excretion of cortisol and its metabolites and this process is impaired in chronic kidney disease patients 48 and reviewed in 49 In healthy subjects, aldosterone and cortisol exhibit time dependent increases in the early morning. In anephric patients, the correlation between plasma aldosterone and plasma cort isol is lost: daily variation in plasma aldosterone is not present whereas plasma cortisol rhythms remain 50 Rhythmic changes in urinary cortisol and aldosterone excretion are disrupted in kidney transp lant patients as well 51 Renal hand ling of cortisol and aldosterone may affect adrenal hormone production through disruption of known negative feedback loops. For example, synthesis of the pituitary hormone adrenocorticotropic hormone (ACTH), which stimulates cortisol production by the


29 adre nals, is inhibited by high levels of cortisol in the circulation. Thus, decreased renal function, or perhaps, disruption of the kidney clock, negatively affects cortisol handling and may subsequently alter glucocorticoid production inappropriately. Althoug h central and peripheral circadian clocks certainly contribute to regulation of the hypothalamic pituitary adrenal (HPA) axis 52, 53 the role of the molecular kidney clock in these processes remains poorly understoo d. The Kidney Clock The core circadian clock proteins regulate almost half of all expressed genes and do so in a tissue specific manner 8 As illustrated in Figure 1 2, Bmal1 Clock Per1 and Cry2 exhibit clear cir cadian variation in expression in the whole kidney over a 48 hr period (CircaDB 14 ). Rodent models have proven invaluable to our understanding of the mechanism of the renal circadian clock and its impact on BP regulation. An animal model of CKD can be produced by removing one entire kidney and 2/3 of the other kidney, removi ng about 5/6 of the total kidney mass and hence providing the name for this model, the 5/6 nephrectomy. In mice, this causes an increase in MAP of about 30 mmHg two weeks after the procedure 54 In rats, this caused peak Bmal1 expression in the remnant kidney to shift 4 hours earlier than sham operated rats 55 Using immunohistochemistry to determine localization of protein, D site binding protein ( DBP ) localization changed fr om mainly glomerular in the sham controls to much more widely dispersed in the CKD model rats. PER2 protein expression was high in the corticomedullary junction in controls and decreased in CKD rats. Conversely, expression was highly increased in the corte x of CKD rats compared to controls. Thus, not only can circadian proteins mediate changes in BP changes in BP can affect localization of circadian proteins in the kidney.


30 The link between the kidney and maintenance of normal BP was further illustrated usi ng spontaneously hypertensive rats (SHRs). Kidneys transplanted from normotensive rats into SHRs lead to a decrease in mean arterial pressure of over 50 mmHg compared to sham operated controls 56 Conversely, when kidneys from SHRs were transplanted into normotensive rats, the MAP increased by around 40 mmHg. The fact that a hypertensive phenotype tracks with the kidney to such a large degree highlights the magnitude of the renal contribution to BP homeostasis. Whil e the SHRs display daily rhythms in plasma sodium concentration 57 closer investigation into the contribution of the kidney clock to this phenotype remains to be done. Animal models with disruption of circadian gen es of interest also provide evidence for the roles of specific clock genes. However, it is important to keep in mind that most of these models have disruption of clock genes systemically, so while the phenotypes observed certainly support heavy renal invol vement, participation from other systems cannot be ruled out. Global knockout (KO) of Bmal1 results in complete loss of circadian rhythmicity measured via wheel running activity 58 These mice are also sterile 59 and have shorter lifespans than wild type (WT) mice 60 They have a loss of circadian rhythmici ty in HR and BP and are also hypotensive compared to the WT mice 61 Whether a renal phenotype exists in global Bmal1 KO mice has yet to be determined. The circadian proteins are important for BP control through m echanisms involving multiple tissues. Loss of Bmal1 specifically in the forebrain leads to mice with a loss of a circadian pattern of activity in constant light or constant dark conditions but


31 normal rhythms in LD (12 hours light, 12 hours dark) and otherw ise healthy, fertile mice 62 In the first cell type specific KO of a clock gene in the kidney, Firsov and colleagues generated mice lack ing BMAL1 in renin producing cells 63 Expression of BMAL1 was lost in cells of the juxtaglomerular apparatus (see Figure 1F) and in some cells of the CD. These kidney specific Bmal1 KO mice have decreased plasma al dosterone compared to control mice and also have significantly lower BP compared to controls. Thus, the loss of BMAL1 only in certain cells of the kidney was profound enough to cause systemic changes in aldosterone and BP Clock KO mice are hypotensive as well, yet they retain the normal 24 hour rhythmic variation in BP 18 Expression of 20 HETE (20 hydroxyeicosatetraenoic acid), which is a regulator of BP is also altered in these mice 64 If acting on preglomerular arterioles in the kidney, 20 HETE leads to an increase in BP through constriction of the arterioles 64 However, 20 HETE also inhibits some of the sodium transporters foun d in the PT and TAL of the nephron which leads to less sodium reabsorption and a consequent decrease in BP Therefore, the dysregulation of 20 HETE has been proposed as a contributory mechanism to the hypotensive phenotype shown in Clock KO mice. While the se mice have similar mean levels of plasma aldosterone compared to WTs, they have significantly lower levels at ZT12 64 which stands for Zeitgeber Time 12 12 hours after the lights have been turned on and on a 12 :12 LD cycle, the onset of darkness. Interestingly, ZT12 is the time that Per1 mRNA expression peaks in mouse kidney (CircaDB 14 ).


32 In 2005, the first evidence for circadian control of a renal gene was provided by Okamura and colleagues 13 They showed that NHE3 (encoded by the gene Sl c9a3 ) mRNA expression is circadian and furthermore is regulated by CLOCK:BMAL1 heterodimer binding to an E box element on Slc9a3. The circadian expression of NHE3 mRNA is severely blunted in Cry1/2 KO mice 13 furth er supporting the circadian control of this gene. These mice also exhibit salt sensitive hypertension due to increased levels of aldosterone 65 Sglt1 (encoded by Slc5a1 ) and its expression is also circadian: SGLT1 mRNA expression peaks around ZT12 in the mouse kidney while NHE3 mRNA expression peaks about two hours earlier (data derived from CircaDB, see 14 ). Dbp/Hlf/Tef triple KO mice lack three circadian clock regulated genes: DBP, hepatic leukemia factor (HLF), and thyrotroph embryonic factor (TEF) 66 Mice lacking one or two of these factors have mild phenotypes which may be due to the fact that DBP, TEF and HLF have well conserved amino acid sequences and thus they likely compensate for each other in the single or double KO mice. Mice lac king all three usually do not live longer than 1 year, with symptoms such as seizures occurring during the first 3 months of life 67 In the kidney of the triple KO mice, Northern blot analysis revealed significantl y less mRNA from potential target genes involved in detoxification and drug metabolism. This may be related to the mechanism of variable drug effectiveness depending on the time of administration. Later, this group showed that the triple KOs had low BP de creased aldosterone levels and cardiac hypertrophy 68 Another useful model of circadian gene disruption is the PER2 mutant mouse 69 The blunted PER2 protein expressed in t hese mice is missing dimerization sites (PAS B and PAC domains) 70 These domains have been implicated in the ability of PER2 to


33 interact with other proteins and possibly act as a light sensor through FAD (Flavin Ad enine Dinucleotide) binding 71, 72 Indeed, mice with mutant Per2 (and also Per1 ) genes have altered responses to light 73 This group found that the mutant mice have higher heart rate during the light period than WT mice but have similar locomotor activity. In normal LD conditions, PER2 mutant mice had circadian periods of mean arterial pressure (MAP), heart rate (HR) and activity similar to WT mice. However, in the absence of light (DD), the mutant mice had shorter periods with a trend of decreased amplitude of MAP, HR and activity compared to WT controls. The mutant mice also were unable to maintain day night differences in MAP, HR and activity when put into constant dark conditions for 8 days, but showed im proved differences when returned to LD. In addition to CRY and PER, DEC1 and DEC2 are also involved in the negative arm of the transcriptional circadian feedback loops 74 Gene expression of both Dec1 and Dec2 exhib its a circadian pattern of expression in the rat heart while only Dec2 shows a comparable rhythm in the kidney 75 Since rats are nocturnal, they normally eat at night. When food intake was restricted to the light c ycle/rest phase for seven days, Dec1 and Dec2 mRNA expression in the heart was shifted by about 8 hours. In the kidney, however, Dec1 expression became rhythmic and the peak of Dec2 expression was shifted 4 hours earlier than controls. The authors also rev ersed both feeding time and the LD schedule. After 7 days, the peak of Dec1 expression was shifted only in the heart while the peak of Dec2 expression was shifted in both the heart and kidney. This provides evidence that the timing of food cues can actuall y induce rhythmic expression of clock controlled genes in the rat kidney.


34 Contrary to the rats from the previous experiments, Dec1 exhibits a circadian rhythm of expression in the mouse kidney and liver, and this rhythm is disrupted in mice expressing a mu tant CLOCK protein resulting from deletion of exon 19 76 In the kidney, mutant CLOCK expression was associated with decreased total Dec1 expression. However, rhythmic expression of Dec1 mRNA was not affected in LD or DD, suggesting that loss of WT CLOCK expression was not sufficient to alter circadian expression. Loss of DEC1 does not appear to negatively affect mice compared to WT controls 77 Since DEC1 and DEC2 have simila r DNA binding domains, it seems that DEC1 may be redundant in the kidney with DEC2 able to compensate for loss of DEC1. This possible redundancy suggests the importance of maintaining the circadian clockwork in the kidney. PER1 A ction in the K idney Due to the important role that ENaC plays in maintaining sodium balance, it is tightly regulated at multiple levels. PER1 has been a focus of investigation in the kidney because it is a direct target gene of the sodium and BP regulating hormone aldosterone 78 which regulates ENaC via multiple mechanisms 79 Aldosterone mediated transcriptional regulation of the Scnn1a mineralocorticoid receptor (MR). PER1 appears to be involved in this regulation 80 In one of the first reports to link the molecular clock to renal sodium handling, Gumz and colleagues demonstrated that knockdown of PER1 in several renal CD cell lines resulted in decreased expression of 20 Furthermore, Per1 KO mice exhibited increased urinary sodium excretion, again supporting a role for the molecular clock in the regulation of renal sodium handling. In order for PER1 to get into the nucleu s and


35 (PF670462) prevents PER1 nuclear entry in a murine cell line model of the CCD, mpkCCD c14 cells 81 If PER1 is unable to enter th e nucleus, it is therefore unable to associate with E kidney 19 Per1 levels were decreased using siRNA in mpkCCD c14 cells which led to a decrease in mRNA expression of Fxyd5. Conversely, Ubiquitin Conjugating Enzyme E2E3 (Ube2e3), Caveolin 1 (Cav 1) and Endothelin 1 (ET 1, encoded by Edn1 ) expression increased following PER1 knockdown. FXYD5 inc reases activity of the Na, K ATPase which is responsible for pumping sodium reabsorbed from the filtrate back in to the blood 82 UBE2E3 is an E3 ubiquitin ligase, an enzyme which adds ubiquitin molecules to proteins, a common signal for proteasomal degradation. When UBE2E3 ubiquitinyl ates ENaC, it is removed from the membrane, thus decreasing sodium reabsorption 83 Reduction of PER1 levels lead to increased expression of this ligase, potentially leading to increased ENaC degradation. CAV 1 is a lipid raft protein associated with removal of ENaC from the membrane as well 84 leading to decreases in sodium reabsorption like UBE2E3. Finally, ET 1 leads to decreased ENaC open probability through a mechanism involving nitric oxide and the Endothelin type B (ET B ) receptor 85, 86 Following PER1 knockdown, ET 1 expression increased almost four fold 19 PER1 knockdown lead to


36 decre increased amount of ET 1, CAV 1 and UBE2E3 81 Per1 KO mice on a 129/sv background exhibit BP s about 18 mmHg lower than WT mice but maintai n normal BP rhythms 19 Although the extra renal contributions to this phenotype have not been evaluated, many pieces of evidence support role of the renal clock. Per1 aC mRNA in the medulla compared to WT mice 20 These mice exhibit increased levels of ET 1 in the cortex and medulla of the kidney. While known as a potent vasoconstrictor in the majority of the body, ET 1 acts to d ecrease BP and increase natriuresis (sodium excretion) in the kidney through inhibition of ENaC 85, 87 Furthermore, ET 1 peptide levels are increased in ex vivo IMCD cells from Per1 heterozygous mice (which have app roximately 50% less PER1 expression than WT mice) 88 Increased ENaC inhibition by ET 1 coupled with de creased basal ENaC levels may contribute to the low BP phenotype observed in the global Per1 KO mice. In healthy humans, plasma ET 1 levels decrease at night when BP dips, whereas patients with CKD have increased night time plasma ET 1 levels that correlat e with loss of night time BP dipping 89 Normally, people experience a 10 20% decrease in BP non dippers. Non dipping has been linked to increased risk of not only cardiovasc ular events 90 but also chronic kidney disease 91, 92 .Together these results suggest that ET 1 may contribute to the circadian profile of BP Per1 heterozygous mice also hav e decreased levels of plasma aldosterone relative to WT 129/sv mice 93 Aldosterone regulation of ENaC expression is mediated at least in part by PER1. Treating mpkCCD c14 cells with aldosterone leads to increased


37 interaction of PER1 with E 80 Increased binding of RNA POLII to the ENaC prom oter in the presence of aldosterone wa s observed as well, further validated by the observation that PER1 knockdown in the presence of ldosterone alone 20 Similar results were observed following nuclear blockade of PER1 using a 81 In addition to PER1 regulation by aldosterone, it seems t hat aldosterone may be regulated by PER1. In addition to lower plasma aldosterone levels, Per1 heterozygous mice do not exhibit the normal increase in plasma aldosterone during their active phase that occurs in WT mice 94 One explanation for this phenotype may be that Per1 HSD (3 hydroxysteroid dehydrogenase) HSD is an enzyme produced in zona glomerulosa cells of the adrenal glands, which are responsible for the production of aldosterone, among other hormones. This enzyme catalyzes the synthesis of progesterone, which is a precursor to aldosterone in the steroid hormone biosynthesis pathway 95 The time dependent HSD during the active phase observed in WT mice i s blunted in Per1 heterozygous mice. PER1 knockdown in a human adrenal cell line (NCI H295R) causes HSD mRNA levels 96 A similar result was obtained in vivo in the adrenal gland in WT 129/sv mi Cry1/2 HSD which correlates with the observed increase in their plasma aldosterone levels 65 These mice exhibit salt sensitive hyp ertension. Interestingly, this phenotype is nearly opposite that of Per1 KO


38 mice which exhibit reduced BP and that of Per1 heterozygous mice which have HSD 19, 94 Evidence s uggests that PER1 and 94 Decreased expression of PER1 expression in vitro and in vivo in the liver and the kidney was associated with an increase in CRY2 protein levels. This is further supported by the observation that increases cytosolic and nuclear CRY2 expression 96 Further supporting opposing roles of CRY2 and PER1, mice lacking both Per1 and Cry2 have a normalization of their free running period compared to mice lacking Per1 or Cry2 alone 97 The thiazide sensitive NCC is also regulated by PER1 16 Either PER1 knockdown or pharmacological blockade of nuclear entry in a model of the DCT (mDCT15 cells) resulted in decreased NCC expre ssion. Furthermore, nuclear blockade of PER1 via levels exhibited time of day dependent changes with higher expression during the mouse active period. Consistent with the in vitro data from mDCT15 cells, NCC expression levels were also decreased in the cortex of Per1 heterozygous mice expression compared to vehicle treated controls. These data s uggest that the circadian clock is critical for regulation of many important aspects of renal sodium reabsorption. Development The fetal SCN is synchronized by the mother and after birth, maternal care helps to keep the clock entrained 98 Eventually, the neonatal SCN matures enough to take over its job as the central clock 98 To investigate developmental changes in the


39 circadian clock in several tissues, transgenic rats expr essing a PER2 LUCIFERASE fusion protein were utilized 99 These rats are transgenic for the mouse Per2 promoter fused to a destabilized luciferase (luciferase fused with a modified PEST sequence 100 ) reporter gene ( Per2 dLuc ) 101 This allows Per2 promoter activity to be visualized by measuring luminescence. Significant differences were shown in Per2 dLuc activity between developmental stages E20 (embryonic day 20), P5 (postnatal day 5), P19 and adult rats. Per2 dLuc activity varied in total luminescence units as well as amplitude of change over time between the different developmental stages in the SCN, lung, kidney and liver in cultur e. In addition, other tissues were studied and similar tissue to tissue variability in Per2 dLuc expression was found. The peak phase of Per2 dLuc advanced after birth to adulthood by about 12 hours in the kidney and lung but not in the liver and only slig htly in the SCN between E20 and adult. The SCN was one of the tissues in which Per2 dLuc activity did not vary or shift as much during development even though other tissues displayed significant changes, supporting the idea that peripheral clocks are not solely entrained by the SCN. One reason for the differential entrainment of the SCN and peripheral clocks could be due to the fact that between 2 and 3 weeks after birth, pups switch from drinking milk to eating chow 99 The different nutrient composition may give different cues to the pups regarding entrainment. Another explanation is that pups usually feed while mothers rest, so they are eating during the day, or what should be their inactive period. The switch fr om milk to chow would be accompanied by a reversal of feeding time, resynchronizing the peripheral clocks. The different expression of PER2 may also be because organs are not fully mature at birth. The function changes as the pups age


40 and this may also acc ount for the different expression of PER2 due to changing developmental needs 102 In order to investigate embryonic stages of development more thoroughly, gene expression from embryonic days E10 E19 was evaluated in PER2::LUC mice 103 Similar to the Per2 dLuc rats, these mice express a PER2 LUCIFERASE fusion protein. These mice are on a C57BL/6J background and have the luciferase gene added in frame to the end of the Per2 gene 104 Real time PCR measurements of clock gene expression in the liver showed little to no circadian rhythms during this time even though maternal rhythms were robust. However, if these tissues were excised and cultured in vitro large circadian rhythms in PER2::LUC protein were observed. Although this could be an artifact o f cell culture, these results do not rule out the possibility that individual cells have rhythms and they are just not yet synchronized at embryonic stages. Circadian variation during development of other clock controlled genes and kidney specific clock co ntrolled genes was studied in the kidneys of Sprague Dawley rats. While small amplitude rhythms of expression of Bmal1 Rev Per2 and Cry1 mRNA were seen at E20, the rhythms became more robust with increasing peak to trough differences from 1 week, 4 weeks and 12 weeks after birth 105 By 12 weeks after birth, p eak expression levels of Bmal1 Per2 and Cry1 were observed during the dark period while peak expression of Rev occurred during the day. Kidney specific clock controlled genes Scnn1a Sgk1 (serum and glucocorticoid inducible kinase 1), Slc9a3 (NHE3) and Avpr2 (arginine vasopressin receptor 2) showed some rhythmic expression at E20 and these rhythms became more robust one week after delivery in addition to shifting the peak of expression for all four genes by about 12


41 hours. One week after birt h, the peak expression levels of these genes occurred during the day but this again shifted three weeks later and peak expression times shifted closer between ZT3 7, t he time of their highest feeding behavior, for one week after birth. This lead to a 12 hour shift in Bmal1 expression and caused most other genes that were studied to lose their rhythm of expression, possibly due to conflicting light and feeding cues. Regu lation of the Kidney C lock After food deprivation for one day and in total darkness, male Wistar rats were allowed access to food for 30 minutes. This feeding stimulus lead to a four hour shift in peak expression of a classic circadian gene, Dbp (D site al bumin binding protein), in the heart but had no effect on expression in the kidney 106 ). The same study also showed the 30 minute feeding stimulus decreased levels of expression of Bmal1 Cry1 Dbp Per1 and Per2 in the heart while only Per1 mRNA was significantly decreased in the kidney. Feeding time also seems to be able to synchronize some peripheral clocks in the absence of a central clock. Bmal1 forebrain KO mice crossed with PER2::LUC mice had no circadian rhy thm of PER2::LUC in the kidney in constant darkness but time restricted feeding restored this rhythm in the kidney as well as in the liver 62 This is further supported by the finding that feeding mice only during the day (when they would normally be inactive) shifted the peak of DBP protein levels by 12 hours in the kidney, liver and heart 107 Rever sed feeding shifted PER1 and PER2 levels in the liver by 12 hours while there was no effect on either of these proteins in the SCN.


42 In addition to the time of feeding, specific composition of diets may affect entrainment as well. The type of diet can alte r gene expression in order to adapt to changing requirements 108 Specifically, a high salt (HS) d iet causes increased Dbp mRNA expression while decreasing Bmal1 expression in the liver and kidney 109 HS diet also advanced the time of peak expression of enzymes involved in glycogen synthesis ( Gys2 ), gluconeogen esis ( G6pc ) and the synthesis of fatty acids, cholesterol and bile acid ( Fasn, Hmgcr and Cyp7a1 respectively). Even though HS diet affected many genes important to metabolism, activity, feeding and drinking behavior were not affected. Timing of food intak e and food content both appear to be important cues with respect to the entrainment of peripheral clocks. Although it is clear that food is a dominant Zeitgeber for peripheral clock entrainment, the dominant cue for the central clock is light. The effects of reversing the LD cycle, restricting time of feeding or changing both the light cycle and feeding times were investigated specifically in the kidney 110 LD reversal did not alter the circadian pattern of express ion of Bmal1 Cry1 Clock or Per2 mRNA but did delay the peak expression of Per1 by 4 hours. In addition, the amplitude of expression of Per1 Cry1 Clock and Bmal1 were altered only with the reversal of the LD cycle. Shifting feeding time by 12 hours caus ed 8 12 hour shifts in the peak expression of Clock Cry1 and Bmal1 after 7 days while causing 4 hour shifts in Per1 and Per2 After 7 days of LD and feeding time reversal, a total inversion of expression of all 5 genes occurred. Thus, separately, light an d food cues partially entrain the circadian clock while both synchronize the clock throughout the whole body. Together these results suggest that food cues are a critical synchronizing signal for the kidney clock.


43 Transgenic PER2::LUC mice were further us ed to study the effects of temperature on time dependent entrainment 111 These mice were placed in w ater baths at 35C, 37C and 41C. The treatment at 41C led to phase advancement by about four hours in the kidney and liver after 1 2 days but peak PER2::LUC expression started to return to normal after repeated treatments for more than two days. After a 2 hour treatments at 41C, PER2::LUC expression levels were also increased in the kidney and liver. Thus, temperature appears to be another important cue for peripheral clock entrainment. The circadian clock is classically considered to be temperature co mpensated, meaning that the free running period, associated with translation and degradation of clock proteins, does not change much with changes in temperature. This is an intriguing piece of the circadian clock puzzle as the rate of enzymatic reactions s hould increase with temperature 112 However, other experi ments in PER2::LUC mice suggest that the SCN is not affected by temperature while peripheral clocks are more susceptible to temperature induced changes in PER2::LUC expression and this may be mediated by the heat shock pathway 113 In a more recent study, this same mouse model was used to investigate the effect of sleep deprivation on PER2. Sleep deprivation for 6 hours led to increased PER2::LUC levels in the brain, liver and kidney 114 Thus, even relatively short disruptions in the sleep pattern can lead to disrupted circadian rhy thms of PER2::LUC protein levels in peripheral tissues including the kidney. Potassium Potassium is another urinary electrolyte that has been shown to be regulated by the circadian clock and potassium excretion oscillates with sodium in multiple species


44 (r eviewed in 115, 116 ). In the first study of its kind, Firsov and colleagues performed microarray analysis to characterize circadian gene expression in the kidney, revealing that the expression of many renal genes ex hibited time dependent changes in expression 18, 64 including many genes involved in potassium handling (reviewed in 115 ). Also like sodium, potassium plays an important role in BP In addition to disrupted patterns of potassium excretion in diseases such as CKD, potassium supplementation is beneficial in the treatment of hypertension 117, 118 It has been shown t hat potassium supplementation leads to increased sodium excretion and therefore decreased BP In addition to decreasing BP potassium may also be beneficial in restoring a night time dip in BP thus reducing the risk of cardiovascular events. Crambert and colleagues investigated the mechanism of regulation of potassium reabsorption using WT mice, Clock gastric H,K Atp12a ) 119 significantly higher amounts in the luminal membrane at the end of the rest period while there were no sufficient differences in Clock more potassium compared to WTs during the rest pe riod but they had no differences in mRNA expression peaked about 9 hours later than controls and amplitude of expression increased by 45%. This diet also affected expression of Nrf2 a transcription factor wh mRNA mRNA expression by about 4 hours. On a low potassium diet, peak Nrf2 expression was delayed by about 10 hours, mRNA.


45 specific to the TAL which regulates NKCC2 15 mRNA expression is circadian in male CD1 mice with expression peaking at ZT4. Furthermore, this rhythm is lost in mice without Clock Na/K urinary excretion ratio and decreased NKCC2 mRNA expression and activity, sugge sting a positive regulation of NKCC2. NKCC2 expression is also circadian in mouse kidney, with a peak early in the light period (CircaDB 14 ), suggesting that it is under transcriptional regulation by the molecular clock. In addition to complex regulation of sodium balance in the kidney, the circadian clock is also integral t o maintenance of potassium levels. Clinical Implications Many widely used treatments for hypertension such as ACE inhibitors and Ang II receptor blockers (ARBs) aim to inhibit the RAAS. Due to their demonstrated regulation of renal sodium transporters and BP clock genes may represent novel targets for hypertension treatment. Another potential hypertension treatment target is the elevated HSD observed in the Cry1/2 KO mice that exhibit salt sensitive hypertension. In addition to targeting clock genes to tr eat disease, another strategy is to work with the clock. The aim of chronotherapy is to administer medicines at certain times of the day to maximize efficacy and reduce side effects. Many hypertension medications are usually taken in the morning, however c onvincing evidence concerning the effects of chronotherapy shows the benefit of taking at least one hypertension medication at night (for an excellent review, see 120 ). The MAPEC study followed over 2000 hypertensive patients over the course of a median of 5.6 years 91 The study divided the volunteers into two groups, one taking all BP medications u pon waking and the other group taking at least one medication at night. The night time dosing group showed significant


46 decreases in nocturnal BP and an increase in dipping prevalence of almost 30% compared to the morning dosing group. At the follow up of the study, the night time dosing group had a lower risk of cardiovascular events. They also had a significantly lowered morbidity and mortality due to cardiovascular disease. Similar to timing the treatment of hypertension, timing of dialysis treatment was also shown to be important with respect to the overall well being of patients. People undergoing dialysis have been observed to lose a normal night time peak of melatonin production 121 This hormone is important to the maintenance of the circadian cycle of sleep 122 Normally, melatonin shows a circadian rhythm of expression with high production at nigh t that is suppressed by light. This helps to keep the body in sync with day and night. The loss of this rhythm would imply a dysregulation of the sleep cycle. It has been shown that patients undergoing dialysis during the day experience sleep disturbances and daytime tiredness 123 while night time dialysis helps t o reduce these effects. The night time dialysis patients also have a restored melatonin peak at night 124 It has also been shown that lack of sleep can cause disturbances in kidney function and BP patterns. Compared to people who slept 8 10 hours at night, those who were sleep deprived (no sleep f or the night of the experiment) had a greater than 50% increase in nocturnal urine output 125 While potassium and sodium excretion were increased in both genders in the sleep deprived group, the effect was greater in men. The sleep deprived group exhibited an attenuation of the normal dipping that was observed in the control group. These observations indicate that disruption of circadian rhythms via sleep deprivation can affect kidney function which has important im plications for maintaining overall health.


47 The circadian clock appears to be an important player in the regulation of the cell cycle and disruption of the molecular clock has been associated with cancer 126 Renal cell carcinoma is known to be associated with hypoxic signaling pathways and these pathways have recently been linked with the circadian clock 127 In an important human study linking dysregulation of circadian clock gene expression to renal cell carcinoma, Mazzoccoli et al. demonstrated that several core clock genes were differen tially expressed in tumor samples compared to matched normal tissue 128 These findings likely have important implications for the treatment of kidney cancer. Further studies exploring the role of the molecular cloc k in mechanisms underlying renal cell carcinoma are needed.


48 Figure 1 1. Diagram of Two Nephrons and Representative Cell Types. A. Blood enters the nephron and is filtered at the glomerulus. B. Filtrate then moves to the PT (PT). This segment of the n ephron contains the sodium glucose linked transporter isoform 1 (SGLT1) and the sodium hydrogen exchanger isoform 3 (NHE3), both of which are located on the apical membrane (facing the filtrate) and are regulated by the circadian clock. Sodium reabsorbed f rom the filtrate is pumped back into the blood by the basolateral Na/K ATPase, found on each of the cell types illustrated here. C. Filtrate then moves to the Loop of Henle. Cells in the Thick Ascending Loop (TAL) contain the sodium potassium chloride cotr ansporter isoform 2 (NKCC2) which is regulated by potassium channel (ROMK), responsible for pumping K+ back into the filtrate. Again, these cells have a basolateral Na, K ATPase. D. The DCT (DCT) cells contain apically located sodium chloride cotransporters (NCC) which reabsorb Na+ and Cl from the filtrate. This co transporter is regulated by the circadian clock and well as with no lysine (WNK) kinases. E. The last part of the nephr on filtrate enters is the CD (CD). The ENaC (ENaC) reabsorbs Na+ from the filtrate and is regulated by the clock. FXYD5, CAV 1 and ET 1 are regulated by the circadian clock as well and in turn FXYD5 positively regulates the Na/K ATPase, while CAV 1 negativ ely regulates ENaC. F. Juxtaglomerular apparatus: Cells in the macula densa communicate with the glomerulus to modulate GFR in a process known as tubule glomerular feedback (TGF). Glomeruli, PTs and DCTs are located in the cortex of the kidney while the L oop of Henle and part of the CD span into the medulla. Multiple CD s converge to ultimately transport urine out of the kidney.




50 Figure 1 2. Circadian Expression of Core Clock Genes in the Kidney. Relative expression levels are plotted on the y axis with Zeitgeber time on the x axis. Shaded areas represent time when lights are off. Peak Expression of Bmal1 and Clock are in phase and approximately antiphase to Cry2 and Per1 mRNA expression. Data derived from CircaDB 14


51 CHAPTER 2 TRANSCRIPTIONAL REGULATION OF NHE3 AND SGLT1 BY THE CIRCADIAN CLOCK PROTEIN PER1 IN PT CELLS Background The circadian clock is an important regulator of multiple physiological functions including BP the immune response, sleep wake cycle, metabolism, vascular function, and renal function (reviewed in 129, 130 ). Urinary sodium excretion, renal blood flow, glomerular filtration rate, and plasma aldosterone levels have been shown to undergo rhythmic fluctuations (reviewed in 26 ). The circadian clock on the molecular level consists of four core proteins, which interact with one anot her to regulate expression of circadian target genes 3 These four core circadi an proteins are: CLOCK, Bmal1, Per, and Cry. CLOCK and Bmal1 heterodimerize and interact with E box response elements to transcriptionally up regulate circadian target genes, including Per and Cry. Per and Cry interact and then repress the transcriptional activity of CLOCK and Bmal1 131 The circadian clock also undergoes post translational modifications through the phosphorylation of the Per proteins by the circadian kinases, Casein Kinase 1 isoforms 132 134 We have previously demonstrated that Per1 positively regulates the basal and aldosterone mediated expression of 19, 20, 133 135 We also recently demonstrated that Per1 coordinately regulates the expression of multiple genes involved in the regulation of renal sodium reabsorption in CD cells 19 It was initially assumed that Per1 behaves primarily as a repressor of CLOCK and Bmal1 _________________________ *Reprinted with Permission from : Solocinski K, Richards J, All SC, Cheng KY, Khundmiri SJ, Gumz ML. Transcriptional regulation of nhe3 and sglt1 by the circadian clock protein per1 in PT cells. Am J Physiol Renal Physiol 2015:ajprenal 00197 02014


52 activity. However, we and others have shown that Per1 appears to activate gene expression in a manner that appears to be gene and tissue specific 19, 97, 134 137 Concordantly, we proposed a putative mechanism for Per1 action involving repression of the circadian repressor Cry2 134 These data predict that loss of Per1 should result in decreased renal sodium reabsorption, with subsequent decreased plasma volume and decreased BP. Consequently, we have demonstrated that Per1 KO mice have significantly lower BP compared to WT controls 19 and increased urinary sodium levels 20 Consistent with these results, we have recently shown that mice with re duced Per1 expression exhibit reduced plasma aldosterone levels and renal sodium wasting 96 that modulate renal sodium reabsorption. We have also recently demonstrated that Per1 regulates NCC and the WNK kinases in a model of the murine distal convolute tubule 138 However the role of Per1 i n the PT has not been investigated. The PT is the major site of sodium reabsorption in the nephron. A majority of sodium is reabsorbed in this segment through the actions of the sodium hydrogen exchanger NHE3 (reviewed in 139 ). The sodium glucose co transporters (SGLT1 and SGLT2) also play an important role in the reabsorption of sodium and glucose (reviewed i n 140 ). Previous work has demonstrated that NHE3 mRNA oscillates with a circadian pattern in mouse renal medulla 13, 141 Likewise, SGLT1 is regulated in a similar manner in the colon 142, 143 However, it is unknown if ex pression of NHE3, SGLT1, or SGLT2 are regulated by Per1. The goal of this study was to test whether Per1 contributes to the regulation of sodium transporters in the PT via transcriptional modulation of NHE3 and SGLT1.


53 Pharmacological blockade of nuclea r Per1 entry in vivo led to decreased mRNA expression of NHE3 and SGLT1 in the renal cortex of mice. Using several additional, independent methods in the human PT cell line HK 2, we demonstrated a role for Per1 in the transcriptional regulation of NHE3 and SGLT1 but not SGLT2. Importantly, pharmacological blockade of Per1 nuclear entry resulted in decreased intracellular and membrane protein levels of NHE3 and SGLT1. Taken together, these data demonstrate a potential role for the circadian protein Per1 in the regulation of NHE3 and SGLT1 in human PT cells. Methods Animals All animal use protocols were approved by the University of Florida and North Florida/South Georgia Veterans Administration Institutional Animal Care and Use Committees in accordance wit h the NIH Guide for the Care and Use of Laboratory Animals. WT and Per1 KO mice (129/sv) were originally provided by Dr. David Weaver (University of Massachusetts 144 ). WT and Per1 heterozygote (het) mice were bred in house by UF Animal Care Services Staff and maintained on a normal 12 hours light:dark cycle. For in vivo PF670462 ( experiments, weight matched male WT 129/sv mice were either given vehicle (20% hydroxypropyl b cyclodextrin) or 30 mg/kg PF670 462 subcutaneously every 12 hours for 2.5 days starting at noon and sacrificed at midnight 12 hours after last i njection as previously described 96, 138 Mice were anesthetized by inhalant isoflourane and tissues were collected and snap frozen in liquid nitrogen. Kidneys were later dissected and cortex removed for RNA isolat ion or protein collection.


54 Cell Culture HK 2 cells were kindly provided by Kirstan Meldrum (University of Florida, Gainesville, FL) 145, 146 HK 2 cells were maintained in DMEM F 12, 10% FBS, and 1% penicillin st reptomycin at 37C. For RNA silencing experiments, Non Target and Per1 2 cells (PF670462) experiment s were performed as described previously 133, 134 For the dose and time course, HK vehicle (water) for 24, 48, or 72 hours as previous described 133, 138 For nuclear entry experiments, HK RNA Isolation and quantitative real time PCR Total RNA was isolated and DNaseI treated using Direct RNA Miniprep (Zymo were used as template for reverse transcription with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The resulting cDNAs (20 ng) were then used as template in quantitative real time PCR (qPCR) reactions (Applied Biosystems) to evaluate changes in Per1, NHE3, SGLT1, SGLT2, and Actin mRNA levels. Cycle actin and relative quantification was pe 147 Fold change values were calculated as the change in mRNA expression levels rel ative to the control. Mouse and human TaqMan primer/probe sets were purchased from Applied Biosystems. Membrane Protein Analysis, Nuclear Protein Isolation, and Western Blot Analysis. Membrane protein was isolated using the Cell Surface Protein Isolation Kit (Pierce) 2 cells were grown to confluence and


55 isolated using the NE PER kit (Pierce) according to the manufacturer's instructions. For the Per1 nuclear entry experiments, protein concentrations were then quantified by BCA assay (Pierce). Western blots were performed as previously describe d 96, 133, 134 Proteins were separated o n a 4 20% Tris HCl Ready Gel (BioRad), and transferred to a PVDF membrane. The membrane was blocked with 2% non fat dry milk in TBS S (TBS plus Per1 (1:500) (Pierce), anti NHE3 (Mill ipore), anti SGLT1 (Millipore), or anti actin (1:500) Actin was used as a loading control. The membrane was washed with 2% non fat dry milk in TBS S for 15 minutes and then incubated with horseradish peroxidase conjugate anti rabbit secondary antibody and incubated in 2% non fat dry milk in TBS S for 1 hour at 4 C. After incubation, the blot was washed with TBS S for 15 minutes. Detection was performed using Novex ECL Chemiluminescent Substrate reagents (Invitrogen). Densit ometry was performed using ImageJ ( hnRNA Analysis. hnRNA analysis was performed as previously described 20, 135, 138 Primers were designed to amplify regions spanning the intron/exon boundary of the NHE3, SGLT1, and GADPH genes. GAPDH was used as a cDNA loading control. Sequences and exon numbers are shown in Table 2. Total RNA was isolated from vehicle and CKinh treated cells, DNase I treated, and converted to cDNA as described above. PCR re actions were performed using 40 ng of cDNA as template. Reactions were heated to 95C for 15 minutes to activate the Taq polymerase. 35 amplification cycles were performed using


56 the following parameters: 95C for 30 seconds, 55C for 30 seconds, 72C for 1 minute, followed by a final 10 minute extension at 72C. Chromatin Immunoprecipitation (ChIP). HK 2 cells were grown to ~80% confluency and then treated with vehicle (water) or Enzy previously described 80, 138 Chromatin concentrations were calculated and equal amounts of vehicle omatin were used per pull Per1 (Pierce), anti CLOCK (Pierce), anti Pol II (Santa Cruz), or rabbit IgG (Bethyl). Complexes were incubated overnight at 4C with end over end rotation. Immunoprecipitat ed DNA was amplified by End Point PCR. Band intensities were quantitated using densitometry, which was performed using ImageJ ( Bands were relativized to the relevant vehicle or PF670462 treated 10% input. Putative E boxes in the NHE3 an d SGLT1 promoters we re analyzed using ALGGEN PROMO ( bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3 ) 148, 149 For each gene, three primer sets were designed as shown in Table 1. Ouabain sensitive 86 Rb uptake Human kidney PT cells, HK 2 (ATCC) or HKC11 (provided by Dr. Racusen, Johns Hopkins University) were treated with 10 M PF670462 for 24 h in se rum free DMEM:F12 containing 5 mM glucose. Ouabain sensitive 86 Rb uptake was measured as an index of Na + /K + ATPase mediated ion transport as described previously 150 Briefly, circuit Na channels so as to measure Na + /K + ATPase mediated ion transport at V max A trace amount of 86 Rb ( 1


57 86 RbCl) was added to the cells. Uptake was carried out for 10 minutes, after which the cells were washed five to six times with ice cold PBS. One half of the cells were treated with 1 mM ouabain along with monensin for 30 min. The cells were lysed overnight in 0.5 N NaOH containing 0.1% Triton X 100 at 37C. An aliquot (100 the lysate was used to measure radioactivity or to measure protein. The difference between 86 Rb uptake measured in the presence of 1 mm ouabain and the absence of ouabain was used as a measure of Na + /K + ATPase mediated transport activity. Uptake dat a are calculated as nanomoles of 86 Rb accumulated per milligram of protein per 10 min and expressed as percent difference from the vehicle treated cells. Statistical Analysis. All data are represented as mean standard error of the mean (SEM) with n=3 or more. Statistical analyses were performed using Graphpad Prism v6. All graphs/plots t test was used to compare differences between control and treated groups. All P values less than 0.05 were consid ered significant. R esults Per1 must be phosphorylated by 132 Our lab has previously shown that pharmaco ENaC activity 133, 134 To determine if Per1 regulates NHE3, SGLT1, and SGLT2 in vivo, WT mice were tre inhibitor PF670462 as previously described 96 Kidneys were harvested and cortex dissected. mRNA levels of NHE3, SGLT1, and SGLT2 were measured by qPCR. PF6704 62 treatment resulted in significantly decreased levels of NHE3 (Figure 2 1A) and SGLT1 (Figure 2 1B), when


58 compared to vehicle treated mice. SGLT2 mRNA levels did not appear to be affected by PF670462 treatment (Figure 2 1C). We have previously demonstrat ed that PF670462 treated mice had significantly reduced levels of nuclear Per1 in kidney cortex 138 To further investigate our in vivo results, the human PT ce ll line HK 2 was employed for subsequent studies 151, 152 Per1 was knocked down using siRNA in HK 2 cells and mRNA levels of NHE3, SGLT1, and SGLT2 were measured by qPCR. As expected, Per1 knockdown resulted in sig nificantly decreased mRNA expression of Per1 (Figure 2 2A) as we have previously demonstrated 19, 134, 135 Consistent with our in vivo results, Per1 knockdown resulted in significantly decreased expression of NHE3 (Figure 2 2B) and SGLT1 (Figure 2 2C), but not SGLT2 (Figure 2 2D). To further explore the potential role of Per1 in the regulation of NHE3 and SGLT1, HK 2 cells were treated with PF670462 and NHE3, SGLT1, and SGLT2 mRNA expression was determined by qPCR. After 24 hours, treatment with 10 resulted in significant reduction of NHE3 and SGLT1 mRNA (Figure 2 3A and B). SGLT2 expression was not affected at any time point or dose tested (Figure 2 3C). To insure that uclear entry, HK 2 cells were treated with vehicle or blotting was performed to assess nuclear Per1 levels. As expected, and as we have shown previously in other cell types 133, 134 nuclear Per1 levels were significantly decreased with 2 4A; quantified in 2 4B). Measurement of short lived heterogeneous nuclear RNA (hnRNA) is a measure of transcriptional activity 153, 154 knockdown on NHE3 and SGLT1 was transcriptional, hnRNA levels were assessed by


59 PCR amplification of intron exon junctions using cDNA templates from HK 2 cells treated with either PF670462 for 24 hours or Per1 siRNA for 48 hours. Per1 siRNA mediated knockdown or blockade of Per1 nuclear entry led to significantly decreased hnRNA expression of both NHE3 (Figure 2 5A) and SGLT1 (Figure 2 5B). As mentioned above, regulation of target genes by circadian clock proteins is mediated through interaction of these proteins with E box response elements in the promoters of target genes. Therefore, to assess if Per1 and CLOCK interact with the endogenous promoters of NHE3 a nd SGLT1, ChIP was performed in HK 2 cells treated with either vehicle or designed to amplify short regions containing putative E box sites upstream of the transcription start site (Panel A in Fi gures 2 6 and 2 7). Loading was demonstrated by performing the PCR on 10% input (Panel B in Figures 2 6 and 2 7). IgG was used as a negative control (Panel F Figures 2 6 and 2 7). decreased binding of RNA Pol II at al l three sites in the NHE3 promoter, indicative of decreased transcription, further corroborating our mRNA and hnRNA data (Figure 2 and third promoter regions, but neither CLOCK nor Per1 appeared to interact with the promoter region amplified by the first primer set (Figure 2 6D and E). On the SGLT1 promoter regions (Figure 2 7C). A decrease of CLOCK and Per1 interaction with the second and third promoter regions, but there was no apparent interaction with the promoter region amplified by the first primer set (Figure 2 7D and E).


60 T extended to the level of protein, plasma membrane and intracellular fractions were isolated from HK 2 cells treated with vehicle or blot analys is was performed to determine changes in NHE3 and SGLT1 protein levels from intracellular and membrane fractions. of both NHE3 and SGLT1 in both the membrane and intracellular fraction (Figure 2 8A and B), con sistent with the hypothesis that inhibition of Per1 nuclear entry leads to decreased transcription of NHE3 and SGLT1 134 20, 96 96, 138 + /K + ATPase activity was measured as ouabain sensitive 86 Rb uptake in two cell lines representative of human PT cells (HK 2 and HKC11 155 ). Ouabain sensitive Na + /K + ATPase activity (F igure 2 decreased Na + /K + ATPase activity indirectly provides support for the hypothesis that D iscus sion The present study demonstrates a role for Per1 in the transcriptional regulation of NHE3 and SGLT1 in PT cells. Pharmacological blockade of Per1 nuclear entry in vivo and in vitro resulted in decreased mRNA expression of NHE3 and SGLT1. ChIP and hnR NA assays performed in HK 2 cells consistently demonstrated that the effect of Per1 on NHE3 and SGLT1 appears to involve a transcriptional mechanism. Importantly, we demonstrated that the regulation of NHE3 and SGLT1 by Per1 extends to the protein level. Pharmacological blockade of nuclear Per1 entry resulted in decreased protein expression of NHE3 and SGLT1 at the membrane and in the cytosol.


61 Consistent with decreased expression of NHE3 and SGLT1 at the apical membrane, which would predict decreased api cal sodium reabsorption, pharmacological inhibition Na + /K + ATPase activity in two different PT cell lines, HK 2 and HKC11. Together, these data suggest a role for Per1 in the positive regulation of genes that function in sodium reabsorption in PT cells; this model is entirely consistent with our previously proposed model for Per1 action on ENaC in cortical CD cells 19 and on NCC in distal convoluted cells 138 Previous work has demonstrated that NHE3 is a potential circadian target in mouse renal medulla 13, 141 while others studies have demonstrated that SGLT1 is sim ilarly regulated in the colon 142, 143 However, it was unknown if NHE3 and SGLT1 were regulated by Per1 in the kidney. For the first time, we have demonstrated that both NHE3 and SGLT1 are Per1 targets in a cell mo del of the human PT Along with our previous data on Per1 regulation of transporters in the cortical CD 19, 20, 80, 133 135 and in the DCT 138 These data predict that loss of Per1 should result in decreased renal sodium reabsorption, leading to decreased plasma volume and decreased BP. Consistent with these predictions, we have recently shown that mice with reduced level s of Per1 expression exhibit renal sodium wasting 96 and that Per1 KO mice display lower BP compared to WT controls 19 Increasing evidence supports an important role for the circadian clock in the regulation of BP control and renal sodium handling (Reviewed in 26, 129, 130, 156 ). Per2 KO mice display decreased 24hr diastolic BP, increased heart rate, and reduced diurnal dipping 69 Bmal1 KO mice have reduced BP in the active phase, leading to a blunting of circadian BP variation 61 CLOCK KO mice are hypotensive and display dysregulated


62 urinary sodium excr etion with mild diabetes insipidus 18, 64 Cry1/2 KO mice display salt sensitive hypertension partially due to increased aldosterone production and serum aldosterone levels 65 This was potentially due to increased expression of 3 beta dehydrogenase isomerase, an enzyme in the aldosterone synthesis pathway. We have shown that Per1 KO mice have significantly lower BP than WT controls 19 and excrete more sodium in their urine 20, 96 Mice with reduced levels of Per1 also have decreased plasma levels of aldosterone and decreased renal sodium reabsorption 96 While a multitude of factors contribute to the BP phenotypes of these animals (including, but not limited to, the heart (reviewed in 157 ), sympathetic activity 158, 159 the vasculature 160 and/or nitric oxide 161, 162 ), accumulating data from multiple labs suggests that salt handling by the kidney is likely to play an important role. Consistent with our findings, a recent human study demonstrated that Per1 was overexpressed in the renal medulla of human hypertensive pa tients compared to normotensive controls 163 Given the magnitude of the BP and renal sodium phenotypes in Per1 KO or Per1 heterozygous mice, it is perhaps not surprising that multiple sodium transporter genes along the nephron are regulated by Per1. Taken together, the data presented here demonstrates a novel role for Per1 in the transcriptional regulation of NHE3 and SGLT1 in the kidney in vivo and in PT cells in vitro Determining the functional effects of this r egulation will require further investigation. Reduced NHE3 expression may lead to reduced GFR 164 which was not tested in the present study. On the other hand, knockout of NHE3 in a rodent model was associated with reduced BP without alteration in the autoregulation of GFR 165 Per1 appears to regulate gene expression in a manner consistent with driving sodium reabsorption in cortical CD


63 cells 19, 20, 80, 133 135 and DCT cells 138 Now for the first time, we have demonstrated a role for Per1 in the regulation of sodium transporter gene ex pression in PT cells.


64 Table 2 1. Sequences and exon numbers Gene Spanning Exon Forward Primer Reverse Primer NHE3 7 AC SGLT1 3 C GAPDH 4 G NHE3, Na+/H+ exchanger 3; SGLT1, Na+ glucose tran s porter 1. Table 2 2 Primer sets for each gene Forward Primer Reverse Primer NHE3 Primer set 1 Primer set 2 CTTGTGAGCGA Primer set 3 SGLT1 Primer set 1 Primer set 2 Primer s et 3 NHE3, Na+/H+ exchanger 3; SGLT1, Na+ glucose tran s porter 1.


65 Figure 2 1. Pharmacological blockade of Per1 nuclear entry in vivo results in decreased NHE3 and SGLT1 expression, but not SGLT2 express ion in the renal cortex. Weight matched male WT 129/sv mice were injected subcutaneously with vehicle (20% hydroxypropyl b cyclodextrin) or 30 mg/kg PF670462 every 12 hrs for 2.5 days starting at noon and euthanized at midnight 12 hours after last injectio n as previously described 96 Kidneys were harvested, cortex dissected, and NHE3 (Panel A), SGLT1 (Panel B), or SGLT2 (Panel C) mRNA expression was measured by qPCR, n=4 *p<0.05 compared to WT. Values are represent ed as the mean SEM. Figure 2 2. Per1 knockdown results in decreased NHE3 and SGLT1 expression, but not SGLT2 expression in HK 2 cells in vitro HK 2 cells were treated with Non Target or Per1 siRNA for 48 hours. qPCR was used to evaluate changes in Per1 (Panel A), NHE3 (Panel B), SGLT1 (Panel C), or SGLT2 (Panel D) mRNA expression in Per1 siRNA versus non target siRNA control, n=3. *p<0.05, **p<0.01, Values are represented as the mean SEM.


66 Figure 2 3 Pharmacological blockade of Per1 nuclear e ntry results in decreased NHE3 and SGLT1 expression, but not SGLT2 expression in vitro A B. HK 2 cells were treated with the Casein Kinase 1 / inhibitor PF670462 with either 0.1, in NHE3 (Panel A), SGLT1 (Panel B), or SGLT2 (Panel C) expression in PF670462 treated cells versus vehicle (water) treated cells, n=3. *p<0.05. Values are represented as the mean SEM. CKinh Casein Kinase 1 / inhibitor.


67 Figure 2 4. Pharmacologica l blockade of Per1 nuclear entry results in decreased nuclear Per1 in vitro A. Nuclear extracts were collected from HK 2 cells / inhibitor PF670462 or water for 24 hours. Western blot analysis was performed using anti P er1 (nuclear Per1 ~50 kDa) or anti actin (~42 kDa) antibodies as a loading control. Data are representative of three independent experiments. B. Densitometry analysis was used to quantitate the level of Per1 in panel A, n=3 **p<0.01 Values are represent ed as the mean SEM. CKinh Casein Kinase 1 / inhibitor.


68 Figure 2 5. Pharmacological blockade of Per1 nuclear entry results in decreased transcription of NHE3 and SGLT1. Primers were designed to amplify regions of hnRNA spanning the intron/exon bo undary of the NHE3 (Panel A) and SGLT1 (Panel B). GAPDH was used as a cDNA loading control. Arrows are representative of primer location. Bands were quantitated using ImageJ densitometry ( Signal strength was normalized to the relevant vehicle or CKinh treated GAPDH control, n=3 *p<0.05 values are represented as the mean SEM. CKinh Casein Kinase 1 / inhibitor.


69 Figure 2 6. Pharmacological blockade of Per1 nuclear entry leads to decreased occupancy of CLOCK and Per1 on the NHE3 promoter in HK 2 cells. Chromatin immunoprecipitation experiments were performed using HK 2 cells treated with either PCR was performed using primer sets flanking three putative E boxes in the NHE3 promoter (Panel A). Chromatin immunoprecipitations were performed using anti Pol II (Santa Cruz) (Panel C), anti CLOCK (Pierce) (Panel D), anti Per1 (Pierce) (Panel E), or rabbit IgG (Bethyl) (negative control) (Panel F) antibodies. Bands were quantitated using densitometry, which was performed using ImageJ ( Signal strength was normalized to the releva nt vehicle or CKinh treated input control, n=3. *p<0.05, values are represented as the mean SEM


70 Figure 2 7. Pharmacological blockade of Per1 nuclear entry leads to decreased occupancy of CLOCK and Per1 on the SGLT1 promoter in HK 2 cells. Chromat in immunoprecipitation experiments were performed using HK 2 cells PCR was performed using primer sets flanking three putative E boxes in the SGLT1 promoter (Panel A). Chromatin i mmu no precipitations were performed using anti Pol II (Santa Cruz) (Panel C), anti CLOCK (Pierce) (Panel D), anti Per1 (Pierce) (Panel E), or rabbit IgG (Bethyl) (negative control) (Panel F) antibodies. Bands were quantitated using densitometry, which was performed using ImageJ ( Signal strength was normalized to the relevant vehicle or CKinh treated input control, n=3. *p<0.05, values are represented as the mean SEM.


71 Figure 2 8. Pharmacological blockade of Per1 nuclear entry resu lts in decreased membrane and intracellular protein expression of NHE3 and SGLT1 in HK 2 cells. Membrane and intracellular extracts were collected from HK 2 cells / inhibitor PF670462 or water for 24 hours. Western blot analysis was performed using anti NHE3 (~85 kDa) (Panel A), anti SGLT1 (~75 kDa) (Panel B) or anti actin (~42 kDa) antibodies as a loading control. Data are representative of three independent experiments. Densitometry analysis was used to quantitate the level of NHE3 and SGLT1 in panel A B, n=3 *p<0.05 Values are represented as the mean SEM. CKinh Casein Kinase 1 / inhibitor.


72 Figure 2 9. Na + /K + ATPase activity is reduced in HK 2 and HKC11 cells following 2 (Panel A) and HKC11 (Panel B) cells both show significantly reduced Na + /K + ATPase activity following PF670462 for 24 h in serum free DMEM:F12 containing 5 mM glucose. They we re then treated with 5 M monensin to short circuit Na channels and thus measure Na + /K + ATPase transport at V max Half of the cells were treated with 1 uM ouabain for 30 min followed by addition of 86 RbCl for 10 min to determine Na + /K + ATPase specific acti vity. Cells were then lysed and protein was measured to normalize uptake to total protein. Values are calculated as nmoles of 86 Rb accumulated per milligram of protein per 10 min and expressed as percent activity relative to the vehicle treated control cel ls. n=6 for Panel A, n=12 for Panel B, **p<0.01. Values are represented as the mean SEM.


73 CHAPTER 3 DOCP SALT TREATMENT LEADS TO NONDIPPING HYPERTENSION IN PER1 KNOCKOUT MICE Background unctions to synchronize our daily activities, on a physiological and molecular level, with the time of day and certain behaviors that usually occur around the same time each day 2 Many physiological functions exhibit circadian rhythms, including BP Healthy individuals experience a 10 20% decrease in BP at night. Individuals who do not exhibit dipping is associated with an increased risk of chronic kidney disease 91, 92 adverse cardiovascular events 90 166 and can also be used to predict future cardiovascular events 167 These risks are further increased by chronic hypertension, or high BP, which is also a risk factor for CVD C VD is the leading cause of death of Americans 12 accounting for nearly 1 million deaths/year 168 A subset of hypertensive patients exhibit salt sensitive hypertension in wh ich BP is abnormally elevated after high salt intake, implicating aberrant sodium handling in the pathogenesis of the disease. The underlying mechanisms of these pathologies remain incompletely understood. A central clock located in the suprachiasmatic nu cleus of the brain is entrained by light signals from the retina 21 These signals are then relayed through neuronal and humoral pathways to peripheral clocks located in other tissues and organs of the body. The importance of circadian proteins for BP control is illustrated by clock KO mice. _________________________ *Reprinted w ith Permission from: Solocinski K, Holzworth M, Wen X, Cheng KY, Lynch IJ, Cain BD, Wingo CS, Gumz ML. Desoxycorticosterone pivalate salt treatment leads to non dipping hypertension in per1 knockout mice. Acta Physiol (Oxf) 2017;220:72 82


74 Every clock gene KO mouse that has been tested exhibits a BP phenotype 169 Global Bmal1 KO mice lose circadian rhythmicity of BP and are hypotensive 61 Loss of Bmal1 in renin producing cells in the kidney also leads to a significant decrease in BP compared to controls 63 Loss of Clock causes mice to become hypotensive yet reta in the circadian rhythm of BP 18 Cry1/Cry2 global KO mice exhibit salt sensitive hypertension 13 65 Mice lacking all three Period isoforms lose their BP dip on a low salt diet 170 Thus, the clock is a major regulator of baseline BP and rhythmic BP. The kidney is a critical regulator of BP and many aspects of renal function oscillate in a circ adian manner including sodium excretion, renal blood flow 24 and glomerular filtration rate 23 Aldosterone, a critical regulator of renal sodium handling, i s also released in a circadian manner 53 Gumz et al. identified Per1 as a novel aldosterone target gene in renal CD cells 78 Circadian clock mediated regulation of a renal sodium transporter was first reported by Okamura and colleagues who demonstrated that the Na + ,H + exchanger NHE3 exhibited time dependent changes in expression 13 Gumz et al. provided the link between the clock and the ENaC ENaC with 20 Subsequent studies provided further evidence that PER1 coordinately regulates seve ral genes encoding proteins that function in renal sodium reabsorption 16, 88, 171 Global loss of Per1 on the hypertensive, salt sensitive 129/sv mouse strain resulted in significantly reduced BP compared to WT (WT ) controls on a normal diet 19 The goal of the present study was to characterize the role of PER1 in the normotensive C57BL/6J background strain. Small but significant differences in mean arterial pressure (MAP) w ere observed between WT and PER1 KO mice at baseline.


75 Since Per1 is an aldosterone target gene and coordinately regulates many renal sodium transport genes, we subjected WT and Per1 KO mice to a high salt (HS) diet in combination with desoxycorticosterone pivalate (DOCP) injection as a model for evaluating salt sensitive changes in BP. DOCP is a long acting aldosterone analog that bypasses the sodium excreting effects of Renin Angiotensin Aldosterone System (RAAS) suppression normally seen with high salt in take (reviewed in 172 ) and leads to increased renal sodium reabsorption. Interestingly, loss of PER1 in C57BL/6 mice subjected to HS/DOCP treatment resulted in significantly elevated BP accompanied by disruption of the normal BP rhythm. Thus, PER1 appears to be an important regulator of baseline and rhythmic BP in C57BL/6J mice. Methods Mice Male WT C57BL/6 J mice and C57BL/6J Per1 KO between the ages of 14 18 weeks were used for these experiments (original heterozygous mice were the kind gift of Dr. Shin Yamazaki, Vanderbilt 173 ). WT and Per1 KO mice were litter mates derived from mating mice heterozygous for Per1 Mice were maintained on a normal 12:12 L:D cycle at the NF/SG VA Healthcare animal facility. Experiments were perfor med in accordance with the NIH Guide for the Care and Use of Laboratory Animals and with the approval of UF and VA Medical Center IACUCs. Mice were implanted with telemetry devices from Data Sciences International through the carotid artery and extended in to the aortic arch following established protocol 174 and as described previously 19 Mice were allowed at least seven days to recover. Mice were then placed on control diet (0.2% NaCl, Harlan) for at least 3 days, during which time baseline BP recordings were made. Mice were then placed on a high salt (4% NaCl, Harlan) diet for 3 more days, followed by injection


76 with75 g/g bw DOCP. Three days after the injection and after si x total days on a high salt diet, telemetry recordings for the HS/DOCP period were made. Tissue dissection and quantitative real time RT PCR (qPCR) Tissue dissection and QPCR were performed as described previously in 171 Flame Photometry Flame photometry was used to determine urine and plasma Na and K concentrations. Aldosterone ELISA Plasma aldosterone was measured by ELISA as described in 96 Statistics Day values are the 3 da y averages of MAP between 6am (ZT0) through 5pm (ZT11). Night values are the 3 day averages of MAP recorded between 6pm (ZT12) and 5am (ZT23). Paired t tests and standard and repeated measures 2 way ANOVAs were performed as appropriate using GraphPad Prism software (version 6.07 for Windows, GraphPad Software, La Jolla California USA, ). Cosinor analysis was done using ClockLab software (Actimetrics) and the generous help of Dr. Joshua Speed (Universit y of Alabama Birmingham). p<0.05 was considered significant. JTK Cycle was used as described 175 The program cosinor was downloaded from (last accessed 09/01/2016). Values are shown as mean SEM. Results Given our previous findings that Per1 regulates a number of genes related to renal sodium reabsorption in vitro and in vivo in hypertensive 129/sv mic e, we investigated the expression of candidate genes in WT normotensive C57BL/6 mice compared to Per1 KO mice. Real time quantitative PCR (QPCR) was used to assess


77 relative differences in gene expression in the renal cortex between WT and Per1 KO mice at t wo distinct time points in the circadian cycle. Kidneys were collected at noon and midnight in order to capture time points at the midpoint of the rest phase and the active phase, respectively. We investigated time and PER1 dependent changes in steady sta te mRNA expression for genes known to contribute to renal sodium reabsorption or the regulation of this process. The Angiotensin II receptor (AT 1 R) leads to increased sodium reabsorption and BP through the RAAS and has positive effects on ENaC 176 and expression displays a significant change over time due to the interaction of time and genotype (p=0.0075) (Figu re 3 1A). UBE2E3, a ubiquitin E3 ligase, has been previously shown to regulate the alpha subunit of the ENaC 83 and is regulated by PER1 16 As shown in Figure 3 1B, there was a significant effect of time on UBE2E3 expression (p=0.015) with a trend towards a genotype effect as well (p=0.065). Nitric 177 (Figure 3 1C) and trended towar ds an interaction between genotype and time. With no lysine kinase 1 (WNK1) is a regulator of the sodium chloride cotransporter 178 and trended towards a genotype time interaction as well (Figure 3 1D). Figure 3 1E significantly with time (p=0.024) and a possible trend for an interaction between time and genotype was observed (p=0.12) (Figure 3 1E). In contrast to these changes, PCSK9, an ENaC regulating protease, did not undergo a ny changes in expression relative to time or Per1 KO (Figure 3 2 A). Likewise, mRNA levels for the Endothelin A Receptor, a regulator of renal sodium balance and BP did not change with time or Per1 KO (Figure 3 2 B). Therefore, Per1 appears to selectively r egulate only certain genes which govern sodium transport and reabsorption.


78 Given our previous findings that PER1 acts as a coordinate regulator of several genes in the kidney that participate in the regulation of renal sodium reabsorption, we hypothesized that Per1 KO mice would exhibit altered BP. Due to the altered gene expression patterns observed in Figure 3 1, we compared MAP between WT and KO mice under baseline conditions. The mice were implanted with telemetry devices and after recovery MAP was moni tored over a 3 day period. During the day when mice are inactive, WTs have a MAP of 102.1 1.7 mmHg which increases to 112.5 1.08 mmHg during the night when they are active, an inc rease of 10.41.5 mmHg (Figure 3 3 A). Per1 KO mice exhibit an inactive ph ase MAP of 103 1.4 mmHg which rises to 119.8 .9 during the active phase. This is a change of 16.8 1.7 mmHg which is significantly higher than the inactive/active phase difference observed in WT mice (p < 0.05). In Figure 3 3 B, hourly MAP data are sho wn. The 12 hour period of the inactive phase is active phase is indicated by the shaded areas between ZT12 and ZT 0. The dip in MAP during the inactive phase is easily v isualized, as well as the slightly higher MAP in the Per1 KO mice seen during the dark/active period. Under these baseline conditions, significant differences between WT and Per1 KO mice in terms of plasma aldosterone levels, plasma Na or K, or body weight were not observed (Table 3 1). Given the subtle but significant differences in MAP between WT and Per1 KO mice under control conditions, we examined the response of both groups to an established model of salt sensitivity: high salt diet plus mineralocort icoid treatment. Following the baseline recordings presented in Figure 3 3 the same mice were given a high salt diet (4% NaCl) in addition to injection of DOCP. Figure 3 4 A shows day and


79 night averages taken the same way as described in Figure 3 3 WT mic e exhibit a significant inactive/active phase difference in BP with an increase from 101.5 1.92 mmHg during the inactive phase to 114.5 1.1 mmHg during the active phase. This is a 13 2.2 mmHg increase which is not different from the inactive/active d ifference in BP of WT mice observed under control conditions. In contrast, Per1 KO mice exhibited an inactive phase average MAP of 118.1 1 mmHg that increased to 125.3 1.5 mmHg during the active phase. This is a difference of only 7.2 1.8 mmHg, which is significantly different from the WT and, importantly, classifies these mice as non dippers. Panel B shows the hourly BP measurements, clearly illustrating the attenuated dip of the KO mice during the inactive phase. In order to analyze the dipping patt ern of the mice, we compared inactive/active phase differences in MAP of WT and Per1 KO mice under control and HS/DOCP conditions (Figure 3 5 ). It is clear that Per1 KO mice have a greater decrease in MAP from the active to inactive phase compared to WT on control diet (Figure 3 5 A). The Per1 KO dip of 14 1.4% is significantly larger than the WT 9.2 1.3% dip (p < 0.05) under baseline conditions. On HS/DOCP, WT mice maintain their dip in BP whereas Per1 KO mice exhibit an attenuated inactive/active phase difference in BP (panel B). WT MAP decreases by 11.4 1.9% from night to day while Per1 KO mice decrease only 5.7 1.4% (P<0.05). Diastolic and systolic BP changed in parall el to MAP (Figure 3 6 ). In contrast to these differences in BP, inactive/active phase differences in activity patterns were higher in WT mice compared to Per1 KO mice under control and HS/DOCP conditions ( F igure 3 7 ). Thus, activity levels do not account for the higher MAP observed in Per1 KO mice.


80 Finally, in order to determine if th ere was a change in the 24hr rhythm of BP, we subjected the hourly telemetry data to cosinor analysis. This allowed us to examine changes in circadian parameters that may have occurred in response to HS/DOCP and loss of PER1. Table 3 2 lists the values fo r amplitude (difference between the highest and lowest BP values), mesor (mean value for MAP across all time points), and phase (Zeitgeber time at which MAP peaks) 179 Consiste nt with our findings in Figure 3 4 there was a significant change in mesor between WT (107.6 5) and Per1 KO (121.2 5.3) on HS/DOCP. Per1 KO mice on HS/DOCP also had a significant phase shift (22.3 1.3) compared t o WT mice on HS/DOCP (19 0.3, p=0.018) as well as KO mice on control diet (18.2 0.1, p=0.013). We also u se d JTK cycle (Table 3 3) and cosinor analysis (Table 3 4) to evaluate circadian rhythms demonstrated significant rhythms in MAP (p<0.0001 for each mouse in every group). Discussion The most striking finding of this study was that loss of a single gene, Per1 in combination with high salt and mineralocorticoid treatment resulted in a non dipping phenotype. Importantly, these significant results were observed in a relatively mild HS/DOCP model without surgical nephrectomy. This is also accompanied by a significant phase shift in the peak of MAP in Per1 KO compared to WT mice. To the best of our knowledge, this is the first demonstration of a non dippin g, phase shifted BP phenotype in a single clock gene KO mouse in response to a model of salt sensitive hypertension. We have previously demonstrated a role for PER1 in the regulation of renal sodium handling with accompanying effects on BP 16, 80, 96, 171, 180 The results of the present study further implicate PER1 as an important regulator of BP. When challenged


81 with HS/DOCP, Per1 KO mice undergo increased BP as well as a disruption of the normal 24 hr rhythm of B P. Our previous work in the hypertensive 129/sv mouse strain demonstrated a protective effect of Per1 KO on BP. This is not surprising given that strain dependent differences in BP regulation are an established phenomenon, with wide ranging baseline BP 181 sodium sensitivity 182 183 and susce ptibility to renal damage 184 and diabetic nephropathy 185 Thus, the differences in baseline BP between Per1 KO mice on 129/sv and C57BL/6J backgrounds are likely due to str ain dependent differences in background genetics. Even though non dipping is a recognized problem with estimates of non dipping prevalence as high as 45% 186 the need for better treatments and thus better animal models remains. Other animal models of non dipping hypertension have been identified using chronic models 187 or ablation of multiple genes 170, 188 In depth testing of Period isof orm KOs in 129/sv mice by Rudic and colleagues further supports the importance of the PER proteins in BP regulation (Pati et al., 2016). In this study, Angiotensin II infusion into Per2 KO mice in total darkness for 3 weeks elicited a non dipping phenotype Interestingly, a low salt diet resulted in non dipping in Per1/2/3 KO mice in normal light conditions as well. It is difficult to directly compare these results due to the differences in background strain and treatment conditions. However, it is clear th at the PER proteins are important regulators of BP. Thus the novel model presented in this study is the first single gene knockout model to produce a hypertensive non dipping phenotype which develops quickly under normal light/dark conditions. In addition, WT C57Bl/6J mice are quite resistant to the current study regimen and usually require some additional insult to see a phenotype 183, 189 making the non dipping exhibited by Per1 KO


82 mice even more remarkable. Moreo ver, C57BL/6J Per1 KO mice are slightly hypertensive at baseline, which is a common characteristic of many non dippers 190 191 Non dipping in humans can likely be attributed to many factors including kidney damage and salt sensitive hypertension (reviewed in 192 ). The model presented here is pertinent to human disease as it has been dem onstrated that high salt intake can blunt the nocturnal dip in humans 193 and conversely, restricted salt intake can help increase this dip 194 The changes observed in these mice likely involve the contribution of multiple organ systems since the current model is a global KO. It is e vident that the vasculature and heart contribute to rhythms in cardiovascular function (reviewed in 195 ) and the contribution of these systems to the regulation of BP rhythms is an active area of investigation. For example, d b/db mice displayed a nondipping phenotype along with altered expression of clock gene mRNA levels in vascular smooth muscle tissue, which likely contributed to the nondipping phenotype observed in this mouse model of diabetes 196 While the actions of PER1 in the vasculature remain largely unknown, the vasculature is an important mediator of BP regulation through changes in vessel compliance. Decreased compliance is accompanied by an increase in SBP and no change or a decrease in DBP 197 The parallel changes in DBP and SBP observe d in these mice suggest a largely volume dependent mechanism of hypertension which likely involves the action of the kidneys. Diuretics have been shown to reverse non dipping in humans 198 suggesting a volume depen dent mechanism as well. The kidney is further implicated in the etiology of non dipping through the association of renal transplantation with an improvement in dipping status in humans 199 While certain SNPs in Bmal1 have been associated with hypertension 200 to the best of our knowledge


83 there have been no studies large enough to i dentify SNPs in Per1 which could be attributed to salt sensitivity or non dipping in humans. Taken together with the results of the present study, these findings support a role for the circadian clock and the kidney in the maintenance of baseline BP and rh ythmic BP. In conclusion, Per1 KO C57BL/6J mice on HS/DOCP regimen may represent an important new model for understanding mechanisms underlying non dipping hypertension. Limitations of this study include the fact that the mice used in this study are globa l KO. Thus, tissue specific contributions to the non dipping phenotype cannot be assessed. These studies were also limited to male mice. The experiments presented here were performed in a normal 12:12 light/dark cycle. Since a truly circadian process is de fined as one that persists in the absence of light cues, determination of a true circadian defect would require the use of total darkness, an experimental maneuver that is beyond the scope of the present report. To place these findings in a clinical perspe ctive, accumulating evidence suggests that chronotherapy, or dosing of medication at a specific time of day, can be helpful in treating non dipping in certain patients 201 20 2 While chronotherapy or lifestyle changes can decrease non dipping prevalence in some patients, not all are helped by these interventions. Since non dipping increases the risk of cardiovascular events, there is a need for better models and treatments. O ur novel model of Per1 KO mice treated with a HS/DOCP regimen models a diminished dipping phenotype similar to that seen in humans and may provide a new way to test interventions. PER1 is again implicated in BP control and thus presents as a putative targe t for modulation of dipping status.


84 The correlation between non dipping and cardiovascular risk highlights not only the importance of normal circadian rhythms to overall health but also the way in which BP measurements are taken. 24 hour ambulatory BP moni toring (ABPM) is a much more accurate predictor of dipping status and therefore future risk as it markedly reduces white coat hypertension and placebo effects 203 204 205 Somewhat similar to white coat hypertension experienced by humans, mice can become stres sed by tail cuff BP measurements 206 207 ABPM is a powerful meth od for BP studies since recordings are taken continuously as opposed to the snapshots generated from tail cuff or office measurements. In summary, here we present a new model of non dipping hypertension: Per1 KO mice on a HS/DOCP regimen. This is the first report of a single clock gene KO resulting in a non dipping phenotype in response to high salt plus mineralocorticoid. The data implicate PER1 in the control of a normal dip in BP during the inactive period. This is especially significant given that C57BL /6J mice are often resistant to perturbations in BP. The new model of non dipping proposed here ( Per1 KO mice on HS/DOCP) can also serve as a means of discovering new ways to treat non dipping. In contrast to WT mice, global C57BL/6J Per1 KO mice are relat ively hypertensive at baseline and are sensitive to a high salt/mineralocorticoid model of hypertension. These Per1 KO mice display attenuated dipping of BP which is similar to non dipping hypertension in humans that is associated with increased risk of ad verse cardiovascular events.


85 Figure 3 1. Differences in renal gene expression between WT and Per1 KO mice at noon and midnight on control diet. Kidney cortex from WT (black bars) and Per1 KO (checkered bars) mice collected at noon (inactive phase) and midnight (active phase) time points was analyzed by qPCR for differences in genes related to renal sodium transport. (A) AT 1 R, (B) UBE2E3, (C) NOS1, (D) WNK1 and (E) ENaC. Significance was calculated by two way ANOVA. n=3 for KO at noon, n=4 for all others. p<0.05 was considered significant.


86 Figure 3 2. There are no significant differences in expression of Pcsk9 or ETA in WT or Per1 KO mice at noon and midnight. Kidney cortex was collected at noon and midnight time points and made into cDNA. Gene expression measured by qPCR is not significantly different between WT and Per1 KO mice at either noon or midnight for Pcsk9 (A) or ETA (B). Significance was calculated b y two way ANOVA. n=4 animals per group. Figure 3 3 Inactive/active averages and 72 hour telemetry recordings of MAP in WT and Per1 KO mice on control diet. A. Day and night MAP averages. Day values are the 3 day averages of MAP between 6am ZT0 ZT11. N ight values are the 3 day averages of MAP recorded between ZT12 ZT23. B. Telemetry recording of MAP with lights on from ZT0 ZT12 depicted with light areas and ZT12 ZT0, the time when lights are off, shaded gray. n=8 for WT, n=6 for Per1 KO. Significance wa s calculated by two way ANOVA. p< 0.01, ** p<


87 Figure 3 4 Inactive/active averages and 72 hour telemetry recordings of MAP in WT and Per1 KO mice on HS/DOCP diet. MAP recordings from da y 3 to day 6 on HS/DOCP. A. Day and night MAP averages. Values were calculated as in Figure 2. B. Telemetry recording of MAP depicted the same as in Figure 2. n=8 for WT, n=6 for Per1 KO. Significance was calculated by two way ANOVA. p< 0.01, ** p< 0.001 compared to same time. Figure 3 5 Per1 KO mice lose the inactive/active difference in MAP on HS/DOCP diet. A. The change in MAP from night to day is shown for WT mice and Per1 KO mice on control diet. B. The chang e in MAP from night to day is shown for WT and Per1 KO mice on HS/DOCP diet for 6 days. Percent dip is given for each group. Significance was calculated using a t test. *p<0.05. n=8 for WT, n=6 for Per1 KO.


88 Figure 3 6 Parallel changes in DBP, SBP and MAP. DBP, SBP and MAP as measured by telemetry change in parallel in WT mice as well as Per1 KO mice on both diets. n=8 for WT, n=6 for Per1 KO.


89 Figure 3 7 WT and Per1 KO mice maintain inactive/active activity patterns on control and HS/DOCP diet. Activity patterns measured by telemetry were higher during the night than day in both genotypes on both control (A) and HS/DOCP (B) diets. Activity was significantly lower for Per1 KO on HS/DOCP compared to WT and thus does not account for the higher MAP o bserved. Statistics were calculated by two way ANOVA. n=8 for WT, n=6 for Per1 KO. Table 3 1 Baseline characteristics of WT and Per1 KO mice. WT mice Per1 KO mice Significance Noon Midnight Noon Midnight Time Genotype Plasma Aldosterone (pg/mL) 5 51.6193 292.147.9 384.229.2 260.89476.5 NS Plasma Na + (mM) 150.71.1 152.50.62 149.91.38 1530.72 NS Plasma K + (mM) 4.540.14 4.710.07 4.980.21 4.690.07 NS NS Body weight (g) 27.60.56 27.30.35 NS Plasma concentrations of aldosterone, sod ium (Na) and potassium (K) were determined from samples taken at noon and midnight from WT and Per1 KO mice. Significance was determined by two way ANOVA (excluding body weight). Data are presented as mean SEM. *p<0.05. n=4 mice per group.


90 Table 3 2. Cosinor analysis of HS/DOCP effects on MAP in WT and Per1 KO mice. WT Control Per1 KO Control WT HS/DOCP Per1 KO HS/DOCP Amplitude 15.8 1.8 22.4 3.4 21.1 2.7 23.3 4.5 Mesor 106.9 4 111.4 1.3 107.6 5 121.2 5.3 Phase 18.9 0.56 18.2 0.1 19 0.3 22.3 1.3* Using ClockLab software, we determined amplitude, mesor and phase data from MAP measurements. All values were calculated separately for each animal and the mean is given SEM. Significance was calculated by t test. *p<0.05 comp ared to same genotype, p<0.05 compared to same treatment. n=6 for KO, n=8 for WT. Table 3 3 Analysis of MAP by JTK Cycle. WT Control KO Control WT HS/DOCP KO HS/DOCP Phase 24.63 0.76 23.33 0.49 24.81 0.45 27.08 1.66* Amplitude 7.26 0.93 1 9.53 1.95 Period 24 24 24 24 Amplitude and phase data from MAP measurements were calculated using JTK Cycle. All values were calculated separately for each animal and the mean is given SEM. Significance was calculated by t t Table 3 4 Analysis of MAP by Cosinor Analysis. WT Control KO Control WT HS/DOCP KO HS/DOCP Period 24.82 0.44 24.02 0.29 24.05 0.25 23.55 0.63 Mesor 108 4.04 111.72 1.78 109.31 4.89 Amplitude 8.03 0.82 12.11 0.96 10.91 1.25* 11.51 1.9 Amplitude and phase data from MAP measurements were calculated using Cosinor. All values were calculated separately for ea ch animal and the mean is given SEM. Significance was calculated by t test. *p<0.05 effect of treatment, p<0.05 effect of KO, n=8 for WT.


91 CHAPTER 4 L OSS OF THE CIRCADIAN CLOCK PROTEIN PER1 DOES NOT LEAD TO NONDIPPING IN FEMALE C57BL/6 MICE Background Hypertension, is the greatest risk factor for death around the world 208, 209 It is also the number one risk factor fo r stroke and CVD which is the leading cause of death of Americans 12 claiming 1/3 of the total deaths in the U.S. annually 168 While about 30% of U.S. adults have hypertens ion, nearly half of them do not have adequate BP control (less than 140/90 mmHg) 210 Clearly, better awareness and treatments are needed, not only for the sake of patients but because of the direct and indirect costs of CVD and stroke. The American Heart Ass ociation estimates this to be $320 billion annually and predicts an increase to $918 billion by 2030 168 While the primary cause of the majority of hypertension is not known, table salt (NaCl) is known to increase sensitivity to BP dysregulation. Salt sensitivity affects people across a spectrum but it is known to be more prevalent in African Americans, affecting almost of hypertensive patients, compared to Caucasians, in which about half of hypertensive patients are also salt sensitive 211 Salt sensitivity has also been associated with increased risk of cardiovascular events 212 which is possibly independent of high BP 213 The kidney is the organ responsible for water and ion (including Na + Cl ) homeostasis via filtration by the functional unit of the kidney: the nephron. The DCT and the CD reabsorb the final 10 15% of filtered sodium and are the most highly regulated segments. The kidney has been implicated in the pathogen esis of hypertension in cross transplantation studies in mice 214 and human kidney transplant recipients 215 Similar experiments in rats 216 showed that salt sensitivity tracks with the kidney as well.


92 Many of the sodium transporters located along the nephron are regulated by the circadian clock. This ancient mechanism for energy conservation and physiological synchronicity can be tr aced all the way back to archaebacterial (reviewed in 2 ). Very briefly, on a molecular lev el the clock is comprised of four core proteins (CLOCK, CRY, PER and BMAL1) whose expression patterns oscillate over a ~24 hour period (for a review on this mechanism, see 4 ). Through their actions as transcription factors, these core clock components regulate over 40% of all expressed genes 8 including NCC and the WNK pathway in the DCT 16, 17 he CD 18 20 evidence that the gamma su bunit of ENaC is regulated as well (CircaDB ) 14, 217 N umerous circadian gene knockout mice have been created and all of them develop a BP phenotype, highlighting the close relationship between the clock and BP regulation 18, 19, 61, 63, 65, 68, 69 However, all of thes e studies have focused only on male mice. Previously, we investigated the BP response of C57BL/6 male mice with global loss of PER1 on a HS diet in combination with DOCP a long acting aldosterone analog 218 Normal ly on high salt diet, aldosterone production is suppressed (reviewed in 172 ) so through the addition of DOCP, we are able to increase sodium reabsorption and thus create a model of hypertension similar to that seen in salt sensitive African Americans 219 with low renin yet high aldosterone 220, 221 In o rder to investigate sex differences in this model, we used the same experimental technique with female mice. In contrast to male mice on HS/DOCP, female Per1 KO mice maintain a normal circadian rhythm of BP and appear to be protected from the harmful non d ipping


93 phenotype seen in males. To our knowledge, this is the first investigation of this treatment in a clock gene KO model in female mice. Methods Mice Female WT and Per1 KO C57BL/6J mice between the ages of 14 24 weeks were used for these experimen ts (original heterozygous mice were obtained from Dr. Shin Yamazaki, Vanderbilt 173 ). Mice were housed as described in 218 and telemetry surgery was performed as described in 19, 174 After at least a week of recovery after surgery, mice were given control diet for 3 days and baseline recordings w ere done. They were then given 0% NaCl diet for 3 days, 4% NaCl (Harlan) diet for 3 more days, followed by injection with 75 g/g bw DOCP. HS/DOCP recordings were taken 3 days after injection. All animal use was in compliance with the American Physiologica Guiding Principles in the Care and Use of Laboratory Animals, and animal use protocols were approved by the Institutional Animal Care and Use Committees of the University of Florida and the North Florida/South Georgia Veterans Administration. T issue dissection and Real Time PCR Dissection and PCR were performed as previously described in 171 Flame Photometry Flame photometry was used to determine urine and plasma Na and K concentrations. Aldosterone ELISA Pl asma aldosterone was measured by ELISA as described in 96


94 Statistics Graphpad was used to perform t tests and 2 way ANOVAs. All values are presented as mean SEM. Results Figure 4 1 shows a representative tracing of mean arterial pressure recordings of the mice over 4 days of control diet. Time is given as Zeitgeber time (ZT), indicating lights on between ZT0 and ZT12 and lights off between ZT12 and ZT0 (grey bars). Both WT and KO mice display a normal circadian r hythm of BP with an increase in the night when they are active and a decrease during the day when mice are inactive. On control diet, Per1 KO mice have a MAP about 4 5 mmHg higher than WT mice. This is in contrast to male mice who exhibit little to no diff erence in MAP between WT and Per1 KO s at baseline 218 The mice were challenged with a series of different diets to test their ability to regulate BP with different salt loads. Mice on each diet maintained circadian rhythms similar to that shown in Figure 4 1 but for simplicity, the results in Figure 4 2 are given as an average of 3 days of diet shown as one cycle. KO mice maintain consistently higher MAP than WTs on all diets, but the difference between the two grou ps is amplified by HS/DOCP This figure also highlights the progressive increase in BP in both groups over the course of the experiment. In addition, it is important to note that both groups of female mice maintain a day time dip in BP unlike the male mic e 218 In order to quantify the results in Figure 2, MAP was calculated as day (ZT0 ZT11, 6am 5pm) and night (ZT12 ZT23, 6pm 5am) values for each diet. A similar trend of increasing MAP with increasing salt is seen in WT and KO mice. KO mice also have a consistently higher MAP than WT, averaging about 5 mmHg higher at all times. While


95 on control diet, the difference in MAP between WT and KO was 4 mmHg at night and on HS/DOCP this difference rose to about 7.5 mmHg at night. This again is in contrast to male mice who only show differences in BP on HS/DOCP diet 218 Cosinor analysis was done on MAP to investigate differences in the diurnal pattern of MAP on control, 0% NaCl, 4% N aCl and 4% NaCl + HS/DOCP There were no significant differences in mesor (the MAP hallway between the highest and lowest MAP recorded) or the amplitude (the difference between the highest and lowest MAP recorded) except for a small difference in amplitude on control diet between WT and Per1 KO mice (Table 1). This may be due in part to the fact that the animals were on a normal light/dark cycle and not in total darkness. Since there seemed to be little difference between how WT and PER1 KO mice react to th e different diets, we wanted to compare baseline characteristics of these mice to attempt to explain the higher MAP observed in KOs. We compared many effectors of BP between WT and KO mice (summarized in Table 4 1), focusing on parameters likely to be affe cted by changes in renal regulation. We chose to focus on the kidney because changes in BP that we do observe occur in parallel between diastolic and systolic measurements (Figure 4 4 ), suggesting a volume dependent mechanism which points to the kidney. Is olated increases in S BP suggest increased arterial stiffness 222 which leads to decreased D BP 223 Isolated changes in diastolic BP occur due to increased peripheral resistance 224 Thus, the parallel changes in SBD and DBP suggest increased blood volume, which is regulated by the kidney. No sig nificant differences in baseline plasma Na, K or aldosterone were observed at either noon or midnight time points. However, there was a trend of increased plasma aldosterone at noon for each


96 group though significance was not reached due to high animal to a nimal variability, following the trend of varying levels of aldosterone over the course of a day 96 64 There was also no difference in urine Na or K as well as mouse age o r hematocrit. There was a small but significant difference in mouse weight. To consider a mechanism, expression levels of genes involved in renal sodium handling were assessed Kidneys were obtained from WT and Per1 KO mice at noon, the midpoint of their i nactive period to match the experiments done in males. Kidneys were roughly dissected into cortex and medulla and isolated RNA was used to make cDNA for Real Time PCR analysis. Selected results are shown in Figure 4 5 In general, female Per1 KO mice show higher expression of renal genes associated with sodium reabsorption relative to WT females. Expression is higher in the cortex and medulla of the gamma subunit of the ENaC 5 A and D). The sodium potassium chloride cotransporter is more highly expressed in the cortex (Figure 4 5 B), but not medulla (Figure 4 5 E) of KOs. The Endothelin A (ETA) receptor and is also significantly upregulated in both cortex and medulla in KOs (Figure 4 5 C and F). There were no ndothelin 1 (in the medulla) or the ETB receptor, however (Figure 4 6). Discussion It is known that sex differences in BP and cardiovascular parameters exist but the cause for these differences is not well understood. Even less explored is the role of the circadian clock in cardiovascular sex differences. The aim of this study was to explore the role of a core circadian clock protein, PER1, in BP regulation disparities between male and female mice and compare the results to what we have previously observed in male mice. We found that while male mice on a HS/DOCP diet develop


97 harmful non dipping hypertension 218 females are protected from this phenotype and retain normal circadian BP rhythms. In contrast to the male p henotype, female Per1 KO mice maintain slightly higher MAP over a range of sodium intake compared to WTs. It has been well established that there are sex (used here to refer to the genetic/phenotypic contribution of sex chromosomes and hormones) differenc es in the prevalence of hypertension in humans with women having lower rates until the age of menopause (reviewed in 225 ) (with exceptions to this general rule between different races). Females of other species also have slightly lower BP than males including dogs 226 birds 227 and rats 228, 229 at baseline as well as experimental rodent models of hypertension 230 234 though some studies do show similar baseline BP 230 235 236 In general, females are protected compared to males with respect to cardiovascular morbidities. In animal models thes e include but are not limited to the response to Angiotensin II 230, 232, 237 239 DOCA salt (similar to DOCP) 236, 240 242 and excretion of a salt load 235 In humans, male sex generally increases the risk for cardiovascular disease, chronic kidney disease and hypertension 168 It has also been suggested that postmenopausal women have a higher i ncidence of non dipping 243 and this may be lessened by the use of hormone replacement therapy 24 4 The role of hormones, specifically estradiol and progesterone, has been investigated as a source of reported sex differences. In addition to different expression patterns between sexes of ETA R and ETB R in the nephron, it has been shown that ENaC is reg ulated by estrogen and progesterone 217, 245 Ovariectomy reduced ETA R and ETB R expression in kidney cortex and this result was abolished by supplementation with estrogen 246 In addition, salt sensitiv ity was shown to be increased


98 by ovariectomy in spontaneously hypertensive and dahl s alt sensitive rats 247, 248 Many more instances have been demonstrated and are reviewed in great detail in 225 Although it is tempting to specul ate that ovarian hormones are the underlying mechanisms of protection, a limitation of our study is that we did not perform ovariectomy. This is a likely future direction of this line of research Our lab is particularly interested in the first homolog of P eriod, PER1, in the kidney. This was initiated by the discovery that the sodium and BP regulating hormone aldosterone regulates PER1 expression 78 Subsequent work in our lab and that of collaborators solidified t he role of PER1 in the kidney by showing that it regulates 20 Endothelin 1 19 which acts to promote sodium excretion 87 NCC 16 and NHE3 171 PER1 global KO mice on a 129/sv background have a mean arterial BP 18mmHg lower than WT controls, at least in part likely due to reduced plasma aldo sterone 96 as well as reduced expression of the sodium channels and transporters mentioned above. Conversely PER1 global KO mice on a C57BL/6J background have slightly increased BP compared to WTs. Although our und erstanding of the role of background strain genetics is limited in rodent cardiovascular studies, there are reports of differences in diabetic nephropathy 185 baseline BP 181 susceptibility to renal damage 184 and salt sensitivity 182 183 If we think in terms of each mouse strain being representative of a different human being and realize that SNPs, environment, genetics and many other factors influence the different responses to stimuli and disease seen in hum ans, it follows that two mouse strains would react differently to the same to the same treatment.


99 One of the predominant regulators of BP is Endothelin 1 which signals through A and B receptors. In the kidney, ETA receptors promote sodium reabsorption whi le ETB receptors lead to sodium excretion through the blockade of ENaC 85, 249 Since the ratio of ETA R /ETB R is higher in males 250 suggesting sex specific regulation and the Endothelin axis is regulated by PER1 88 we hypothesized that this could be a contributing factor to the higher MAP in KOs. Indeed, the female KO mice do have higher expression of ETA in the cortex and medulla of the kidney as measured by r eal time PCR. Higher expression of ENaC and NKCC2 can also help explain the increased BP through increased sodium reabsorption. Further experiments are needed to delineate a functional role for these transporters and signaling pathways. The fact that there is a much less dramatic phenotype seen in females compared to male Per1 KO mice indicates that between sexes, PER1 may have different roles or be regulated differently. Our lab has previously demonstrated the important role of PER1 in the regulation of B P and renal sodium handling 16, 19, 80, 96, 171, 180 However, these studies were conducted in male mice exclusively. This is the first demonstration of a BP phenotype in female mice lacking a core circadian clock g ene. That they are protected from non dipping hypertension relative to male Per1 KO mice is consistent with human literature that pre menopausal women are less likely to exhibit non dipping hypertension. This is an important first step in understanding sex differences in the regulation of cardiovascular function by the circadian clock. Future studies are clearly needed to ascertain the precise role of sex hormones and the kidney specific role of PER1 in BP regulation.

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100 Figure 4 1. Circadian Rhythm of BP on Cont rol Diet Recordings of mean arterial pressure from implanted telemetry devices in WT and Per1 KO female mice are shown. Time on the x axis is presented as Zeitgeber time, denoting the time of lights on (ZT0, white bars) and lights off (ZT12, grey bars). B oth WT and Per1 KO mice maintain a normal circadian rhythm of BP on control diet, with MAP peaking at night when mice are active. n=6WT, 4KO. Figure 4 2. Mean Arterial Pressure Averaged Over Multiple Days of Diets. Three days of diet are shown as an a verage of one cycle starting at midnight (ZT18). MAP is consistently higher in the KOs (red lines) compared to WTs but each group shows an increase in MAP with increasing salt and the addition of DOCP. n=6WT, 4KO.

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101 Figure 4 3. Average MAP for All Diet s. MAP was calculated for the day (ZT0 ZT11, 6am 5pm) and night (ZT12 ZT23, 6pm 5am) periods on the different diets. In all groups and diets, MAP is lower during the inactive period when lights are on. There is also a trend of increasing MAP from control t o 0% NaCl to 4% NaCl to HS/DOCP diet in both WT and KO animals. n=6WT, 4KO. ***p Figure 4 4. Changes in DBP and SBP on Control and HS/DOCP Diets. Parallel changes can be seen in DBP an d SBP in both genotypes and on control and HS/DOCP diets, suggesting a volume dependent mechanism of BP changes. Percentage dip is given next to each corresponding line.

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102 Figure 4 5. Real Time PCR Expression of Selected Genes in Kidney Cortex and Medu lla. Real time PCR expression of selected renal genes involved in sodium transport is shown. KO mice have higher expression of genes responsible for sodium reabsorption including the gamma subunit of the ENaC in the cortex and medulla (A and D), the sodium potassium chloride cotransporter in the cortex (B) but not the medulla (E) and the Endothelin A

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103 Figure 4 6. Additional Real Time PCR Expression of Selected Genes in Kidney Cortex and Medulla. Real time PCR expression of selected renal genes involved in sodium transport is shown. There were no differences observed in EDN1 expression in medulla (D), the Endothelin B receptor in cortex and medulla (B and E) or compared to WT.

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104 Table 4 1. Cosinor a na lysis of m ean a rterial BP on d ifferent d iets Genotype Diet Mesor Amplitude WT Control 103.78 3.17 11.4 0.4 KO Control 108.93 0.86 9.67 0.58 WT 0% NaCl 103.63 3.05 11.61 0.51 KO 0% NaCl 109.63 0.95 12.5 0.95 WT 4% NaCl 106.67 3.88 13.03 0.71 KO 4% NaCl 111.56 1.61 13.48 1.05 WT 4% NaCl & DOCP 109.13 4.2 8.64 0.59 KO 4% NaCl & DOCP 114.82 0.78 8.3 0.64 Cosinor analysis was done to determine changes in circadian parameters of MAP. Mesor is the MAP halfway between the highest and lowest MAP recorded. Amplitude is the difference between the highest and lowest MAP recorded. n=6 WT, 4 PER1 KO. test. Table 4 2 Baseline Characteristics of WT and KO Mice. WT KO Plasma Na (noon)(mM) 157.33 2 .38 (6) 152.23 2.09 (6) Plasma Na (midnight) (mM) 163.54 3.78 (6) 155.88 2.77 (6) Plasma K (noon) (mM) 4.49 0.25 (6) 4.30 0.13 (6) Plasma K (midnight) (mM) 4.18 0.19 (6) 4.56 0.24 (6) Urine Na, 48h average (mEq/g BW) 0.01233 0.00122 (12 ) 0.01282 0.00049 (12) Urine K 48h average (mEq/g BW) 0.01834 .000292 (12) 0.01934 0.000412 (12) Weight (g) 21.72 0.38 (12) 20.31 0.43* (12) Age (weeks) 16.5 0.90 (12) 17.24 1.02 (12) Hematocrit 41.5 0.59 (12) 42.46 0.90 (12) Plasma Aldosterone (pg/ul) (noon) 504.05 46.78 (5) 530.88 40.61 (6) Plasma Aldosterone (pg/ul) (midnight) 417.76 79.02 (6) 390.00 109.46 (6) test.

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105 CHAPTER 5 IDENTIFICATION AND INITIAL CHARACTERIZATION OF A lncRNA ANTISENSE TO THE EDN1 GENE: EDN1 AS Background Endothelin 1 (ET 1, encoded by the Edn1 gene) is one of the most potent vasoconstrictors known 251 When first translated, prepro Endothelin 1 is a 212 amino acid protein that gets progressively cleaved to a 21 amino acid, mature ET 1 peptide (reviewed in 252 ). Though each of the cleavage steps are likely regulated, the major regulation of Endothelin 1 occurs at a transcriptional level (reviewed in 253 ). ET 1 binds to two different receptors (ETAR and ETBR) which are expressed by almost every cell in the body and generally produce opposite effects (reviewed in 252 ). For example, stimulation of ETAR produces vasoconstriction, kidney damage and increased oxidative stress 254, 255 (reviewed in 256 ) while ETBR stimulation generally promotes BP lowering and antifibrotic effects (reviewed in 252 ). These two receptors also bind the two other isoforms of Endothelin, Endothelin 2 and 3, but these isoforms are not as abundant or widespread as ET 1 257 The inner medulla of th e kidney is the site of the highest levels of ET 1 production in the body 258 and almost every cell in the kidney synthesizes ET 1 and contains ET receptors (reviewed in 252 ). The renal vasculatur e is also highly sensitive to the actions of ET 1 259 While ET 1 is generally viewed as a vasoconstrictor, in the kidney tubule ET 1 has natriuretic effects, promoting the excretion of sodium and subsequent fall in blood volume and BP The natriuretic effec ts of ET 1 appear to be perpetuated via ETBR and possibly ETAR and subsequent blockade of ENaC open probability 85, 87, 260 through a mechanism likely involving nitric oxide 2 61 Localization of

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106 ETBR to the basolateral membrane of tubular cells in the kidney suggest an autocrine or paracrine mechanism of regulation by ET 1 in the CD 252 Due to the almost ubiquitous expression of ET 1 throughout the body, dysregulation of ET 1 has been implicated in the etiology of numerous diseases including cancer (reviewed in 262 ), hypertension 263, 264 and chronic kidney disease (CKD) 265 ET receptor antagonists have been used in the treatment of pulmonary arterial hypertension 266 a life threatening disease that involves aberrant ET actions. However, there are not many successful treatments targeted at the kidney. A small clinical trial of CKD patients showed benefits of ETAR block ade 267 However in diabetic CKD patients, this was harmful 268 The ASCEND trial was prematurely terminated due to increased congestive heart failure outcomes in patients wi th type II diabetes taking an ETAR blocker (Avosentan) compared to placebo groups 268 With estimates of over 10% of the populations of the United States and Europe diagnosed with CKD 269 better therapies are needed. Since the renal ET system is activated in almost all CKD cases 256 it represents a promisin g target. Indeed, a number of human clinical studies suggest the possibility that ETAR antagonists in combination with other therapies may be beneficial in the treatment of CKD, with careful dosing and mindfulness of patient populations with increased like lihood of fluid retention 256 Our lab has recently discovered a long, non coding RNA (lncRNA) antisense to the EDN1 gene, providing a potential new target in the endothelin axis for therapeutic treatment. lncRNAs are a relatively new class of RNAs that have been implicated in gene regulation. These >200 bp species arise from parts of the genome that does not code

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107 DNA 270 However, largely due to the ENCODE Project 271 it is now clear that this DNA is anything but junk. lncRNAs have been shown to regulate many aspects of the genome with perhaps one of the most well known being Xist. Xist is the lncRNA responsible for female X chromosome inactivation and is itself regulated by a ncRNA transcribed antisense to itself, Tsix 272 Many examples of lncRNAs are k nown and have been shown to possess a vast assortment of regulatory methods including epigenetic regulation 273 trafficking and transcription (reviewed in 274 ). While most lncRNAs regulate their targets in a negative manner, t here are instances of positive regulation as well (reviewed in 275 ). It has also been suggested that RN As could provide a bridge for the interaction of histone modifying enzymes and DNA 275 since histone mo difying enzymes lack DNA binding domains 276 A specific class of lncRNAs, antisense lncRNAs, are transcribed from the opposite strand of DNA as an mRNA and thus are capable of regulating their complimentary strand of DNA or mRNA (reviewed in 277 ). Antisense lncRNAs can also regulate other genes and transcripts as well depending on post translation al processing. When acting in a cis manner, antisense lncRNAs regulate genes close to their site of synthesis, as in the case of Xist which is transcribed from the soon to be inactivated X chromosome 278 COOLAIR is an antisense lncRNA which silences its sense gene (FLC) through polycomb complex recruitment in response to cold stimulus in Arabidopsis 279 When acting in trans antisense RNAs act on distal genes and can regulat e many targets due to imperfect complementarity 280 HOTAIR is the first lncRNA discovered to regulate gene expression in this manner. HOTAIR downregulates HOXD locus genes while it is actually transcribed in the HOXC gene locus. It has also been implicated in

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108 regulation of a number of genes related to cell cycle progression and metastases and therefore has also been implicated in cancer etiology (reviewed in 281 ) Experiments to verify the existence of EDN1 AS and start initial characterization are detailed in this chapter. EDN1 AS was shown to be expressed in multiple cell lines of mouse and human origin as well as authentic human kidney RNA. Preliminary experime nts suggest that EDN1 AS is expressed in a time dependent manner which may be due in part to Per1 residence in the promoter region of END1 AS as shown by chromatin immunoprecipitation (ChIP). Further studies using CRISPR/Cas9 to delete regions of the promo ter region of END1 AS have resulted in heterozygous knockout (KO) mIMCD3 and HK 2 cell lines and 2 homozygous KO HK 2 lines. One ET 1 ELISA of media from these cells did not show significant differences in ET 1 levels from WT cells. Experiments using a dif ferent method of CRISPR/Cas9 KO will be undertaken in the near future. Methods Exploration of EDN1 Chromatin State and Regulation with the UCSC Genome Browser Human EDN1 chromatin state was assesse d using the UCSC Genome Browser 282 at Using primarily the Genome Segments, Transcription Factor ChIP, Layered H3K2Ac, DNase Clusters, Broad Chrom HMM and Broad Histone Modification tracks, it was obser ved that there was open chromatin with multiple indicated to be a predicted promoter region with high confidence by the genome browser (Figure 5 1). This ~800 bp region was term AS. Further investigation into the promoter region showed binding of multiple

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109 transcription factors that suggest active transcription and regulation including TFIID, USF1 and GR (Figure 5 2). Strand Specific Primer Design H uman Strand Specific primers (SS primers) were designed by two previous lab members (Lauren Jeffers and Dr. Sarah Barilovits). SS Primers (LJ3, LJ4, LJ5) were (illustrated in Figure 3) for use in reverse transcriptase reactions. Oligo dT primers were used as a positive control for any polyA tailed mRNA. Primers have a complimentary sequence to the EDN1 sense strand so they will only anneal to antisense RNA. PCR primers (LJ7 and LJ8) were designed to amplify the same region of cDNA regardless of the SS Primer used. Mouse SS Primers were designed by the same method and in roughly homologous locations (e.g. mEDN1 AS3 lays down in a homologous location to LJ3). RNA Isolation and DNase Treatment RNA was isolated from cells and tissue using Trizol (Ambion) per manufacturer instructions. In general, 1ml Trizol was used per well in a 6 well plate. 0.5 ml Trizol was used for inner medulla tissue, 1ml for outer medulla and 2 3 ml for co rtex tissue. Tissue was homogenized by hand in Trizol and processed to total RNA (tRNA). Tissue and cells were treated with DNase (Ambion) per manufacturer instructions. cDNA Conversion and PCR Reverse Transcriptase (RT) reactions were carried out using a kit from Thermo Fisher. Random hexamers were used as primers to make cDNA (final concentration 4ng/l) to assess EDN1 mRNA levels. SS Primers were used +RT and RT to assess EDN1 AS levels (+RT) and DNA contamination ( RT). Any sample which had a band in

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110 RT lanes was considered contaminated and not used. Preliminary experiments used all SS Primers but subsequent experiments to compare multiple samples used LJ4 or mEDN1AS4 as SS Primers. Most RT reactions were brought down to 5 l total volume due to the pr ecious nature of the RNA. LJ7 and LJ8 (or mouse equivalent) were used in a PCR reaction for all samples including random hexamer RT samples. PCR was done at 35 cycles for all reactions to better visualize bands, however PCR using 30 cycles still showed the presence of EDN1 AS. Real time PCR for EDN1 was done using TaqMan probes and mastermix. Cell Culture mIMCD3 FBS and 0.5% Gentamycin. Cells were passaged when they reached 80 100% confluenc e. HK 2 10% FBS and 1% Penicillin/Streptomycin (and after 7/2017 with 0.5% Gentamycin). Cells were passaged when they reached about 80% confluence. Aldosterone Treatment mIMCD3 cells were trea ted with 100 n M aldosterone for 4 hours in 6 well dishes in 3 separate experiments. RNA was then isolated and SS RT PCR was done to check antisense expression. Dexamethasone Treatment HK 2 cells were grown to ~80% confluence in 6 well cell culture plates. 100 nM Dexamethasone was added for 30 minutes and then media was changed. This allows the circadian clock to be synchronized in all cells (Figure 5 4). After 24 hours, cells were trypsinized and RNA was isolated every 2 hours for 24 hours.

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111 Chromatin Immuno precipitation Chromatin Immunoprecipitation (ChIP) was done using the Active Motif ChIP Kit following manufacturer instructions. Mice Male and Female 129sv/s1s4 and Male 129sv/s2 mice were housed in normal 12:12 L:D conditions and fed a normal diet with ad libitum access. Animals were euthanized with 5% isoflurane and cardiac puncture. Kidneys were collected and flash frozen in liquid nitrogen. All animal use was in compliance with the American Physiological of Laboratory Animals, and animal use protocols were approved by the Institutional Animal Care and Use Committees of the University of Florida and the North Florida/South Georgia Veterans Administration. CRISPR/Cas9 gRNA Design Guide RNAs (gRNAs) were des ign ed using the online tool CC Top 283 from https://crispr.cos.uni A sequence of ~150 bp of the promoter region of (mouse or human) EDN1 AS was used as input and the program produced m ultiple possible gRNAs. The region was selected based on identified (in human, Figure 5 5A) or predicted (in mouse, Figure 5 5B) transcription factor binding sites hypothesized to be important to initiation of transcription. gRNAs were selected based on lo cation and predicted off target sites. If possible, only gRNAs with no off target sites or those with sites only in regions between genes were chosen. None were used if the gRNA had predicted off target sites in exonic or intronic regions of any gene to mi nimize possible off target effects. Originally gRNAs were designed to remove ~100 bp of sequence but all combinations were used in the end.

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112 Creation of the gRNA Plasmid gRNA design from CC Top included an overhang for insertion into the plasmid. Cloning wa s carried out according to the pro tocol provided by the Zhang Lab 284, 285 Briefly, the LentiCRISPR v2.0 plasmid (a gift from Dr. Kevin Brown (Addgene plasmid # 52961)) was digested with BsmbI to remove a 2 kb fragm ent with overhangs complimentary to the sites added to the gRNAs. The larger fragment was gel purified using the Qiagen kit and was ligated to phosphorylated and annealed gRNA oligos with Quick Ligase (NEB). AT NO POINT IS CIP (Calf Intestinal Phosphatase) TO BE USED. The ligated plasmid and gRNA was transformed into Stbl3 bacteria following manufacturer instructions. Puromycin Kill Curve Kill curves were obtained for HK 2 and mIMCD3 cells with puromycin. Initially, 80 100% confluent cells in a 24 well plat e were subjected to puromycin concentrations of 0 g/ml, 0.25 g/ml, 0.5 g/ml, 1 g/ml, 3 g/ml, 5 g/ml, 8 g/ml and 10 g/ml in triplicate. This was subsequently narrowed down to a range of 0 3 g/ml to confirm that 2 g/ml puromycin was an optimal conc entration, killing all cells after 72 hours but not before 48h. Creation of Virus and Infection of Cells With the gracious help of Dr. Kevin Brown, HEK293 cells were transfected with the LentiCRISPR + gRNA 1, 2, 3, and 4 plasmids in addition to pCMV VSV G (Addgene plasmid # 8454) for viral envelope proteins and psPAX2 (Addgene plasmid # 12260) for lentiviral packaging. After sufficient time, media containing virus particles with the LentiCRISPR plasmids encapsulated was collected and spun down to remove ce ll debris. The respective virus containing human or mouse gRNA/LentiCRISPR plasmid

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113 was then added onto HK 2 and mIMCD3 cells in every combination (e.g. gRNA 1+2, 1+3, 1+4 etc.). 2 g/ml puromycin was used for both lines to select for cells successfully in fected with virus. 96 well cloning First attempts to clone CRISPR cells were done in 96 well plates. Cells were plated into the first of 4 columns of a 96 well plate in 200 l of media. 20 l of cells were then diluted using a multichannel pipette into the 3 subsequent columns of wells containing 180 l of media in order to have single cells in the third and fourth columns. After one to two weeks, when media started to indicate acid build up in columns 1 and 2, wells were inspected for the presence of one t o three colonies of cells (illustrated in Figure 5 6). These wells were then trypsinized with 1 drop of 0.25% Trypsin per well and plated into a 6 well plate. Then gDNA was isolated using the Zymo gDNA isolation spin kit and cells were genotyped. Depending on the genotyping results, cells were replated into 96 well plates and the process was repeated to attempt to obtain homozygous cell lines. 10 cm dish cloning When 96 well cloning did not result in homozygous cell lines (either due to mixed cell populatio ns or heterozygous KO of single cells), cloning was attempted in 10 cm dishes. Cells were plated in order to obtain a plate with ~100 cells total. These were allowed to grow for over a week undisturbed. Colonies were observed as small cloudy points on the bottom of the plate. They were marked with a dot and then examined under the microscope. If it was clear to be one colony sufficiently separated from others, colonies were removed from the plate. Cloning rings were dipped in vacuum grease to create a seal and pressed down over the colony. Media was aspirated and 1 drop of Trypsin was added. After ~5 minutes, trypsinized cells were removed and plated in 6 well plated for

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114 genotyping. Depending on genotyping results, cells were replated and the process repeate d to obtain homogenous cell lines. Nomenclature Cell line names were derived based on the well they were removed from in a 96 well plate and the gRNAs used. For example, if a cell line is named 23A8A11, these cells were subjected to gRNAs 2 and 3 and clone s were isolated first from well A8 and a subsequent round of cloning produced a cell colony from well A11. Note: after sequencing, many of the gRNA combinations noted in the cell line name appear to be in error. However, to avoid even more confusion, names remain unchanged. CRISPR Genotyping Primer Design Genotyping primers were designed using Primer3 software 286 Initially, two sets of p rimers were designed to amplify the region containing hEDN1AS CRISPR 1 and 2 (hCRISPRpcr1F/R) and hEDN1AS CRISPR 3 and 4 (hCRISPRpcr2F/R). Subsequently, hCRISPRpcr1F and hCRISPRpcr2R were used for genotyping because they worked better. WT/ Het/ KO cells we re determined by subtracting the calculated Cas9 cut size from the WT amplicon size (Table 5 1). Sequencing Gel purified PCR products from hCRISPRpcr1F and hCRISPRpcr2R amplification were sent to GENEWIZ for sequencing and analyzed with the help of Dr. Kev in Brown. Mycoplasma Decontamination and Detection Mycoplasma was detected with the VenorGeM OneStep PCR Kit (Minerva Biolabs) following manufacturer instructions. gDNA as well as cell culture media was used in reactions. Mycoplasma was successfully elimi nated from infected cell lines ( HK 2 23G7H3 and 23A8A11) using Mynox Gold (Minerva Biolabs).

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115 ET 1 ELISA Media from HK 2 and mIMCD3 cell lines was taken, spun down to remove cell debris and then frozen at 80 C until all samples were ready to use. When re ady, media was thawed and concentrated using a speed vac on medium heat setting (no heat re froze the samples). ELISA for ET 1 was done with the Human Endothelin 1 QuantiGlo ELISA Kit (R&D Systems) according to manufacturer instructions. Luminescence was a ssessed using the Synergy HT Microplate reader with the following settings: emission through hole in filter wheel (no set wavelength), 1 min lag time (time after plate is inserted into reader), 0.5 sec integration time (time each well is measured) and auto matic gain. Summation mode is recommended in the kit instructions but not available on the Synergy. R&D customer service said it is not critical. Results In order to confirm the presence of EDN1 AS predicted from the genome browser, strand specific reverse transcriptase followed by PCR (SS RT PCR) was done on multiple cell lines (Figure 5 7). DNase treated RNA was used as a template in reverse transcriptase (RT) reactions using strand specific (SS) primers (LJ3, LJ4, LJ5, SB1, SB2 and SB3). As a control for DNA contamination, each sample was also ran without RT. This cDNA was then amplified with PCR primers (LJ7 and LJ8). Absence of bands in RT lanes confirms that bands in +RT lanes are due to EDN1 AS and not DNA contamination. EDN1 AS expression was shown in s9 (human bronchoendothelial) cells (Figure 5 7A), HMEC (human primary mammary endothelial cells) (Figure 5 7B), HK 2 (human kidney PT epithelial cells) (Figure 5 7C) and total human kidney RNA (Figure 5 7D).

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116 Since all of the samples shown to express ED N1 AS were of human origin, we investigated the expression of EDN1 AS in mice. Kidneys from 129sv/s2 mice sacrificed at midnight were dissected into cortex and medulla and processed to cDNA with mouse SS primers in the same way as human samples. As shown i n Figure 5 8, EDN1 AS is expressed in mouse cortex (Figure 5 8A) and outer medulla (Figure 5 8B). Even though the gel cannot be quantified, it does appear that expression of EDN1 AS is more highly expressed in outer medulla compared to cortex. We also chec ked the aorta of C57BL/6J mice on control and HS/DOCP diet and found that while there were multiple bands in RT lanes, two mice did not show bands in RT yet there were bands in the +RT lanes, confirming expression of EDN1 AS in aorta as well (Figure 5 9) Next, we wanted to investigate sex differences in expression. Following the same protocol for the 129sv/s2 mice, male and female kidneys from 129sv/s1s4 mice were examined for EDN1 AS expression (Figure 5 10). EDN1 AS expression was detected but was much more variable than in the 129sv/s2 strain. Female estrus cycle was not synchronized, possibly accounting for variation as well. Since Edn1 is regulated by aldosterone, we wanted to see if the same was true of EDN1 AS. We used a model of mouse inner medull ary CD cells (m mIMCD3 ) treated with 100 nM aldosterone in three separate experiments (Figure 5 11). It appears that expression varies from passage to passage but within passages, EDN1 AS expression is higher in aldosterone treated samples compared to vehic le (100% ethanol). Edn1 is also regulated by the circadian clock. In order to see if EDN1 AS is similarly regulated, we treated human kidney PT cells ( HK 2 ) with 100 nM dexamethasone for 30 minutes to synchronize their circadian clocks (Figure 5 12). 24

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117 ho urs later, RNA was isolated every 2 hours for 24 hours and SS RT PCR was done. In addition to using SS primers, random hexamers were used in RT reactions to assess EDN1 expression. Figure 5 11A shows that EDN1 AS expression varies over 24 hours and is in p hase with EDN1 expression as measured by real time PCR (curve overlay on panel A). GAPDH expression as measured by semi quantitative PCR did not change. This is a preliminary experiment with n=1. To determine if the mineralocorticoid and circadian regulati on was occurring at the expected promoter region of EDN1 AS, chromatin immunoprecipitation was done on an area of the promoter shown to have glucocorticoid and clock binding sites (Figure 5 13, experiment done by Dr. J. Richards of the Cain lab). Indeed, i n a preliminary experiment, Per1 and the glucocorticoid receptor were shown to bind at the promoter region. Since EDN1 AS expression is conserved and appears to be regulated in multiple ways, we wanted to investigate the regulatory effect EDN1 AS has on ED N1. In order to accomplish this, CRISPR/Cas9 was used to delete parts of the EDN1 AS promoter region selected based on shown (in human) or predicted (in mouse) binding of important transcription factors. With the aid of Dr. Kevin Brown, HEK293 cells were t ransfected with plasmids containing guide RNAs for Cas9 as well as viral assembly proteins. The viruses were then used to infect HK 2 and mIMCD3 cells as human and mouse models, respectively. After multiple rounds of cloning, genotyping and sequencing, it was determined that all mIMCD3 clones were either heterozygous cell populations or had heterozygous deletion in individual cells. HK 2 cells comprised a mix of homozygous KO, heterozygous KO and heterozygous cell populations. A

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118 representative genotyping ge l is shown in Figure 5 14 with examples of A) heterozygous KO, B) WT and C) homozygous KO HK 2 cells as well as D) mIMCD3 cells, displaying bands of multiple sizes most likely due to heterozygous cell populations of heterozygous KOs. After almost a year of cloning and genotyping, two HK 2 cell lines were genotyped as homozygous KOs and this was confirmed by sequencing. These cells along with six different mIMCD3 lines including WT, CRISPR WT and heterozygous lines were grown and media was taken to use in an ET 1 ELISA (Figure 5 15). The preliminary data appears to show increased ET 1 levels in CRISPR cells, however, all of the high data points are from media collected after 3 days compared to the other points which were taken after 1 day on the cells. Thus, more experiments are required to determine if changes are due to promoter disruption or simply the time that media was on the cells. Discussion The work summarized here has shed light on a possible new mechanism of EDN1 regulation EDN1 AS. We have show n that this antisense transcript is expressed in multiple human cell lines with apparent differences in expression level (measured by semi quantitative PCR) depending on the origin of the cells. In addition, EDN1 AS is expressed in mouse kidney and aorta, again with seemingly different levels of expression based on location. Preliminary experiments indicate that expression of EDN1 AS is regulated by the circadian clock and aldosterone and this is further supported by preliminary ChIP data showing the presen ce of Per1 protein and the glucocorticoid receptor at the EDN1 AS promoter region. Studies of EDN1 AS promoter activity KO in cell lines have resulted in ambiguous data so far. However, the presence

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119 of EDN1 AS in human and mouse as well as its apparent reg ulation by multiple means are highly suggestive of the importance of this transcript. Endothelin 1 is an integral part of normal development throughout the entire body (reviewed in 253 ). Dysregulation of Endothelin 1 and its receptors has been i mplicated in many disease states including diabetic nephropathy 287 pulmonary arterial hypertension (PAH) 288 preeclampsia 289, 290 atherosclerosis 291, 292 cancer 293 and coronary artery disease 294 Endothelin receptor antagonists have not had great success at treating various diseases in which ET 1 is implicated. ETA receptor antagonists have shown some efficacy in treating PAH, howev er it seems that often the side effects are worse than the treatment (reviewed in 295 ). ET 1 plays a role in the regulation of almost every part of the kidney including the renal vasculature, glomerular mesangial ce ll proliferation, matrix production and contraction, nerve activity, acid/base handling and sodium and water transport in the nephron (reviewed in 296 ). Increa sed levels of ET 1 have been correlated with chronic kidney disease, diabetic nephropathy as well as increased renal cell proliferation and inflammation (reviewed in 297 ). While ETA receptor antagonists appear to be useful in opposing the unwelcome actions of ET 1 298, 299 none are yet approved for use in humans to treat any of the conditions in the kidney. Our recent discovery of a lncRNA antisense to the Endothelin 1 gene (EDN1 AS) may provide another route of treatment and regul ation of ET 1 levels. Antisense lncRNAs can regulate genes in a discordant manner, negatively regulating expression of their sense RNA, or in a concordant manner, leading to increased levels of corresponding sense RNA 300 It appears that most antisense regulation is of the

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120 discordant category 301 Many natural an tisense transcripts (NATs) have been implicated in diseases including breast cancer 302 renal cancer 303 schizophrenia 304 305 leukemia 306 307 to name a few. It has also been demonstrated that targeting antisense transcripts with strand specif ic oligonucleotides can be therapeutically beneficial. Wahlestedt et al. were the first group to demonstrate this in vivo 308 This group investigated Brain Derived Neurotrophic Factor (BDNF) which is responsible for neuronal growth, differentiation and memory and is regulated by a NAT (BDNF AS). They infused AntagoNATs (small oligonucleotides which antagonize sense antisense interaction) by minipump into mice and found that after 28 days BDNF mRNA and protein levels were increased 50 100%. This lays the foundation for the proposal that EDN1 AS may also be a practical target for disease therapy in the future. Since lncRNAs also seem to be expressed and regulated in a tissue specific manner 301 tissue specific delivery could decrease the risk of side effects compared to chemical compounds. Preliminary data suggests that EDN1 AS expression does vary b etween tissue (outer medulla and cortex of kidney) as well as between mouse strain (129sv/s2and 129sv/s1s4). Additionally, antisense transcripts tend to be low abundance and are not as extensively spliced as sense transcripts 309 EDN1 AS again seems to fall into this category based on the fact that EDN1 AS is difficult to amplify, especially with RNA that is over 2 weeks old, but when it is it is amplified regardless of which SS primer is used. Thus, it appears that EDN1 AS is likely UTR of EDN1) and is not extensively spliced or processed, but further ex periments are needed to confirm this.

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121 Further experiments are also needed to investigate sex differences in EDN1 AS expression and regulation. We know that EDN1 AS and EDN1 are both regulated by the clock and expression is in phase with each other and that both are positively regulated by aldosterone. Thus, it follows that since EDN1 expression is different between the sexes, the expression of EDN1 AS would differ as well. Preliminary experiments concur with this prediction, showing that in one experiment f emale mice seem to have higher expression levels of EDN1 AS in the kidney compared to males. Tantalizingly, the estrous cycle of the females was not synchronized, which could explain the high variability of expression between female mice. ET 1 protein as w ell as EDN1, EDNRA and EDNRB mRNA levels vary in corpus luteum over the course of the bovine estrous cycle 310 In two a small studies of 10 and 16 participants, ET 1 plasma levels were shown to vary over the course of the menstrual cycle in humans 311, 312 Another possibility is that EDN1 AS is not a lncRNA but an enhancer RNA (eRNA). eRNAs are transcripts produced from enhancers but their role is not yet clear (reviewed in 313 ). The first evidence of eRNAs was produced about 25 years ago while investigating the globin locus control region 314, 315 However, eRNAs were not named until 2010 with two papers confirming the production of transcripts from enhancers 316, 317 Thus, since the idea of an eRNA is relatively young, it is not clear what their function is. Li et al. have proposed three possible classes of eRNAs: those which are transcriptional noise, having no function, those which function to recruit transcription machinery to aid in transcription of other genes or those which actually act in cis or trans as an RNA to regulate other genes 313 The authors have also listed a few ways to differentiate lncRNas from eRNAs including the presence of H3K36me3 (not present at

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122 eRNA but present at lncRNA) and whether splicing has occurred (it is uncommon with eRNAs howeve r lncRNAs have a bias for containing 2 exons). However, unpublished data which I was able to see recently suggests that EDN1 AS is actually a long, intergenic non coding RNA (lincRNA) which starts thousands of bases further downstream than we thought and t hus the area we have been targeting with CRISPR was very incorrect. No one can deny the importance of Endothelin 1 as evidenced by its ubiquitous expression and conservation across species. What is also undeniable is the implication of ET 1 in many diseas es, most of which cannot be adequately treated by targeting Endothelin 1 binding to its receptors EDN1 AS provides a new possibility for treatment and although it is not well characterized yet, the data we have gathered shows promise for EDN1 AS an import ant regulator of EDN1. Expression in multiple cell lines, authentic human kidney RNA as well as mice in addition to multiple means or regulation and possible sex dependent changes in expression all suggest that this transcript is important and worth the tr ouble of conserving over the course of evolution Only future studies will be able to address this prediction.

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123 Figure 5 1. The UCSC genome browser predicted chromatin state of EDN1 in HUVEC (human umbilical vein endothelial cells). There is a predic ted promoter at the end of the EDN1 sense transcript. Figure 5 2. Transcription Factor Binding Sites at the EDN1 AS Predicted Promoter Region. These transcription factor binding sites were shown by ChIP assay. The cluster of DNase sensitivity as wel l as confirmed binding of many transcription factors known to be important in transcription suggest a Dr. J. R. Richards.

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124 Figure 5 3. Diagram of strand specific primer placem ent. Strand specific primer used to identify EDN1 AS is illustrated here (not to scale). Each primer from LJ3 to the same piece of cDNA regardless of the strand specific primer us ed. Therefore, all bands in the PCR gel are the same size though they represent different sizes of EDN1 AS detected. Figure 5 4. Dexamethasone Treatment of HK 2 Cells and RNA Isolation. HK 2 cells were plated in 6 well plates and grown to 80% confluen cy. Dexamethasone was added to media and removed after 30 minutes. 24 hours later, when cells were synchronized, RNA was isolated every 2 hours for 24 hours and used to make cDNA for later experiments.

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125 Figure 5 5. Rationalization for gRNA Design in H uman EDN1. CRISPR gRNAs were designed in order to attempt to remove important transcription factor binding sites from the promoter region. Two gRNAs were spaced about 100bp apart. A) Human sites were confirmed by ChIP assay and obtained from ENCODE data th rough the UCSC Genome Browser. B) Mouse sites are predicted and were obtained from ENCODE data through the UCSC Genome Browser.

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126 Figure 5 6. 96 well Cloning Method for CRISPR/Cas9 Knockout Cells. Multiple cells ated along a column of a 96 well plate in 200 l of media. 20 l was taken and diluted 1:10 in 3 subsequent columns, producing a final dilution of 1:1000. Using media color as an indication of growth, once the first one or 2 columns showed significant acid ification, columns 3 and 4 were observed for colony growth. Individual wells with one to three colonies were isolated with trypsin and replated in a new 96 well plate to attempt to homogenize populations.

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127 Figure 5 7. EDN1 AS is expressed in several h uman cell lines and in human kidney. EDN1 AS is expressed in A) In s9 (human bronchoendothelial) cells, B) HMEC (human primary mammary endothelial cells), C) HK 2 (human kidney PT epithelial) cells and D) total human kidney RNA. The first five lanes in A, B, and C correspond to water, oligo d(T), LJ3, LJ4 and LJ5 strand specific primers, respectively, with no reverse transcriptase (RT) as negative controls. The last five lanes in A, B and C correspond to oligo d(T), LJ3, LJ4 and LJ5 strand specific primers and gDNA as a positive control. In D, lanes correspond to gDNA, LJ3, LJ4, LJ5 and SB1, SB2 and SB3 (which are strand specific primers even more distal to the LJ primers) with RT and the next lanes correspond to the same primers with no RT and the last lane has water as a negative control. The presence of the 165 bp band in the +RT lanes indicates the presence of EDN1 AS.

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128 Figure 5 8. EDN1 AS expression is increased in the murine renal outer medulla. Male 129/svs2 mouse kidneys were harvested at midnight and dissected into cortex and outer medulla. RT PCR was performed as described except with mouse EDN1 AS strand specific and PCR primers. EDN1 AS was detected in both A) cortex and B) outer medulla. Each lane on the cortex gel corresponds to the same lane and mouse on the outer medulla gel. Lanes 1 8 on each gel are RT controls and lanes 9 16 on each gel are +RT. A single strand specific RT primer (mEDN1AS 4) was used so each band represents a different mouse. Levels of EDN1 AS appear to be increased in t he outer medulla compared to the cortex for each mouse.

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129 Figure 5 9. EDN1 AS Expression in Mouse Aorta. Aorta collected from C57BL/6J mice on control diet and HS/DOCP was processed and subjected to SS RT PCR to probe for EDN1 AS expression. While there were multiple mice that had bands in the RT lanes (panel A, lanes 4, 8, 9, 10), two did not have bands in RT (panel A, lanes 3, 6) but showed bands +RT (panel B, lanes 3, 6) (blue boxes), confirming the presence of EDN1 AS in aorta. Panel B lane 11 is a negative water control, lane 12 is a positive gDNA control. The extreme exposure (as indicated by the ladder and gDNA) suggest low expression levels of EDN1 AS in aorta or poor RNA quality.

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130 Figure 5 10. EDN1 AS expression may exhibit sex differences i n murine renal cortex. Male and female 129/svs1s4 mouse kidneys were harvested at midnight and cortex was dissected and processed for EDN1 AS as described. A) RT controls were negative while B) +RT samples showed varying amounts of EDN1 AS. In both panels lanes 1 7 are male mice and lanes 8 14 are female. It appears that females may have higher levels of EDN1 AS in kidney cortex, however the estrous cycles were not synchronized which may account for some variation. Each band represents an individual mouse

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131 Figure 5 11. EDN1 AS expression is regulated by aldosterone in m mIMCD3 cells. m mIMCD3 (a model of the murine inner medullary CD ) cells were treated with 100 nM aldosterone in three separate experiments. Cells were harvested and RNA was collected wit h Trizol. After DNase treatment, strand specific RT was performed as previously described. On the left side of Figure 6 are RT controls, which have no visible bands. The samples on the right had +RT and bands are clearly visible. Vehicle (ethanol) and ald osterone treated samples alternate and it is apparent that there is an increase in EDN1 AS expression with all three aldosterone treatments. Thus, it appears that EDN1 AS expression is positively regulated by aldosterone in vitro.

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132 Figure 5 12. EDN1 AS is regulated by the circadian clock. HK 2 cells were synchronized with 100 nM dexamethasone for 30 minutes, after which media was changed. 24 hours later, RNA from these cells was isolated every 2 hours for 24 hours starting at noon (12:00). A) Strand sp ecific RT using LJ4 and PCR reactions using LJ7 and LJ8 was done to assess EDN1 AS expression. cDNA was also made using random hexamers to assay EDN1 expression (real time curve, panel A). Expression of EDN1 AS is in phase with EDN1 mRNA. B) GAPDH expressi on did not change over time.

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133 Figure 5 13. RNA polymerase II, GR and Per1 are present at the EDN1 AS promoter Preliminary ChIP data show the binding of RNA polymerase II, the glucocorticoid receptor (GR) and the circadian clock protein Per1 to the E DN1 AS promoter region. Chromatin/protein interactions were fixed in HK 2 cells and sheared chromatin was immunoprecipitated with antibodies against Pol II, GR, Per1 and rabbit IgG (as a negative control). PCR with primers encompassing the EDN1 AS promoter (indicated by green arrows) gave positive signals for all three antibodies and no signal for the negative control. Data provided by the Cain lab, courtesy of J. Richards.

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134 Figure 5 14. Representative Genotyping Gel for CRISPR/Cas9 Knockout. HK 2 cell s were genotyped for EDN1 AS promoter region deletion in the gel on the left side showing A) heterozygous KO, B) WT and C) homozygous KO cells. On the right side gel, mIMCD3 cells were genotyped, displaying bands of multiple sizes (D). Figure 5 15. ET 1 ELISA of Media from CRISPR Cell Lines. ET 1 ELISA of media from HK 2 and mIMCD3 cells was done to assess differences due to KO of the promoter region of EDN1 AS. A) Levels of ET 1in WT and homozygous KO HK 2 lines. B) Levels of ET 1 in WT (1 3) or heter ozygous KO (4 6) mIMCD3 cell lines.

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135 Table 5 1. Cut size and expected amplicon size for all gRNA combinations Human Primers 1F and 2R Mouse Primers 1F and 2R gRNAs cut size amplicon size gRNAs cut size amplicon size none 0 689 none 0 621 1 and 2 77 612 1 and 2 65 556 1 and 3 366 323 1 and 3 394 227 1 and 4 489 200 1 and 4 513 108 2 and 3 289 400 2 and 3 329 292 2 and 4 412 277 2 and 4 448 173 3 and 4 123 566 3 and 4 119 502

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136 CHAPTER 6 CONCLUSION S AND FUTURE DIRECTIONS Conclu sions The contribution of Per1 to the regulation of salt and water reabsorption by the nephron is clear from previous work in our lab. The work presented in Chapter 2 showing that Per1 regulates sodium transport in the PT added to the previous work and sho wed that Per1 regulates sodium transport throughout the entire nephron. Additionally, the work suggested that this regulation was done in a coordinate manner, with Per1 positively regulating positive regulators of sodium reabsorption and negatively regulat ing negative regulators of sodium reabsorption. All of the cell culture data fit nicely with experiments in WT and Per1 KO mice on a 129/svs2 background. However, when telemetry data was analyzed from WT and Per1 KO mice on a C57BL/6J backg round, the same baseline phenotypes were not observed as the mice on a 129sv/s2 strain. While Per1 KO mice on a 129sv/s2 background had considerably lower BP (BP) compared to WT mice, Per1 KO mice on a C57BL/6J background had significantly higher BP than W T mice. After an initial panic, it became clear, and after some thought not all that surprising, that strain differences in background genetics of the mice could be at play. After all, two different humans could display two different phenotypes after the s ame insult and nobody would bat an eye because humans are not inbred like the laboratory mice that are used. Furthermore, these mice who were at first a problem became a great source of information when their BP on a HS/DOCP diet was examined. Normally, C 57 mice are hardy little creatures and are difficult to stress. Hypertension is usually induced in this mice not only by a high salt diet plus mineralocorticoid (like DOCP) but also by infusion

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137 of Angiotensin II or uninephrectomy. Without this, the mice sh ow no change in BP, as we see in the male WT mice in Chapter 3. However, without Per1 these mice develop harmful nondipping on a HS/DOCP diet. Thus, simply by removing Per1 and without the need for nephrectomy or infusion of a hypertensive agent, a signifi cant phenotype was created in these mice. This is a powerful example of the importance of Per1 to sodium and BP regulation. To compliment these studies, we examined the role of Per1 in HS/DOCP diet in female mice, discussed in Chapter 4. In general, female s are more protected from harmful effects of protocols like this and indeed, the female mice did not display a nondipping phenotype like the males. This suggests a protective effect in females which may be linked to sex hormones as many of the general prot ection seen in females is lost after menopause. While this was a limited study, both by the number of animals used and the experimental procedures performed, it was an important first step in the analysis of sex differences in the regulation of BP by the c lock. There is a decided lack of studies investigating sex differences in general but also specifically with regard to the circadian control of BP The last chapter of this dissertation deals with antisense lncRNAs and Endothelin 1 which seem to be unrelat ed to the rest of the work at first glance. However, Endothelin 1 is the most potent natural vasoconstrictor known and has diuretic effects in the nephron. It is also regulated by Per1. Early evidence also indicates that Per1 is involved in the regulation of EDN1 AS as well, shown by Per1 residence at the promoter region of EDN1 AS. Thus, if EDN1 AS is later shown to have regulatory function an d be regulated itself, there is a high likelihood that Per1 will be strongly

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138 involved. Although there are many piec es of evidence supporting the importance and regulation of EDN1 AS, many of the data is preliminary and needs to be confirmed with more experiments. Thus, Per1 is clearly an important modulator of BP through regulation of salt and water reabsorption by the nephron. Not only is Per1 implicated in normal regulation of BP, but this work has shown it may also play a role in pathological states as well. Additionally, preliminary but convincing data suggest that Per1 also regulates EDN1 AS which in turn may regul ated Endothelin 1. Future Directions There appears to be different regulatory mechanisms of Per1 based on model system and strain as well as sex. This provides numerous avenues of exploration. Unfortunately, both strain differences and sex differences are not very broadly studied even though the human race is made up of largely non inbred humans, about half of which are female. In order to have a representative body of knowledge, this gap needs to be filled. Cursory examination of the Jackson Lab Mouse Phen ome Database shows a wide range of baseline systolic BP from 75 mmHg to 130 mmHg in 23 strains 318 320 Thus, it is not all that surprising that C57BL/6J mice have an opposite phenotype from 129sv/s2 mice and that mi ce on a mixed substrain 129sv/s1s4 background have a slightly different phenotype than both. While at first these conflicting results are frustrating, in reality they provide a way to study very specific aspects of Per1 action since the genetic differences between the strains will point the way. Ideally, genome wide analysis of Per1 binding sites would provide a list of genes to investigate first. If there are any promising candidates, further study of expression differences in WT and

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139 Per1 KO mice of each s train could suggest a mechanism for the observed strain differences. This could eventually be applied to personalized medicine based on genotype or certain single nucleotide polymorphisms (SNPs). Sex differences provide a very similar avenue to explore spe cific aspects of Per1 regulation. There is a reason that males develop nondipping and females do not. Exploration and elucidation of this reason could provide very useful information when it comes to personalized treatments for conditions in which Per1 is not regulated properly. Repeating the HS/DOCP experiment with sham and ovariectomized mice would be the first step in understanding the role of sex hormones in the observed phenotypes. If ovariectomy shows no difference, mice could be allowed to age natura lly in order to determine if age itself plays a role. It would also be interesting to see if there are any estrogen receptor binding sites on any genes related to sodium regulation in the nephron and see if mutation of these sites produces an observable ph enotype. It is also important to continue work on nondipping in the male mice. This is a problem in humans and is associated with increased risk of cardiovascular events. There is a need for more information on the etiology and risk conferred by nondipping Simultaneously, there is a need to modulate nondipping and restore a proper circadian rhythm to BP in nondippers. These mice represent a novel way to approach this problem and further study of differences not only between WT and KO males but between fema les as well could shed light on the mechanism of nondipping. Finally, EDN1 AS shows promise as yet another target o f treatment of various diseases. While all of the data gathered so far is quite preliminary, when taken together it becomes more convincing. If EDN1 AS is subsequently shown to regulate EDN1

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140 expression, it could be a major target not only in the kidney but in multiple organs and tissues since EDN1 is so ubiquitously expressed. All of the experiments presented here should be repeated to add con fidence. CRISPR/Cas9 cloning is planned to be repeated in the lab shortly with a lipofectamine method instead of the viral method shown here. It is the hope that this method will be more effective and produce clones in a shorter amount of time in order to reduce passage number and the time that cells are alive before experiments can be done. Once clones are created, ET 1 expression levels can be assed at baseline between WT and KO cells as well as in response to different treatments like aldosterone, glucos e or albumin. In the future, if EDN1 AS is proven to be all that is hoped, a mouse model can be made with KO of EDN1 AS to determine the tissue specific effects of this lncRNA.

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141 LIST OF REFERENCES 1. Solocinski K, Gumz ML. The circadian clock in the regulation of renal rhythms. J Biol Rhythms 2015;30:470 486 2. Richards J, Gumz ML. Advances in understanding the peripheral circadian clocks. FASEB J 2012;26:3602 3613 3. Dibner C, Schibler U, Albrecht U. The mammalian cir cadian timing system: Organization and coordination of central and peripheral clocks. Annu Rev Physiol 2010;72:517 549 4. Partch CL, Green CB, Takahashi JS. Molecular architecture of the mammalian circadian clock. Trends in cell biology 2014;24:90 99 5. Badura L, Swanson T, Adamowicz W, Adams J, Cianfrogna J, Fisher K, Holland J, Kleiman R, Nelson F, Reynolds L, St Germain K, Schaeffer E, Tate B, Sprouse J. An inhibitor of casein kinase i epsilon induces phase delays in circadian rhythms under free runnin g and entrained conditions. J Pharmacol Exp Ther 2007;322:730 738 6. Lee JW, Hirota T, Peters EC, Garcia M, Gonzalez R, Cho CY, Wu X, Schultz PG, Kay SA. A small molecule modulates circadian rhythms through phosphorylation of the period protein. Angewandt e Chemie 2011;50:10608 10611 7. Koike N, Yoo SH, Huang HC, Kumar V, Lee C, Kim TK, Takahashi JS. Transcriptional architecture and chromatin landscape of the core circadian clock in mammals. Science 2012;338:349 354 8. Zhang R, Lahens NF, Ballance HI, Hug hes ME, Hogenesch JB. A circadian gene expression atlas in mammals: Implications for biology and medicine. Proc Natl Acad Sci U S A 2014;111:16219 16224 9. Moore Ede MC. Physiology of the circadian timing system: Predictive versus reactive homeostasis. Am J Physiol 1986;250:R737 752 10. Ede MC, Faulkner MH, Tredre BE. An intrinsic rhythm of urinary calcium excretion and the specific effect of bedrest on the excretory pattern. Clin Sci 1972;42:433 445 11. Palmer LG, Schnermann J. Integrated control of na transport along the nephron. Clin J Am Soc Nephrol 2015;10:676 687

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168 BIOGRAPHICAL SKETCH Kristen attended St. Peter the Apostle and St. Joan of Arc for middle and elementary school. She attended South Lake High School in St. Clair Shores, MI. She completed her first two years of undergraduate wo rk at Macomb Community Co llege and finished her Bachelor of Science with a double major in Biochemistry and Biology at Western Michigan University. She then moved to Gainesville, Florida to attend graduate school. She graduated with her Doctor of Philosoph y in Biomedical Sciences in December 2017.