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1 IMPACT OF EXERCISE ON RENAL NITRIC OXIDE AND ANTIOXIDANT SYSTEMS By NATASHA CELINE MONINGKA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEG REE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011
2 2011 Natasha Celine Moningka
3 To my Opa (grandfather) Pinardi Rorong
4 ACKNOWLEDGMENTS First and foremost, I must thank my f amily for their unconditional love To my Opa who inspire d me to study renal physiology I have turned his loss to ren al disease into a labor of love To Deddy, Mama, and Oma for providing the support only good parents and a grandmother can provide. Your personal sacrifices have not gone unappreciated. I al so thank my little sister, Nathalie, for never letting me forget how special I am and my cousins, Windy and Gita, for their support as well. I am eternally indebted to my mentor Dr. Chris Baylis. I would not be the person I am today without her. The wo rk entailed in this dissertation would not have been possible with out her dedication and guidance. I thank h er for teaching me how to be a critical thinker and renal physiologist, for being a champion of my success and for her genuine care of my well bei ng in graduate school and in life Her passion for science is inspiring and I look forward to applying the knowledge gained under her mentorship in my future endeavors. To my committee members, Dr. Judy Delp, Dr. Christiaan Leeuwenburgh, and Dr. Segal, I thank y ou each for not just your expertise and generous counsel in my dissertation work but for the work you each do in your research I would also like to thank all my collaborators, and in particular, Dr. Judy Delp and Dr. Brad Behnke and their resp ective lab members for the o pportunity to do great science amid talented individuals I also extend thanks to my undergraduate research mentor, Dr. Kenneth Baldwin and members of his lab, f or introducing me to the exciting world of research I am etern ally grateful for their encouragement I would also like to thank my lab mates for their helping hand moral support, and life long friendship. They have made this journey so enjoyable. In particular, I thank
5 Jenny for being the exemplar of an inspiring scientist and mother and my big sister Mark for always believing in me and for being an inspirer of all good things Harold for his help in all my projects and Bruce for his encouragement I will never forget the laughs we shared together. I must also ackn owl edge past lab members who have in some way shape or form contributed to my dev elopment as a graduate student and/or completion of this body of work: Dr. You Lin Tain, Dr. Peter Chen, Dr. Tatsiana Tsarova Dr. Laszlo Wagner, Dr. Andrea Fekete, Oladel e Akinsi ku, Stefan Shaw and Myrline Sterling. It was a privilege to work with this team known as the Baylis lab. To all my friends back home in California and to the ones I have made here at the University of Florida, I thank them for always uplifting me Last but not least, I would like to thank my best friend Isaac for being my rock in practically everything He is by far the best thing that has happened to me and I am the luckiest person in the world to have him in my life.
6 TABLE OF CONTENTS p age ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 INTRODUCTIO N ................................ ................................ ................................ .... 14 Cardiovascular Responses to Exercise ................................ ................................ .. 15 Cardiovascular Adaptations to Exercise ................................ ........................... 15 Endothelial Ada ptations to Exercise: The Role of Nitric Oxide ......................... 16 Renal Responses to Exercise ................................ ................................ ................. 18 Exercise Reduces Renal Blood Flow ................................ ............................... 18 Exercise Reduces Urinary Sodium Excretion ................................ ................... 19 Exercise In duced Proteinuria ................................ ................................ ........... 20 Renal Adaptations to Chronic Exercise ................................ ............................ 21 Exercise and Renal Disease ................................ ................................ ................... 22 Chronic Kidney Disease and NO Deficiency ................................ .................... 22 The Aging Kid ney: A Model of CKD ................................ ................................ 24 Exercise and Chronic Kidney Disease ................................ ............................. 25 Exercise and Acute Kidney Injury ................................ ................................ ..... 27 Summary and Objectives ................................ ................................ ........................ 28 2 IMPACT OF VOLUNTARY EXERCISE ON RENAL NITRIC OXIDE AND ANTIOXIDANT STATUS: A STRAIN DIFFERENCE COMPARISON ..................... 30 Background ................................ ................................ ................................ ............. 30 Methods ................................ ................................ ................................ .................. 31 Animal Procedures ................................ ................................ ........................... 31 Immunohistochemistry ................................ ................................ ...................... 32 Preparation for Telemetry ................................ ................................ ................. 33 Western Blot ................................ ................................ ................................ ..... 33 Analytical Methods ................................ ................................ ........................... 34 Statistical Analyses ................................ ................................ .......................... 34 Results ................................ ................................ ................................ .................... 34 Discussion ................................ ................................ ................................ .............. 36 3 EXERCISE EX ACERBATES ISCHEMIA REPERFUSION INDUCED ACUTE KIDNEY INJURY IN THE SPRAGUE DAWLEY BUT NOT FISHER 344 RAT ....... 48
7 Background ................................ ................................ ................................ ............. 48 Methods ................................ ................................ ................................ .................. 49 Animal Procedures ................................ ................................ ........................... 49 Renal Pathology ................................ ................................ ............................... 52 Western Blot ................................ ................................ ................................ ..... 52 Analytical Methods ................................ ................................ ........................... 53 Blood Flow Measurements ................................ ................................ ............... 53 Statistical Analyses ................................ ................................ .......................... 55 Results ................................ ................................ ................................ .................... 56 Discussion ................................ ................................ ................................ .............. 58 4 TWELVE WEEKS OF TREADMILL EXERCISE DOES NOT REVERSE AGE DEPENDENT CHRONIC KIDNEY DISEASE IN THE FISHER 344 MALE RAT ..... 71 Background ................................ ................................ ................................ ............. 71 Methods ................................ ................................ ................................ .................. 73 Animal Procedures ................................ ................................ ........................... 73 Renal Pathology ................................ ................................ ............................... 74 Western Blot ................................ ................................ ................................ ..... 75 Analytical Methods ................................ ................................ ........................... 76 Statistical Analyses ................................ ................................ .......................... 76 Results ................................ ................................ ................................ .................... 76 Discussion ................................ ................................ ................................ .............. 79 5 PROTECTION AGAINST AGE DEPENDENT RENAL INJURY IN THE F344XBROWN NORWAY MALE RAT IS ASSOCIATED WITH MAINTAINED NITRIC OXIDE SYNTHASE ................................ ................................ ................... 90 Background ................................ ................................ ................................ ............. 90 Methods ................................ ................................ ................................ .................. 92 Animal Procedures ................................ ................................ ........................... 92 Renal Pathology ................................ ................................ ............................... 93 Western Blot ................................ ................................ ................................ ..... 93 Analytical Methods ................................ ................................ ........................... 94 Statistical Analyses ................................ ................................ .......................... 94 Results ................................ ................................ ................................ .................... 95 Discussion ................................ ................................ ................................ .............. 97 6 CONCLUSIONS ................................ ................................ ................................ ... 108 Summary of Findings ................................ ................................ ............................ 108 Genetic Background Determines Renal Response to VWR EX ..................... 108 Genetic Background Determines Susceptibility to IR induced AKI ................. 109 Chronic TM EX Does Not Alter Age Related Renal Injury .............................. 109 Protection Against Age Related Renal Damage Associates with Intact NO System ................................ ................................ ................................ ........ 110 General Discussion ................................ ................................ ............................... 110
8 Limitations and Future Directions ................................ ................................ ......... 117 Clinical Perspectives ................................ ................................ ............................. 122 LIST OF REFERENCES ................................ ................................ ............................. 124 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 138
9 LIST OF TABLES Table page 2 1 Voluntary wheel running activity presented as average daily km run per day. ... 42 2 2 Renal functional responses in SD and F344 after 6 weeks vol untary exercise .. 42 3 1 Sprague Dawley Renal hemodynamic responses ................................ ........... 64 3 2 Fisher 344 Renal hemodynamic responses. ................................ .................... 64 4 1 Characteristics of male F344 rats obtained from Harlan, NIA. ............................ 84 4 2 Characteristics of male F344 rats obtained from Taconic Farms, N IA. ............... 84 5 1 A ging and chronic R AS blockade on pla sma ADMA, L Arginine, L Arginine:AD MA, and SDMA in male F344xBN rats. ................................ ......... 102 5 2 A ging on kid ney cortex ADMA, L Arginine, L Arginine:ADMA, and SDMA in male F344xBN rats. ................................ ................................ .......................... 102
10 LIST OF FIGURES Figure page 2 1 BW in SD and F344 after 12 weeks VWR EX ................................ .................... 43 2 2 Kidney cortex eNOS and EC SO D abundance after 3, 6, or 12 weeks VWR EX ................................ ................................ ................................ ....................... 44 2 3 Immunohistochemical analyses of eNOS after 6 weeks VWR EX ..................... 45 2 4 Kidney corte x CuZn SOD and Mn SOD abundance after 12 weeks VWR EX ... 46 2 5 Kidney cortex p22phox and nitrotyrosine abundance after 12 weeks VWR EX .. 47 3 1 BW in SD and F344 after 12 weeks VWR EX for IR studies .............................. 65 3 2 Acute renal structural injury after 12 weeks VWR EX and UNX IR ..................... 66 3 3 Kidney cortex eNOS, EC SOD, and p22phox abundance after 12 weeks VWR EX and IR induced AKI. ................................ ................................ ........... 67 3 4 Lung eNOS and EC SOD abundance after 12 weeks VWR EX and IR induced AKI ................................ ................................ ................................ ....... 68 3 5 Weight data for SD and F344 after 10 12 weeks TM EX ................................ .... 69 3 6 Strain difference responses in blood flow measurements during T M EX ........... 70 4 1 Aging and chronic TM EX impacts on renal structural injury ............................. 85 4 2 young and old, SED and TM EX rats ................................ ................................ 86 4 3 Kidney cortex and kidney medulla SOD enzyme abundance in young and old, SED a nd TM EX rats ................................ ................................ .................. 87 4 4 Kidney cortex and kidney medulla oxidative stress markers in young and old, SED and TM EX rats ................................ ................................ ......................... 88 4 5 Aortic eNOS and EC SOD in young and old, SED and TM EX rats ................... 89 5 1 Glomerular structu ral injury in aging F344xBN rat ................................ ........... 103 5 2 Kidney cortex NOS enzyme abundance in aging F344xBN rat. ...................... 104 5 3 Kidney cortex DDAH, DDAH2, and PRMT1 abundance in aging F344xBN rat 105 5 4 Kidney cortex oxidative stress markers in aging F344xBN rat ......................... 106
11 5 5 Kidney cortex SOD enzyme abundance in aging F344xBN rat ....................... 107
12 Abstract of Dissertation Presented to the Graduate School of the Uni versity of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy IMPACT OF EXERCISE ON RENAL NITRIC OXIDE AND ANTIOXIDANT SYSTEMS By Natasha Celine Moningka December 2011 Chair: Chris Baylis Major: Me dical Sciences Physiology and Pharmacology Exercise has numerous cardiovascular benefits including increased blood flow which increases shear stress leading to induction of the endothelial nitric oxide (NO) synthesizing enzyme (eNOS). The antioxidant, ex tracellular superoxide dismutase (EC SOD) is also induced and both enzymes are required for optimal vascular health. In the kidney, however, exercise reduces blood flow, which might lead to falls in NO and antioxidant bioavailability. This dissertation f ocuses on the impact of exercise on renal NO and antioxidants in health and in injury. In the cardiovascular injury prone Sprague Dawley (SD) rat, low intensity voluntary exercise reduced renal eNOS and EC SOD, and this was associated with increased susce ptibility to acute kidney injury (AKI) induced by ischemia/reperfusion (IR). In contrast, Fisher 344 (F344) rats, a strain resistant to cardiovascular injury, voluntary exercise increased renal eNOS/EC SOD in association with protection against AKI. Real time assessment of renal blood flow (RBF) revealed that exercise training reduces resting RBF and that the fall in RBF seen with acute exercise in sedentary rats is blunted by training in both strains. Studies in the aging F344 revealed that chronic trea dmill exercise did not alter age related renal injury. We also confirmed that protection against age related renal injury is associated
13 uncovered the mechanism for the dif ferent renal eNOS/EC SOD responses to exercise in the SD vs. F344. However, these data indicate that the renal response to exercise and age are influenced by genetic background and that exercise influences the status of pre existing renal endothelial heal th which determines the severity of IR induced AKI. Consideration of these factors is required for optimal exercise benefit for patients of endothelial dysfunction including but not limited to renal disease.
14 CHAPTER 1 I NTRODUCTION According to the 2010 annual report by the US Renal Data System, renal fai lure afflicts an estimated 11.5% of the adult population and creates a tremendous economic burden on the healthcare system. These numbers are predicted to increase as a result of th e rising incidence of cardiovascular disease, a co morbidity of chronic kidney disease (CKD). Advancing age also increases the risk of developing renal failure, a concern since the aging population continues to grow. Lifestyle modifications involving p hysical activity have proven effective in reducing the risk for cardiovascular mortality which is high in patients with renal failure. Metabolic benefits of exercise include reductions in plasma triglycerides, increases in the high density lipoprotein to low density lipoprotein ratio, and improved insulin sensitivity and overall cardiac function (Sasaki & Gisele, 2005). Also important are the direct vascular effects of exercise. In vascular beds where blood flow increases during exercise, shear stress di rectly interacts with the endothelium to up regulate essential factors required for optimal endothelial health. However, in the kidney, blood flow is reduced during exercise and few have thoroughly investigated exercise induced endothelial adaptations. I ndeed, there are reports of exercise induced acute kidney injury (AKI) in man (Seedat et al. 1990; Yan et al. 2010; Bosch et al. 2009) and exercise induced exacerbation of age associated renal structural injury in mice ( Lichtig et al 1987). Certainly, th e impact of exercise on the aging kidney and on the susceptibility to AKI is of considerable clinical relevance. The purpose of this section is to provide background regarding the cardiovascular and renal responses to exercise, with an emphasis on exercis e induced endothelial adaptations in both circulations. It
15 will also explore literature pertaining to the impact of exercise on the kidney in the presence of underlying renal injury, and will end with the objectives of this work Cardiovascular Responses to Exercise Cardiovascular Adaptations to Exercise Exercise stresses the regulatory ability of the cardiovascular system. It increases heart rate, stroke volume, and the total systemic arteriovenous oxygen difference. The ability for each of these varia bles to increase determines maximal oxygen uptake (VO 2 max), the functional capacity of the cardiovascular system. Oxygen uptake and exercise intensity increase linearly but eventually oxygen uptake plateaus despite further increases in exercise intensity (Rowell 1993). Thus, VO 2 max is attained at submaximal rates of exercise intensity. In normal young individuals, VO 2 max ranges from 45 to 53 mL/kg/min and up to 85 mL /kg/min in endurance athletes (Rowell 1993). Cardiac function improves as a consequenc e of physical conditioning, a term to describe repeated exposure to exercise training. Part of this adaptation involves greater cardiac output mainly due to increases in stroke volume and oxygen extraction despite a decrease in heart rate. While some stu dies report the requirement of both mechanisms, others demonstrate that greater maximal cardiac output can occur with minimal increases in oxygen extraction (Clausen et al. 1977; Saltin et al. 1969). Improvements in stroke volume are mainly due to changes that enhance ventricular contraction as dictated by the Frank Starling law of the heart: End diastolic volume increases due to a combination of increased blood volume, thickening of the left ventricular wall, quicker ventricular filling, and increased myo cardial contractility (Rowell 1993). Improvements in oxygen extraction are partly dictated by the regional vasoconstriction of the renal and splanchnic circulation. Shunting of blood from these
16 vascular beds provides greater blood supply to active muscl es. In addition, in the muscle where metabolic demand is high during exercise, oxygen extraction increases due to increased skeletal muscle vascular conductance which provides greater capillary blood volume. In turn, this reduces diffusion distance betwe en the microcirculation and the muscle fiber, resulting in increased efficiency of oxygen delivery (Rowell 1993). Mechanisms of vascular conductance will be described in greater detail in the Endothelial adaptations to exercise: The role Physical conditioning also reduces cardiovascular risk factors. It decreases blood pressure, body fat, plasma triglycerides, total cholesterol, and increases insulin sensitivity (Sasaki & Gisele Dos Santos, 2005). Beneficial metaboli c actions of exercise have been proven effective in treatment of hypertension (Fagard 2011), diabetes (Nishida et al. 2004), and obesity (Savage et al. 2004). Finally, the benefits of physical conditioning extend to the endothelium. Endothelial Adaptatio ns to Exercise: The Role of Nitric Oxide A large body of literature supports efficacy of exercise training in improving endothelial function via a nitric oxide (NO) mediated mechanism (Delp et al. 1993; Delp and Laughlin, 1997; McAllister et al. 2009; Sind ler et al. 2009; Mora et al. 2007; Green et al. 2004). Using the inhibitor of NO formation, N G nitro L arginine methyl ester (L NAME), Delp et al. demonstrated that enhanced acetylcholine mediated dilation with training was due to an increase in NO produc tion (Delp et al. 1993). In rats, this response is present by four weeks of endurance treadmill training (Delp & Laughlin, 1997). Nitric oxide (NO) is an essential signaling molecule that regulates vasomotor tone (Ignarro et al. 1987) and protects the e ndothelium against development of
17 atherosclerotic lesions via inhibition of lipid oxidation, vascular smooth muscle cell proliferation and platelet aggregation (Harrison et al. 2006). NO is produced by NO synthase (NOS) using the substrates L Arginine and oxygen. Three NOS isoforms exist: endothelial (e) NOS, neuronal (n) NOS, and inducible (i) NOS. All require the cofactors tetrahydrobiopterin (BH 4 ), calmodulin (CaM), flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN). Shear stress, or the frictional force exerted on a vessel wall caused by blood flow, stimulates production of nitric oxide (NO). Within seconds, shear stress increases intracellular calcium levels resulting in binding of calmodulin to eNOS, thus resulting in eNOS activat ion (Harrison et al. 2006). In addition, protein kinase A phosphorylates eNOS on serines 635 and 1177 after the onset of shear, resulting in increased enzyme activity. In situations of prolonged shear stress, eNOS mRNA stability increases (Davis et al. 2 001). This in turn increases eNOS transcription and translation. Shear stress also up regulates the expression of the extracellular superoxide dismutase (EC SOD) (Fukai et al. 2000), an antioxidant that protects against oxidative stress by scavenging for the highly reactive superoxide molecule. Two other SOD isoforms have been characterized, the cytosolic CuZn SOD and mitochondrial Mn SOD; however, EC SOD is the major superoxide scavenger in the vascular extracellular space (Harrison et al. 2006). Mice deficient in c Src, a tyrosine kinase required for the shear stress response, failed to increase their aortic eNOS and EC SOD protein levels with exercise training (Davis et al. 2003), confirming the role of blood flow and shear stress in exercise induced endothelial adaptations. Furthermore, eNOS knock out mice failed to show increases in EC SOD, despite exercise training, suggesting that increased NO is
18 required for EC SOD up regulation (Fukai et al. 2000). Thus, increased shear stress improves endothel ial function in circulations where exercise increases blood flow. However, little is known about the impact of exercise training in areas where blood flow is reduced. Renal Responses to Exercise Exercise Reduces Renal Blood Flow In contrast to other vas cular beds, exercise reduces renal blood flow (RBF). Peripheral vasoconstriction in the renal and splanchnic circulations is required to supply working muscles with increased blood flow. In humans, falls in RBF and renal plasma flow (RPF) occur immediate ly and are dependent on exercise intensity (Poortmans 1985). Paraminohippurate (PAH) clearance measured in healthy young men revealed dose dependent falls in RPF with increasing exercise intensity and that the declines remained even after 40 minutes of re covery following exercise (Chapman et al. 1948). Using radioactive microspheres, exercise trained miniature swine reduced their RBF (Bloor et al. 1986). In the dog, while some studies report a reduction in RBF with exercise (Delgado et al. 1975; Musch et al. 1987), others find the opposite response (Sadowski et al. 1981). It is likely that the dog is relatively resistant to exercise induced falls in RBF but nevertheless exhibit declines at severe intensity. Clearly, species and possibly strain differenc es are present. Despite exercise induced falls in RBF in human and swine, glomerular filtration rate (GFR) is relatively well maintained at low and moderate exercise intensities due to increases in filtration fraction (FF) (Castenfors 1967). However, at high intensities (>50 60% VO 2 max), GFR declines (Merrill & Cargill 1948; Kachadorian & Johnson 1970).
19 Various mechanisms mediate exercise induced falls in RBF including increased sympathetic nervous outflow and the participation of several hormone syst ems. Renal et al. 1993). In the denervated rabbit kidney, exercise failed to cause renal vasoconstriction and a fall in RBF, confirming the participation of the renal nerve s (Mueller et al. 1998). Angiotensin II, endothelin 1, and vasopressin also mediate the decline in RBF during exercise (Stebbins et al. 1995, Ahlborg et al. 1995; Maeda et al. 2004; Stebbins et al. 1993). Indeed, plasma renin activity increases due to in creases in renal nerve activity (Lifschitz & Horwitz 1976). In man, there is also a role for prostaglandins since the non selective cyclooxygenase 2 inhibitor indomethacin exaggerated the reduction in RBF after 30 minutes of strenuous exercise (Walker et al 1994). Thus, several mechanisms are involved in the reduction of RBF during exercise. Exercise Reduces Urinary Sodium Excretion Renal handling of sodium is crucial in establishing total body fluid homeostasis, especially during exercise where volume depletion due to water and sodium loss through sweating occurs. In an attempt to conserve sodium, free water clearance and urinary sodium excretion significantly falls with exercise (Baker et al. 2005). This response is primarily due to increased tubular reabsorption (Castenfors 1977). Interestingly, a decrease in urinary sodium excretion is not entirely attributable to hormones of the renin angiotensin system since pharmacological inhibition of angiotensin II had no effect on the anti natriuretic respo nse of exercise (Mittelman 1996; Wade 1987). Treatment with an aldosterone antagonist also failed to alter the decrease the urinary sodium excretion with exercise (Zambraski 1990). However, these findings may reflect an issue with dosage. Future stud ies are required to define
20 the role of the renin angiotensin system in the response of reduced urinary sodium excretion with exercise. It is likely that t he exercise induced renal sodium retention may also reflect increased sympathetic nerve activity to d irectly enhance tubular sodium reabsorption. Exercise Induced Proteinuria Another renal response of exercise training is the appearance of proteins in the urine. Although proteinuria is a hallmark of renal injury, exercise induced proteinuria is transien t, lasting for only 24 hours after exercise, and is considered normal (Coye & Rosandich 1960). Various urinary proteins have been identified including albumin, transferrin, ceruloplasmin, and immunoglobulin G (Rowe & Soothill 1961). As with falls in RB F, post exercise proteinuria is directly related to exercise intensity and may be secondary to increased glomerular permeability and/or reduced proximal tubular protein reabsorption (Poortmans & Labilloy 1988; Poortmans & Vanderstraten 1994; Poortmans 1 990). Urinary excretion of heparan sulfate proteoglycan, components of the glomerular basement membrane responsible for anionic sites, significantly increased from 248 to 23577 ng/min after 45 minutes of mild bicycle exercise in normotensive subjects (H eintz et al. 1995). Furthermore, intravenous injections of the NADPH oxidase inhibitor, diphenyleneiodonium chloride, four days before exercise, attenuated exercise induced increases in urinary excretion rates of protein and the oxidative stress markers, thiobarbituric acid reactive substance and protein carbonyl contents (Kocer et al. 2008). These studies suggest that causes of increased glomerular permeability include transient loss of glomerular charge and increased oxidative stress (Fox JG et al. 1993 ; Poortmans & Vanderstraten 1994). Mittleman et al. also showed that indomethacin ameliorated the urinary excretion of protein after 30
21 minutes of strenuous exercise in human subjects (Mittelman et al. 1992), suggesting a direct or indirect role for pros taglandins. Since exercise induced proteinuria occurs in the normal kidney, it will be crucial to determine the compounding impact of renal disease. Renal Adaptations to Chronic Exercise Chronic exercise results in an attenuation of renal vasoconstrictio n and therefore RBF during acute exercise. Using the microsphere technique to determine regional blood flows in rats, Armstrong and Laughlin demonstrated that chronic exercise reduced the magnitude of the decrease in RBF after treadmill exercise (Armstron g & Laughlin 1984). These adaptations were despite differences in baseline values between trained and untrained groups. The study also reported that exercise training led to blunted falls with acute exercise in blood flows to organs of the splanchnic ci rculation (i.e. spleen, liver, stomach, duodenum, and colon). This adaptation is likely due to a combination of training induced reductions in sympathetic outflow, angiotensin II, vasopressin, and norepinephrine (McAllister 1998). The type of exercise t raining also plays a role since in rats endurance treadmill confers this adaptation but not high intensity sprint training (Musch et al. 1996). Although less understood, local endothelial changes may also contribute to the reduced renal vasoconstrictor r esponse to exercise in the trained setting. In isolated perfused kidneys from New Zealand White rabbits, De Moraes et al. showed that 12 weeks of treadmill exercise enhanced acetylcholine induced vasodilation and that L NAME blunted this response, suggest ing that the adaptation of exercise training was due to increased NO bioavailability (De Moraes et al. 2004). However, studies from Miyauchi et al. in rats suggest otherwise. These investigators observed a reduction in
22 renal eNOS mRNA, protein, and enzym e activity after acute treadmill exercise, whereas in the lung these measurements increased (Miyauchi et al. 2003). Their findings argue that exercise reduces shear stress in the kidney and that in organs where blood flow is increased with exercise, incre ased shear stress follows, resulting in NO up regulation. On the other hand, Padilla et al. postulate that despite reductions in RBF during exercise, the combination of increased cardiac output and renal vasoconstriction may lead to increases in shear str ess and therefore increases in NO production (Padilla et al. 2011). To date, the levels of shear stress in the renal circulation during exercise have not been measured and will be extremely difficult to determine in vivo given the complexity of the renal circulation (Moffat & Fourman 1963). Thus, renal endothelial adaptations in response to exercise training require further study. In addition, it will be important to determine how exercise influences development of kidney disease. Exercise and Renal D isease Chronic Kidney Disease and NO Deficiency Nitric oxide (NO) deficiency contributes to the progression of chronic kidney disease. Several mechanisms cause NO deficiency including decreased protein abundance of the NO synthesizing enzyme, NO synthase (NOS), deficiency of the NOS substrate, L arginine, and increased levels of the endogenous NOS inhibitor, asymmetric dimethylarginine (ADMA) (Baylis 2008). A reduction in total NO production, as measured by urinary nitrite and nitrate (stable metabolites of NO) levels, is evident in various rodent models of CKD including the 5/6 ablation/infarction, chronic glomerulonephritis, chronic puromycin aminonucleoside nephrosis, and the aging rat (Baylis 2009). Moreover, chronic NOS inhibition leads to hypertens ion, proteinuria, and structural injury in the form of glomerular sclerosis, tubulointerstitial injury, and
23 glomerular ischemia (Zatz & Baylis 1998). Oxidative stress as a result of increased reactive oxygen species and decreased antioxidants can also re duce NO bioavailability, thus further exacerbating renal injury. In the kidney, superoxide is mainly generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and reacts with NO to form peroxynitrite, a highly reactive nitrogen species inv olved in lipid peroxidation (Gill & Wilcox 2006). Increases in renal p22phox, a subunit of NADPH oxidase, up regulates NADPH oxidase activity, leading to oxidative stress (Chabrashvili et al. 2003 ) Indeed, p22phox silencing prevents oxidative stress in the angiotensin II induced model of hypertension (Modlinger et al. 2006). Additional studies have also shown increased oxidative stress in CKD (Vaziri, 2004; Dounousi et al. 2006). Oxidative stress also mediates development of acute kidney injury (AKI). The most common cause of AKI is an ischemic insult followed by reperfusion. This leads to recruitment of local inflammatory signals which up regulate reactive oxygen species resulting in reductions in NO bioavailability and therefore development of rena l dysfunction (Le Dorze et al. 2009). Morphological changes accompanied with ischemia/reperfusion induced AKI include disruption of cell cell junctions, endothelial cell swelling, and alterations in the glyocalyx proteins of the renal endothelium (Sutton et al. 2003). Thus, endothelial injury characterized by increased oxidative stress and reduced NO bioavailability is the main mechanism by which ischemia/reperfusion induced AKI propagates. Protection afforded against development of renal injury associate s with having a maintained NO system and genetic background plays a role. For example, in the Wistar Furth rat, resistance to CKD induced by puromycin administration parallels with
24 preserved NO production (Erdely et al 2004). Furthermore, minimal age re lated renal injury in the Fisher 344XBrown Norway rat associates with increased renal NOS protein abundance (Moningka et al. 2011). In contrast, in the Sprague Dawley rat, development of significant renal injury after 11 weeks of 5/6 ablation/infarction i s accompanied with falls in renal and total NO production (Erdely et al. 2003). Male gender is another risk factor since female Sprague Dawley rats exhibit maintained renal NOS protein abundance with age and are protected against age dependent kidney dama ge compared to males (Erdely et al. 2003; Baylis 2009). The sexual dimorphism seen with NO deficiency as it relates to aging is presumably due to sex hormones. Thus, genetic background influences the progressive renal injury related to renal NOS deficien cy, with females and rat strains such as the Wistar Furth and Fisher 344xBrown Norway protected. The Aging Kidney: A Model of CKD The aging kidney is a model of slowly developing CKD and is also associated with NO deficiency. Although not inevitable, GFR declines with age, and more rapidly in males compared to females (Lindeman et al. 1985; Wesson 1969). Falls in GFR are due to structural damage of the renal blood vessels caused, in part, by increased extracellular matrix accumulation and expansion of th e glomerular mesangium (Leon et al. 2003). This leads to development of glomerular sclerosis and ischemia, and tubulointerstitial injury (Neugarten et al. 1999; Thomas et al. 1998). The culmination of structural injury eventually results in loss of funct ioning nephrons. Functional declines are due to increased renal vasoconstriction and falls in RPF (Baylis 2009). In aged rats, micropuncture studies revealed increased glomerular pressure due to a loss in afferent arteriolar resistance (Anderson et al. 1994). A sustained increase in glomerular
25 pressure eventually leads to glomerular injury (Brenner et al. 1996). However, age related declines in renal function and structural injury can occur without development of glomerular hypertension (Remuzzi et al. 1988; Baylis 1994). A mechanism of age related renal injury is endothelial dysfunction, mainly a result of NO deficiency. Causes of NO deficiency are similar to those in CKD: loss of NOS protein abundance/activity, NOS substrate deficiency, oxidative st ress and increased presence of endogenous NOS inhibitors (Baylis et al. 2009). Furthermore, several cardiovascular risk factors can accelerate the rate of age related loss of renal function including male gender, hypertension, dyslipidemia, and accumulati on of advanced glycosylation end products (Weinstein & Anderson, 2010). Exercise and Chronic Kidney Disease In the clinical setting, there are both risks and benefits with physical exercise in CKD. Risks include renal ischemia, dysrhythmia, and musculos keletal injury but these are usually associated with high intensity exercise (Johansen 2007). There are numerous benefits including improved blood pressure control, mental health, and muscle strength (Johansen 2007). However, in patients with establish ed CKD, exercise has been reported as of no benefit in slowing down the rate of decline in renal function (Eidemak et al. 1997). Concern has been expressed that because of the transient post exercise proteinuria, exercise might actually worsen the rate of progression with established CKD (Poortmans & Vanderstraeten 1994). Several rat studies suggest that the cardiovascular benefit of exercise does not necessarily guarantee renal benefit in the setting of CKD where both cardiovascular and renal complicati ons are present. Voluntary exercise effectively ameliorated the increase in several cardiac proinflammatory markers associated with CKD in the female rat;
26 however, the study did not assess these markers in renal tissue (Bai et al. 2009). Using the renal mass reduction model of chronic kidney disease (CKD), Heifets et al. reported that exercise was beneficial since it increased GFR and reduced proteinuria and the level of glomerular sclerosis (Heifets et al. 1991). In contrast, Adams et al. found that vol untary exercise did not ameliorate the hypertension associated with the 5/6 nephrectomy model (Adams et al. 2004). In the same CKD model, Bergamaschi et al. reported that 60 days of treadmill exercise did not prevent CKD induced proteinuria and glomerular slcerosis despite normalization of hypertension (Bergamaschi et al. 1997). Others have also demonstrated both renoprotective and anti hypertensive effects with exercise in rat CKD models (Lu et al. 2006; Kanazawa et al. 2006). Therefore, it remains unce rtain whether exercise training can reverse progression of CKD. A key factor in the renal response to exercise relates to the impact on the renal eNOS and ECSOD since intrarenal NO deficiency is associated with both experimentally induced and age depende nt CKD. The findings by Miyuachi et al. that exercise reduces renal eNOS in normal rats raises the possibility that the NO deficiency associated with CKD could be exacerbated by exercise (Miyauchi et al. 2003). Exhaustive exercise in the young rat also decreased renal cortical NOS and SOD activity (Lin et al. 2010). Furthermore, four weeks of treadmill exercise worsened renal injury in rats with chronic NOS inhibition (Kuru et al. 2005). Despite a blood pressure lowering effect, exercise magnified the arteriolar wall thickening, focal tubular atrophy, and interstitial inflammatory infiltration associated with chronic NOS inhibition. This is of particular concern in the aging kidney where falls in eNOS abundance and NOS activity (Erdely et al 2003) and increased oxidative stress occur (Gomes et al.
27 2009) in conjunction with slowly developing glomerular and tubulointerstitial injury (Baylis and Corman,1998). Additional deficits in renal NO due to exercise could exacerbate renal age dependent injury. Indeed, six weeks of exercise in old C57BL/6J mice magnified the age associated renal structural injury (Lichtig et al 1987). Poortmans and Ouchinsky sought to determine the impact of maximal exercise on post exercise proteinuria in aging man. Their findings suggest no harmful effect of exercise training on urine albumin excretion in the elderly; however, aged participants had no past history chronic disease of any type, smoking, or evidence of kidney/liver dysfunction so were considered a rela tively healthy elderly population (Poortmans & Ouchinsky, 2006). It is likely that genetic background plays a role in dictating the renal response to exercise, whether in CKD or aging. Exercise and Acute Kidney Injury Even in the absence of pre existing injury, renal injury due to exercise has been reported. Exercise induced AKI reflects renal ischemia caused by rhabdomyolysis (the breakdown of muscle fibers) and/or severe volume depletion (Seedat et al. 1990; Yan et al. 2010; Bosch et al. 2009). Cases are usually associated with high intensity exercise such as marathon running (Clarkson 2007). The increased oxidative stress accompanied by reperfusion injury following ischemia is likely to contribute to exercise induced AKI. Furthermore, there is like ly to be even greater susceptibility to renal injury by exercise if pre existing oxidative injury exists as in the case of patients with renal hypouricemia, a condition where uric acid, an antioxidant, is low in plasma. Indeed, these patients frequently d evelop exercise induced acute renal failure (Yan et al. 2010; Saito et al. 2011; Ishikawa 2002). Together, these studies suggest that exercise may
28 increase susceptibility to an oxidative stress mediated insult such as ischemia/reperfusion induced acute r enal failure. Summary and Objectives Exercise improves cardiovascular function and provides benefit in several diseases including hypertension, diabetes, and obesity. One important mechanism by which exercise benefits the vasculature is through shear str ess, the frictional force of blood flow on the endothelium. During exercise, shear stress increases as a result of increased blood flow to some parts of the circulation. This stimulates production of NO which has vasodilatory and anti atherosclerotic pro perties and EC SOD which is an antioxidant that protects against oxidative stress. Several studies have characterized the benefits of exercise induced endothelial adaptations but mainly in hyperemic tissue during exercise (i.e. skeletal muscle). Few, how ever, have thoroughly studied the impact of exercise on the kidney. Overall renal function declines during exercise since blood flow and GFR are reduced due to renal vasoconstriction. Exercise also leads to transient proteinuria, a hallmark of renal inju ry. In the literature, efficacy of exercise training in CKD and on age related kidney damage is controversial. Furthermore, studies by Miyauchi et al.demonstrate that acute exercise decreases renal NO production in the healthy, normal rat, presumably due to falls in renal shear stress ( Miyauchi et al. 2003). This is of particular concern since NO deficiency is a cause and consequence of CKD and age related renal injury. Therefore, the following studies were conducted to determine impact of exercise on t he renal NO and antioxidant systems in the normal, healthy kidney and in models of the acute and chronic kidney injury (i.e. aging). This dissertation presents the work in four separate chapters which are listed below with their respective objectives.
29 To characterize the impact of chronic voluntary exercise on renal cortical eNOS and EC SOD protein abundance. We determined whether duration of training influences the renal endothelial responses to 6 and 12 weeks of voluntary exercise in two different strai ns with differing cardiovascular risk profiles; the Sprague Dawley (SD) rat which is susceptible to experimentally induced hypertension and the Fisher 344 (F344) which is resistant. To characterize the impact of chronic voluntary exercise on susceptibility to acute renal injury. We determined whether chronic voluntary exercise exacerbates susceptibility to ischemia/reperfusion induced acute renal failure. To determine the role of genetic background, we compared the SD and F344. We also used the radiolabel ed microsphere technique to detect strain difference responses in renal blood flow during exercise. To characterize impact of chronic treadmill exercise on progression of age dependent renal injury. We determined whether chronic treadmill exercise exacer bates age dependent renal injury in the male F344 rat. We assessed the renal NOS and SOD enzymes as well as the role of oxidative stress. To determine if protection against age dependent renal injury in the Fisher 344xBrown Norway rat is associated with maintained renal nitric oxide synthase. To further examine the role of genetic background on renal NOS, we investigated the impact of aging on various determinants of NO production in the F344xBrown Norway rat, a model of healthy aging. We also investig ated whether there would be any beneficial effect of chronic renin angiotensin system (RAS) blockade in these
30 CHAPTER 2 IMPACT OF VOLUNTARY EXERCISE ON RENAL NITRIC OXIDE AND ANTIOXIDANT STATUS: A STRAIN DIFFERENCE COMPARISON B ackground One mechanism by which the vasculature benefits from exercise is by increases in blood flow. This increases shear stress which induces the nitric oxide (NO) generating enzyme, endothelial NO synthase (eNOS), and the antioxidant, extracellular superoxide dismutase (EC SOD) (Fukai et al. 2000; Harrison et al. 2006; Davis et al. 2003). As a result, endothelium dependent vasodilation improves and the risk for developing cardiovascular disease is reduced ( DeSouza et al. 2000 ). In the kidney, however, renal blood flow (RBF) falls during exercise (McAllister 1998; Tidgren et al. 1991; Mueller et al. 1998 ; Musch et al. 2004 ) and this could lead to falls in NO and antioxidant bioavailability ( Miyauchi et al. 2006; Middlekauff et al. 1997 ). The impact of chr onic exercise on the intrarenal NO and antioxidant systems are poorly understood. This is clinically relevant since chronic kidney disease (CKD) is associated with a high level of cardiovascular risk (Hostetter 2004; Anavekar & Pfeffer, 2004), and exerci se is often prescribed to combat cardiovascular related complications. Since NO deficiency contributes to CKD progression (Baylis 2009), any intervention that impairs renal NO production could accelerate the underlying kidney disease. Therefore, the mai n objective of this study was to investigate the impact of chronic voluntary exercise on the renal cortical abundance of eNOS and EC SOD. In order to determine whether the duration of training influences the renal endothelial responses we compared 6 and 12 weeks of voluntary exercise. We also conducted the study in two different rats strains with differing cardiovascular risk profiles; the Sprague Dawley rat which is susceptible to development of experimentally induced, as well as age
31 dependent hypertensio n (Erdely et al. 2003), and the Fisher 344 (F344) rat which is resistant (Hall et al. 1976; Goldstein 1988). In addition to renal eNOS and EC SOD, we also determined the abundance of other anti and pro oxidant proteins, the eNOS localization using immun ohistochemistry and also measured indices of oxidative stress, NO production and renal function. Methods Animal Procedures All aspects of animal handling were approved and in accordance with the mmittee. Male Sprague Dawley (38) and Fisher 344 (31) rats at 10 12 weeks of age were purchased from Harlan (Indianapolis, IN). Rats from each strain, SD and F344, were either maintained as sedentary (SED) controls or were given 24 hour ad libitum access to voluntary wheel running (VWR; Lafayette Instruments, Lafayette, IN) exercise (EX). Running wheels were attached to odometers that calculated distance run and data was acquired using the Activity Wheel Monitor Software (Lafayette Instruments, Lafayette IN). All rats were singly housed in a temperature and light controlled environment with access to standard rat chow and water. The following groups were studied: In the 1 st series of studies SD rats were run for 3 weeks (n=3) and compared to controls ( n=3) or 6 weeks (n=7) and compared to controls (n=5). The 6 week VWR and SED rats had been implanted with telemetry probes ~10 days prior to randomization into groups and mean blood pressure (BP) and heart rate (HR) were measured at baseline and then once /week for 6 weeks with continual recording over a 24h period. Rats were removed from running wheels and
32 sacrificed under isoflurane anesthesia within 30 min and the soleus muscle and kidneys were harvested, and the cortex separated and flash frozen in liq uid nitrogen. In the 2 nd series, SD (n=3) and F344 (n=3) were allowed access to VWR for 6 weeks and were compared to SED SD (n=5) and SED F344 (n=4), respectively. At the end of the 6 week period and to allow determination of total NO production (from u rinary NO 2 + NO 3 = NO x ) all rats were placed on a nutritionally complete, low nitrate (AIN 76C, MP Biomedicals, Solon, OH) diet for 24 hours, and then housed in metabolic cages for overnight collection of urine. The following day rats were anesthetized wi th isoflurane and sacrificed which in the case of the EX rats meant ~24h after cessation of VWR. The abdomen was opened and the aortic bifurcation cannulated, a blood sample withdrawn, and then the kidneys perfused with cold PBS and the left kidney was re moved and the cortex flash frozen in liquid nitrogen. The perfusate was then switched to a 2% paraformaldehyde lysine periodate (PLP) and the right kidney perfused for 5 minutes. A slice of the perfused kidney was placed in the same fixative for 24 hours at 4C, and then transferred into cold PBS where it remained at 4C for further analyses (see below). In the 3 rd series SD (n=6) and F344 (n=12) rats were allowed 12 weeks access to VWR and compared to SED SD (n=6) and SED F344 (n=12), respectively. In t hese rats the right kidney cortex was removed and flash frozen in liquid nitrogen for later analysis. Immunohistochemistry PLP perfused kidneys were embedded in polyester wax, sectioned at a thickness of 5 m, and then mounted onto glass slides pre treated with gelatin. Sections were dewaxed, peroxidase blocked for 45 minutes then washed with distilled water. To reduce background, sections were steamed in antigen retrieval solution (DAKO) for 30
33 minutes, cooled for 20 minutes, and then blocked with protei n blocker (DAKO) for 15 minutes. Next, sections were placed in a humidified tray and incubated overnight with the mouse monoclonal eNOS antibody (BD Transduction; 1:5000) at 4C. The following day sections were washed with PBS, incubated for 30 minutes w ith one drop of MACH2 mouse HRP polymer secondary antibody (Biocare Medical), washed again with PBS, and then incubated with diaminobenzidine for 5 minutes. Sections were then dehydrated with xylene, mounted onto cover slips using Eukitt mounting medium ( Sigma), and allowed to dry flat prior to observation by light microscopy. Preparation for Telemetry In a preliminary operation, under isoflurane anesthesia and using full sterile technique, a catheter was fed under the skin by trocar and introduced into t he left femoral artery. The catheter was tied into position, and the C40 transmitter unit was sutured to the internal abdominal wall. Rats were singly housed and allowed to recover for ~10 days. Blood pressure (BP) and heart rate (HR) were measured using the DSI equipment and software (St Paul, MN). Western Blot The relative protein abundance of eNOS (BD Transduction; 1:250), SOD isoforms (Stressgen Reagents; EC SOD 1:250, CuZn SOD 1:2000, and Mn SOD 1:2000), p22phox (Santa Cruz Biotechnology, Inc.; 1:5 0), and nitrotyrosine ( Millipore; 1:500) in renal cortical tissue were measured by Western Blot as previously described (Moningka et al. 2011). Briefly, 200 g of homogenized kidney cortex were separated by electrophoresis (7.5% or 12% acrylamide gel, 140 V, 65 min), and then transferred onto nitrocellulose membranes (GE Healthcare) for 60 min at 0.18 A. Membranes were stained with Ponceau Red (Sigma) to check for transfer efficiency/uniformity and equal
34 loading, incubated in blocking solution for 6 0 min, and washed in TBS + 0.05 % Tween before overnight primary antibody incubation at 4C. Membranes were then incubated with the appropriate secondary antibody for one hour, washed and developed with enhanced chemiluminescent reagents (Thermo Scientific). Ba nds were quantified by densitometry using the VersaDoc Imaging System and One Analysis Software (BioRad). Protein abundance was calculated as integrated optical density (IOD) of the protein of interest (after subtraction of background), factored for Ponc eau Red stain (total protein loaded) and positive control, then expressed as a % change from the respective SED control. Analytical Methods As previously described, plasma and urine creatinine concentrations were measured by HPLC, and urine protein level s were detected using the Bradford method (Sasser et al. 2009) Citrate synthase activities in soleus tissue in units of uM/min/g wet tissue weight were based on methods adapted by Srere ( Srere 1969 ) Statistical Analyses Data are presented as mean SE and analyzed with the unpaired Student t test between SED and VWR of each strain using GraphPad Prism software (San Diego, CA) Significance was defined as p<0.05. Results Running activity in both strains is given in Table 2 1 for the series 3 experiments where rats were exposed to 12 weeks of VWR. Running increased gradually over the first 3 weeks of exposure to VWR and by week 4 a maximum value was reached that remained steady thereafter (Table 2 1). The majority of activity occurre d during the wake ( data not shown ) cycle in b oth strains. As shown in Fig. 2 1, BW increased with
35 age in all rats but in both strains the rate of rise was attenuated with VWR when compared to SED rats. In the 1 st series of rats, running activity showed a similar gradual increase as compared to SD rats of the 3 rd series in Table 2 1 (from 1.10.3, to 2.1 0.4, to 3.3 0.7 km/day at weeks 1, 2, and 3). This level of activity was not sufficient to produce an increase in soleus muscle citrate synthase activity (20.13.4 vs. 21.92.7 M/min/g wet weight in SED and 3 weeks VWR, respectively), and as shown in Fig. 2 2 A there was no difference in the kidney cortex abundance of either eNOS or EC SOD in SED vs. EX. In the rats allowed 6 weeks of VWR, running activity had reached a plateau of ~4.7 1.6 km/day after week 4 and there was a clear elevation in sol eus muscle citrate synthase activity (19.11.2 vs. 24.30.6 M/min/g wet weight in SED and EX, respectively; p<0.05). We also observed that HR fell significantly as a result 6 weeks VWR (3485 and 3199 bpm, SED and EX, respectively; p<0.05), although BP was not altered (1055 vs. 979 mmHg, SED and EX, respectively ). As shown in Fig. 2 2 B both eNOS and EC SOD abundance in kidney cortex were markedly reduced after 6 weeks of VWR. In the 2 nd series we compared SD and F344 rats ex posed to 6 weeks VWR vs. SED. Both running activity and BW changes followed the same patterns shown in Table 2 1 and Fig 2 1(data not shown). In the SD rats both eNOS and EC SOD were again reduced in EX vs. SED (Fi g. 2 2 C ) although the magnitude of the fall in eNOS was blunted compared to the 1 st series 6 week VWR rats (Fig 2 2 B ); perhaps reflecting the 24h break from exercise prior to sacrifice. In contrast, in the F344 rat, 6 weeks VWR led to increased renal cortex eNOS while EC SOD was unchanged with EX (Fig
36 2 2 C ). For bo th rat strains, eNOS localized predominately to the endothelial lining of vessels of the kidney (Fig 2 3) and 6 weeks of VWR did not change eNOS localization in either strain. However, eNOS staining decreased in intensity in the SD with VWR (Fig 2 3 A B ), whereas in the F344 rat, eNOS staining increased (Fig 2 3 C D ), in accordance with the Western blot data. No significant differences in PCr, CCr, and UpV values were detected between SED and 6 wk VWR groups within each strain (Table 2 2). In the 3 rd se ries 12 weeks of VWR again significantly decreased kidney eNOS and EC SOD in SD in contrast to the F344 rat where marked increases in eNOS and EC SOD occurred (Fig 2 2 D ). In SD, 12 weeks of VWR had no effect on renal CuZn SOD but increased Mn SOD, while abundance of both enzymes was increased with VWR in F344 (Fig 2 4). Interestingly, absolute values for renal eNOS in the SED SD rat were significantly greater than the SED F344 rat (5.7 1.10 vs. 2.3 0.30 IOD/Ponceau/Positive Control, SD vs. F344, respec tively; p<0.05). Moreover, absolute values for renal EC SOD in the SED SD rat were significantly lower compared to the SED F344 rat (10.21.92 vs. 16.30.72 IOD/Ponceau/Positive Control, SD vs. F344 respectively; p<0.05). Strain differences were detected in the renal oxidative stress response to exercise (Fig 2 5); 12 weeks VWR decreased p22phox abundance and had no effect on H 2 O 2 or nitrotyrosine levels in the SD rat. In contrast, the F344 rat exhibited increases in both kidney cortex p22phox and H 2 O 2 while nitrotyrosine was unchanged with 12 weeks of VWR. Discussion The main novel finding in this study is that the impact of chronic exercise on eNOS and EC SOD in the kidney is variable and influenced by genetic background. Despite
37 comparable running pr ofiles and equivalent renal functional responses to 6 12 weeks of between the young adult male SD and F344 rat. In the SD rat, VWR significantly decreased kidney cortex eNOS EC SOD and p22phox whereas in the F344, VWR increased these variables. Immunohistochemical studies confirm that the strain dependent changes in eNOS occur exclusively in the vascular endothelium. These directionally opposite changes in eNOS and EC SOD abundance between the two rat strains suggest that while chronic mild exercise may have beneficial renal vascular effects in the F344, it could be damaging to the SD. Shear stress is the frictional force of blood against a vessel wall and is considered a primary signal for exercise induced endothelial adaptations. With increases in blood flow and therefore increases in shear stress, the endothelium responds by producing NO, an essential vasodilator that has anti atherogenic properties (Harrison et al. 20 06). In addition, endothelial NO directly up regulates the production of the antioxidant EC SOD (Fukai et al. 2000). The importance of shear stress in mediating exercise induced increases in eNOS/EC SOD is emphasized by the finding that mice deficient in c Src (a tyrosine kinase required for the shear response), failed to increase aortic eNOS and ECSOD protein levels following exercise (Davis et al. 2003). The increased production of vascular EC SOD during exercise is critical to offset one potentially ne gative effect of exercise, namely increased metabolism leading to increased generation of reactive oxygen species (ROS) in the vasculature. The net result of these increases in vascular eNOS and EC SOD will be an improvement in endothelial function.
38 In deed, numerous studies report improved endothelial dependent vasodilation with exercise training (Green et al. 2009; Jasperse et al. 2006; Laughlin 1995). However, in the kidney, blood flow falls with exercise. The exercise induced renal vasoconstrictio n occurs rapidly and probably involves increased renal sympathetic nerve activity as well as activation of various vasoconstrictor agents (Mueller et al. 2004; Stebbins et al. 1995, Ahlborg et al. 1995; Maeda et al. 2004; Stebbins et al. 1993). T he degre e of renal vasoconstriction is exercise intensity dependent, for example, in male SD rats, renal blood flow falls ~ 50% during mild exercise, ~75% with moderate and <90% with severe treadmill running ( McAllister 1998 ). During moderate exercise, glomerular filtration rate (GFR) is well maintained despite falls in RBF, probably due to increased glomerular blood pressure. As exercise intensity increases and further reductions in renal blood flow occur, GFR also falls. Transient, exercise induced proteinuria occurs, possibly secondary to increased glomerular BP ( Poortmans, 1988). Of note, both glomerular hypertension and increased protein excretion can le ad to kidney damage (Brenner et al. 2006; Abbate et al 2006). In addition, when renal vasoconstriction occurs during exercise, shear stress may be reduced within parts of the renal vasculature, leading to reduction in local eNOS and ECSOD. Therefore, al to exercise could have adverse effects on the kidney. As discussed in a recent article by Padilla et al., exercise induced endothelial adaptations in vascular beds not involved in the hyperemic response warrant further study (Padilla et al 2011). In the Wistar rat, Miyauchi and colleagues reported decreased renal eNOS mRNA, protein, and enzyme activity in previously exercise trained rats subjected to 45 min at 25m/min of acute treadmill exercise, whereas in the lung, where blood flow increases
39 with exercise, these variables increased (Miyauchi et al. 2003 ). Our observations in the SD extend these findings and show that mild chronic exercise in the SD led to a sustained decrease in eNOS and EC SOD in kidneys harvested ~30 min after 6 weeks VWR. This effect persisted 24 hours after exercise after both 6 and 12 weeks, and an increase in soleus muscle citrate synthase nor any change in renal eNOS and ECSOD. We chose to use the low intensity VWR exercise protocol since it does not require the use of air jet stress or electric shock to motivate animals to run. A limitation of VWR is that running activity is variable among rats since tota l distance and intensity of exercise cannot be controlled. In this study, despite variation in running activity in distance run was similar in both strains. Despite the similarity in VWR activity between the rat strains, there were profound differences in the renal responses since in the F344 rat, exercise increased eNOS and EC SOD abundance. These are surprising differences in the renal response to exercise between norm al young adult males of the two strains. Since the strain difference is seen with VWR it is unlikely to reflect different stress responses, which could be a concern for forced exercise. Both SD and F344 exhibit intensity dependent falls in RBF with tread mill running (McAllister 1998) although there has been no direct comparison between the two strains. If the F344 have a more efficient cardiac output response to low intensity exercise vs. SD they may not exhibit a fall in RBF with low intensity VWR but this remains to be determined. Alternatively, both strains may undergo renal vasoconstriction and falls in RBF with VWR but the pattern of intrarenal shear stress
40 may vary, since shear stress will depend on flow but also on vessel radius and local viscosi ty. The renal circulation is very complex with intricate branching patterns (Moffat & Fourman, 1963) and there may be architectural differences within the renal vasculature of the two strains that create different local shear responses. These are intrigu ing possibilities that merit further study since genetic bac kground may also determine the renal eNOS and EC SOD responses to exercise in man. If so, knowledge of the renal exercise phenotype would be important in determining recommended exercise intensit y and modality. It is certainly true that at high exercise intensities, acute kidney injury due to rhabdomyolysis and/or dehydration can develop in normal individuals (Clarkson 2007; Seedat et al. 1990; Yan et al. 2010; Bosch et al. 2009). It may be tha t exercise induced loss of renal eNOS and EC SOD predisposes to acute kidney injury. In addition to the strain dependent differences in renal EC SOD in response to exercise, we also observed that while renal Mn SOD increased with exercise in both strains CuZn SOD abundance increased only in F344. Further, abundance of renal p22phox (NADPH oxidase subunit) fell with exercise in SD but rose in F344. These findings suggest that in the SD reductions in eNOS and EC SOD were counterbalanced by antioxidant ef fects (increased MnSOD and falls in p22phox). In the F344 where p22phox increased, there was also an increase in the CuZn SOD and in both strains the unchanged renal nitrotyrosine level suggested no net alteration in renal oxidative stress. In conclusion this study provides evidence of a strain difference in the renal response to voluntary exercise. The loss of eNOS and EC SOD seen in the SD rat could render the kidney vulnerable to superimposed AKI. The cause for the strain
41 difference is currently unk nown but may be related to different intrarenal hemodynamic responses to exercise.
42 Table 2 1. Voluntary wheel running activity presented as average daily km run per day. Sprague Dawley (n=6) Fisher 344 (n=12) Week 1 0.890.31 1.920.32 Week 2 3.601. 19 2.790.52 Week 3 4.031.27 3.160.48 Week 4 4.791.67 3.330.53 Week 6 4.231.57 2.420.34 Week 9 4.051.68 3.370.54 Week 12 2.051.11 2.590.34 Table 2 2 Renal functional responses in SD and F344 after 6 weeks voluntary exercise SD F344 SE D (n=6) EX (n=3) SED (n=4) EX (n=3) PCr (mg/dl) 0.100.01 0.080.00 0.080.01 0.070.01 CCr ( mL /min/100g BW) 2.470.24 2.970.14 2.690.35 3.070.27 UpV (mg/day/100g BW) 8.151.35 5.991.47 6.090.14 6.880.22 SD, Sprague Dawley; F344, Fisher 344; SED, sedentary; EX, exercise; PCr, plasma creatinine; CCr, creatinine clearance; UpV, urinary protein excretion. *p<0.05 vs. respective SED; +p<0.05 vs. SED EX.
43 Figure 2 1. Body weight (BW) measurements in the (A) Sprague Dawley (SD) and (B) Fisher 344 (F 344) rat. Voluntary exercise effectively reduced BW throughout the 12 week training period in both the SD and F344 rat.
44 Figure 2 2. Kidney cortex endothelial nitric oxide synthase (eNOS) and extracellular superoxide dismutase (EC SOD) abundance after 3, 6, or 12 weeks of voluntary wheel running (VWR) in the Sprague Dawley (SD) and Fisher (F344) rat. In the SD rat, no changes in eNOS or EC SOD were detected after (A) 3 weeks of VWR, whereas significant falls were observed after (B & C) 6 and (D) 12 we eks of VWR. In the F344 rat, both (C) 6 and (D) 12 weeks of VWR increased eNOS and EC SOD. Relative density units were expressed as a % from respective SED controls. *Denotes a statistical significance of p<0.05 between the two groups.
45 Figure 2 3 Effect of 6 weeks voluntary wheel running on the immunoreactivity of endothelial nitric oxide synthase (eNOS) in the kidney of (A,B) Sprague Dawley and (C,D) Fisher 344 rats. For all groups, eNOS predominately localizes to the endothelium of vessel walls (indicated by arrowheads). Localization was unchanged after 6 weeks of VWR in either strain. However, in the SD, eNOS immunoreactivity decreased with exercise, whereas in the F344, it increased.
46 Figure 2 4 Impact of 12 weeks voluntary wheel runnin g (VWR) on CuZn superoxide dismutase (CuZn SOD) and manganese SOD (Mn SOD) in the kidney cortex of the (A) Sprague Dawley (SD) and (B) Fisher 344 (F344) rat. 12 weeks of VWR increased CuZn SOD in the F344 only and increased Mn SOD for both strains. Relat ive density units were expressed as a % from respective SED controls. *Denotes a statistical significance of p<0.05 between the two groups.
47 Figure 2 5 Impact of 12 weeks voluntary wheel running (VWR) on oxidative stress; p22phox protein and nitrotyr osine protein in the kidney cortex of the (A) Sprague Dawley (SD) and (B) Fisher 344 (F344) rat. In the SD rat, 12 weeks VWR decreased p22phox, whereas in the F344 rat it increased. No differences were detected in nitrotyrosine abundance in either strain Relative density units were expressed as a % from respective sedentary (SED) controls. *Denotes a statistical significance of p<0.05 between the two groups.
48 CHAPTER 3 EXERCISE EXACERBATES ISCHEMIA REPERFUSION INDUCED ACUTE KIDNEY INJURY IN THE SPRAGUE DAWLEY BUT NOT FISHER 344 RAT B ackground In vessels of skeletal muscle, lung, and heart, exercise increases blood flow which imparts increased shear stress on the endothelium leading to increases in vasodilatory molecules such as nitric oxide (NO). In addition to increased NO, exercise also stimulates expression of the antioxidant, extracellular superoxide dismutase (EC SOD) (Fukai et al. 2000). Applied in the disease setting, exercise significantly improves impaired endothelial dependent vasodilation associated with aging (Spier et al. 2007), diabetes (Sakamoto et al. 1998), and hypertension (Higashi et al. 1999). Thus, one mechanism by which exercise is beneficial is through the induction of endothelial eNOS and EC SOD in the vasculature. Despite i ncreasing evidence for the beneficial role of NO in exercise hyperemia, its role in tissues where blood flow reduces is less understood. For instance, in the kidney, blood flow is markedly decreased, a process dependent on intensity and increased sympathe tic outflow (Poortmans 1990; Tidgren et al. 1991; Mueller et al. 1998). With an acute bout of high intensity treadmill exercise, Miyauchi et al. reported significant decreases in renal endothelial NO synthase (eNOS) mRNA, protein, and enzyme activity. Al though not measurable the authors concluded that exercise decreased shear stress in the kidney leading to falls in eNOS. Furthermore, exercise trained mice deficient in c Src, a critical component of the shear stress signaling cascade, failed to increase aortic eNOS and EC SOD protein compared to exercise trained wild type mice (Davis et al 2002). Altogether, these studies suggest the importance of blood flow in dictating increased NO production and EC SOD with exercise.
49 As previously reported in Chapter 2, we found that in the Sprague Dawley (SD) rat VWR EX leads to falls in renal eNOS and EC SOD which may render the kidney vulnerable to ischemia/reperfusion (IR) induced acute kidney injury (AKI), an injury associated with reduced NO production and incre ased oxidative stress (Noiri et al. 2001). Thus, the aim of the present study was to investigate if exercise exacerbates IR induced AKI in the SD. Strain differences were also explored since predisposition to chronic kidney disease (CKD) varies between t he Sprague Dawley and Fisher 344, with the latter being more protected (Erdely et al. 2003; Moningka et al. 2011). Based on our finding reported in Chapter 2 that in the F344 VWR EX results in increased renal eNOS and ECSOD, we hypothesized that the exerc ised F344 might be protected against IR induced AKI. Finally, in an effort to define a hemodynamic mechanism for the different renal eNOS/EC SOD responses to VWR exercise training in the two strains, we also employed the radiolabeled microsphere method to determine renal blood flow during exercise. Because of technical issues we used mild treadmill exercise (TM EX) rather than VWR EX in these studies. Methods Animal Procedures All animal handling was in accordance with and approved by the University of Fl Fisher 34 (F344) rats 10 12 weeks of age were purchased from Harlan (Indianapolis, IN). All rats were singly housed in a temperature and light controlled environment with a ccess to standard rat chow and water. For each strain, rats were randomly divided into a sedentary control (SED; SD n=12; F344 n=15) or voluntary exercise (VWR EX; SD n=13; F344 n=15) group. For 12
50 weeks SED rats led a sedentary lifestyle, whereas the V WR EX rats were given individual running wheels with 24 hour ad libitum access (Lafayette Instruments). VWR activity was measured by attached odometers and acquired using the Activity Wheel Monitor Software (Lafayette Instruments, Lafayette, IN). At the end of the 12 weeks, rats were prepared for right uninephrectomy (UNX) and ischemia/reperfusion (IR) induced acute kidney injury (AKI) to the left kidney, ~24 hours after running cessation. All surgeries were with full sterile technique. While under gener al anesthesia using isoflurane (5% induction and 1 2% maintenance dose), the left renal pedicle was clamped for 35 minutes. In order to compare injury susceptibility between SED and VWR EX groups, we chose a clamping period that did not cause maximal inju ry, and based on preliminary studies 35 minutes met these criteria. To maintain tissue moisture, 0.9% sterile saline solution was applied over the ischemic kidney. During the 35 minute ischemic period, we performed UNX of the right (normal) kidney. Cort ical sections of the right kidney were immediately flash frozen in liquid nitrogen and stored at 80C for further analyses. Incisions were closed with Vicryl silk (internal suture) and stapled (external suture). The analgesic buprenorphine (0.05 mg/kg b ody weight) was injected subcutaneously and the rat recovered for the next 24 hours. This allowed the kidney to reperfuse 24 hours prior to a renal function study. Additional studies were performed in F344 SED (n=9) and F344 VWR EX (n=9) and SD SED (n=10 ) and SD VWR EX (n=7) with right UNX only. All rats were then prepared for terminal inulin clearance studies 24 hours after UNX IR or UNX surgery for determination of glomerular filtration rate (GFR) and renal plasma flow (RPF). Rats were anesthetized wi th Inactin (intraperitoneal injection, 120
51 mg/kg BW) and placed on a heating table to maintain a body temperature of 371C. The trachea was cannulated with PE 240 tubing and exposed to a constant flow of oxygen. Using PE 50 tubing filled with heparinize d saline, the femoral artery was cannulated for measurement of blood pressure (BP) and for collection of blood samples. A baseline BP was taken as well as a blood sample of ~250 for measurement of creatinine. The femoral vein was then cannulated and a 0.5 mL bolus of pre dialyzed FITC Inulin (final concentration 2 mg/ mL 0.9% NaCl; Sigma) was infused; thereafter, FITC inulin was infused at a rate of 1.2 mL /100g BW/hr. To ensur e that the rat was globulin was infused at a rate of 1% 100g BW/hr for the first fifteen minutes, and then at a rate of 0.15% 100g BW/hour for the remainder of the study. Next, the abdominal cavity was opened to expose the left kidney and bladder. A non occluding catheter was placed in the left renal vein to obtain an arterial venous inulin extraction for calculation of RPF. The bladder was cannulated with flanged PE 50 tubing for collectio n of urine and for gravemetric determination of urine volume and flow rate. The level of anesthesia, BP, and body temperature were monitored throughout. After a 60 minute stabilization period, two x 20 minute urine collections with mid point collections of femoral arterial and renal venous blood were taken for FITC Inulin measurement. All blood samples were centrifuged for collection of plasma which was analysed for FITC inulin content and together with urine samples were used to calculated .glomerular f iltration rate (GFR, from inulin clearance), renal plasma flow (RPF, from (Urine inulin concentration / plasma A V inulin extraction)/urine flow) and filtration fraction (FF, from GFR/RPF). The rat was sacrificed by exsanguination and the lung and left ki dney (with IR induced AKI)
52 were removed. A portion of the kidney was prepared for histology (see below), and the remaining divided into sections of cortex. Lung and kidney tissue were flash frozen in liquid nitrogen and stored at 80C for further analys es. Renal Pathology Histology related procedures were performed in the Gainesville Veterans Affairs formalin (Sigma) for 48 hours, processed, embedded in paraffin wax, and then cut into 5 m sections. All sections were stained with periodic acid schiff (Sigma) and counterstained with hematoxylin and eosin (Sigma). B.P. Croker, an expert renal pathologist, scored all tissue sections for acute tubular necrosis while blinded by the treatment groups. He evaluated the tubules of the renal cortex for evidence of cell swelling, brush border loss, nuclear condensation, karyolysis (dissolution of the nucleus), regeneration, capillaritis (endothelial inflammation), and cell sloughin g. Each were involved, 1 = 2 = 11 25 %, 3 = 26 50 %, 4 = 51 75 %, 5= 76 100% were involved. Western Blot The protein abundance of endothelial nitric oxide synthas e (eNOS), extracellular superoxide dismutase (EC SOD), cytosolic copper zinc SOD (CuZn SOD), mitochondrial manganese SOD (Mn SOD), and p22phox were detected in whole kidney cortex tissue homogenates as previously described (Moningka et al. 2011). Whole lu ng tissue were homogenized similarly and detected for eNOS and EC SOD abundance only. Kidney cortex and lung samples were run on 7 or 12% acrylamide gels (BioRad) and then semi dry transferred (BioRad) onto nitrocellulose membranes (Biorad). To
53 confirm e qual loading, membranes were stained with Ponceau Red (Sigma), a dye that stains for protein. Primary antibody concentrations were as follows: 1) eNOS (BD Transduction; 1:250), 2) EC SOD (Abcam; 1:250), 3) CuZn SOD (Stressgen; 1:2000), 4) Mn SOD (Stressg en; 1:2000), and 5) p22phox (Santa Cruz; 1:50). Membranes were developed with chemiluminescent reagents and bands at the expected molecular weight were analyzed by densitometry using the VersaDoc Imaging System and One Analysis Software (BioRad). Analytical M ethods FITC Inulin concentrations (mg/ mL ) in urine and plasma were measured by fluorescence (excitation and emission wavelengths, 485 nm and 530 nm, respectively) as previously described (K night et al. 2007). Creatinine concentrations (mg/dl) in plasma were measured by HPLC (Sasser et al. 2009). Blood Flow Measurements Blood flow measurements to the kidney, mesentery, and select muscles of the hindlimb were conducted on a separate set of male SD and F344 rats (10 12 weeks of age; SD n=13; F344 n=9) using the reference sample microsphere method (Laughlin et al. 1982; Behnke et al. 2006). A subset of sedentary F344 rats were provided from a previous study (Dominguez et al. 2010) for at rest RBF measurements (unpublished) only (n=10). For these studies, training and acute exercise was by TM EX since injection of microspheres through indwelling catheters would interfere with the voluntary wheel apparatus. Initially, all rats were habituated on a motor driven rodent treadmill. During this period, rats walked/ran at an initial intensity of 5 m/min for 5 min/day and at zero degree incline for 5 days. Intensity progressed to 15 m/min by day 5. Rats were then randomly separated into SED (SD; n= 7 and F344; n=3) and TM EX (SD; n=6 and
54 F344; n=6) groups. SED rats remained sedentary for 10 12 weeks while TM EX rats were gradually conditioned to run at an intensity of 15 m/min, 5 days/week, and 15 incline for 20 minutes at week 1, 30 minutes at wee k 2, 40 50 minutes at week 3, 50 60 minutes at week 4, and then 60 minutes at week 5. TM EX rats continued to run for 60 minutes at the same intensity and incline for the remainder of the study. This training protocol effectively increases skeletal muscl e citrate synthase activity (Delp et al 1993). Training adherence was prompted with small bursts of compressed air and/or a low voltage electric grid at the back of the treadmill; however, these tactics were used mainly during the first three weeks and m inimally thereafter. At the end of the 10 12 weeks, all rats were surgically implanted with indwelling catheters in their carotid and caudal arteries as previously described using PE 10 and PE 50 tubing, respectively (Behnke et al. 2006). All surgeries we re performed with sterile technique and while the rat was under general isoflurane anesthesia. Both catheters were exteriorized through the skin, primed with heparinized saline, and then plugged. After incisions were closed, rats were given the analgesic bupivacaine (0.5 mg/ mL ), and then returned to individual cle an cages where they were carefully Next, all rats, including SED, were placed on a motor driven treadmill. Running speed gradually increased to 15 m/min (15 incline) over 30 s and then maintained. While r saline syringe connected to a Harvard infusion withdrawal pump set at a withdrawal rate of 0.25 mL /min. After 3 min of treadmill running, radiolabelled microspheres ( 46 Sc or 85 Sr, rand omly chosen; PerkinElmer NEN
55 Microspheres were sonicated and vortexed prior to injection to prevent clumping and to ensure thorough mixing. The carotid artery catheter was then flushed with 0.5 mL of warm (37C) 0.9% saline, and connected to a pressure transducer for measurement of BP. Withdrawal of the reference blood sample started ~30 s prior to microsphere infusion and continued for ~30 60 s after injection to ensure that all microspheres were cleared from the catheter line. Exercise wa s then terminated. The rat was then allowed to rest for 30 min after which a second microsphere injection (using the other radioactive label) was performed in a similar approach as just described, except that the microsphere injection and withdrawal of the reference sample were conducted while the animal rested quietly. To ensure optimal resting conditions, room lights were dimmed while the rat situated itself in a corner of the treadmill that was topped with a cover. Rats were then euthanized with an ove rdose of sodium pentobarbital (>100 mg/kg) injected into the carotid artery. The kidneys, gastrocnemius, and mesentery were carefully dissected, weighed, and placed into tubes. For the gastrocnemius muscle, the red portion was dissected. All blood and t issue samples were detected for their level of radioactivity with a gamma counter (Packard Cobra II Auto Gamma). Individual tissue blood flows were calculated using the reference sample method (Ishise et al 1980). Adequate microsphere distribution was c onfirmed by demonstrating a <31% difference in blood flow between paired kidneys. Statistical Analyses All data are presented as mean SE and analyzed with the unpaired or paired Student t test, or with ANOVA using GraphPad Prism software (San Diego, CA ). Significance was defined as p<0.05.
56 Results Body weight (BW) responses to VWR EX and running activity were comparable in both strains (Fig. 3 SED and VWR EX. Despite gradual increases in BW in both SED and VWR EX, presumably due to normal rate of growth, VWR EX significantly reduced BW compared to SED in both strains. Running activity steadily increased from weeks 1 to 4, reaching a plateau of ~3 4 km/day, a pattern demonstrated for bo th strains, although the SD rat demonstrated greater variability. As illustrated in Table 3 1, the renal hemodynamic response to 24h UNX in the SD rat was similar between SED and VWR EX groups, showing falls in GFR and RPF by ~ 50% of control (~1 mL /100g BW/min). When IR was superimposed on UNX much greater falls in GFR and RPF occurred in both SED and EX groups and plasma creatinine (PCr) rose markedly. However, the fall in GFR and rise in PCr were greatly exacerbated in the VWR EX SD rats subjected to UNX IR compared to SED, due to greater falls in RPF and FF in the EX group (Table 3 1). In the F344 rat, as seen in the SD rat, the renal hemodynamic response to UNX was similar in SED and VWR EX groups (Table 3 2). Again combination UNX and IR lead to increases in PCr and falls in GFR, RPF, and FF compared to UNX alone. In contrast to SD, there was no difference in the severity of the falls in GFR and RPF and increase in PCr in the EX vs. SED F344. The combination of UNX and IR led to structural chan ges in kidney cortex characteristic of AKI which in the SD tended to be worse in the VWR EX rats and there was significant brush border loss compared to SED (Fig. 3 2 A ). In the F344 rat, UNX
57 IR generally affected SED and VWR EX groups to the same degree, although there was less cell sloughing in the VWR EX rats (Fig. 3 2 B ). VWR EX reduced kidney cortex eNOS (by 16%) EC SOD (by 38%) and p22phox (by 28%) abundance in the normal (right) kidney of the SD rat (Fig. 3 3 A ). IR injury significantly increased eN OS abundance similarly for both SED and VWR EX groups, but had no further effect on EC SOD and p22phox abundance in the SD rat. By contrast, in the F344 rat, VWR EX increased eNOS (by >100%) EC SOD (by >100%) and p22phox (by 62%) kidney cortex abundance in the normal kidney (Fig. 3 3 B ) and IR injury markedly increased eNOS in a similar manner for both SED and VWR EX groups. In F344 rats, EX reduced EC SOD with IR; however, n o further changes were detected in p22phox abundance with IR injury in either gr oup of F344 rats. In the lung, where blood flow increases with exercise, the abundance of both eNOS and EC SOD increased significantly with VWR EX (Fig. 3 4). For determination of RBF during TM EX, additional rats of each strain comparable in age were obt ained, also from Harlan (Indianapolis, IN). In the SD rat, 10 12 weeks of TM EX reduced BW, increased the soleus weight: BW ratio and had no effect on left ventricle (LV):BW ratio (Fig. 3 5 A ). In the F344 rat, 10 12 weeks of TM EX increased LV:BW but had no effect on BW or soleus weight (Fig. 3 5 B ). As shown in Fig. 3 6 A blood flow to the red portion of the gastrocnemius increased in SED SD whereas blood flow to the kidney and mesentery decreased during a ~4 minute bout of TM EX (15 m/min). The chroni cally TM EX trained SD rat showed similar trends but only mesenteric blood flow fell significantly with acute exercise (Fig. 3 6 A ). During the bout of TM EX, SED F344 rats increased red
58 gastrocnemius blood flow and decreased blood flow to the kidney while the decline in mesenteric blood flow was not significant. Whereas the increase in gastrocnemius blood flow persisted with acute exercise in the TM EX trained rats, the fall in RBF was completely abolished (Fig. 3 6 B ). Of note, the resting RBF (factored for BW) was remarkably similar in both SED SD a nd SED F344 (59748 and 59251L /min) and also in VWR EX trained SD and F344 rats (332106 and 37372 L /min). In both strains chronic exercise training lowered the resting value of RBF by ~35 45% (both p<0.0 5). Discussion The main novel findings in this study are that 1) whereas chronic VWR EX causes directionally opposite changes in renal cortical eNOS and EC SOD in SD vs. F344, in the lung, increases in both enzymes are seen in both strains, 2) 12 weeks of VWR EX exacerbates IR induced falls in GFR in the young adult male SD but not in the F344 rat, 3) using the radiolabeled microsphere technique, we found that TM EX leads to significant and similar reductions in resting RBF in both rat strains. In the untr ained SED animals, acute exercise significantly and similarly reduces RBF in both strains while with training the acute RBF response to TM EX is blunted in SD and is absent in the F344. There is substantial evidence provides benefit in skeletal muscle an d coronary blood vessels and protects the heart against IR induced injury (Sindler et al. 2009; Fogarty et al. 2004; Powers et al. 2007). Several mechanisms are involved in the cardiac protection including the up regulation of myocardial antioxidant capac ity which helps combat the increased content of reactive oxygen species, lipid peroxides, protein oxidation, and protein nitration associated with myocardial IR (Powers et al. 2007). The
59 exercise induced increases in antioxidant capacity are through incre ased shear stress which stimulates NO production and in turn, also stimulates production of the EC SOD antioxidant. This was confirmed by a study that discovered a lack of EC SOD up regulation with exercise in aorta (where shear stress increases) in eNOS deficient mice (Fukai et al. 2000). Pulmonary blood flow (and shear stress) increases with exercise and as shown here eNOS and EC SOD abundance in the lung increase with exercise in both SD and F344. However, in the kidney, blood flow is thought to be re duced during exercise. The eNOS mRNA, protein abundance, and enzyme activity decreased with an acute bout of treadmill exercise in trained Wistar rats, suggesting that shear stress is reduced in the kidney (Miyauchi et al. 2000). Exhaustive exercise lead ing to functional and structural kidney damage in the untrained SD rat also decreased renal cortical NOS and SOD activity (Lin et al. 2010). In Chapter 2, we reported that chronic VWR EX reduced renal eNOS and EC SOD in the SD rat, whereas in the F344 rat these beneficial enzymes increased. We confirmed this response of increased eNOS and EC SOD in the F344 rat using chronic TM EX (Moningka et al. 2011, in revision). Falls in NO bioavailability can render the kidney susceptible to injury since NO is cr itical for normal renal function and its deficiency leads to chronic kidney disease, CKD (Baylis 2007). In addition, endothelial injury is a major mechanism by which IR induced AKI propagates. There is recruitment of local inflammatory signals which up r egulate reactive oxygen species leading to reductions in NO bioavailability and therefore development of renal dysfunction (Le Dorze et al. 2009). Several studies have reported cases of renal ischemia leading to exercise induced AKI ( Seedat et al. 1990; Ya n et al. 2010; Bosch et al. 2009) some of which is due to rhabdomyolysis which leads to
60 myoglobin induced AKI (Bosch et al. 2009). Also, when there is pre existing oxidative stress, development of exercise induced AKI is high, for example, in patients wit h renal hypouricemia ( Yan et al. 2010; Saito et al. 2011; Ishikawa 2002 ). Based on these findings that exercise may cause/exacerbate AKI and on our observed strain differences in renal eNOS and EC SOD with exercise training, we conducted the present study to determine whether exercise influences susceptibility to UNX IR induced AKI in the two strains. D espite comparable running activities and BW responses to VWR training, we discovered differences in the impact of VWR EX on susceptibility to IR injury be tween the SD and F344 rat. In the SD rat, 12 weeks of VWR EX reduced renal eNOS and EC SOD, rendering the kidney susceptible to IR induced AKI since VWR EX exacerbated the falls in GFR, RPF, and increased PCr associated with UNX IR. In contrast, we found that in the F344 rat, 12 weeks of VWR EX increased renal eNOS and EC SOD abundance and that the chronically exercised F344 showed some protection against the decline in renal function with IR. We also discovered a strain difference in susceptibility to I R in SED rats with the F344 more vulnerable. UNX IR reduced GFR by 63% and 87% in the SD and F344 rat, respectively. Moreover, there were also greater reductions in RPF with UNX IR in the F344 rat versus SD rat (65% vs. 78%, SD vs. F344, respectively). Despite greater reductions in renal function to UNX IR, exercise afforded protection in the F344 rat. Overall, o ur data suggest that vulnerability to an oxidative stress mediated renal insult such as IR induced AKI is determined by the state of endothelia l health which is influenced by genetic background.
61 It is interesting that UNX IR had no effect on either kidney cortex EC SOD or p22phox in either strain. Moreover, the directional responses of EC SOD, and p22phox to VWR EX were also unchanged by IR, aga in for both strains. These data indicate that despite the decline in renal function with UNX IR seen for both strains, the antioxidant/ pro oxidant EC SOD and p22phox were not influenced by IR injury. We also found that IR increased eNOS abundance in bot h the SED and VWR EX rats of both strains. NOS protein has been shown to increase with IR injury but this is usually the inducible form of NOS associated with inflammation (Goligorsky et al. 2002; Chatterjee et al. 2002); the inducible NOS isoform was not investigated in the present study. While the increased eNOS with IR may represent an attempt at compensation, falls in renal function and histological evidence of injury occur in both strains. Despite a lack of marked differences in each index, we found that in the IR susceptible SD rat, brush border loss was greater in the VWR EX IR compared to the SED EX IR which is in accordance with the greater functional injury with UNX IR seen in the exercised SD. Despite potential injury associated with renal is chemia, a reduction in RBF due to exercise is considered a normal physiological response since it allows diversion of blood to accommodate the increase in oxygen demand by active muscles (McAllister 1998). Evidence points to increased renal sympathetic ne rvous system outflow as the primary mechanism (Tidgren et al 1991). Using exercise trained rabbits, Mueller et al. reported that dose dependent increases and concomitant falls in renal blood flow caused by adrenergic receptors (Mueller et al. 1998). Angiotensin II, endothelin 1, and vasopressin are also involved (Stebbins et al. 1995, Ahlborg et al. 1995; Maeda et al. 2004; Stebbins et al. 1993). Despite fal ls
62 in renal blood flow, glomerular filtration rate is well maintained at low intensities due to compensatory increases in filtration fraction; however, at high intensities, renal blood flow is sev erely compromised (Poortmans & Vanderstraeten, 1994). To d etermine whether there were differences in renal hemodyamic responses to exercise between the two strains, which might account for the differences in renal endothelial enzymes with exercise, we utilized the radiolabeled microsphere method to measure real t ime total RBF during exercise and at rest. We found that in the SD rat, blood flow during exercise increased in the red gastrocnemius but decreased in the kidney and mesentery; however, with chronic TM EX, the magnitude of fall in RBF during exercise decr eased. We also found that in the F344 SED rat, kidney blood flow significantly reduced during acute exercise but that this response was lost in the TM EX group. These findings agree with observations by Armstrong and Laughlin who showed that in SD rats ch ronic exercise reduced the magnitude of the decrease in RBF after acute treadmill exercise (Armstrong & Laughlin 1984). In conclusion, this study provides evidence that genetic background influences susceptibility to IR induced AKI. While running activ ity and BW responses to VWR were comparable between the male SD and F344 rat, their functional responses to UNX IR were different. While UNX IR injury markedly reduced renal function in both SED and EX of both strains, in the SD, VWR EX exacerbated falls in GFR, RPF, and PCr. This is in contrast to the F344 rat, where VWR EX provided some protection against renal UNX IR injury. Indices of acute structural injury in the kidney cortex also support these functional data. These findings align with our obser vations that 12 weeks of VWR EX reduced eNOS and EC SOD renal cortical abundance in the SD rat but increased eNOS
63 and EC SOD renal cortical abundance in the F344 rat. However, these strain differences are not related to different renal hemodynamic respons es to chronic and acute exercise since resting RBF fell similarly with exercise training in both strains, and the robust response to acute exercise seen in SED rats was blunted by exercise training in both strains. Perhaps differences in intrarenal vascul ar architecture and shear stress patterns can account for the different eNOS and EC SOD responses to exerc ise seen in the two rat strains.
64 Table 3 1. Sprague Dawley Renal hemodynamic responses SED VWR EX UNX UNX IR UNX UNX IR GFR ( mL /min/100g BW) 0. 470.08 0.170.04* 0.450.03 0.040.01*+ RPF ( mL /min/100g BW) 2.740.82 0.960.23* 1.830.26 0.300.15*+ FF 0.240.02 0.190.03 0.280.03 0.120.02* PCr (mg/ mL ) 0.280.02 1.070.27* 0.270.03 2.420.35*+ GFR, glomerular filtration rate; RPF, renal plas ma flow; FF, filtration fraction; PCr, plasma creatinine. *p<0.05 vs. respective UNX, +p<0.05 vs. SED UNX IR. Table 3 2. Fisher 344 Renal hemodynamic responses. SED VWR EX UNX UNX IR UNX UNX IR GFR ( mL /min/100g BW) 0.470.03 0.060.01* 0.580.04 0. 160.06* RPF ( mL /min/100g BW) 1.840.23 0.400.10* 2.530.26 1.020.35* FF 0.280.02 0.200.02* 0.250.02 0.210.05 PCr (mg/ mL ) 0.250.04 1.770.19* 0.290.02 1.780.28* GFR, glomerular filtration rate; RPF, renal plasma flow; FF, filtration fraction; PCr, plasma creatinine. *p<0.05 vs. respective UNX.
65 Figure 3 1. Effect of 12 weeks voluntary exercise (VWR EX) on body weight (BW) and running activity in the (A) Sprague Dawley and (B) Fisher 344 rat. Voluntary exercise effectively reduced BW throug hout the 12 week training period in both the SD and F344 rat. For each strain, total average distance (km) run per day steadily increased over the first few weeks and remained relatively constant for the remainder of the study. *Denotes a statistical sign ificance of p<0.05 between the two groups. # Denotes a statistical significance of p<0.05 compared to baseline.
66 Figure 3 2. Effect of 12 weeks voluntary exercise (VWR EX) and renal uninephrectomy (UNX) ischemia/reperfusion (IR) injury on indices of ac ute renal structural injury in kidney cortex of (A) Sprague Dawley (SD) and (B) Fisher 344 (F344) rats. Analyses were conducted on IR kidneys only since minimal to no injury was expected for UNX alone. In the SD rat, EX and IR tended to increase all mark ers of acute injury and significantly increased the level of brush border loss. In the F344 rat, IR affected SED and EX groups to the same degree; however, 12 weeks of VWR EX ameliorated the level of sloughing against IR. *Denotes a statistical significan ce of p<0.05 compared to SED IR.
67 Figure 3 3 Effect of 12 weeks voluntary exercise (VWR EX) and renal uninephrectomy (UNX) and ischemia/reperfusion (IR) injury on endothelial nitric oxide synthase (eNOS), extracellular superoxide dismutase (EC SOD), an d p22phox abundance in kidney cortex of (A) Sprague Dawley (SD) and (B) Fisher 344 (F344) rats. In the SD rat, VWR EX reduced eNOS, EC SOD, and p22phox, whereas in the F344 rat, these proteins increased. For both strains, IR increased eNOS for both SED a nd VWR EX groups but had no further effect on EC SOD and p22phox abundance. *Denotes a statistical significance of p<0.05 between the two groups.
68 Figure 3 4. Effect of 12 weeks voluntary exercise (VWR EX) and renal uninephrectomy (UNX) and ischemia/ reperfusion (IR) injury on endothelial nitric oxide synthase (eNOS), extracellular superoxide dismutase (EC SOD), and p22phox abundance in lung of (A) Sprague Dawley (SD) and (B) Fisher 344 (F344) rats. For both strains, VWR EX significantly increased eNO S and EC SOD in lung. *Denotes a statistical significance of p<0.05 between the two groups.
69 Figure 3 5. Effect of 10 12 weeks treadmill exercise (TM EX) on final BW, soleus weight:BW, and left ventricular (LV) weight:BW in the (A) Sprague Dawley (SD ) and (B) Fisher 344 (F344) rat. In the SD rat, TM EX significantly reduced BW, increased soleus weight but had no effect on LV weight. In the F344 rat, despite no effect of TM on BW and soleus weight, LV hypertrophy was evident. *Denotes a statistical significance of p<0.05 between the two groups.
70 Figure 3 6. Blood flow (Q) measurements in sedentary (SED) and treadmill exercise (TM EX) rats chronically trained for 10 12 weeks taken at rest and during exercise in the (A) Sprague Dawley (SD) and (B ) Fisher 344 (F344) rat. In the SD SED rat, blood flow during exercise increased in the red gastrocnemius (gastroc) but decreased in the kidney and mesentery; however, with chronic TM EX, the changes in red gastrocnemius and kidney blood flow were blunted while mesentery blood flow reduced during exercise. In the F344 SED rat, only kidney blood flow significantly reduced during exercise, a response that was lost in the TM EX group. Further, red gastrocnemius blood flow significantly increased during exer cise in the chronically TM EX trained F344 rat. *Denotes a statistical significance of p<0.05 compared to rest. Note: For red gastroc and mesentery BF in SED F344 n=3 but for RBF in SED F344 n=13; see methods for explanation.
71 CHAPTER 4 TWELVE WEEKS OF T READMILL EXERCISE DOES NOT REVERSE AGE DEPENDENT CHRONIC KIDNEY DISEASE IN THE FISHER 344 MALE RAT Background Exercise reduces morbidity and mortality from various cardiovascular diseases in the elderly population (Larson & Bruce 1987). It has benefic ial metabolic actions including reduction in plasma triglycerides, increases in the high density lipoprotein to low density lipoprotein ratio and insulin sensitivity, and improves cardiac function (Heath 1983; Sun et al 2008) Physical activity may also reduce depression and mental stress thus indirectly improving blood pressure (Paluska & Schewnk, 2000) Another beneficial response to exercise is the stimulation of endothelial nitric oxide synthase (eNOS), the main enzyme responsible for vascular NO production which is essential for optimal vascular health. An increase in endothelial shear stress as a result of increased blood flow results in 1) prolonged eNOS mRNA stability, 2) increased eNOS protein translation, and 3) increased NOS enzyme activity (Harrison et al 2006). In addition, increased shear stress increases the antioxidant extracellular su peroxide dismutase (EC SOD) enzyme by an NO dependent mechanism (Fukai et al 2000). Mice deficient in c Src, a critical component of the shear stress signaling pathway, do not increase their aortic eNOS or EC SOD protein abundance with exercise (Davis et al 2003). The shear stress induced up regulation of eNOS and EC SOD enhances endothelium dependent vasodilation in parts of the circulation where blood flow increases during exercise, such as skeletal muscle, pulmonary and coronary circulations (Spier e t al 2004; Johnson et al 2000; DeSouza et al. 2000; Muller et al 1994). As a result of all these effects exercise has powerful cardiovascular protective actions.
72 In the kidney, blood flow decreases during exercise in an intensity dependent manner to sh unt adequate perfusion to working muscles. At very high exercise intensities renal blood flow can reduce from the normal ~20% of cardiac output to ~1% (Castenfors et al 1967). Under some circumstances a fall in blood flow leads to a reduction in shear s tress, which could lead to reductions in endothelial NOS and EC SOD. Indeed, in the male Wistar rat, acute treadmill exercise in pre trained rats reduced renal eNOS mRNA, protein, and enzyme activity, whereas increases occurred in the lungs where blood fl ow increases with exercise (Miyauchi et al 2003). Blood flow also falls with exercise in the splanchic circulation and mesenteric arteries isolated from trained rats do not show enhanced flow mediated dilation in these vascular beds, whereas in skeletal muscle arteries exercise does improve flow mediated dilation (Sun et al 1998). Thus, one direct endothelial benefit of exercise is shear stress dependent, and in organs such as the kidney where blood flow falls, depending on how intrarenal shear stress i s altered, endothelial function may not be improved, or may even be impaired (Miyauchi et al 2003). This is of particular concern in the aging kidney where falls in eNOS abundance and NOS activity (Erdely et al 2003) and increased oxidati ve stress occur (Gomes et al. 2009) in conjunction with slowly developing glomerular and tubulointerstitial injury (Baylis & Corman,1998). Superimposing additional eNOS and EC SOD deficits could exacerbate age dependent kidney damage. Indeed, 6 weeks of exercise in old C57BL/6J mice magnified the age associated renal structural injury (Lichtig et al 1987). The main purpose of the present study was to determine the impact of chronic treadmill exercise on the progression of age dependent renal injury in the male Fisher
73 344 (F344) rat. We also investigated the effect of exercise on the protein levels of various isoforms of the NOS and SOD enzymes, in kidney cortex and medulla as well as aorta in young and old rats. Furthermore, since reactive oxygen spe cies contributes to the development of age dependent renal changes, we assessed several markers of oxidative stress (Asghar et al 2007). Methods Animal Procedures All animal handling was in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved and monitored by the West Virginia University and University of Florida Institutional Animal Care and Use Committees. Young (3 months; n=16) and old (22 24 months; n=16) male Fisher 344 (F344) rats supplie d from Harlan (Indianapolis, IN) were purchased from the National Institute of Aging (NIA Bethesda, MD). All rats were housed in a temperature/light controlled environment and given access to standard rat chow and water ad libitum For acclimation purpos es, all rats were placed on a motor driven treadmill for 3 day sessions of 5 min training at an intensity of 5 m/min and zero degree incline. Rats were then randomly assigned to either young or old sedentary (Young SED; Old SED; n=8 for all), or young and old exercise (Young EX; Old EX; n=8 for all) groups. EX rats were trained for 10 12 weeks and SED were age matched. After a steady increase in treadmill training during the first 3 weeks, EX rats performed 5 days/week for 60 min/day at an intensity of 1 5 m/min and 15 incline for the remaining weeks. Forty eight hours after their last bout of training, rats were weighed, and anesthetized with isoflurane prior to sacrifice. The kidney, aorta and soleus muscle were dissected and
74 weighed. A transverse sl ice of the left kidney was saved in 10% phosphate buffered formalin for histology (see below) and the remaining tissue separated into cortex and medulla. All tissues were flash frozen in liquid nitrogen, then stored at 80 C until further analyses. For functional measurements, a separate group of young (n=16) and old (n=16) F344 male rats of comparable age, supplied from Taconic Farms (Hudson, NY), were purchased from the NIA. Some rats were treadmill trained (Young EX, n= 10; Old EX, n=8) according to the same protocol described above, while others remained SED (Young SED, n=6; Old SED, n=8). Seven 12 days prior to sacrifice, all rats were placed on a low nitrate, complete diet for 24h (AIN 76C, MP Biomedicals, Solon, OH), then placed in metabolic cage s for overnight (~16h) collection of urine. While in the metabolic cages, rats were fasted but were given access to distilled water. Metabolic cage collections did not interfere with the daytime training. Rats were then returned to daily training and re gular diet and were sacrificed as described above, and blood was drawn from the aorta, centrifuged, and plasma collected and stored at 80 o C. The left kidney was removed and weighed and a section of the kidney was fixed for histology. Renal Pathology Fi xed kidney tissue was paraffin embedded and 5m sections were cut and stained with periodic acid schiff (PAS, Sigma, St Louis, MO) followed by hematoxylin as the secondary stain. All glomeruli in the section (>100) were scored, blinded, as follows: 0=heal thy glomeruli, +1=<25% damage, +2=25 50% damage, +3=51 74% damage, +4=>75% damage. The glomerulosclerosis index score (GSI) was calculated using the following equation: (#of+1)+ 2(#of+2)+ 3(#of+3)+ 4(#of +4)/ total glomeruli observed.
75 Western Blot The p roteins measured by Western blot, with their specific primary antibody and concentration given in parenthesis, were: 1) endothelial nitric oxide synthase (eNOS; BD Transduction; 1:250), 2) neuronal (n)NOS (Santa Cruz; 1:50), 3) nNOS (Thermo Scientific, formerly ABR; 1:500), 4) extracellular superoxide dismutase (EC SOD; Abcam; 1:250), 5) cytosolic copper containing SOD (CuZn SOD; 1:2000), 6) mitochondrial manganese containing SOD (Mn SOD; 1:2000), 7) p22phox (Santa Cruz; 1:50), and 8) nitrotyrosine (Millipore; 1:500). Homogenized samples of kidney cortex, kidney medulla, and aorta, standardized by protein concentrations (50 separated by electrophoresis (7.5% or 12% acrylamide gel, 200 V, 65 min) and transferred onto n itrocellulose membranes as previously described (Sasser et al 2009). Membranes were stained with Ponceau Red (Sigma) to check for transfer efficiency/uniformity and equal loading, blocked and washed then incubated overnight while on a rocker with primary antibody at 4C. Membranes were then incubated with the appropriate secondary antibody for one hour at room temperature, and developed with enhanced chemiluminescent reagents (Thermo Scientific). Bands were quantified by densitometry using the VersaDoc Imaging System and One Analysis Software (BioRad). Protein abundance was calculated as integrated optical density (IOD) of the protein of interest (after subtraction of background), factored for Ponceau Red stain (a marker of total protein loading) and an internal positive control value (to allow for quantitative comparisons between different membranes). To compare values between kidney cortex and kidney medulla, the protein abundance is represented as
76 IOD/Ponceau/Control relative to the appropriate con trol group of the kidney cortex or medulla. Analytical Methods As previously described, plasma and urine creatinine concentrations were measured by HPLC, and urine protein levels were detected using the Bradford method (Sasser et al 2009) Hy drogen peroxide (H 2 O 2 ) levels in homogenates of kidney cortex, kidney medulla, and urine were measured using the Amplex Red Kit (Molecular was confirmed by incubation with 2000 units of catalase (Sigma). Tissue H 2 O 2 mL ) and expressed as nmol/mg protein. Citrate synthase activities in soleus tissue in units of uM/min/g wet tissue weight were based on methods adapted by Srere ( Srere P, 1969 ) Statistical Analyses Data are presented as means SEM and analyzed using SigmaPlot soft ware (San Jose, CA). The effects of age and exercise training on functional, biochemical and protein abundance data were analyzed by two way ANOVA, and if found significant, followed by Newman Keuls post hoc analyses. Renal pathology was analyzed with no n parametric analyses. Significance was defined as p<0.05. Results The first series of studies on young and old, sedentary and exercise trained rats were conducted on NIA colony F344 rats obtained from Harlan (Indianapolis, IN). Body weight was higher i n old when compared to young rats and exercise reduced body weight in both age groups (Table 4 1). Kidney weight (factored to body weight) revealed hypertrophy with age in the EX group only. As reported previously (Sindler et
77 al 2009), training efficacy was confirmed with increased activities of citrate synthase in soleus skeletal muscle in both young and old (by ~18% in young rats and by ~20% in old rats). Young rats exhibited minimal glomerular injury and this was not altered by exercise (Fig. 4 1). With age, the index of glomerular sclerosis (GSI; Fig. 4 1 A ) and the % age of total damaged glomeruli (Fig. 4 1 B ), increased substantially and exercise had no effect on renal pathology. In the kidney cortex, exercise significantly increased eNOS protein abu ndance in the young rat but had no effect in old (Fig. 4 2 A ). There were no differences in kidney cortex nNOS abundance due to either age or exercise (Fig. 4 2 B ) and nNOS was low and unaffected by EX in young rats while there was a profound increase in nNOS abundance in both SED and EX old rats (Fig. 4 2 C ). In the kidney medulla, eNOS protein abundance was higher than in cortex and was similar in old and young rats and not altered by exercise (Fig. 4 2 A s unchanged by either exercise or age; however, in the kidney medulla, significance increases were seen with exercise in the old (Fig. 4 2 B ). Age related increases in nNOS protein abundance in both kidney cortex and medulla were unaffected by exercise (F ig. 4 2 C ). The abundance of the nNOS in medulla was higher than in cortex in both young groups and was further elevated in the old (Fig. 4 2 C ). As with eNOS, exercise increased the EC SOD abundance in the kidney cortex of young but not old rats (Fig. 4 3 A ). There were no exercise effects on the other SOD isoforms in kidney cortex or medulla (Fig. 4 3 B and C ). With age there were falls in EC SOD and CuZn SOD in both kidney cortex and medulla whereas for Mn SOD, there was no impact of age or exercise in the kidney. Values of EC SOD were higher in medulla
78 while CuZn and Mn SOD abundance values were lower in medulla vs. cortex in all groups. The p22phox subunit of the NADPH oxidase increased with age in both kidney cortex and medulla but exercise was w ithout effect (Fig. 4 4 A ). Tissue H 2 O 2 levels (Fig. 4 4 B ) and nitrotyrosine abundance (Fig. 4 4 C ) increased with age in both kidney cortex and medulla but were unaffected by exercise. In the aorta of young rats exercise training significantly increased t he abundance of eNOS and age increased these levels even further (Fig. 4 5 A ); however, no differences were found between Old SED and Old EX (Fig. 4 5 A ). Aortic EC SOD abundance tended to increased with exercise in the young rats but due to high variabilit y, this did not reach statistical significance (Fig. 4 5B). No further change occurred in aortic EC SOD with age or exercise (Fig. 4 5B). Nitrotyrosine abundance in the aorta significantly rose with age in both groups and EX was again without effect (Fig 4 5 C ). For additional functional measurements, later groups of young (n=16) and old (n=16) F344 rats of similar age were obtained from Taconic Farms, also from the NIA colony. Increased BW and renal hypertrophy occurred with age (Table 4 2) as also seen in the Harlan NIA rats (Table 4 1), and the responses to exercise in BW were similar in young and old (Table 4 2). Plasma creatinine (PCr) increased with age in both SED and EX groups, while creatinine clearance (CCr) fell significantly with age in SED o nly (Table 4 2). Urinary protein excretion markedly increased with age and exercise was without effect in either young or old groups (Table 4 2). To determine total body NO production, we measured the urinary excretion of the stable NO oxidation products NO 2 and NO 3 (NOx; UNOxV). With age, UNOxV tended to fall; however,
79 UNOxV significantly increased with exercise in both young and old rats (Table 4 2). No changes in urinary H 2 O 2 excretion were detected among any of the groups (3.810.56, 5.690.99, 4.2 60.80, 2.900.75 nmol/24hr/100g BW for Young SED, Young EX, Old SED, and Old EX, respectively). Furthermore, an age dependent increase in glomerular injury was again observed and this was not improved by exercise (GSI scores: 0.040.02, 0.030.01, 0.930 .14 + and 0.780.18 + for Young SED, Young EX, Old SED, and Old EX, respectively; + p<0.05 vs. respective Young). Discussion The main finding in the present study was that 10 12 weeks of treadmill exercise increased both eNOS and EC SOD abundance in the kidn ey cortex of the young rat, despite an expected exercise induced fall in renal blood flow. There was no change in renal eNOS and ECSOD with exercise. Rats developed age dependent renal damage and exercise was not able to reverse it. In contrast to other strains, there was also no loss of renal eNOS or nNOS kidney cortex increased >100x due to aging, and exercise did not prevent this. The age dependent loss of the kidney cortex antioxidants EC SOD and CuZn SOD, and age dependent increase in p22phox were not influenced by exercise. The second series of rats indicated that with age, F344 rats develop significant proteinuria with some loss of renal function and a tendency for total NO production to fall, and that exercise do es not reverse these decremented changes. F344 rat, which develops mild moderate kidney damage with advancing age without systemic hypertension (Wei et al. 1986). There are differences in the renal response to aging, with some strains such as the Sprague Dawley being very vulnerable while
80 others show mild moderate injury (Munich Wistar and F344), and yet others are resistant (Wag/Rij and F344/ Brown Norway cross) (Baylis & Corman, 1998; Moningka et al. 2011) There are also sex differences in the rate of loss of kidney function and development of structural damage in aging man and rats, with the male being most vulnerable (Baylis 2009; Baylis & Corman, 1998). We have previously reported that development o f severe chronic kidney disease (CKD) is invariably associated with loss of renal cortical nNOS abundance, irrespective of animal model (Baylis 2008). This includes the aging male Sprague Dawley rat where marked glomerular injury occurs (Erdely et al. 2 003). The present study demonstrates that the mild to moderate renal injury exhibited by the aging F344 rat is not associated with a loss of eNOS or nNOS protein. In contrast, there are large age dependent increases in the renal cortical abundance of th induced forms of CKD, including renal mass reduction (Smith et al. 2009; Tain et al. 2011) and chronic allograft nephropathy (Tain et al ation in CKD was a secondary, compensatory response to the loss of other NOS isoforms in the damaged precedes loss of the other NOS isoforms, suggesting that this may in fact be involved in causing th e early, mild moderate renal damage. There are also antioxidant and pro oxidant changes in the aging F344 kidney cortex which include loss of both EC SOD and CuZn SOD protein abundance, increased p22phox (NADPH oxidase subunit), increased nitrotyrosine, a nd increased H 2 O 2 levels. These changes may also contribute to the early renal damage.
81 A main aim of this study was to determine the impact of exercise training on the kidney of the F344 rat. Here, we report that in the young adult F344, exercise incr eased both eNOS and EC SOD protein in the renal cortex which confirms earlier preliminary findings by us in this strain (Moningka et al. 2011 ). Exercise also increased aortic eNOS indicating that in the F344, these beneficial endothelial enzymes are enhan ced by both increased blood flow (to aorta) and decreased blood flow (to kidney). Although not measured by us, rats of various strains including the F344 do reduce their renal blood flow with exercise (Musch et al 2004; Kregel 1995; La ughlin & Armstrong, 1982). Presumably in the young exercise trained F344 increased intrarenal resistances oppose the decreased flow and lead to increased intrarenal shear stress in some locations, and hence eNOS and EC SOD activation. This is particularly interesting since we previously observed exactly opposite intrarenal effects of exercise in the young male Sprague Dawley rat, where falls in eNOS and EC SOD were seen (Moningka et al. 2010 ). Marked falls in renal blood flow also occur in this strain (Laughlin & Armstrong 1982). We speculate that in the Sprague Dawley kidney the exercise induced renal vasoconstriction leads to net falls in intrarenal shear stress. The directional effect of exercise on renal endothelial enzymes has profound consequences since loss of the se enzymes renders the Sprague Dawley kidney more susceptible to acute kidney injury, while the F344 strain is protected (Moningka et al. 2011). In the present study, however, since the young sedentary F344 has minimal spontaneous injury we did not see an y histologic effect of exercise. Our primary goal was to establish the effect of exercise on renal endothelial enzymes as well as overall renal structure and function in the aging F344 rat. We have
82 already reported that similar exercise has significant g eneral cardiovascular benefits in the aging F344 leading to enhanced endothelial function in skeletal muscle (Spier et al. 2004; Sindler et al. 2009). Despite these cardiovascular improvements, we report here that the structural and functional kidney dama ge in the aging F344 is not reversed by exercise, suggesting that the beneficial effects of the same type/duration of exercise are not inevitably transmitted to the kidney. In fact, there is considerable variability in the reported response of the aging k idney to exercise. In the old (23 months) male Sprague Dawley rat, life long voluntary wheel running reduced kidney structural damage and was as effective as lifelong caloric restriction (Loupal et al 2005). Of note, in the same study, treadmill running over the same period had no beneficial effects in the Sprague Dawley rat (Loupal et al 2005), similar to our present findings in the old F344 subjected to 10 12 weeks of treadmill running. In contrast, in the old C57BL/6J mouse which develops significant kidney damage, only 6 weeks of forced wheel running considerably worsened the age associated renal structural injury (Lichtig et al .1987). The aged F344 rat shows an exaggerated fall in renal blood flow during exercise which we speculate causes unchanged intrarenal shear stress, accounting for the lack of exercise induced renal eNOS and EC SOD. This lack of activation of these endothelial enzymes, together with loss of CuZn SOD in the aging kidney, which is not restored by exercise, probably contributes t o the lack of exercise induced protection against kidney damage and dysfunction. Oxidative stress, defined as the imbalance between oxidants and antioxidants, is reported to increase with age, and can be combated with exercise. Moderate exercise reduced age associated increases in mitochondrial oxidative stress in some organs
83 (including kidney) of old male mice, although beneficial effects declined in senescent animals (Navarro et al 2004). The same study found that exercise did not prevent the age dep endent decline in kidney CuZn SOD and catalase activity levels (Navarro et al 2004). Here, we report no net effect of exercise on age related increases in oxidative stress. The protein abundance of p22phox, a subunit of the superoxide generating NADPH o xidase enzyme was up regulated in both the kidney cortex and kidney medulla with age, and was unchanged with exercise. H 2 O 2 levels, an additional marker of oxidative stress, also increased with age in the kidney medulla only, but again, was not affected b y exercise. Furthermore, nitrotyrosine, also increased with age in renal cortex, medulla and aorta and was not affected by exercise. Overall, this study demonstrates that in the young male F344 rat, 12 weeks of treadmill exercise increases kidney cortex eNOS and EC SOD abundance, and that with age, this response is lost. We observed several other age related changes in the kidney including worsening of renal structural injury, increased renal oxidative stress as detected by increase protein abundance in p22phox, H 2 O 2 content and nitrotyrosine, and decreased antioxidant defenses reflected by loss of EC and CuZn SOD. Interestingly, exercise did not prevent any of these adverse changes in the kidney. Therefore, we conclude that 10 12 weeks of chronic tread mill exercise was ineffective in reversing the age associated declines in renal function and renal antioxidant status in the male F344 rat.
84 Table 4 1. Characteristics of male F344 rats obtained from Harlan, NIA. Young SED Young EX Old SED Old EX BW ( g) 3678 3455* 4099 + 3779* + Kidney wt/100g BW 0.320.01 0.300.0 0.350.02 0.360.01 + Sedentary, SED; exercise, EX; body weight, BW. *p<0.05 vs. respective SED. + p<0.05 vs. respective Young. Table 4 2. Characteristics of male F344 rats obtained fr om Taconic Farms, NIA. Young SED Young EX Old SED Old EX BW (g) 40312 38710 45617 + 41723* Kidney wt/100g BW 0.260.01 0.280.01 0.410.04 + 0.340.02 PCr (mg/dl) 0.110.02 0.120.01 0.220.01 + 0.200.03 + CCr ( mL /min/100g BW) 1.980.22 1.860.12 0.7 40.15 + 1.090.16 UpV (mg/24hr/100g BW) 2.40.2 3.80.1 88.821 + 65.117 + UNOxV (umol/24 hr/100g BW) 0.760.01 1.210.08* 0.460.15 0.880.10* + Sedentary, SED; exercise, EX; body weight, BW; plasma creatinine, PCr; creatinine clearance, CCr; urinary pro tein excretion, UpV; urinary nitrate/nitrite excretion, UNOxV; *p<0.05 vs. respective SED. + p<0.05 vs. respective Young.
85 Figure 4 1. Glomerular structural changes in young and old, sedentary (SED) and exercise (EX) trained rats. Whole kidney sections were stained with periodic acid schiff, followed by a hematoxylin counterstain prior to blind histological grading. (A) The gomerular sclerosis index (GSI) significantly increased with age and was unaffected by 12 weeks of treadmill exercise (5 days/week 60 min/day at 15 m/min, 15 degree incline). (B) The total percentage of damaged glomeruli also rose with age and exercise was without effect. *Denotes a statistical significance of p<0.05 between the two groups.
86 Figure 4 2. Protein levels of the n itric oxide synthase (NOS) enzymes in the kidney cortex and kidney medulla. Total homogenates of kidney cortex and kidney medulla from exercise (EX) trained and sedentary (SED) rats were run using Western blot and probed for (A) endothelial nitric oxide s ynthase (eNOS), (B) Relative density units were expressed as a % from Young SED controls of the kidney cortex. *Denotes a statistical significance of p<0.05 between the two groups.
87 Figu re 4 3. Protein levels of the superoxide dismutase (SOD) enzymes in the kidney cortex and kidney medulla. Total homogenates of kidney cortex and kidney medulla from exercise (EX) trained and sedentary (SED) rats were run using Western blot and probed for (A) extracellular superoxide dismutase (EC SOD), (B) cytosolic localized copper/zinc SOD (CuZn SOD), and (C) mitochondrial localized manganese SOD (Mn SOD). Relative density units were expressed as a % from Young SED controls of the kidney cortex. *Denot es a statistical significance of p<0.05 between the two groups.
88 Figure 4 4. Oxidative stress markers: p22phox protein, H 2 O 2 levels, and nitrotyrosine protein in kidney cortex and kidney medulla. Total homogenates of kidney cortex and kidney medulla f rom exercise (EX) trained and sedentary (SED) rats were prepared for (A) p22phox Western blot detection, (B) H 2 O 2 concentration levels, and (C) nitrotyrosine Western blot detection. All values are expressed as a % from Young SED controls of the kidney cor tex. *Denotes a statistical significance of p<0.05 between the two groups.
89 Figure 4 5. Aortic protein levels of eNOS (endothelial nitric oxide synthase), EC SOD (extracellular superoxide dismutase), and nitrotyrosine. Total homogenates of aorta from exercise (EX) trained and sedentary (SED) rats were run using Western blot and probed for (A) endothelial nitric oxide synthase (eNOS), (B) extracellular superoxide dismutase (EC SOD), and (C) nitrotyrosine Relative density units were expressed as a % from Young SED controls of the kidney cortex. *Denotes a statistical significance of p<0.05 between the two groups.
90 CHAPTER 5 PROTECTION AGAINST AGE DEPENDENT RENAL INJURY IN THE F344XBROWN NORWAY MALE RAT IS ASSOCIATED WITH MAINTAINED NITRIC OXIDE SYNT HASE B ackground In man, the kidney develops structural damage with age that is associated with thickening of the glomerular basement membrane, expansion of glomerular mesangium, increases in extracellular matrix proteins and appearance of tubulointerstit ial injury (Kasiske, 1987; McLachlan et al 1977) In addition, glomerular filtration rate (GFR) falls secondary to both glomerular injury and to falls in renal plasma flow because of renal vasoconstriction ( McLachlan et al. 1977 ). Even in the absence of primary kidney disease, a decline in renal function is expected although not inevitable, as demonstrated by the Baltimore Longitudinal Study on A ging (Lindeman et al. 1985) Age dependent kidney damage and dysfunction are also seen in the aging rat, with some strains showing rapidly developing, age dependent chronic kidney disease (CKD), while others maintain excellent renal function and structure even when very old (Baylis & Corman, 1998) All forms of CKD are associated with nitric oxide (NO) deficiency, which is both a result of CKD and a contributing factor to progression (Baylis 2008; Baylis 2009 ) In the Sprague Dawley (SD) rat where renal disease progresses rapidly, age dependent kidne y damage is related to decreased abundance and activity of the NO synthesizing enzyme, NO synthase (NOS) in the kidney cortex (Erdely et a l 2003 ) Plasma levels of the endogenous inhibitor of NOS, asymmetric dimethylarginine (ADMA) are also elevated in elderly humans and rats (Boger et al 2000; Kielstein et al 2003; Xiong et al. 2001) providing an additional mechanism of NO deficiency in aging.
91 It is evident that genetic background plays a critical role in how organ function changes with age. In fact, age dependent changes in humans can be attributed to more than 600 genes, about 100 of which contain expression associated single nucleotide polymorphisms (Wheeler et al. 2009) In contrast to the injury prone Sprague Dawley, age dependent CKD develops slowly in the Munich Wistar rat and is minimal in WAG/Rij (Baylis & Corman, 1998) and Fisher 344xBrown Norway (F344xBN) strains (Lipman et al. 1996) With a life span of ~36 months and relatively preserved renal function, the F344xBN is considered 2008). When compared to another commonly utilized aging model, the F344, which interestingly has increased insulin resistance and glome rular nephropathy but no systemic hypertension with age, the F344xBN has fewer glomerular lesions and a greater mean age at which 50% mortality occurs (F344: 103 wks. vs. F344xBN: 145 wks; Lipman et al. 1996). It is clear that genetic differences dictate outcome of age related declines so further characterization of the various aging models is critical for aging investigations. In the present study, we investigated the impact of aging on various determinants of NO production in the F344xBN rat. Determin ants included 1) abundance of the NO synthesizing protein, NOS; 2) NOS inhibitor levels; 3) abundance of enzymes that regulate NOS inhibitors; 4) abundance of anti oxidant and oxidative markers. The primary goal was to test the hypothesis that in the abse nce of significant age dependent damage, renal NOS abundance is maintained. We also investigated whether there would be any beneficial effect of chronic renin angiotensin system (RAS) blockade in
92 Methods Animal Procedures All ani mal procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals ( Principles of Laboratory Animal Care ; NIH publication No. 86 23, revised 1985) and approved and monitored by the University of Florid a Institutional Animal Care and Use Committee. Young (3 months; n=8) and old (24 months; n=24) male F344xBN rats were purchased from the National Institute of Aging colony (Harlan Sprague Dawley, Indianapolis, IN) and singly housed in a temperature/light controlled environment and given access to standard rat chow/water ad libitum Old rats were divided into three groups (n=8/group) and used to compare 6 months of placebo (normal aging) with RAS blockade with either an angiotensin converting enzyme inhibi tor (ACEI; enalapril; 40 mg/kg body weight) or angiotensin II receptor blocker (ARB; candesartan; 10 mg/kg body weight). Bacon flavored tablets (BioServ #F05072) were given with or without the drug compounded into them, and old rats were sacrificed for st udy at 30 months of age by rapid decapitation. Young rats (n=8) were maintained on the same ad lib diet and daily bacon flavored tablets without drug for two weeks and then sacrificed at ~3 months of age under isoflurane anesthesia. Blood was taken eithe r by aortic puncture or from the trunk in young and old, respectively, and then spun for collection of plasma. The kidneys were removed and while one kidney was prepared for histological analyses (see below), the other was separated into cortical and medu llary sections, and then flash frozen in liquid nitrogen. However, all analyses were conducted in cortical tissue only. All samples were stored at 80C for further analyses. Plasma creatinine levels were measured by HPLC as previously described (Sasser et al. 2009)
93 Renal Patholo gy One kidney was cut along the transverse axis and fixed in 10% buffered formalin for 48 hours at 4C, paraffin wax embedded, cut into 5 micron thick sections and stained with periodic acid schiff, followed by a hematoxylin counterstain (Sigma). Sections were then examined, blind, for the level of glomerular sclerosis, glomerular ischemia/atrophy, tubular atrophy, and interstitial fibrosis. Each category was scored (0=none, 1=10%, 2=10 25%, 3=25 50%, 4=50 75%, 5=75 100%) based on the percentage of struct ures that displayed the described injury. Western Blot Relative protein abundances of endothelial nitric oxide synthase (eNOS; BD 1:500), dimethyldiaminohydrolase (DDAH) isoforms (Santa Cruz; DDAH1 1:250 and DDAH2 1:100), protein methyl transferase (PRMT1; Millipore; 1:2000), superoxide dismutase (SOD) isoforms (Stressgen Reagents; EC SOD 1:250, CuZn SOD 1:2000, and Mn SOD 1:2000), and p22phox (Santa Cruz; 1:50) were measured by Western Blot. Homogenized samples of kidney cortex standard ized by protein concentrations (50 200 ug) were separated by electrophoresis (7.5% or 12% acrylamide gel, 200 V, 65 min) and transferred onto nitrocellulose membranes (GE Healthcare) for 60 min at 0.18 A as previously described (Sasser et al. 2009) Membranes were stained with Ponceau Red (Sigma) to check for transfer efficiency/uniformity and equa l loading, incubated in blocking solution for 60 min, and then washed in TBS + 0.05 % Tween before overnight primary antibody incubation at 4C. Membranes were then incubated with the appropriate secondary antibody for one hour at room temperature, with a series of washes before and after, and developed with enhanced chemiluminescent reagents
94 (Thermo Scientific). Bands were quantified by densitometry using the VersaDoc Imaging System and One Analysis Software (BioRad). Protein abundance was calculated as integrated optical density (IOD) of the protein of interest (after subtraction of background), factored for Ponceau Red stain (total protein loaded), and normalized w ith an internal positive control value. This allowed for quantitative comparisons between different membranes. The specific protein abundance is represented as IOD/Red Ponceau/Control relative to the appropriate control group. Analytical Methods ADMA conc entrations in plasma and renal cortical tissue homogenates were measured by reverse phase HPLC using the Waters AccQ Fluor fluorescence method as previously described (Sasser et al. 2009) Renal cortical tissue ADMA mL ) and therefore expressed as mol/mg. Renal cortical concentration of hydrogen peroxide was measured using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Mol ecular Probes) cortical tissue was homogenized with 1x phosphate buffered saline (PBS; MediaTech, Inc.; PBS), diluted 1:3, incubated at room temperatu re with Amplex Red reagent and HRP for 45 minutes, and then read at a wavelength absorbance of 560 nm. Assay specificity was confirmed using 2000 units of catalase (Sigma). Renal cortical H 2 O 2 (mg/ mL ) and therefore expressed as nmol/mg. Statistical Analyses Data are presented as mean SEM and analyzed by either one way ANOVA followed by post hoc analysis, or the non parametric Kruskal Wallis test (for histology
95 only) using GraphPad Prism 4 software (San Diego, CA). Significance was defined as p<0.05. Results Body weight (BW) increased with age (young: 337 8 vs. old: 560 24 g) and plasma creatinine fell (young: 0.11 0.01 vs. old: 0.06 0.00 mg/dl). Histological analyses revealed no difference in th e percentage of glomeruli with sclerosis in old vs. young rats although there were increases in glomerular ischemia/atrophy, tubular atrophy, and interstitial fibrosis with age (Fig. 5 1). RAS blockade had no impact on the age dependent rise in BW (old: 5 60 24 vs. old ACEI: 542 17 vs. old ARB: 546 18 g) but plasma creatinine significantly rose with both ACEI and ARB treatment (old ACEI: 0.09 0.01 vs. old ARB: 0.09 vs. 0.01 mg/dl) in comparison to untreated old rats. The only impact of RAS inhibition on his tology was that ARB treatment significantly reduced both tubular atrophy and interstitial fibrosis compared to untreated old rats a nd those given ACEI (Fig. 5 1). NOx (stable metabolites of NO=NO3 + NO2) levels in the plasma were unchanged with age (youn g: 12 1 vs. old: 12 4 M) and ARB treatment (9 2 M), although ACEI increased plasma NOx (30 1 abundance in the kidney cortex of old rats (Figs. 5 2 A and B ), whereas nNOS abundance (Fig. 5 2 C ) remained unchanged. Neither ACEI nor ARB treatment in old rats affected kidney cortex eNOS or nNOS abund ance (Figs. 5 2 A and C ); however, ARB did increase nNOS abundance compared to old untreated rats and marked 5 2 B ).
96 Plasma levels of ADMA, L Arginine, and SDMA were unchanged with ag ing although the plasma L Arginine:ADMA ratio increased, favoring increased NO production (Table 5 1). Similarly, in the kidney cortex there were also no differences in ADMA and L Arginine levels in young vs. old, although the L Arginine:ADMA ratio increa sed (Table 5 2). There was no effect of RAS blockade on plasma levels of ADMA, L Arginine, L Arginine:ADMA ratio, or SDMA compared to old untreated rats (Table 5 1). Kidney cortex PRMT 1, the enzyme responsible for ADMA production was increased with age (Fig. 5 3 A ) whereas the ADMA degrading enzyme, DDAH1 tended to increase in abundance (Fig. 5 3 B ) and there was no change in DDAH2 abundance in the kidney cortex (Fig. 5 3 C ). ACEI and ARB treatment reduced kidney cortex PRMT 1 abundance (Fig. 5 3 A ) but had no effect on abundance of either DDAH isoform compared to kidneys from old untre ated rats (Figs. 5 3B and C ). We also assessed indices of oxidative stress in the F344xBN rodent model of aging and found significant increases in p22phox abundance in the ki dney cortex of old vs. young rats (Fig. 5 4 A ) but no change in the H 2 O 2 con tent of the kidney cortex (Fig. 5 4 B ). Old rats showed marked increases in the kidney cortex abundance of EC SOD and Mn SOD with no change in CuZn SOD abundance (Fig. 5 5). RAS blockade had no impact on kidney cortex p22phox abundance or H 2 O 2 content compared to old untreated rats (Figs. 5 4 A and B ), although ACEI reduced kidney cortex EC SOD abundance to levels that fell further with ARB (Fig. 5 5 A ). In contrast, ACEI increased CuZn SOD abundance whereas ARB decreased it (Fig. 5 5 B ). No statistically significant changes were detected for the Mn SOD abundance among any of the old groups (Fig. 5 5 C ).
97 Discussion The main finding of this study is that the protection against age dep endent glomerular sclerosis seen in the F344xBN rat is associated with preserved eNOS and dependent increase in d with age and apparent ly balance d the increased oxidant generating NADPH oxidase (indicated by increased p22phox). Although this rat strain does not develop glomerular sclerosis with age, tubulointerstitial injury increased, which was prevented by RAS inhibition with the ARB, whereas ACEI had no protective effect. It is always difficult to determine what changes occur due to normal aging and what is due to increased susceptibility to acquired disease. The renal response to aging is extremely variable in humans and in rats (Baylis & Corman, 1998; Lindeman et al. 1985) and of note, the female of many strains, including the SD, show marked protection compar ed to the male (Baylis & Corman, 1998; Erdel y et al. 2003 ) In the aged male SD rat where glomerular sclerosis is severe (>60% of glomeruli damaged at 24 months), renal eNOS and nNOS protein abundance falls with age (Erdely et al. 2003 ) In contrast, in the present study we report that in the aged male F344xBN, where only ~ 2.5% of glomeruli show sclerotic damage by 30 months of age, the renal eNOS and nNOS isoforms are preserved. We have suggested that falls in renal NOS protein abundance are both a consequence and a cause of progression of several forms of CKD (Baylis 2009 ) The p resent study expands this by showing that normal aging is not inevitably accompanied by loss of renal NOS, reinforcing our hypothesis that there is a causal association between kidney injury and loss of renal NOS protein (Baylis 2009 )
98 In addition to NOS protein abundance, NO production is controlled by the local and circulating concentrations of the endogenous NOS inhibitor, ADMA. Several studies report that plasma ADMA increases in normal aging hum ans and that an increase, although delayed, also occurs in aging women (Kielstein et al. 2003; Schulze et al 2005) This may be associated with the age d ependent development of endothelial dysfunction (Bode Boger et al. 2003) Increased plasma ADMA has also been reported in the aging male SD rat (Xiong et al. 2001) Control of plasma ADMA level is mainly by degradation by the DDAH1 enz yme, which is abundant in liver and kidney (Palm et al. 2007; Sasser et al. 2009) ADMA is made by PRMT1, a class of enzymes that methylate amino acids (including arginine) while they are incorporated into intact protein s (Nicholson et al. 2009) The free (active) ADMA is released by proteolysis. In this study, we found that in the 30 month old male F344xBN, neither circulating nor renal cortical ADMA levels were changed with age. The renal abundance of the enzyme responsible for ADMA pr oduction, PRMT 1, was unchanged with age and the ADMA degrading enzyme, DDAH1 (main renal enzyme) tended to increase and DDAH2 was unchanged. Thus, an increase in plasma ADMA with age is not inevitable and in fact we observed an increase in the ratio betw een L Arginine/ADMA, an effect that favors NO production and enhanced endothelial function. This was due to a non significant tendency for increased plasma L Arginine with age, also reported by us previously in the aging male SD (Mistry et al. 2002) Furthermore, in spi te of a lifetime (30 months) of oxidative metabolism, the F344xBN shows no obvious signs of increased oxidative stress. DDAH activity and abundance is reported to be redox sensitive and it is widely believed that exposure to
99 oxidative stress will inhibit DDAH and increase ADMA (Palm et al. 2007) Activity of PRMT1 is also increased by oxidative stress; however, as noted above, the abundance of these enzymes w as not affected by aging in the F344xBN. Ang II induces oxidative stress in the kidney (Gill & Wilcox, 2006; Modlinger et al. 2006) and when kidney injury develops with age, there is also activation of the intrarenal angiotensin II (Ang II) system; however in the F344/BN rat where no glomerular damage devel ops, there is no increase in intrarenal ANGII (Kasper 2008). We did observe an increased abundance of the renal NADPH oxidase subunit, p22phox, with age, which presumably implies increased superoxide production. However, we also found increases in EC SO D and Mn SOD, a likely compensatory response to scavenge the increased superoxide production. Since renal cortical H 2 O 2 is also unchanged with age there is presumably also increased renal catalase activity. Chronic RAS blockade is used for treatment of C KD and/or hypertension. Chronic ACEI protects against glomerular sclerosis, reduces proteinuria, and lowers blood pressure in aging male Munich Wistar rats, treated from 3 to 30 months of age (Anderson et al. 1994) Lifetime treatment with ACEI in the male WAG/Rij rat also reduces blood pressure, urinary protein excretion, and expansion of the mesangial matrix (Heudes et al. 1994) although WAG/Rij rats show minimal age dependent injury (Baylis & Corman, 1998) In the present study, male F344xBN rats show little evidence of glomerular sclerosis and yet some beneficial effects of RAS blockade were observed. It is interesting that even in the absence of glomerular sclerosis, significant tubulointerstitial injury develops in these rats by 30 months of age, and 6 months of treatment with the ARB protected against tubulointerstiti al injury. Tubulointerstitial injury
100 develops as peritubular capillaries are lost in various forms of CKD and is particularly prominent in slowly evolving age dependent damage (Lombardi et al. 1999) Although not reported in the present study, there is no age dependent proteinuria in the F344xBN rat, and long term ACE inhibition with enalapril had no effect on ur inary protein excre tion (Kasper 2008). Thus, tubulointerstitial injury, in the absence of glomerular injury, does not cause proteinuria in this model. In the aging Wistar rat, where substantial glomerular damage occurs, endothelin receptor blockade reversed proteinuria an d prevented glomeruloslcerosis, while tubulointerstitial injury remained (Ortmann et al. 2004). Thus, it is likely that the development of proteinuria in response to kidney damage is primarily glomerular, rather than tubular in origin. Tubulointerstitial injury has also been suggested to be an NO deficiency mediated event in the aging kidney (Lombardi et al. 1999) Although there is no evidence of renal NO deficiency in the 30 month old F344xBN male, both ARB and ACEI lead to marked increases in renal cortical nNOS in the present study. However, in contrast to the beneficial effect of ARB, 6 months of ACEI treatment gave no reductio n in tubulointerstitial injury, suggesting that the protection against tubulointerstitial injury produced by the ARB is not due to nNOS stimulation. It is interesting that in this setting the ARB is superior to ACEI, whereas ACEI outperforms ARB in heart failure (Berry et al. 2001) ARB and ACEI are also thought to have equivalent efficacy in treating patients with a wide range of cardiovascular risk (Baumhakel & Bohm, 2009; Schindler 2008) The fall in plasma creatinine in the old untreated rat most likely relates to the well known loss of lean body mass that occurs with age (Griffiths 1996) It is interesting that
101 both methods for RAS blockade raised plasma creatinine to the young (still very low) value. This may reflect RAS blockade induced changes in body composition and/or activity level. It is unlikely to reflect loss of renal function since chronic intra renal RAS blockade is associ ated with beneficial effects. Further, the plasma SDMA is unaffected by aging or RAS blockade and SDMA normally increases as GFR falls (Marcovecchio et al. 2009 ) In summary, we conclude that in the aged male F344xBN rat, in contrast to our previous findings in the aged male SD rat, renal N OS is preserved and there is minimal age dependent glomerular sclerosis (Erdely et al. 2003 ) Both anti oxidant and oxidant systems in the kidney are activated with age and there is no net effect on circulating or renal cortical ADMA concentrations. The tubulointerstitial injury seen with aging is rev ersed with 6 months of ARB but not ACEI, and is not associated with renal NOS. Our data highlight the complexity of the aging process in that factors such as genetic background can dictate histological outcomes that are associated with the renal NO system
102 Table 5 1. The effect of aging and chronic RAS blockade on plasma levels of ADMA, L Arginine, L Arginine:ADMA, and SDMA in young (~3 month), old (30 month), old ACEI, and old ARB male F344xBN rats. Group ADMA (M) L Arginine (M) L Arginine:ADMA SDMA (M) Plasma Young 0.26 0.02 89 7 346 14 0.18 0.02 Plasma Old 0.26 0.04 102 9.0 430 34* 0.20 0.02 Plasma Old ACEI 0.30 0.13 106 30 442 30 0.22 0.07 Plasma Old ARB 0.21 0.03 94 26 374 33 0.19 0.04 RAS, renin angiotensin system; ADMA, asymmetric dimethylarginine; SDMA, symmetric dimethylarginine; ACEI, angiotensin converting enzyme inhibitor; ARB, angiotensin receptor blocker. ACEI and ARB treatment lasted for 6 months in old only. *p<0.05 vs. young. Table 5 2. The effect of aging on kidney c ortex levels of ADMA, L Arginine, L Arginine:ADMA, and SDMA in young (~3 month) and old (30 month) male F344xBN rats. Group ADMA (nmol /g) L Arginine (nmol/mg) L Arginine: ADMA SDMA (nmol/g) Kidney Cortex Young 9 1 2.00 0.32 216 25 28 2 Kidney Cortex Old 8 1 2.00 0.18 298 24* 22 2* RAS, renin angiotensin system; ADMA, asymmetric dimethylarginine; SDMA, symmetric dimethylarginine; ACEI, angiotensin converting enzyme inhibitor; ARB, angiotensin receptor blocker. ACEI and ARB treatment lasted for 6 mon ths in old only. *p<0.05 vs. young.
103 Figure 5 1. Representative images of periodic acid Schiff stained sections that were examined, blind, for the level of glomerular sclerosis, glomerular ischemia/atrophy, tubular atrophy, and interstitial fibrosis. (A) A healthy, normal cortex in the young rat (~3 months). (B) A 30 month old, untreated rat, with mild glomerular sclerosis (left glomerulus) and tubular atrophy. (C) A 30 month old rat given 6 months of angiotensin converting enzyme inhibition (old ACEI) with focal interstitial fibrosis (center of image). (D) A 30 month old rat given 6 months of angiotensin receptor blockade (old ARB) showing some mesangial matrix expansion but otherwise with preserved glomerular and tubular architecture. (E) Qu antification of the histological data. Each category was scored (0=none, 1=10%, 2=10 25%, 3=25 50%, 4=50 75%, 5=75 100%) based on the percentage of structures that displayed the described injury. *p<0.05 vs. Young; +p<0.05 vs. Old; **p<0.05 vs. Old ACEI; n=8/group for all.
104 Figure 5 2 We assessed the kidney cortex protein abundance of the nitric oxide synthase (NOS) enzymes in young (~3 month), old (30 month), old ARB, and old ACEI male F344xBN rats: (A) endothelial (e)NOS (A), (B) neuronal (n) NOS isoform (nNOS ), and (C) nNOS isoform young; +p<0.05 vs. old; n=8/group for all. Relative densities in protein abundance were determined by Western blot and normalized to Red Ponceau staining a nd an internal positive control.
105 Figure 5 3 We assessed the kidney co rtex protein abundance of the enzymes that regulate asymmetric dimethylarginine (ADMA) synthesis and catabolism in young (~3 month), old (30 month), old ARB, and old ACEI male F344xBN rats: (A) Protei n methyltransferase 1 (PRMT 1), (B) dimethyldiaminohydr olase1 (DDAH1), and (C) DDAH2. *p<0.05 vs. young; +p<0.05 vs. old; n=8/group for all. Relative densities in protein abundance were determined by Western blot and normalized to Red Ponceau staining and an internal positive control.
106 Figure 5 4 Oxidat ive stress in the kidney cortex as assessed by determining the protein abundance of p22phox (A) and content H 2 O 2 (B) in young (~3 month), old (30 month), old ARB and old ACEI male F344xBN rats. *p<0.05 vs. Young; n=8/group for all. Relative densities in protein abundance were determined by Western blot and normalized to Red Ponceau staining and an internal positive control. Kidney cortex H 2 O 2 content was confirmed with catalase and normalized to mg of protein.
107 Figure 5 5. We assessed the kidney c ortex protein abundance of the antioxidant enzymes in young (~3 month), old (30 month), old ARB, and old ACEI male F344xBN rats: (A) Extracellular superoxide dismutase (EC SOD), (B) cytosolic located CuZn SOD, and (C) mitochondrial located Mn SOD (Mn SOD) *p<0.05 vs. Young; +p<0.05 vs. Old; **p<0.05 vs. Old ACEI; n=8/group for all. Relative densities in protein abundance were determined by Western blot and normalized to Red Ponceau staining and an internal positive control.
108 CHAPTER 6 CONCLUSION S This p rimary purpose of this body of work is to characterize the impact of exercise on the renal NO and antioxidant systems in states of health and injury. The overall hypothesis is that the reduction in blood flow to the kidney during exercise decreases the sh ear stress dependent enzymes eNOS and EC SOD which are required for optimal vascular health. This may put the kidney at risk for falls in NO and antioxidant bioavailability. Indeed, it has been shown that exercise reduces renal eNOS and/or NOS activity ( Miyauchi et al. 2003; Lin et al. 2010). The studies previously described in this body of work demonstrate that the renal response to exercise and age are influenced by genetic background. Further, exercise also influences the status of pre existing renal endothelial health which determines severity of IR induced AKI. Consideration of these factors is required for optimal exercise benefit for patients with endothelial dysfunctional including but not limited to renal disease. In this section, a summary of findings directly taken from each study will be presented followed by a general discussion. Summary of Findings Genetic Background Determines Renal Response to VWR EX The protein abundance of eNOS and EC SOD in response to 12 weeks of VWR EX is variable a nd influenced by genetic background. Despite comparable running profiles and equivalent renal functional responses to 6 and 12 weeks of VWR, we young adult male SD and F344 rats. In the SD rat, VWR significantly decreased kidney cortex eNOS, EC SOD and p22phox whereas in the F344, VWR increased these
109 variables. Immunohistochemical studies confirmed that the strain dependent changes in eNOS occur exclusively in the vascular endothelium. These directionally opposite changes in eNOS and EC SOD abundance between the two rat strains suggest that while chronic mild exercise may have beneficial renal vascular effects in the F344, it could be damaging to the SD. Genetic Background Determines Susceptibility to IR induced AKI Despite differences in renal eNOS and EC SOD responses to VWR EX between the SD and F344 rat, lung eNOS and EC SOD for either strain both increased with VWR EX and IR injury. We also found that 12 weeks of VWR EX exacerbated susceptibility to IR induced AKI in the SD but not the F344 rat. In both strains, the reduction in RBF with acute exercise in the untrained SED rat was lost in the trained rat (10 12 weeks of TM EX, 15 m/min). Resting RBF values fell simil arly in the trained rats of each strain. Our data indicate that vulnerability to an oxidative stress mediated renal insult such as IR induced AKI is determined by the state of endothelial health which is influenced by genetic background. These strain dif ferences are not attributable to renal hemodynamic responses to TM EX. Chronic TM EX Does Not Alter Age Related Renal Injury 10 12 weeks of TM EX increased both eNOS and EC SOD abundance in the kidney cortex of the young rat, despite an expected exercise induced fall in RBF. There was no change in renal eNOS and EC SOD with exercise. Exercise did not reverse age effect on either eNOS or nNOS in the kidney cortex with age. TM EX did not influence an age dependent loss of the kidney cortex antioxidants EC SOD and CuZn SOD and age dependent increase with p22phox. Aging F344 rats also developed significant
110 proteinuria with some loss of renal function and a tendency for total NO product ion to fall, and that exercise does not reverse these decremented changes. These data suggest that long term TM EX does not alter age associated renal injury in the male F344 rat. Protection Against Age Related Renal Damage Associates with Intact NO Syst em In the F344xBN rat, a lack of development of glomerular sclerosis with age increase in th e p22phox subunit of the superoxide generating NADPH oxidase. Although this rat strain did not develop glomerular sclerosis with age, tubulointerstitial injury increased, which was prevented by RAS inhibition with the ARB candesartan, whereas the ACEI ena lapril had no protective effect. These data suggest that protection against age related renal injury associates with an intact NO system. General Discussion Although kidney function is critical for body fluid, electrolyte, and acid base homeostasis, it is an organ system that is much less investigated in the field of exercise physiology. Many of the studies outlining the major functional responses were reported in very early investigations. Therefore, it is a main purpose of this work to contribute new u nderstanding on the physiological responses of the kidney to exercise in states of health and disease. Vascular benefits of exercise are mainly due to shear stress dependent mechanisms. Shear stress, or the frictional force exerted on a vessel wall, poten tly stimulates NO production by increasing eNOS mRNA stability, eNOS transcription and translation, and eNOS enzyme activity (Harrison et al. 2006). NO is a critical regulator
111 of vasomotor tone and contains several anti atherosclerotic properties includin g inhibition of vascular smooth muscle proliferation and platelet aggregation. In, addition, shear stress can also up regulate several antioxidants including EC SOD (Harrison et al. 2006). Mice deficient in c Src, a tyrosine kinase critical for the shear stress response, failed to increase their aortic eNOS and EC SOD protein levels with exercise training (Davis et al. 2003), demonstrating that the importance of blood flow, and therefore shear stress, dictates the vascular benefits of exercise. However, d espite increasing evidence for the beneficial role of NO in exercise hyperemia, its role in tissues where blood flow reduces is less understood. Reductions in RBF are required to provide working muscles increased blood flow during exercise and are determi ned by intensity which is further controlled by several factors including the renal sympathetic nervous system and vasoconstricting hormones such as angiotensin II and endothelin 1 (McAllister 1998). At high intensities, GFR is severely compromised due t o marked falls in RBF. However, physical conditioning reduces the magnitude of reduction in RBF (Armstrong & Laughlin 1984). Nonetheless, it has been shown that in SD rats, exhaustive exercise leads to development of renal structural injury and to falls in renal NOS activity (Lin et al. 2010). In Wistar rats, acute treadmill exercise also significantly reduced renal eNOS mRNA, protein, and NOS activity, opposite to the increased renal eNOS protein content found in the lung (Miyauchi et al. 2003). Stud ies reported in Chapter 2 and 3 demonstrated that VWR EX reduced renal eNOS and EC SOD in the SD rat, whereas in the F344 rat, these enzymes significantly increased. The responses in the F344 were not unique to VWR EX since we validated
112 increased renal eN OS and EC SOD with 10 12 weeks of TM EX ( Chapter 4). Interestingly, we also detected decreased renal p22phox abundance, a marker of oxidative stress, in the SD rat, which again contrasted findings in the F344 rat. These were unexpected since we predicted falls in NO and antioxidant capacity in the SD to increase oxidative stress, and vice versa for the F344 rat. Nonetheless, findings of Chapter 3 where we superimposed AKI, revealed the functional importance of these findings. It is well known that NO i s critical for normal renal function and its deficiency is a cause and consequence of CKD (Baylis 2007). Insults that promote falls in NO bioavailability can therefore render the kidney susceptible to injury, as in the case of our VWR EX trained SD rats. Indeed, there are reports of renal ischemia leading to exercise induced AKI ( Seedat et al. 1990; Yan et al. 2010; Bosch et al. 2009). In addition, when there is pre existing oxidative stress, development of exercise induced AKI is high, for example, in patients with renal hypouricemia ( Yan et al. 2010; Saito et al. 2011; Ishikawa 2002 ). As described in Chapter 3, we discovered that severity of IR induced AKI was exacerbated in the VWR EX trained SD rat compared to SED SD controls. R eductions in GFR an d RPF due to IR injury were more severe with VWR EX in the SD rat. In contrast, VWR EX conferred protection against development of IR injury in the F344 rat. Even without the influence of exercise, we discovered a strain difference in susceptibility to I R in SED rats with the F344 more vulnerable. UNX IR reduced GFR by 63% and 87% in the SD and F344 rat, respectively. Moreover, there were also greater reductions in RPF with UNX IR in the F344 rat versus SD rat (65% vs. 78%, SD vs. F344, respectively). However, d espite greater reductions in renal function
113 to UNX IR, exercise afforded protection in the F344 rat. These findings underscore the influence of genetic background in determining the impact of exercise on the kidney. There are several known diffe rences between the SD and F344 rat. However, to our knowledge, we are the first group to directly compare their renal responses to exercise training. Both strains have contrasting cardiovascular risk profiles with the SD highly susceptible to development of hypertension (Erdely et al. 2003) and the F344 as relatively resistant (Hall et al. 1976; Goldstein 1988). Both strains are also susceptible to age related renal injury, although the SD progresses more rapidly (Erdely et al. 2003; Lipman et al. 1996 ) We sought to investigate if differences in renal hemodynamic responses to exercise between the strains may account for the differences in renal endothelial enzymes with exercise. However, using the radiolabeled microsphere method to measure real time t otal RBF during exercise and at rest, we detected similar responses between the strains ( Chapter 3). In untrained SED SD and SED F344 rats, RBF significantly fell during acute exercise compared to at rest; however, this response was lost in the chronicall y TM EX trained rats for either strain. In addition, resting RBF reduced with chronic TM for both strains. Our findings do align with those by Armstrong and Laughlin who showed that in SD rats chronic exercise reduced the magnitude of the decrease in RBF after acute treadmill exercise (Armstrong & Laughlin, 1984). Therefore, differences in the renal eNOS and EC SOD response to exercise and susceptibility to IR induced AKI are not related to differences in handling of RBF. We can speculate that there are differences in intrarenal shear stress patterns which may depend on vessel radius and local viscosity in addition to vessel radius. Given the complexity of the renal circulation, it is also plausible that architectural differences within
114 the renal vascul ature also contribute. This is a particular concern for the SD rat where there is reduced renal eNOS and EC SOD with chronic exercise despite maintained RBF. Perhaps the attenuation of decreased RBF with traini ng is a maladaptive response. We can specul ate that systems governing reductions in RBF can also play a role. For example, the renal sympathetic nervous system or hormones such as angiotensin II, endothelin 1, and vasopressin which have been previously reported to dictate exercise induced falls in RBF (Mueller et al. 1998; Stebbins et al. 1995, Ahlborg et al. 1995; Maeda et al. 2004; Stebbins et al. 1993). In taking an evolutionary perspective it is likely that the role of exercise was to respond to threat. According to Walter Cannon, the fight o r flight response describes the increased sympathetic outflow which results in an attempt to prime an animal for preparation of combat (i.e. fight) or for preparation of fleeing as to avoid the combat (i.e. flight). We can speculate that endurance running in hominids evolved as a by product of enhancing walking capabilities and was beneficial for effective scavenging as well as for survival tactics. The stress exerted during exercise served benefit for the original hominids; however, in present t imes, do es the risk of exercise serve the same purpose? Thus, the physiological responses we describe herein must take into account the evolutionary perspective of exercise. This work heavily focuses on impacts of exercise on the kidney but we must also acknowled splanchnic circulation where there is decreased metabolic demand. For example, acetylcholine induced vasodilation in arterioles of the spinotrapezius, a skeletal muscle that does not ex hibit increased blood flow during exercise, has been shown to increase
115 with training (Lash et al. 1997; Xiang et al. 2005). In humans, several studies have demonstrated that lower limb exercise training improved brachial artery flow mediated dilation, a r egion that would be considered nonworking during the exercise (Walsh et al. 2003; Watts et al. 2004). These benefits may be due to the actions of retrograde and oscillatory shear that are increased in the brachial artery as a result of increased resistanc e in the lower limbs (Simmons et al. 2011). Further, s ome but not all studies also reported improved mesentery vascular reactivity via acetylcholine induced or flow mediated vasodilation in mesentery of rats with exercise (Chen et al. 2001; Sun et al. 199 8). Thus, vascular benefits of exercise can extend to regions of decreased metabolic demand. Perhaps there are strain differences in the response to exercise in these areas as well. It is likely that shear stress independent mechanisms may also contribu te to the vascular benefits of exercise. Studies described herein also suggest that the renal response of exercise is influenced by age. Instead of using an acute model of renal injury as in Chapter 3, we sought to determine the superimposing impact of th e aging kidney with exercise since there is NO deficiency and increased oxidative stress in this model as well (Erdely et al. 2003; Gomes et al. 2009). As described in Chapter 4, using young (16 months) and aged (22 24 months) male F344 rats only, we disc overed that 10 12 weeks of TM EX did not alter the age related renal injury. Rats developed age dependent renal damage and exercise was not able to reverse it. We also observed an age dependent loss of the kidney cortex antioxidants EC SOD and CuZn SOD, and age dependent increase in p22phox which were not influenced by exercise. There were also no significant changes in renal function with exercise in the aged F344 rats. These findings do not
116 align with Lichtig et al. who demonstrated that 6 weeks of ex ercise magnified age associated renal structural injury in old C57BL/6J (Lichtig et al. 1987). From data in Chapter s 2 4, it is evident that the F344 background is a strain that is protected against any potential negative influence of exercise on the kidne y. Our lab has previously shown that protection against development of renal injury associates with having a maintained NO system which is influenced by genetic background. For example, in the Wistar Furth rat, resistance to CKD induced by puromycin admi nistration parallels with preserved NO production (Erdely et al 2004). In contrast, in the SD rat, development of significant renal injury after 11 weeks of 5/6 ablation/infarction is accompanied with falls in renal and total NO production (Erdely et al. 2003). Moreover, age dependent CKD develops slowly in the Munich Wistar rat and is minimal in WAG/Rij (Baylis and Corman, 1998) Studies in Chapter 5 extend these findings since we found minimal age related renal injury in the F344XBrown Norway rat that was associated with increased renal NOS protein abundance and no change in renal cortical ADMA levels (Moningka et al. 2011). These data are not surprising since it has been shown that the F344xBN rat has fewer glomerular lesions and a greater mean age at which 50% mortality occurs compared to the regular F344 (F344: 1 03 wks. vs. F344xBN: 145 wks; Lipman et al. 1996). Further, we discovered that t he tubulointerstitial injury seen with aging in the F344xBN rat is reversed with 6 months of ARB but not ACEI and is not associated with renal NOS Altogether, our findings i n the aging F344XBN confirm that protection against age related renal injury is associated with a preserved renal NO system. These studies will provide important essential information for aging investigations where various aging models are employed.
117 Limit ations and Future Directions There are several limitations to the studies presented in this body of work The majority of our studies used VWR EX which circumvented use of additional stimuli (i.e. electric shock or air jet stress) for motivation to run a s seen with TM EX. However, in using VWR we could not control the intensity or adherence of exercise. This limitation was reflected in the high variability of running activity for all rats. Regardless, voluntary running activity was comparable between b oth strains. TM EX served its purpose of achieving a set exercise intensity of 15 m/min but we cannot rule out the possibility of stress induced responses since electric shock or jet stress which was applied in our studies. To minimize this, TM EX was co nducted with vigilant care to ensure that rats were properly acclimated so that they would not require the use of additional stimuli. In addition, rats were not on a reverse light:dark cycle when trained on the TM which may have confounding effects on the ir circadian rhythm. We did note that with VWR EX, both strains predominately ran in the dark As for our microsphere studies, we were limited by the assumption that there was adequate distribution of microspheres upon injec tion. We assessed this by comparing right and left kidney blood flows and excluding rats with a percent difference greater than ~30. It was evident that greatest variability between left and right kidney blood flows was during the exercise period. It is not certain whether this high variability was reflecting a physiological response of the kidney with exercise, or as a result of inadequate microsphere distribution which can be due to clumping or microsphere entrapment. Despite these limitations, the mi crosphere technique in determining real time blood flow during exercise remains very powerful and informative. To date, intrarenal shear stress levels have never been characterized given the complex and
118 intricate structure of the kidney, and it is not cer tain if this will ever be achieved. Without technological advancements, there will always be inherent difficulties in determining real time changes in the kidney during exercise. Nonetheless, these assessments may help uncover factors for why we detected differences in the renal eNOS and EC SOD response to exercise between strains despite their similarities in RBF handling. An indirect approach would be to measure c Src levels in the kidney since it has been previously shown that this protein tyrosine ki nase is a required signaling component of the shear stress response ( Davis et al. 2003). Where shear is reduced, c Src levels will also fall. One can then determine if c Src levels in the kid ney are altered with exercise. There are also transcriptional f actors that can bind to shear stress response elements located on promoters of specific genes related to shear which can be measured. These include NF kappa B and Egr 1 (Gimbrone et al. 2000) There is likely considerable transcriptional regulation of ge nes involved in the shear stress response. We must also acknowledge the limitations of basing our interpretations on protein abundance only. Our observations represent a snap shot of what is occurring in the kidney and do not take into time and location. Measurements of enzyme activity could have also strengthened our findings. However, enzyme activity assays are limiting in that they lack the in vivo milieu and provide conditions of optimal enzyme co factors and substrate supply. Since we used whole ti ssue homogenates we could not determine the structure or location of the changes seen with exercise. We did conduct immunohistochemical approaches to determine eNOS localization but these were done with 6 weeks of VWR only. In those studies, we also did not assess vessel diameter
119 changes in response to VWR EX or between strains. Again, the key will be to determine location of change by characterizing intrarenal shear stress patterns. Another limitation in this study is the correlative and not causative relationship between renal eNOS and EC SOD status and susceptibility to IR induced AKI. Indeed, we detected strain difference responses in severity of IR induced AKI but we cannot ascertain if these were directly due to differences in renal eNOS and EC SO D. One approach to address this is to determine impact of exercise and IR injury in either eNOS or EC SOD kidney specific knock out mice. Increased severity to IR injury with exercise training in either mouse model would confirm our findings and directly implicate the role of either protein. In hindsight, there are several improvements to our IR studies that could have improved our findings. In our UNX IR rats, it is uncertain whether conducting the UNX during the IR has any influence. It is possible that clamping of the left the kidney creates hyperfiltration to th e contralateral, right kidney, and therefore serves as a potential c onfounding factor. Secondly, our protein data in the IR studies compared control, contralateral kidneys taken ~24 hours a fter cessation of VWR versus UNX IR kidneys taken ~48 hours after cessation of VWR. To circumvent this, future studies should include an additional group of SED and VWR rats who will not have UNX. In our aging studies, exercise was not efficacious in al tering age related renal injury. However, it is possible that benefits of TM EX were masked since onset of training was at old age. Furthermore, in these studies, as described in Chapter 4, we studied the inbred F344 rat obtained from both the original N ational Institute of Aging vendor, Harlan, and the current vendor, Taconic Farms. We acknowledged the different
120 vendors but it is a limitation of our study that we also detected disparities between F344 rats obtained from either vendor (i.e. significant b ody weight differences between young F344 supplied from Taconic and Harlan). In this body of work we did not explore sex differences. It is well known that male gender is another risk factor for development of age associated renal injury. Female SD rat s exhibit maintained renal NOS protein abundance with age and are protected against age dependent kidney damage compared to males (Erdely et al. 2003; Baylis 2009). The sexual dimorphism seen with NO deficiency as it relates to aging is presumably due to sex hormones. Few studies have investigated the role of sex differences in impact of exercise on the kidney. It is likely that males will parallel our SD findings in that they will be at increased risk of developing IR induced AKI. We must also not exclu de the potential influencing role of metabolic pathways. Exercise has numerous metabolic actions including increased insulin sensitivity and decreased plasma triglycerides (Sasaki & Gisele, 2005). W e did not consider the roles of angiotensin II and/or va sopressin which are known to influence exercise induced falls in RBF (McAllister, 1998). However, in our studies, we did not make an y assessment of these factors. It is plausible that the marked strain differences in the renal eNOS and EC SOD response to exercise were due to metabolic actions on the endothelium. Indeed, we failed to detect a lack of hemodynamic difference between the strains with exercise. Moreover, although body weight significantly reduced, we found that 3 weeks did not affect renal e NOS and EC SOD in the SD rat. We expected marked changes in renal eNOS and EC SOD since training adaptations were detected (i.e. body weight significantly reduced). Therefore, it is likely that control of renal eNOS and EC SOD is
121 heavily influenced by sy stems other than shear stress such as various metabolic signaling pathways. To study the impact of reduced RBF without influence of the metabolic changes due to exercise, future studies can incorporate the use of the Goldblatt model of renal artery sten osis where there is physical obstruction of the renal artery by clipping. It would be informative to use this model to study the im pact of intrarenal eNOS prote in Several rat studies suggest that the cardiovascular benefit of exercise does not necessarily guarantee renal benefit in the setting of CKD where both cardiovascular and renal complications are present. Bergamaschi et al. reported that 60 days of treadmill exercise did not prevent C KD induced proteinuria and glomerular slcerosis despite normalization of hypertension (Bergamaschi et al. 1997). In contrast, Adams et al. found that voluntary exercise did not ameliorate the hypertension associated with the 5/6 nephrectomy model (Adams e t al 2004). These findings will be highly clinically relevant since cardiovascular co morbidities are high in renal disease patients. To address this, future studies should exploit the differences between the 5/6 ablation and the 5/6 ablation/infarction models of CKD. Both are models of renal mass reduction; however, in the 5/6 ablation/infarction only, there is significant systemic hypertension associated its disease progression (Ibrahim & Hostetter, 1998). It may be the case that exercise has substan tial systemic cardiovascular benefits (i.e. reduces the Altogether, t hese studies would determine whether there is a significant systemic component to the benefit of exercise in a setting where renal injury is present.
122 Extending our findings in rats to humans also has its limitations. Similar to humans, rats exhibit declines in RBF with exercise; however, the mechanisms involved in sympathetic nervous system activation or VO 2 max regulation may uncover species differences. It is also difficult to speculate which rat studied in this body of work is more it is likely there are those that wil l respond like the SD, while others will respond like the F344. Clinical Perspectives Renal failure afflicts a growing population in the United States and according to the 2010 U.S. Renal Data System Annual Report these numbers are expected to grow. Benef its of physical activity are critical for this population given the numerous ca rdiovascular related benefits. For patients with kidney disease, t he National Kidney Foundation recommends to exercise at least three days a week for ~30 minutes a session, and that it must consist of either continuous movement of large muscle groups (i.e. walking, swimming, bicycling, and aerobic dancing) or low level weight bearing exercise (i.e. high repe tition of low weight lifting). They also recommend that participation i n any sort of exercise program must consider type, duration, frequency, and intensity of exercise. Indeed, potential risks with exercise should be considered. The data in this body of work are provoking in that we have discovered certain settings where e xercise can exacerbate renal injury. Genetic background is clearly a major factor which is highly clinically relevant since there is huge influence of genetics in development of renal injury in humans. The purpose of these studies was not to support disc ontinuation of exercise prescription for the renal disease population, but to
123 contribute new knowledge so that maximum benefit is obtained with exercise of optimal modality and intensity.
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138 BIOGRAPHICAL SKETCH Natasha Moningka was born in Jakarta, In donesia in 1983. She and her family emigrated from Indonesia to California where she attended and graduated from Redlands High School in 2002 a nd the University of California at I rvine in 2006 with a degree in b iol ogical s ciences. As a result of her unde rgraduate research work under the mentorship of Dr. Kenneth Baldwin in the Department of Physiology & Biophysics studying responses of skeletal muscle hypertrophy to L Arginine suppl e mentation she Sc hool of Biological Sciences Excel lence in Research In fall of 2006, Natasha entered the Interdisciplinary Program in Biom edical Sciences at the University of Florida where she joined the laboratory of Dr. Chris Baylis in the Physiology and Functional gen omics a year later. As a result of her dissertation work on impact of exercise on the renal nitric oxide and antioxidant systems, she attended and presented at conferences yearly, received the University of Florida Medical Guild Research Incentive Award i n 2008, received the Young Investigator Award in 2010 by the S ociety for Experimental Biology and published se veral abstracts and manuscripts. Natasha also serves on the Water & Electrolyte Homeostasis Section Steering and Trainee A dvisory c ommittees of the American Physiologic al Society. In fall of 2011, she received her Ph.D. from the University of Florida and is currently pursuing her scientific career as a postdoctoral researcher in the laboratory of Dr. Michael Caplan at Yale University where she s tudies mechanisms of polycystic kidney disease