The Role of Rh B Glycoprotein in Renal Ammonia Metabolism

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The Role of Rh B Glycoprotein in Renal Ammonia Metabolism
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english
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Bishop, Jesse M
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Master's ( M.S.)
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University of Florida
Degree Disciplines:
Medical Sciences, Molecular Genetics and Microbiology
Committee Chair:
Weiner, David I
Committee Members:
Reed, Jill V
Snyder, Richard O
Kraft, John

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hypokalemia -- rhbg -- transport
Molecular Genetics and Microbiology -- Dissertations, Academic -- UF
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Abstract:
Renal ammonia metabolism is a critical mechanism involved in the maintenance of acid-base homeostasis.  Renal epithelial cell transport, along with renal ammonia genesis are two major facets of renal ammonia metabolism. Recent studies have identified members of a family of ammonia specific transporters that play a critical role in renal epithelial transport. Rh B glycoprotein (Rhbg) is one member of the large family of ammonia specific transporters.  Rhbgis expressed in the kidney, liver, lung, gastrointestinal tract and skin.   In the kidney, Rhbg is expressed in the basolateral plasma membrane in cells of the distal convoluted tubule (DCT),connecting segment (CNT), initial collecting tubule (ICT) and throughout the collecting duct.  The current study investigates the role of Rhbg in response to conditions that involve altered  renal ammonia metabolism. This includes an investigation into Rhbg’s role in hypokalemia and a preliminary investigation into the renal response to dietary protein restriction.  This study included the use of intercalated cell Rhbg knockout mice (IC-Rhbg-KO) and collecting duct Rhbg knockout mice (CD-Rhbg-KO) and control mice that had normal Rhbg expression. Rhbg protein expression increased in both the cortex and outer medulla in response to hypokalemia. In control mice, hypokalemia increased total urinary ammonia excretion, absolute ammonia concentration and urine pH, whereas in IC-Rhbg-KOmice, urine ammonia concentration did not change significantly from baselineand total urinary ammonia excretion was significantly less than in C mice.  Urine pH, Na+, K+ and volume were unaltered by IC-Rhbg-KO.
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by Jesse M Bishop.
Thesis:
Thesis (M.S.)--University of Florida, 2012.
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Adviser: Weiner, David I.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-08-31

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1 THE ROLE OF RH B GLYCOPROTEIN IN RENAL AMMONIA METABOLISM By JESSE M. BISHOP A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENC E UNIVERSITY OF FLORIDA 2012

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2 2012 Jesse M. Bishop

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3 To my family, friends, teachers, mentors, and team mates. And to all those that stood beside me during my time in the United States Army, as 1 st Ranger Battalion was my first step towar ds a higher education. To all those that never came home to pursue their higher education, your sacrifices have made milestones like this possible for co untless individuals like myself.

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4 ACKNOWLEDGMENTS I thank the chair and members of my supervisory committee for their devotion and commitment to my education over the years. I thank my wife and daughter for their support in every facet of my life, and for the encouragement to complete my studies.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ .......... 7 LIST OF ABBREVIATIONS ................................ ................................ ............................. 8 ABSTR ACT ................................ ................................ ................................ ..................... 9 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 11 2 THE ROLE OF RH B GLYCOPROTEIN IN THE RENAL RESPONSE TO HYPOKALEMIA ................................ ................................ ................................ ...... 13 Methods ................................ ................................ ................................ .................. 13 Animals ................................ ................................ ................................ ............. 13 Antibodies ................................ ................................ ................................ ......... 14 Induction of Hypokalemia ................................ ................................ ................. 14 Electrolyte Measurements ................................ ................................ ................ 14 Tissue Preparation for Immunolocalization ................................ ....................... 15 Immunohistochemistry ................................ ................................ ...................... 15 Double Immunolabeling Procedure ................................ ................................ .. 16 Protein Preparation ................................ ................................ .......................... 16 Immunoblotting Procedure ................................ ................................ ............... 17 Statistical Analysis ................................ ................................ ............................ 18 Results ................................ ................................ ................................ .................... 18 Serum Chemistries ................................ ................................ ........................... 18 Urinary Ammonia Excretion ................................ ................................ .............. 19 Effect of Hypokalemia on Rhbg Expression ................................ ..................... 21 Adaptive Responses to Intercalated Cell specific Rhbg Deletion in Response to Hypokalemia ................................ ................................ ............ 22 Corr elation of Urinary K+ and Ammonia Excretion ................................ ........... 22 Discussion ................................ ................................ ................................ .............. 23 3 CHARACTERIZATION OF COLLECTING DUCT KNOCKOUT RHBG MICE ........ 30 Methods ................................ ................................ ................................ .................. 30 Animals ................................ ................................ ................................ ............. 30 Antibodies ................................ ................................ ................................ ......... 31 Tissue Preparation for Immunolocalization ................................ ....................... 31 Immunohistochemistry ................................ ................................ ...................... 31 Results ................................ ................................ ................................ .................... 32 Recombination in CD Rhbg KO mice ................................ ............................... 32

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6 Rhbg Expression in CD Rhbg KO Mice ................................ ............................ 32 4 WORKS IN PROGRESS AND FUTURE DIRECTIONS ................................ ......... 35 Methods ................................ ................................ ................................ .................. 35 Animals ................................ ................................ ................................ ............. 35 Antibodies ................................ ................................ ................................ ......... 36 Dietary Protein Restriction ................................ ................................ ................ 36 Tissue Preparation for Immunolocalization ................................ ....................... 37 Immunohistochemistry ................................ ................................ ...................... 37 Results ................................ ................................ ................................ .................... 38 Effect of Low Protein Diet on Rhbg Expression ................................ ................ 38 Urine Volume and Urine pH ................................ ................................ .............. 38 Future Directions ................................ ................................ ................................ .... 38 LIST OF REFERENCES ................................ ................................ ............................... 42 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 47

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7 LIST OF FIGURES Figure page 2 1 Effect of hypokalemia on urinary ammonia excretion. ................................ ........ 25 2 2. Effect of hypokalemia on renal Rhbg expression. ................................ ............... 26 2 3 Rhbg expression by immunohistochemistry in response to hypokalemia. .......... 27 2 4 Adaptive responses of proteins involved in renal ammonia metabolism in response to IC Rhbg KO in hypokalemic mice. ................................ .................. 28 2 5 Correlation of changes in urinary K and ammonia excretion in control and IC Rhbg KO mice in response to K free diet. ................................ .................... 29 3 1 Rhbg immunolabel in CD Rhbg KO and control mice. ................................ ....... 34 4 1 Rhbg in the cortex and outer medulla of control and low protein diet mice. ........ 40 4 2 Urine Volume and Urine pH in Response to Low Protein Diet ............................ 41

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8 LIST OF ABBREVIATION S Amt Ammonia transporters B1 Cre Transgenic mice expressing Cre recombinase under control of the H+ATPase B1 subunit promoter CCD Cortical collecting duct CD Rhbg KO Collecting Duct Rh B Glycoprotein Knockout CNT Connecting segment DCT Distal convoluted tubule IC Rhbg KO Intercalated Cell Rh B Glycoprotein Knockout ICT Initial collecting tubule Mep Methylammonium permeases NAE Net acid excretion NEAP Net endogenous acid production OMCD Outer medullary collecting duct PBS Phos phate buffered saline PDG Phosphate dependent glutaminase PEPCK Phosphoenolpyruvate carboxykinase PLP Periodate lysine 2% paraformaldehyde Rhbg Rh B Glycoprotein Rhcg Rh C Glycoprotein

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9 Abstract of Thesis Presented to the Graduate School of the Univers ity of Florida in Partial Fulfillment of the Requirements for the Degree Master of Science THE ROLE OF RH B GLYCOPROTEIN IN RENAL AMMONIA METABOLISM By Jesse M. Bishop August 2012 Chair: I. David Weiner Major: Medical Sciences Translational Biotechnology Renal ammonia metabolism is a critical mechanism involved in the maintenance of acid base homeostasis Renal epithelial cell transport, along with renal ammoniagenesis are two major facets of renal ammonia metabolism. Recent studies have identified members of a family of ammonia specific transporters that play a critical role in renal epithelial transport. Rh B glycoprotein (Rhbg) is one member of the large family of ammonia specific transporters. Rhbg is expressed i n the kidney, liver, lung, gastrointestinal tract and skin. In the kidney, Rhbg is expressed in the basolateral plasma membrane in cells of the distal convoluted tubule (DCT), connecting segment (CNT), initial collecting tubule (ICT) and throughout the collecting duct The current study investigates the role of Rhbg in response to conditions that role in hypokalemia and a preliminary investigation into the renal resp onse to dietary protein restriction. This study included the use of intercalated cell Rhbg knockout mice (IC Rhbg KO) and collecting duct Rhbg knockout mice (CD Rhbg KO) and control mice that had normal Rhbg expression.

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10 Rhbg protein expression increased i n both the cortex and outer medulla in response to hypokalemia. In control mice, hypokalemia increased total urinary ammonia excretion, absolute ammonia concentration and urine pH, whereas in IC Rhbg KO mice, urine ammonia concentration did not change sign ificantly from baseline and total urinary ammonia excretion was significantly less than in C mice. Urine pH, Na+, K+ and volume were unaltered by IC Rhbg KO.

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11 CHAPTER 1 INTRODUCTION Acid base homeostasis is essential for many facets of normal human health. Acid base disorders lead to stunted growth and skeletal disorders in children and in adults it leads to muscle wasting nephrolithiasis, and circulatory disorders (2, 3, 29, 43) Net end ogenous acid production is the amount of net acid produced by the metabolic system. For an individual consuming a typical western diet the endogenous acid production is estimated to be 50 mEq/d (20) Systemic acid base homeostasis is maintained when endoge nous acid production is equivalent to net acid excretion. Net Acid excretion is composed urinary ammonia, titrateable acids and bicarbonate, with renal ammonia metabolism making up 50 70% of the total net acid excretion (32) Renal ammonia metabolism is a critical mechanism involved in the maintenance of acid base homeostasis, it involves integrated ammoniagenesis and renal epithelial (8, 10, 16, 42) cell transport. Recent studies have identified novel mechanisms of renal epithelial cell ammonia transport by members of a family of closel y related ammonia specific transporters (4, 5, 14, 17, 19, 41, 42) Members of this ammonia transporter family include Mep proteins in yeast, Amt prote ins in a wide variety of single cell organisms and plants, and Rh glycoproteins in amphibians, fish and mammals. In mammals, Rh glycoproteins mediate a central role in the regulation of renal ammonia excretion (4, 14, 22, 23, 41, 42) These proteins transport ammonia, but not Na+, K+ or H2O (21, 25 28) and they exhibit polarized expression in the distal convoluted tubule through the inner medullary c ollecting duct. Two members of this family of ammonia specific transporters are Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg). Both Rhbg and Rhcg are present in

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12 collecting duct cells (both intercalated and principal cells), with Rhbg being express ed in the basolateral plasma membrane and Rhcg in the apical and basolateral membranes, in addition to cytoplasmic vesicles (9, 11, 15, 30, 33, 38) The coordinated action of these proteins enables regulated transcellular ammonia transport (40 42) Recent studies in the mouse have shown that Rhbg mediates a critical role in ammonia metabolism under basal conditions and in response to metabolic acidosis. Metabolic acid osis increases Rhbg expression in both the cortex and the outer medulla (5) Moreover, intercalated cell specific Rhbg deletion impaired the normal increase in renal ammonia excretion in response to metabolic acidosis, indicating a key role for Rhbg in renal ammonia transport and the maintenance of acid base homeostasis in re sponse to metabolic acidosis (5) U nder basal conditions, intercalated cell specific Rhbg deletion did not alter either serum HCO3 or urinary ammonia excretion, but did induce adaptive changes in glutamine synthetase expression which appear to compensate for the absence of Rhbg mediated ammonia transport when ammonia excreti on is not stimulated (5)

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13 CHAPTER 2 THE ROLE OF RH B GLYCOPROTEIN IN THE RENAL RESPONS E TO HYPOKALEMIA Hypokalemia is a common condition that increases urinary ammonia excretion and can lead to another acid base disorder, metabolic alkalosis (35, 36) Hypokalemia results in altered ammoniagenesis (36) ammonia transport (6, 7, 39) and increased collecting duct ammonia secretion (7) The purpose of the current study was to Methods Anim als The generation of mice with loxP sites flanking critical exons of the murine Rhbg gene were described previously (5) Transgenic mice expressing Cre recombinase under control of a 6.5 kb portion of the H+ ATPase B1 subunit promoter (B1 Cre) have been described previously (24) IC Rhbg KO mice were bred using floxed Rhbg mice and mice expressing Cre recombinase under con trol of the B1 subunit of H+ ATPase promoter (B1 Cre) as described previously (5) Animal breeding was performed in the University of Florida College of Medicine Cancer and Genetics Transgenic Animal Core Facility by trained personnel. Mice were genotyped using tail clip samples as described previously (15, 18) All mice used in this project were either floxed Rhbg, B1 Cre positive or floxed Rhbg, B1 Cre negative; w e have shown previously that floxed Rhbg, B1 Cre positive mice have intercalated cell specific Rhbg deletion and that floxed Rhbg, B1 Cre negative mice have normal Rhbg expression (5) All animal studies were approved by the Institutional Animal Care and Use Committees of the Gainesville VA Medical Center and of the Universit y of Florida College of Medicine.

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14 Antibodies Affinity purified antibodies to Rhcg and Rhbg were generated in our laboratory and have been previously characterized (15, 17, 21, 38) In particular, we have shown specificity of both the Rhbg and the Rhcg antibodies in studies using heterologous expression in Xenopus oocytes (21) and in studies using genetic deletion of Rhbg and Rhcg (15, 17) Norman Curthoys, Ph.D. (Colorado State Universi ty) provided antibodies to phosphate dependent glutaminase (PDG) and Fiona Karet, Ph.D. (Cambridge Institute for Medical Research, Cambridge, UK) provided antibodies to the a4 subunit of H+ ATPase. Antibodies to glutamine synthetase were obtained from Mil lipore, Billerica, MA, and antibodies to phosphenolpyruvate carboxykinase (PEPCK) were obtained from Cayman Chemical Co., Ann Arbor, MI. Induction of Hypokalemia Hypokalemia was induced by feeding mice a K free diet (TD.88239, Harlan Teklad, Madison, WI) for three days. P owdered foo d was mixed it with H O in a ratio of 6 gm food to 1 ml H O to form a semi solid diet. Adult mice, greater than 8 w ee ks age, were placed into metabolic cages (Tecniplast diures is metabolic cage, Fisher Scientific) and allowed to acclimate for three days whi le receiving control diet. They then received K free or control diet and daily food intake was measured. At all times animals were allowed free access to water. Daily urin e excretion was collected under mineral oil;, urine pH was measured and urine vol ume calculated. Urine samples were stored at 80C until analyzed. Electrolyte Measurements Urine ammonia was measured using a commercially available kit (A7553, Pointe Scien tific, Inc., Canton, MI) modified for use in 96 well plates. Serum bicarbonate was

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15 measured as total CO 2 using a commercially available kit (C750 120, Pointe Scientific, Inc) modified for use with microliter quantities of serum. Urine pH was measured usi ng a micro pH electrode (Thermo Scientific, ROSS semi micro pH, ORION 8115BN). Serum Na+ and K+ concentrations were measured using a flame photometer (Instrumentation Laboratory, Lexington, MA) as described previously (17) Serum and urine creatinine was measured by capillary electrophoresis using techniques described previously (17) Tissue Preparation for Immunolocalization Mice were anesthetized with inhalant isoflurane. The kidneys were preserved by in vivo cardiac perfusion with PBS (pH 7.4) followed by periodate lysine 2% paraformaldehyde (PLP) and then cut transversely into several 2 to 4 mm thick slices and immersed 24 to 30 hours at 4C in the same fixative. For light microscopy, samples of kidney from each animal were embedded in polyester wax (polyethylene glycol 400 distearate (Polysciences, Warrington, PA) with 10% 1 hexadecanol ), and 3 thick sections were cut and mounted on gelatin coated glass slides. Immunohistochemistry Immunolocalization was accomplished using immunoperoxidase procedures detailed previously (15, 17) Tissue sections were dewaxed in ethanol, rehydrated and then rinsed in PBS. Endogenous peroxidase activity was blocked by incubating the sections in Peroxidase Blocking Reagent (DakoCytomation, Carpinteri a, CA) for 45 min. The sections were blocked for 15 min with Serum Free Protein Block (DakoCytomation), then incubated at 4C overnight with primary antibody diluted in Dako Antibody Diluent. The sections were washed in PBS and incubated for 30 min with polymer linked peroxidase conjugated goat anti rabbit IgG (MACH2, Biocare

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16 Medical), again washed with PBS, then exposed to diaminobenzidine for 5 minutes. The sections were washed in distilled water, then dehydrated in a graded series of ethanols and xyle ne, mounted and observed by light microscopy. Comparisons of labeling were made only between sections of the same thickness from the same immunohistochemistry experiment. Sections were examined on a Nikon E600 microscope equipped with DIC optics and phot ographed using a DXM1200F digital camera and ACT 1 software (Nikon). Color and contrast adjustment was performed using Adobe Photoshop CS5 (Adobe Systems, Inc., San Jose, CA). Double Immunolabeling Procedure Double immunolabeling was accomplished using se quential immunoperoxidase procedures described in detail previously (15) Briefly, tissue sections were labeled with the first primary antibody following the procedure described above, using Vector SG (Ve ctor Laboratories) as the chromo gen to produce a blue label. After the Vector SG reaction, sections were washed in PBS t hen blocked using the Peroxidase Blocking Reagent and Serum Free Protein Block as described in the single label procedure. The above procedure was repeated with the substitution of a second primary antibody and the substitution of DAB for Vector SG. The sections were then washed with glass distilled water, dehydrated with xylene, mounted with Permount, and observed by light microscopy. Protein Preparation Animals were anesthetized with inhalant isoflurane and the kidneys were rinsed by in vivo cardiac per fusion with PBS (pH 7.4), rapidly removed, and stored frozen at 80C until used. In some experiments, the right renal vasculature was clamped after in vivo cardiac perfusion with PBS, the right kidney removed, and then the left kidney was

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17 perfused with P LP fixative for immunohistochemistry. Tissues were homogenized using microtube pestles (USA Scientific, Ocala, FL) and proteins were extracted using T PER Tissue Protein Extraction Reagent (Pierce Biotechnology, Inc., Rockford, IL) according to the manufa Rhcg, tissues were homogenized in buffer A (in mM: 50 sucrose, 10 Tris buffer, 1 EDTA, pH 7.4) and then diluted in buffer B (in mM: 250 sucrose, 10 Tris buffer, and 1 EDTA, pH 7.4). Th e sample was then centrifuged at 1,000 g for 5 min at 4C. The pellet was resuspended in buffer B and again centrifuged at 21,000 g for 30 min at 4C. The 21,000 g pellet was finally resuspended in buffer B. An aliquot was used for protein determination with a BCA assay, and the remainder was stored frozen at 80C until used. Immunoblotting Procedure Five to twenty micrograms of renal protein were electrophoresed on 10% PAGE ReadyGel (Bio Rad, Hercules, CA). Gels were then transferred electrophoretical ly to nitrocellulose membranes, blocked with 5 g/dl nonfat dry milk, and incubated for 2 h with primary antibody diluted in Blotto buffer (50 mM Tris, 150 mM NaCl, 5 mM Na2EDTA and 0.05% Tween 20, pH 7.5) with 5 g/dl nonfat dry milk. Loading and transfer equivalence was assessed with Ponceau S staining. After washing, membranes were exposed to secondary antibody (goat anti rabbit IgG; Promega, Madison, WI or goat anti mouse IgG; Upstate, Temecula, CA, conjugated to horseradish peroxidase) at a dilution of 1:5000. Sites of antibody antigen reaction were visualized by using enhanced chemiluminescence (SuperSignal West Pico Substrate, Pierce, Rockford, IL) and a Kodak Image Station 440CF digital imaging system. Band density was quantified using Kodak 1D, ve rsion 3.5.4, software, Kodak Scientific Imaging, New Haven CT.

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18 Band density was normalized such that mean density in the same region (cortex or outer medulla) in C kidneys was 100.0. Statistical Analysis Results are presented as mean SEM. N reflects nu mber of mice examined. test and ANOVA as appropriate. P<0.05 was required for evidence of statistical significance. Results Serum Chemistries In order to study hypokalemia, mice were fed either norma l diet or a K free diet Food intake was determined daily, and did not differ significantly between mice with intact Rhbg expression and IC Rhbg KO mice (data not shown). While on a normal diet, intercalated cell specific Rhbg deletion did not significa ntly alter serum potassium concentration in mice on normal diet; serum potassium averaged 4.1 0.2 mmol/L in mice with intact Rhbg expression and 4.1 0.1 mmol/L in IC Rhbg KO mice (P=NS, N=8 in each group). Following three days of dietary potassium dep letion, serum potassium averaged 3.2 0.1 mmol/L in mice with intact Rhbg expression and 3.7 0.2 mmol/L in IC Rhbg KO mice. Thus, three days of a K free diet induced hypokalemia in mice with intact Rhbg expression and in mice with IC Rhbg KO (P < 0.00 1, N=8 in each group); t he degree of hypokalemia did not differ significantly between mice with intact Rhbg expression versus those with IC Rhbg KO (P=NS, N=8 in each group). Serum bicarbonate averaged 19.1 0.9 and 19.1 0.7 mmol L in control and IC R hbg KO mice on regular di et, respectively (N = 8 in each group) and 19.8 0.8 and 20.2 0.8 mmol L in control and IC Rhbg KO mice on a K free diet, respectively (N = 8 in each group). Serum bicarbonate did not change significantly in response to

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19 hypo kalemia, either in mice w ith intact or with intercalated cell specific Rhbg deletion (P=NS by ANOVA). Also, intercalated cell specific Rhbg deletion did not alter serum bicarbonate significantly, either while on regular diet or on 0 K+ diet (P = NS by ANO VA, N = 8 in each group). A K free diet induced mild polyuria, but neither baseline urine volume nor hypokalemia induced increases in urine volume differed significantly between mice with intact Rhbg expression and intercalated cell specific Rhbg deletion (data not shown). Simila rly, there was no significant difference in serum sodium concentration, either under baseline conditions or with dietary potassium depletion, in either control or IC Rhbg KO mice (data not shown). GFR was estimated using measureme nt of creatinine clearance. While on regular diet, creatinine clearance did not differ significantly between mice with intact Rhbg expression and those with IC Rhbg ; IC Rhbg n=4 in each gr oup, P=NS). After three da ys of a K+ free diet, creatinine clearance did not differ significantly between mice with intact Rhbg expression and those with IC Rhbg ; IC Rhbg n=8 in each group P=NS). In addition, hypo kalemia did not change creatinine clearance significantly, either in mice with intact or with intercalated cell specific Rhbg deletion (P=NS by ANOVA). Thus, IC Rhbg KO did not significantly alter GFR, either under basal conditi ons or in response to a K+ free diet for three days. Urinary Ammonia Excretion Basal ammonia excretion rates did not differ significantly between mice with intact Rhbg expression and with IC Rhbg KO (P=NS, N=8 in each group; Figure 2 1 A ), as we

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20 reported p reviously (5) Hypokalemia increased total urinary ammonia excretion significantly both in mice with intact Rhbg expression and in mice with IC Rhbg KO. However, both total urinary ammonia excretion ( Figure 2 1 A ) and the increase above the basal excretion rate in response to a K+ free diet ( Figure 2 1 B ), a direct measure o f Rhbg KO mice than in mice with intact Rhbg expression (P<0.05 on each day, n=8 in each group at each time point). IC Rhbg KO decreased hypokalemia induced changes in urinary a mmonia excretion by 68 9%, 52 18% and 48 15% on days 1, 2 and 3, respectively (P<0.05 for each comparison, N=8 in each group at each time point). Changes in urinary pH often correlate with changes in urinary ammonia excretion, with, in general, more acidic urine pH associated with greater rates of ammonia excretion and more alkaline urine associated with less ammonia excretion. The most common exception to this correlation is hypokalemia, which typically increases urine pH. We observed in these stud ies that hypokalemia increased urine pH in both control and IC Rhbg KO mice. In contrast to the effects of IC Rhbg KO on ammonia excretion, IC Rhbg KO did not alter urinary pH in hypokalemic mice significantly (Figure 2 1C). Thus, differences in urinary pH do not mediate the differences in urinary ammonia excretion in response to hypokalemia. Changes in total urinary ammonia excretion require either increases in ammonia concentration or increases in urine volume. As noted previously, Rhbg deletion did no t alter hypokalemia induced changes in urine volume. However, there was a significant difference in the effect of hypokalemia on urinary ammonia concentration. In mice with intact Rhbg expression, hypokalemia increased urinary ammonia concentration

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21 signi ficantly ( Figure 2 1D ). In contrast, in mice with intercalated cell specific Rhbg deletion hypokalemia did not alter significantly urinary ammonia concentration (P=NS on each day of hypokalemia, N=8 on each day). Thus, intercalated cell specific Rhbg del etion significantly impairs the expected increase in total urinary ammonia excretion and completely blocks changes in urinary ammonia concentration, and these changes are independent of changes in urine pH. Effect of Hypokalemia on Rhbg Expression The obse rvation that intercalated cell specific Rhbg deletion impairs the response to hypokalemia, but does not alter basal urinary ammonia excretion, suggests that this further, we determined the effect of hypokalemia on renal Rhbg expression using both immunoblot analysis and immunohistochemistry. Immunoblot analysis showed that hypokalemia increased Rhbg protein expression significantly in both the cortex and the outer medulla in mice with intact Rhbg expression ( Figure 2 2 ). I mmunohistochemistry was used to determine whether hypokalemia increased Rhbg expression in intercalated cells, non intercalated cells, or both. Hypokalemia increased basolateral Rhbg immunolabel in inter calated cells, but not in principal cells, in the cortical collecting duct (CCD) and in the outer medullary collecting duct (OMCD). Figure 2 3 shows representative micrographs demonstrating these findings. Thus, hypokalemia induced increases in urinary a mmonia excretion and urinary ammonia concentration is associated with increased Rhbg expression in intercalated cells in the CCD and OMCD.

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22 Adaptive Responses to Intercalated Cell specific Rhbg Deletion in Response to Hypokalemia In a variety of conditions, adaptive responses in other proteins involved in renal ammonia metabolism can often compensate, either partially or completely, for the absence of the Rh glycoproteins, Rhbg and Rhcg (5, 17, 18) In preliminary studies, we confirmed that hypokalemia increased phosphate dependent glutaminase, phosphoenolpyruvate carboxykinase and Rhcg expressio n and decreased glutamine synthetase expression in mice with intact Rhbg expression (data not shown). We then assessed in hypokalemic mice whether intercalated cell specific Rhbg deletion induced alterations in the expression of these critical proteins in renal ammonia metabolism. Figure 2 4 summarizes these results. In hypokalemic mice, IC Rhbg KO did not alter expression of PEPCK, PDG, glutamine synthetase or Rhcg. Adaptive changes in the expression of these proteins does not appear to compensate for the absence of Rhbg mediated ammonia transport in hypokalemic mice. Correlation of Urinary K + and Ammonia Excretion The well known association of increased ammonia excretion and decreased urinary K excretion in response to hypokalemia has sometimes suggested that there may be either direct or indirect NH for K exchange mechanism that regulates urinary K excretion. To begin examining whether hypokalemia induced changes in ammonia and K excretio n are directly related, we examined the effect of IC Rhbg KO, and its effect on urinary ammonia excretion, on urinary K+ excretion. Hypokalemia decreased urinary K excretion rapidly in both control an d IC Rhbg KO mice, and there was no significant differ ence between control mice and IC Rhbg KO mice (Figure 2 5A ). Thus, inhibiting ammonia excretion, by IC Rhbg KO, did not alter net urinary K+ excretion.

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23 Figure 2 5B shows summary data correlating changes in urinary K + and ammonia excretion in response to hypokalemia in control and IC Rhbg KO mice. IC Rhbg KO altered the slope of the correlation between changes in urinary K + and ammonia excretion significantly (P<0.05 by ANOVA). Discussion These studies provide the first detailed assessment of the effect o f Rhbg deletion on the renal response to hypokalemia. In mice with intact Rhbg expression, hypokalemia increased total urinary ammonia excretion, urinary ammonia concentration, and renal Rhbg expression in intercalated cells. In mice with IC Rhbg KO, the expected increase in total urinary ammonia excretion was significantly blunted and the expected increase in urinary ammonia concentration changes was prevented. These changes occurred despite no significant effect of IC Rhbg KO on urinary pH. Thus, incr eased Rhbg expression in intercalated cells mediates a significant role in the increased urinary ammonia excretion observed in response to hypokalemia. Although IC Rhbg KO altered ammonia excretion significantly, it did not alter urinary K+ excretion, ind icating that there is not a direct correlation between ammonia and K+ excretion in response to hypokalemia. The primary finding of this study is that intercalated cell specific Rhbg expression is necessary for the normal hypokalemia induced changes in urin ary ammonia excretion. Dietary potassium restriction in mice with intact Rhbg expression increased Rhbg protein expression in intercalated cells in both the CCD and OMCD and this was associated with increased renal ammonia excretion. In mice lacking Rhbg in intercalated cells the increase in total urinary ammonia excretion was significantly impaired, and there was no significant change in urinary ammonia concentration. Thus,

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24 intercalated cell Rhbg expression is critical for the normal increases in ammoni a excreti on in response to hypokalemia. metabolic acidosis model, show a clear role for Rhbg in renal ammonia metabolism. Mouse studies involving metabolic acidosis and hypokal emia have shown increases of ~ 300% in urinary ammonia excretion for metabolic acidosis and ~ 200% for hypokalemia, when Rhbg is knocked out in intercalated cells, these increase are only ~ 150% and ~ 75%. Because other disease states do not result in cha nges in urinary ammonia excretion as metabolic acidosis and hypokalemia, a more complete knockout of Rhbg will be required in order to study adaptive responses.

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25 F igure 2 1 Effect of hypokalemia on urinary ammonia excretion. Panel A shows baseline each day of a K free diet. Urinary ammonia excretion increased each day in both control and IC Rhbg KO mice, but ammonia excretion was significantly less in IC Rhbg KO mice on each day of h ypokalemia than in control mice (*, P < 0.05, N = 8 in each group). Panel B shows the change in urinary ammonia excretion from baseline rate in response to a K free diet in control and IC Rhbg KO mice. The increase in urinary ammonia excretion was signi ficantly less in IC Rhbg KO mice (N = 8 in each group on each day). Panel C shows urinary pH. Urine pH did not differ between control and IC Rhbg KO mice on any day of the K free diet (N = 8 in each group on each day). Panel D shows changes in the conce ntration of ammonia in the urine in response to hypokalemia. Urinary ammonia concentration increased significantly on each day of the K free diet in control mice, and did not change significantly from baseline urinary ammonia concentration in IC Rhbg KO mice (*, P < 0.05 vs baseline, N = 8 in each group).

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26 Figure 2 2 Effect of hypokalemia on renal Rhbg expression Top Panel shows representative immunoblot analyses and bottom panel shows summary quantitative analyses of Rhbg protein expression in the cortex and outer medulla in response to K free diet for three days. Rhbg protein expression increased significantly in both the cortex and the outer medulla (*, P < 0.05; N = 8 in each group).

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27 Figure 2 3 Rhbg expression by immunohistochemistry in re sponse to hypokalemia. Rhbg immunolabel is shown in brown, and H ATPase immunolabel in blue used to differentiate between principal cells (H ATPase negative collecting duct cells) and intercalated cells (apical H ATPase positive collecting duct cells). In the CCD (Panels A and B), basolateral Rhbg immunolabel intensity increased in response to K free diet in intercalated cells (arrows). There was no detectable change in principal cell basolateral Rhbg immunolabel in the CCD. In the OMCD (Panels C and D), Rhbg immunolabel increased in intercalated cells (arrows), but not in principal cells in response to the K free diet. Images are representative of findings in 6 mice in each group.

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28 Figure 2 4 Adaptive responses of proteins involved in renal ammonia metabolism in response to IC Rhbg KO in hypokalemic mice. This figure summarizes experiments performed to determine whether adaptive changes in the expression of other proteins involved in renal ammonia metabolism compensate partially for the lack of Rhbg expression in intercalated cells. This shows a comparison of expression in hypokalemic mice with intact Rhbg expression to hypokalemic mice with IC Rhbg KO. Panels A D show PEPCK, PDG, glutamine synthetase, and Rhcg protein expression, respectiv ely. There were no significant differences in the expression of any of these proteins, either in cortex or outer medulla, between hypokalemic mice with intact or intercalated cell specific Rhbg deletion (N = 8 in each group).

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29 Figure 2 5 Correla tion of changes in urinary K and ammonia excretion in control and IC Rhbg KO mice in response to K free diet. Panel A shows urinary K excretion in response to K free diet in control and IC Rhbg KO mice. Urinary K excretion decreased rapidly in respon se to the K free diet, and did not differ significantly between control and IC Rhbg KO mice on any day of the K free diet (P = NS, N = 8 in each group). Panel B correlates the change in urinary ammonia with the change in K excretion in response to K f ree diet in control and IC Rhbg KO mice. The correlation differed significantly in control and IC Rhbg KO mice.

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30 CHAPTER 3 CHARACTERIZATION OF COLLECTING DUCT KNOC KOUT RHBG MICE INTRODUCTION animal model has been developed. Knocking out Rhbg in collecting duct intercalated cells has shown that Rhbg is important in overall renal ammonia metabolism, and also specifi cally that principal cell mediated ammonia excretion is important to acid base homeostasis. Here, a collecting duct knockout of Rhbg (CD Rhbg KO) is described. Whereas in the IC Rhbg KO animal model, Rhbg expression remained intact in principal cells, th e goal of the CD Rhbg KO animal model is to study the effect of knocking out Rhbg in the entire collecting duct, and not just in principal cells. Future studies dealing with changes in urinary ammonia excretion of a lower magnitude will be made possible b y CD Rhbg KO mice. M ethods Animals Mice used in this project were the result of mating mice homozygous for floxed Rhbg alleles and expressing Ksp cadherin Cre with mice homozygous for floxed Rhbg alleles b ut not expressing Ksp cadherin C re. The generation of mice with loxP sites flanking critical exons of the murine Rhbg gene have been described previously (5) Transgenic mice expressing Cre recombinase under control of the 1329 bp of the Ksp cadherin promoter have been described previously (13) Animal bre eding was performed in the University of Florida College of Medicine Cancer and Genetics Transgenic Animal Core Facility by trained personnel. Mice were genotyped using tail clip samples as described previously (15, 18) All mice used in this project were either

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31 floxed Rhbg, Ksp Cre positive or floxed Rhbg, Ksp Cre negative. All animal studies w ere approved by the Institutional Animal Care and Use Committees of the Gainesville VA Medical Center and of the University of Florida College of Medicine. Antibodies Affinity purified antib odies to Rhbg have been previously characterized (15, 17, 21, 38) In particular, studies have shown the specificity of the Rhbg antibodies in studies using heterologous expression in Xenopus oocytes (21) and in studies using genetic deletion of Rhbg and Rhcg (15, 17) Tissue Preparation for Immunolocalization Mice were anesthetized with inhalant iso flurane. The kidneys were preserved by in vivo cardiac perfusion with PBS (pH 7.4) followed by periodate lysine 2% paraformaldehyde (PLP) and then cut transversely into several 2 to 4 mm thick slices and immersed 24 to 30 hours at 4C in the same fixativ e. For light microscopy, samples of kidney from each animal were embedded in polyester wax (polyethylene glycol 400 distearate (Polysciences, Warrington, PA) with 10% 1 hexadecanol), and 3 thick sections were cut and mounted on gelatin coated glass sli des. Immunohistochemistry Immunolocalization was accomplished using immunoperoxidase procedures detailed previously (15, 17) Tissue sections were de waxed in ethanol, rehydrated and then rinsed in PBS. Endogenous peroxidase activity was blocked by incubating the sections in Peroxidase Blocking Reagent (DakoCytomation, Carpinteria, CA) for 45 min. The sections were blocked for 15 min with Serum Free P rotein Block (DakoCytomation), then incubated at 4C overnight with primary antibody diluted in Dako Antibody Diluent. The sections were washed in PBS and incubated for 30 min

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32 with polymer linked peroxidase conjugated goat anti rabbit IgG (MACH2, Biocare Medical), again washed with PBS, then exposed to diaminobenzidine for 5 minutes. The sections were washed in distilled water, then dehydrated in a graded series of ethanols and xylene, mounted and observed by light microscopy. Comparisons of labeling wer e made only between sections of the same thickness from the same immunohistochemistry experiment. Sections were examined on a Nikon E600 microscope equipped with DIC optics and photographed using a DXM1200F digital camera and ACT 1 software (Nikon). Colo r and contrast adjustment was performed using Adobe Photoshop CS5 (Adobe Systems, Inc., San Jose, CA). R esults Recombination in CD Rhbg KO mice Transgenic mice with LoxP sites in introns flanking critical exons in the mouse Rhbg gene were bred with transgenic mice expressing Cre recombinase under control of the 1,329 bp Ksp cadherin promoter (13) Mice homozygous for floxed Rhbg alleles and expressing Ksp cadher in Cre were identified. Littermates homozygous for floxed Rh b g alleles but not expressing Ksp cadherin Cre were used as control mice PCR amplification of tail clip DNA was used to confirm recombination in Ksp cadherin Cre mice. There was no recombinat ion in Ksp cadherin Cre negative mice (data not shown). Rhbg E xpression in CD Rhbg KO Mice The Ksp cadherin cre negative mice (control) showed typical Rhbg immunolabel in the kidney with basolateral expression in the distal convoluted tubule (DCT), CNT, initial collecting tubule, and collecting duct (Figure 3 1 A and C). There was nearly comple te loss of Rhbg immunolabel in the CD Rhbg KO mice (Figure 3 1B and D). The only

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33 Rhbg immunolabel found in the CD Rhbg KO mice was found in the CNT and DCT. These results were similar to those published in reports studying collecting duct Rhcg knockout m ice using the same Ksp Cadherin cre promoter (15, 17)

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34 Figure 3 1 Rhbg immunolabel in CD Rhbg KO and control mice. Panel A and C show typical Rh bg expression in the cortex, outer medulla, and inner medulla of control mice Panel B shows that the majority of Rhbg immunolabel has been knocked out, with only some left in the CNT and DCT. Panel D shows that in the outer medulla and inner medulla the re is no Rhbg immunolabel in KD Rhbg KO mice.

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35 CHAPTER 4 WORKS IN PROGRESS AN D FUTURE DIRECTIONS The primary goal of developing CD Rhbg KO mice was to study the role of Rhbg in response to a low protein diet. Is It has been well documented that the typical western diet contains a very high protein content that generates an acid load that over time, can lead to a mild metabolic acidosis (1, 37) The increased intake of protein leads to increased net acid excretion, the predominant component of that being urine ammonia excretion (12, 31) and in studies in both humans and rats, a low protein diet results in decreased urinary ammonia excretion (34) The low grade metabolic acidosis brought on by high protein diets leads to low urine pH, hyocitraturia and hypercalciuria which can also lead to kidney stone disease (1) The purpose of th e current study is to further investigate the renal response to changes in dietary protein intake. To do this, mice were placed on a low protein diet or a calorie matched control diet. Collecting duct knock out Rhbg mice were bred to study the effect of knocking out an ammonia transport protein in response to the low protein diet. Methods Animals Mice used in this project were the result of mating mice homozygous for floxed Rhbg alleles and expressing Ksp cadherin Cre with mice homozygous for floxed Rhbg alleles but not expressing Ksp cadherin Cre. The generation of mice with loxP sites flanking critical exons of the murine Rhbg gene have been described previously (5) Transgenic mice expressing Cre recombinase under control of the 1329 bp of the Ksp cadherin promoter have been described previously (13) Animal breeding was

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36 performed in the University of Florida College of Medicine Cancer and Genetics Transgenic Animal Core Facility by trained personnel. Mice were genotyped using tail clip samples as described previously (15, 18) All mice used in this project were either floxed Rhbg, Ksp Cre positive or floxed Rhbg, Ksp Cre negative. All animal studies were approved by the Institutional Animal Care and Use Committees of the Gainesville VA Medical Center and of the University of Florida College of Medicine. Antibodies Affinity purified antib od ies to Rhbg have been previously characterized (15, 17, 21, 38) In particular, studies have shown the specificity of the Rhbg antibodies in studies using heterologous expression in Xenopus oocytes (21) and in studies using genetic deletion of Rhbg and Rhcg (15, 17) Dietary Protein Restriction Dietary protein intake was controlled through the use of Harlan Teklad custom research diets A 20% protein diet (TD.91532 Harlan Teklad, Madison, WI ) was used as the control diet, or normal protein diet and a 6% protein diet (TD.90016 Harlan Teklad, Madison, WI ) was used as the low protein diet. P owdered foo d was mixed with H O in a ratio of 6 g ram food to 1 ml H O to form a semi solid die t. Adult mice, greater than 8 w ee ks age, were placed into metabolic cages (Tecniplast diuresis metabolic cage, Fisher Scientific) and allowed to acclimate for three days whi le receiving control diet. They received low protein or control diet and daily fo od intake was measured. At all times animals were allowed free access to water. Daily urine excretion was collected under mineral oil;, urine pH was measured and urine vol ume calculated. Urine samples were stored at 80C until analyzed.

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37 Tissue Preparat ion for Immunolocalization Mice were anesthetized with inhalant isoflurane. The kidneys were preserved by in vivo cardiac perfusion with PBS (pH 7.4) followed by periodate lysine 2% paraformaldehyde (PLP) and then cut transversely into several 2 to 4 mm thick slices and immersed 24 to 30 hours at 4C in the same fixative. For light microscopy, samples of kidney from each animal were embedded in polyester wax (polyethylene glycol 400 distearate (Polysciences, Warrington, PA) with 10% 1 hexadecanol), and 3 thick sections were cut and mounted on gelatin coated glass slides. Immunohistochemistry Immunolocalization was accomplished using immunoperoxidase procedures detailed previously (1 5, 17) Tissue sections were dewaxed in ethanol, rehydrated and then rinsed in PBS. Endogenous peroxidase activity was blocked by incubating the sections in Peroxidase Blocking Reagent (DakoCytomation, Carpinteria, CA) f or 45 min. The sections were blocked for 15 min with Serum Free Protein Block (DakoCytomation), then incubated at 4C overnight with primary antibody diluted in Dako Antibody Diluent. The sections were washed in PBS and incubated for 30 min with polymer linked peroxidase conjugated goat anti rabbit IgG (MACH2, Biocare Medical), again washed with PBS, then exposed to diaminobenzidine for 5 minutes. The sections were washed in distilled water, then dehydrated in a graded series of ethanols and xylene, moun ted and observed by light microscopy. Comparisons of labeling were made only between sections of the same thickness from the same immunohistochemistry experiment. Sections were examined on a Nikon E600 microscope equipped with DIC optics and photographed using a DXM1200F digital

PAGE 38

38 camera and ACT 1 software (Nikon). Color and contrast adjustment was performed using Adobe Photoshop CS5 (Adobe Systems, Inc., San Jose, CA). R esults Effect of Low Protein Diet on Rhbg Expression I mmunohistochemistry was used to determine whether low protein diet increased Rhbg expression Preliminary results showed increased Rhbg expression in the cortex and outer medulla (Figure 4 1 ) Further immunohistochemistry and immunoblot studies will be necessary to confirm these results and further identify what cell types these increases occur in. Urine V olume and Urine pH Urine volume remained constant for all four experimental groups thr oughout the study (Figure 4 2 ). Urine pH was increased in both groups of animals that were placed on the low protein diet, while the animals placed on control protein diet remained constant throughout the study. Future Directions Future studies will be r equired to identify other mechanisms involved in the renal response to a protein restricted diet. These experiments will include measurement of renal ammonia excretion in response to the low protein diet to determine if this animal model results in the ex pected decreases in urine ammonia excretion that have been seen in other studies (34) Previous studies have shown that Rhbg expression increases along with increased urina ry ammonia excretion. If the preliminary data is accurate and Rhbg is increased in the low protein diet, then there may be other

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39 mechanisms involved in the regulation of Rhbg in this model that have not been seen previously. It will be necessary to dete rmine if there are altered mechanism of glutamate transport, or changes in renal ammoniagenesis in response to the low protein diet. To study this, glutamate transporter, and glutamate dehydrogenase expression will be quantified by immunohistochemistry an d western blotting.

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40 Figure 4 1 Rhbg in the cortex and outer medulla of control and low protein diet mice. The top two panels show Rhbg expression in the cortex in control protein diet and low protein diet. Bottom panels show Rhbg in the outer medull a. From this first set of preliminary studies Rhbg immunolabel is increased in both the cortex and outer medulla in response to the low protein diet.

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41 Figure 4 2 Urine Volume and Urine pH in Response to Low Protein Diet The top panel shows changes in urine pH during the course of the study. Both groups of control protein animals maintained constant urine pH throughout the protocol. Low protein diet resulted in increased urine pH for both the control and CD Rhbg KO mice. There was no statistical d ifference at any time point. Urine volume remained constant for all four experimental groups.

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42 LIST OF REFERENCES 1. Adeva MM and Souto G Diet induced metabolic acidosis. Clin Nutr 30: 416 421, 2011. 2. Adrogue HJ and Madias NE Management of life threatening acid base disorders. First of two parts. N Engl J Med 338: 26 34, 1998. 3. Adrogue HJ and Madias NE Management of life threatening acid base disorders. Second of two parts. N Engl J Med 338: 107 111, 1998. 4. Avent ND, Madgett TE, Lee ZE, Head DJ, Maddocks DG and Skinner LH Molecular biology of Rh proteins and relevance to molecular medicine. Expert Rev Mol Med 8: 1 20, 2006. 5. Bishop JM, Verlander JW, Lee HW, Nelson RD, Weiner AJ, Handlogten ME and Weiner ID Role of the Rhesus glycoprotein, Rh B glycoprotein, in renal ammonia excretion. Am J Physiol Renal Physiol 299: F1065 F1077, 2010. 6. DuBose TD, Jr. and Good DW Effects of chronic hyperkalemia on renal production and proximal tubule transport of ammonium in rats. Am J Physiol 260: F680 F687, 1991. 7. DuBose TD, Jr. and Good DW Chronic hyperkalemia impairs ammonium transport and accumulation in the inner medulla of the rat. J Clin Invest 90: 1443 1449, 1992. 8. DuBose TD, Jr., Good DW, Hamm LL and Wall SM Ammonium transport in the kidney: new physiological concepts and their clinical implications. J Am Soc Nephrol 1: 1193 1203, 1991. 9. Eladari D, Cheval L, Quentin F, Bertrand O, Mouro I, Cherif Zahar B, Cartron JP, Paillard M, Doucet A and Chambrey R Expression of RhCG, a new putative NH(3)/NH(4)(+) transporter, along the rat nephron. J Am Soc Nephrol 13: 1999 2008, 2002. 10. Hamm LL and Simon EE Roles and mechanisms of urinary buffer excretion. Am J Physiol 253: F595 F605, 1987.

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43 11. Han KH, Croke r BP, Clapp WL, Werner D, Sahni M, Kim J, Kim HY, Handlogten ME and Weiner ID Expression of the ammonia transporter, rh C glycoprotein, in normal and neoplastic human kidney. J Am Soc Nephrol 17: 2670 2679, 2006. 12. HUNT JN The influence of dietary su lphur on the urinary output of acid in man. Clin Sci (Lond) 15: 119 134, 1956. 13. Igarashi P, Shashikant CS, Thomson RB, Whyte DA, Liu Chen S, Ruddle FH and Aronson PS Ksp cadherin gene promoter. II. Kidney specific activity in transgenic mice. Am J Ph ysiol 277: F599 F610, 1999. 14. Javelle A, Lupo D, Li XD, Merrick M, Chami M, Ripoche P and Winkler FK Structural and mechanistic aspects of Amt/Rh proteins. J Struct Biol 158: 472 481, 2007. 15. Kim HY, Verlander JW, Bishop JM, Cain BD, Han KH, Igara shi P, Lee HW, Handlogten ME and Weiner ID Basolateral expression of the ammonia transporter family member Rh C glycoprotein in the mouse kidney. Am J Physiol Renal Physiol 296: F543 F555, 2009. 16. Knepper MA NH4+ transport in the kidney. Kidney Int Suppl 33:S95 102.: S95 102, 1991. 17. Lee HW, Verlander JW, Bishop JM, Igarashi P, Handlogten ME and Weiner ID Collecting duct specific Rh C glycoprotein deletion alters basal and acidosis stimulated renal ammonia excretion. Am J Physiol Renal Physiol 2 96: F1364 F1375, 2009. 18. Lee HW, Verlander JW, Bishop JM, Nelson RD, Handlogten ME and Weiner ID Effect of intercalated cell specific Rh C glycoprotein deletion on basal and metabolic acidosis stimulated renal ammonia excretion. Am J Physiol Renal Phy siol 299: F369 F379, 2010. 19. Lee, H W Verlander, JW, Bishop, JM, Igarashi, P, Handlogten, ME, and Weiner, I D Collecting duct specific Rh C Glycoprotein deletion alters basal and acidosis stimulated renal ammonia metabolism. FASEB J 2008. 20. Lema nn J, Jr. Relationship between urinary calcium and net acid excretion as determined by dietary protein and potassium: a review. Nephron 81 Suppl 1:18 25.: 18 25, 1999.

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44 21. Mak DO, Dang B, Weiner ID, Foskett JK and Westhoff CM Characterization of ammonia transport by the kidney Rh glycoproteins RhBG and RhCG. Am J Physiol Renal Physiol 290: F297 F305, 2006. 22. Marini AM, Boeckstaens M and Andre B From yeast ammonium transporters to Rhesus proteins, isolation and functional characterization. Transfus C lin Biol 13: 95 96, 2006. 23. Marini AM, Soussi Boudekou S, Vissers S and Andre B A family of ammonium transporters in Saccharomyces cerevisiae. Mol Cell Biol 17: 4282 4293, 1997. 24. Miller RL, Lucero OM, Riemondy KA, Baumgartner BK, Brown D, Breton S and Nelson RD The V ATPase B1 subunit promoter drives expression of Cre recombinase in intercalated cells of the kidney. Kidney Int 75: 435 439, 2009. 25. Nakhoul NL, Abdulnour Nakhoul SM, Boulpaep EL, Rabon E, Schmidt E and Hamm LL Substrate specifi city of Rhbg: ammonium and methyl ammonium transport. Am J Physiol Cell Physiol 299: C695 C705, 2010. 26. Nakhoul NL, Abdulnour Nakhoul SM, Schmidt E, Doetjes R, Rabon E and Hamm LL pH sensitivity of ammonium transport by Rhbg. Am J Physiol Cell Physiol 299: C1386 C1397, 2010. 27. Nakhoul NL, Dejong H, Abdulnour Nakhoul SM, Boulpaep EL, Hering Smith K and Hamm LL Characteristics of renal Rhbg as an NH4(+) transporter. Am J Physiol Renal Physiol 288: F170 F181, 2005. 28. Nakhoul NL, Schmidt E, Abdulnour Nakhoul SM and Hamm LL Electrogenic ammonium transport by renal Rhbg. Transfus Clin Biol 13: 147 153, 2006. 29. Neu AM, Bedinger M, Fivush BA, Warady BA, Watkins SL, Friedman AL, Brem AS, Goldstein SL and Frankenfield DL Growth in adolescent hemodialysis patients: data from the Centers for Medicare & Medicaid Services ESRD Clinical Performance Measures Project. Pediatr Nephrol 20: 1156 1160, 2005. 30. Quentin F, Eladari D, Cheval L, Lopez C, Goossens D, Colin Y, Cartron JP, Paillard M and Ch ambrey R RhBG and RhCG, the putative ammonia

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45 transporters, are expressed in the same cells in the distal nephron. J Am Soc Nephrol 14: 545 554, 2003. 31. Remer T Influence of nutrition on acid base balance -metabolic aspects. Eur J Nutr 40: 214 220, 20 01. 32. Sartorius OW, Roemmelt JC, Pitts RF, Calhoon D and Miner P The Renal R e gulation of Acid Base Balance in Man. IV. The Nature of the Renal Compensations in Ammonium Chloride Acidosis. J Clin Invest 28: 423 439, 1949. 33. Seshadri RM, Klein JD, Smith T, Sands JM, Handlogten ME, Verlander JW and Weiner ID Changes in subcellular distribution of the ammonia transporter, Rhcg, in response to chronic metabolic acidosis. Am J Physiol Renal Physiol 290: F1443 F1452, 2006. 34. S prinson DB and R ittenberg D The rate of utilization of ammonia for protein synthesis. J Biol Chem 180: 707 714, 1949. 35. Tannen RL The effect of uncompli cated potassium depletion on urine acidification. J Clin Invest 49: 813 827, 1970. 36. Tannen RL and McGill J Influence of potassium on renal ammonia production. Am J Physiol 231: 1178 1184, 1976. 37. van den Berg E, Hospers FA, Navis G, Engberink MF, Brink EJ, Geleijnse JM, van Baak MA, Gans RO and Bakker SJ Dietary acid load and rapid progression to end stage renal disease of diabetic nephropathy in Westernized South Asian people. J Nephrol 24: 11 17, 2011. 38. Verlander JW, Miller RT, Frank AE, R oyaux IE, Kim YH and Weiner ID Localization of the ammonium transporter proteins RhBG and RhCG in mouse kidney. Am J Physiol Renal Physiol 284: F323 F337, 2003. 39. Wall SM Mechanisms of NH4+ and NH3 transport during hypokalemia. Acta Physiol Scand 179 : 325 330, 2003. 40. Weiner ID and Hamm LL Molecular mechanisms of renal ammonia transport. Annu Rev Physiol 69:317 40.: 317 340, 2007.

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46 41. Weiner ID and Verlander JW Molecular physiology of the Rh ammonia transport proteins. Curr Opin Nephrol Hypert ens 19: 471 477, 2010. 42. Weiner ID and Verlander JW Role of NH3 and NH4+ transporters in renal acid base transport. Am J Physiol Renal Physiol 300: F11 F23, 2011. 43. Workeneh BT and Mitch WE Review of muscle wasting associated with chronic kidney disease. Am J Clin Nutr 91: 1128S 1132S, 2010.

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47 BIOGRAPHICAL SKETCH Jesse Mitchell Bishop was born in 1980 in Cheshire, Connecticut. He attended elementary and middle school in Cheshire, prior to moving to Killingworth, Connecticut where he attended and eventually graduated from high school in 1998. Upon graduation from high school Jess e enlisted in the United States Army. He completed Basic Combat Training and Airborne School in Fort Ben ning Georgia prior to attending The Ranger Indoctrination Program. After successful completion of the Ranger Indoctrination Program Jesse was stationed with The 1 st Battalion of the 75 th Ranger Regiment at Hunter Army Airfield Georgia Jesse spent over four years with 1 st Ranger Battalion, climbing through the ranks from Private to Sergeant, eventually serving as a team leader while deployed for combat operations in Afghanistan in support of Operation Enduring Freedom in 2002 and 2003. After leaving the Army Jesse attended college at both Santa Fe College and the University of Florida for his undergraduate course work, eventually earning a degree in Human Nutrition from the University of Florida in 2007. After graduation Jesse went to work in the Lab of D r. David Weiner in the Nephrology Division at the University of Jesse has been married to Crystal Bishop for 1 year. Th ey have a daughter Kathleen, who is currently 2 months old.