Peter S. Aronson - US grants

The overall aim of the project is to characterize the molecular mechanisms underlying the transcellular transport of Na, Cl and acid-base equivalents (ie. H, OH, HCO3) in the mammalian proximal tubule. One portion of the proposal will continue our efforts to analyze in detail the kinetics of the Na-H exchanger in rabbit cell microvillus (luminal) membrane vesicles. These studies will investigate the kinetics of interaction with internal H, Na, Li and NH4; the transport of NH4; Na-Li exchange; and whether the reaction mechanism is ping-pong or simultaneous. A second portion of the proposal will examine the biochemical features of the Na-H exchanger and will involve the use of group-specific reagents to identify the chemical group(s) at the cation binding site and to label the cation binding site, the development of a method for solubilizing the exchanger and reconstituting its activity in proteoliposomes, and the use of this reconstitution assay to monitor purification of the solubilized Na-H exchanger during protein separation procedures. A third portion of the project will evaluate the transport properties of the Cl-HCO3 exchanger that we have identified in dog renal microvillus membranes and will involve analyzing the possible modifier effects of H, the kinetics of interaction with internal and external Cl and HCO3, the specificity for other anions, and the specificity for inhibitors. A fourth portion of the proposal will examine the transport pathways for Cl, OH and HCO3 in basolateral membrane vesicles isolated from dog and rabbit kidneys. These experiments will screen for conductive pathways, anion exchange mechanisms, K-coupled transport processes and Na-coupled transport processes for Cl, OH and HCO3. Given the common transport mechanisms for Na, Cl, and acid-base equivalents that seem to operate in leaky epithelia, the proposed studies are relevant to understanding the physiology and pathophysiology not only of the proximal tubule but of many other epithelia as well. Moreover, the planned studies on the kinetics and biochemistry of the Na-H exchanger are highly relevant to cell physiology in general, given the important roles that Na-H exchangers in nonepithelial cells play in such important processes as intracellular pH regulation, cell volume regulation and regulation of cell proliferation.

DESCRIPTION (provided by applicant): The present proposal is for continuation of a long-standing research program directed at identifying and characterizing the ion exchangers mediating acid-base and NaC1 transport in the proximal tubule. In the proximal tubule, the principal apical membrane pathway for absorption of Na+ and secretion of H+ is Na+-H+ exchange. Cl- absorption across the apical membrane in this nephron segment occurs by several different C1--base exchange processes including CL--formate exchange. C1--oxalate exchange and Cl- -OH- exchange. During the past project period we have used a variety of experimental approaches to demonstrate that NHE3 is the isoform principally responsible for apical membrane Na+-H+ exchange in the proximal tubule. In addition, we have generated and used anti-NHE3 monoclonal antibodies to discover unexpected aspects of the structure of NHE3 including its association in oligomeric complexes with dipeptidyl peptidase IV, and the fact that epitopes within the C-terminal hydrophilic "cytoplasmic" domain are actually exoplasmic. In the next project period we will pursue studies to define the functional consequences of these two novel aspects of the structure of NHE3. In related studies, we have completed the cloning of a new member of the NHE gene family that is expressed in the kidney. We propose to characterize the functional properties of this new NHE isoform and determine whether it is expressed in the proximal tubule and/or other nephron segments. Finally, we have identified a novel anion transporter that is expressed on the apical membrane of proximal tubule cells and mediates C1--formate exchange (CFEX). We plan to characterize the functional properties of CFEX in more detail and will seek to identify additional transporters that may be responsible for apical Cl--base exchange in the proximal tubule. Taken together, the proposed studies should enhance understanding of the mechanisms of Na+, HCO3-- and CI-- transport in the kidney, which is relevant to clinical disorders caused by alterations in renal acid-base balance (eg. nephrolithiasis, osteoporosis) and NaC1 transport (eg. hypertension, congestive heart failure).

DESCRIPTION: The proposed conference entitled "Structural Biology: Application to the Study of Membrane Transport Proteins" is the third in a series of state- of-the-art workshops aimed at introducing the concepts and the application of protein structure studies to investigators in renal, diabetic, and digestive disease research. The conference, jointly sponsored by the American Society of Nephrology (ASN) and the International Society of Nephrology, is expected to be attended by approximately 125 to 150 investigators and trainees. The conference will include lectures on protein structure, protein folding pathways, evolution, protein stability, oligomerization, structure-function relationships, and selected examples of studies on well-characterized membrane proteins. The invited speakers include four international experts in protein chemistry: Dr. Giuseppi Zaccai from Switzerland, Dr. Jean-Luc Popot from France, Dr. Pieter Cullis from Canada, and Dr. Georg Schulz from Germany.Fourteen domestic speakers also will participate in the program. The program organizers request travel support for five key international speakers and for 40 trainees and junior investigators. To encourage broad participation and attendance, the meeting will be advertised in the Journal of the American Society of Nephrology, Kidney International, the American Journal of Physiology, the Journal of Clinical Investigations, and Nature. Announcements of the meeting also will be sent to NIDDK-supported researchers and trainees.Selection of trainees for travel support will be reviewed by a committee consisting of Dr. Peter Aronson and two members of the ASN Workshop Committee, Drs. Diana Marver and Alan Krensky. Selection will be based on the applicants' commitment to research activities and letters of recommendation. If necessary, a limit of two awards per training program or department will be imposed.

The overall focus of this project continues to be the molecular mechanisms of anion transport in the proximal tubule. Previous work on this project demonstrated the operation of at least three distinct anion exchangers in renal microvillus membrane vesicles (ie. Cl-formate, Cl- oxalate and oxalate SO4=CO3= exchangers), and support a novel model by which Cl-absorption in the proximal tubule involves uphill Cl uptake across the luminal membrane by exchange with formate and oxalate in parallel with organic anion recycling. Microperfusion studies in the proximal tubule carried out in collaboration with project #1 strongly supported this model. Studies are now proposed to test the hypothesis that activities of luminal membrane anion exchangers are appropriately regulated to permit independent regulation of proximal tubule NaHCO3 and NaCI reabsorption. Specifically, it will be determined whether luminal membrane anion exchangers are regulated in response to adaptive states and hormones known to modulate proximal tubule Na+-H+ exchange. Importantly, findings in isolated microvillus membrane vesicles will be correlated with results obtained in project #1 at the level of the microperfused proximal tubule in situ. In a related series of studies, affinity chromatography has been used to identify disulfonic stilbene-binding proteins with properties of anion transporters. In particular, a 71kD stilbene-binding protein with properties of the renal basolateral membrane Na+-HCO3-cotransport system was characterized. Monoclonal antibodies were generated that revealed that this protein is kidney-specific and is expressed on the basolateral membrane of multiple nephron segments where Na+-HCO3- cotransport activity has been described. Studies are now proposed to directly test the hypothesis that this 71kD stilbene-binding protein is in fact the renal basolateral membrane Na+-HCO3-cotransporter. The principal approach will be to isolate and clone a cDNA encoding this protein, and then to use this cDNA to demonstrate functional expression of Na+-HCO3- cotransporter is verified, studies will be performed to isolate cDNA encoding other isoforms of the Na+HCO3-cotransporter. Specific antisera will be generated for use in determining the cell and membrane sites of expression of different isoforms of the Na+-HCO3-cotransporter in the kidney and other regulation of renal C1- and HCO3-transport, and is therefore of relevance for understanding clinical disorders of NaCl and acid-base balance.

One of the most consistent abnormalities in ion transport found in patients with essential hypertension and their first degree relatives is overactivity of the red cell Na+-Li+ countertransporter. Given the fact that this transporter normally mediates Na+-Na+ exchange in red cells under physiologic conditions, its pathophysiologic role in hypertension has remained obscure. The principal goal of this project is to identify both the protein mediating Na+-Li+ countertransport and the gene that encodes it. It will then be possible to characterize the kinetic properties of this transporter, to determine in what other tissues it may be expressed, and thereby to gain new insight into its possible pathophysiologic role in hypertension. Moreover, once the gene encoding the Na+-Li+ countertransporter is identified, we will be able to test directly whether mutations leading to increased transport activity are associated with human hypertension. Our experimental approach will be guided by the hypothesis that the Na+-Li+ countertransporter is an isoform of the NHE (Na+-H+ exchanger) family of monovalent cation exchangers. In addition to characterizing the Na+-Li+ countertransporter and its role in hypertension, we will test whether mutations in NHE isoforms mediating Na+ reabsorption in the kidney (eg. NHE3) are associated with human hypertension. We will pursue the following sequence of studies. First, we will generate suitable antibodies to determine whether NHE4, the only one of the known NHE isoforms for which data have not rendered a role in mediating red cell Na+-Li+ countertransport unlikely, is expressed in red cells of human and rabbit, the latter a species with high countertransporter activity. If NHE4 is expressed in human and rabbit red cells, we will perform functional expression studies to test whether this isoform is capable of mediating amiloride-insensitive Na+-Li+ countertransport, and to evaluate transport properties of more physiologic relevance, such as the ability to mediate Na+-H+ exchange. In parallel with these studies, we will screen human bone marrow libraries to clone cDNAs encoding novel NHE isoform(s). For any novel NHE isoform that is identified, we will generate isoform- specific antisera to confirm its expression in red cells, and to determine the cellular and subcellular sites of its expression in other organs and tissues. In addition, functional expression studies will be performed to test the novel isoform for its ability to mediate Na+-Li+ countertransport, and to characterize its physiologically relevant transport properties. For NHE isoforms found to mediate red cell Na+-Li+ countertransport or to mediate apical Na+ entry into renal tubular cells (eg. NHE3), we will test for linkage to hypertension and will screen for mutations. Functional expression studies will be performed to compare the kinetic properties of wild-type and mutant NHE proteins. We will thereby be able to directly test the hypothesis that NHE mutations causing increased transport activity are associated with hypertension.

The principal focus of this project continues to be the mechanisms of anion transport in the proximal tubule. Studies during the past decade have supported a model by which transcellular CI-absorption in the proximal tubule involves uphill CI-uptake across the luminal membrane by exchange with formate and oxalate. Recycling of formate occurs by H/+- couple formate transport in parallel with Na/+-H/+ exchange, whereas next project period we propose to investigate two aspects of proximal tubule anion transport. First, we plan to complete the cDNA cloning and carry out the physiological characterization of a novel transporter that is likely to play a role in mediating apical membrane anion transport. Specifically, we will isolate, clone and sequence cDNAs encoding the transporter, determine anion specificity and transport modes by functional expression in Xenopus oocytes; general specific antibodies, and determine cell and membrane sites of expression; determine whether different isoforms of the transporter exist; examine structure-function relationships by use of chimeric constructs; and estimate the contribution of the transporter to integrated tubule function in microperfusion studies in mice with targeted disruption of the transporter gene. Second, in collaboration with Gerhard Giebisch (project#1), we propose to continue studies of the mechanisms of regulation of transcellular NaHCO/3 and NaCI reabsorption in the proximal tubule. Specifically, we will measure the activities of both anion exchanges (CI-formate and CI-oxalate) and recycling pathways (H+-coupled formate transport, Na/+- sulfate co-transport, oxalate-sulfate exchange) in renal brush border vesicles isolated from rats subjects to conditions that regulate proximal NaHCO/3 and NaCI reabsorption (eg. metabolic acidosis, hypokalemic alkalosis, furosemide-induced volume contraction). Activities of these pathways in membrane vesicles will be correlated with rates of transtubular HCO/3- and CI- reabsorption in the intact tubule under similar conditions. We will thereby test the hypothesis that activities of luminal membrane anion transporters are appropriate altered tot permit independent regulation of proximal tubule NaHCO/3 and NaCI reabsorption. The proposed project will provide new information on the molecular mechanisms and regulation of renal CI- transport, and is therefore of relevance for understanding clinical disorders of NaCI balance.

The general focus of this project continues to be the structure, function and physiologic roles of the protein(s) mediating Na+-H+ exchange across the plasma membranes of proximal tubule cells. cDNAs encoding four Na+-H+ exchanger isoforms (NHE1-4) have been cloned from mammalian cells. Transcripts for all four isoforms are expressed in the kidney. During the past project period we generated isoform-specific antisera to demonstrate that NHE1 is expressed on the basolateral membrane whereas NHE3 is expressed on the brush border membrane of proximal tubule cells. Based on this initial identification of NHE isoforms expressed in proximal tubule cells, we will pursue three interrelated aims to characterize the structure and function of NHE proteins in the proximal tubule and elsewhere along the nephron. The first general aim is to map the cellular and subcellular sites of expression of NHE isoforms. For this purpose we will continue to generate isoform-specific polyclonal and monoclonal antibodies. These antibodies will be used to determine the cell and membrane sites of expression of each isoform in the proximal tubule and along the nephron by use of immunofluorescence and immunoelectron microscopy, and by Western blot analysis of membrane fractions. We will also assess the temporal and spatial sites of expression of NHE isoforms in the developing nephron of the neonatal kidney. The second general aim is to characterize NHE isoforms with respect to important functional properties. Transport will be studied in stably transfected lines of LAP1 cells that express each isoform (NHE1-4), and in Sf9 cells infected with recombinant baculovirus encoding each isoform. The third general aim is to assess the relationship of structure to function of NHE isoforms. We will confirm that NHE1 and NHE3 are each components of oligomeric complexes, and will characterize the composition of these complexes and the steps involved in their biosynthesis and assembly. The topology of NHE3 will be assessed by localization of anti-peptide antibodies and by vectorial proteolysis of brush border membrane vesicles. Structure- function relationships within the amphipathic domains of NHE proteins will be studied by use of chimeric constructs to identify subdomains that determine functional properties found to differ between isoforms, and by mutation of specific amino acid residues likely to participate in binding and/or transport of cations and H+. Information about both the molecular properties of NHE isoforms and their sites of expression along the nephron will provide insight into the physiologic roles of NHE isoforms in such integrated kidney functions as HCO3 - reabsorption and acid secretion.

In Autosomal Dominant Polycystic Kidney Disease (PKD) there is progressive formation and enlargement of fluid-filled cysts in the kidney. Cyst enlargement and fluid accumulation result from net solute and fluid secretion from the reabsorption that prevails along most segments of the nephron normally. Taking advantage of the generation by Somlo and colleagues of mice and cell lines with genetically altered expression of polycystins, the overall design of this project is to test the hypothesis that deficient expression of polycystins alters the transport phenotype of renal tubular epithelial cells. To accomplish this goal we will pursue the following specific aims: 1. Characterize the renal physiological phenotype of mice generated by Project 1 with altered polycystin expression. As a function of age or time after induction of inactivation of the Pkd2 gene, measure blood pressure, GFR (inulin clearance), plasma electrolytes and acid-base parameters (Na+, K+, C1-, HC03-, Ph, pCO2), fractional excretion of electrolytes, urinary pH and ammonium excretion, and urinary concentration ability. 2. For mouse models in which fluid and electrolyte disturbance is detected before overt cyst formation, evaluate function of relevant nephron segments by microperfusion techniques. 3. By immunocytochemistry, identify sites of expression of major transporters that participate in NaCl reabsorption or secretion (Na+, K+, ATPase, Na+-H+ exchanger isoforms, Na+-K+-Cl-co-transporter isoforms, thiazide-sensitive co-transporter, EnaC subunits, ROMK, CFTR) in renal tubular cells and cyst lining in mouse PKD models. 4. Characterize transport phenotype of renal tubular cell lines generated by Project 1 that are genetically identical except for presence or absence of functional polycystin-2. Evaluate ion flux pathways and perform immunocytochemical localization of transporters that participate in NaCl reabsorption or secretion.

Na-H exchanger NHE3 is responsible directly and indirectly for the majority of NaHCO3 and NaCl reabsorption in the proximal tubule. Phosphorylation sites mediating NHE3 inhibition in response to PKA (S552 and S605), and stimulation in response to SGK1 (S661) and CK2 (S716) have been described. We have recently identified an additional novel phosphorylation site (S791) that stimulates NHE3 activity. The general goal of this project is to elucidate how combinatorial patterns of NHE3 phosphorylation integrate to alter NHE3 trafficking and activity. To accomplish this goal, we will pursue the following specific aims: Aim 1. Generate and confirm specificity of new phosphospecific polyclonal and monoclonal anti-NHE3 antibodies raised against S661, S716, and S791 containing phosphopeptides. Together with our previously generated antibodies to the two PKA sites, these new antibodies will provide us with a unique set of reagents to understand the integrated regulation of NHE3 by phosphorylation in response to physiologic stimuli that regulate proximal tubule transport. Aim 2. Use this panel of phosphospecific antibodies to evaluate the combinatorial patterns of phosphorylation of endogenous NHE3 at multiple residues in native rat kidney in vivo at baseline, and in response to stimuli that either inhibit (e.g. dopamine, PTH) or enhance (e.g. dexamethasone, angiotensin II) NHE3 activity. Compare subcellular localization of phosphorylated NHE3 and total NHE3 in response to these stimuli by immunoblotting of subcellular membrane fractions and by immunofluorescence microscopy. The data from these studies will allow us to generate mechanistic hypotheses concerning the roles of phosphorylation at specific sites or combinations of sites in governing activity and trafficking of NHE3 in vivo. Aim 3. Use OKP proximal tubule cells as a model system to refine and test the hypotheses generated in the preceding aim concerning the roles of phosphorylation at specific sites or combinations of sites in regulating NHE3. Verify changes in patterns of phosphorylation of endogenous NHE3 in OKP cells in response to those stimuli found to alter phosphorylation of NHE3 in vivo. Evaluate temporal sequence of phosphorylation at different sites in NHE3. Compare patterns of phosphorylation of surface and intracellular NHE3 by use of biotinylation studies. Correlate the kinetics of phosphorylation changes with changes in transporter surface expression and activity. Perform mutagenesis studies using transfected NHE3 to test the roles of phosphorylation at specific sites or combinations of sites in governing NHE3 intrinsic activity and trafficking. Aim 4. Identify downstream effector proteins that associate with NHE3 phosphorylated at specific sites or combinations of sites and that mediate changes in NHE3 intrinsic activity and trafficking. Perform co-precipitation studies with antibodies against known NHE3- associated proteins (e.g. megalin, NHERF1, ezrin, PP2A) to determine possible selectivity for NHE3 phosphorylated at specific combinations of residues. Use affinity chromatography and mass spectrometry to identify novel proteins that associate with NHE3 phosphorylated at specific sites or combinations of sites.

[unreadable] DESCRIPTION (provided by applicant): The present proposal is for continuation of a long-standing research program that had been directed at studying the ion exchangers mediating acid-base and NaCI transport in the proximal tubule. As part of this program, the applicants identified and characterized a novel anion exchanger (SLC26A6) that was named CFEX based on its ability to mediate Cl-formate exchange. They also found that a second related transporter, SLC26A7, is also expressed on the apical membrane of proximal tubule cells. Their recent studies using CFEX null mice have revealed that CFEX, by virtue of its activity as a Cl-oxalate exchanger, plays essential roles in proximal tubule NaCI absorption and intestinal oxalate secretion. They demonstrated that the latter process is critical to limiting net intestinal absorption of oxalate and preventing hyperoxaluria and calcium oxalate urolithiasis. Although the applicants plan to continue to examine the roles of CFEX and SLC26A7 in proximal tubule NaCI transport, a major new translational research effort will focus on the use of mouse models to elucidate the roles of CFEX and related transporters in the integrative physiology of oxalate homeostasis and the pathogenesis of hyperoxaluria and urolithiasis. Thus, the specific aims are to: 1) Evaluate the contribution of CFEX to mediating proximal tubule NaCI transport in vivo, and also assess the role of SLC26A7 in mediating components of proximal tubule Cl transport not attributable to CFEX; 2) Evaluate the role of CFEX in mediating proximal tubule oxalate transport, and also assess the role of SLC26A7 in mediating components of proximal tubule oxalate transport not attributable to CFEX; 3) Evaluate the role of CFEX in mediating intestinal oxalate transport, and also assess the role of other SLC26 transporters in mediating components of intestinal oxalate transport not attributable to CFEX; 4) Evaluate the potential role of CFEX mutations in causing hyperoxaluria in patients with urolithiasis; and 5) Evaluate the roles of CFEX-associated proteins in regulating intestinal oxalate transport and oxalate homeostasis. By enhancing understanding of the molecular mechanisms affecting urinary oxalate excretion, the proposed studies may provide new insight into genetic causes of increased stone risk, and may identify novel therapeutic targets to reduce oxalate excretion and thereby decrease stone risk. The proposed studies are also relevant to clinical disorders of NaCI homeostasis such as hypertension and congestive heart failure. [unreadable] [unreadable] [unreadable]

DESCRIPTION, OVERALL (provided by applicant): The overarching goal of the George M. O'Brien Kidney Center at Yale is to facilitate translational and clinical research that will advance the prevention and treatment of kidney diseases. Major themes of the Center are the development and phenotyping of mouse models of renal dysfunction, the identification of simple and complex genetic causes of kidney disease in humans, and the bidirectional exchange of information between mouse and human disease models in order to advance knowledge of human kidney diseases. A critically important benefit of the Yale Center will be to provide renal investigators both at Yale and across the country with access to highly specialized services not otherwise routinely available to support their research. To this end, three Center Cores will be established whose specific objectives are to provide small animal physiology services to allow detailed characterization of renal function at the level of the tubule, the kidney, and the intact organism in normal animals and in animal models of renal injury and kidney disease;provide mouse genetics and cell line services to develop new animal models and kidney cell lines to elucidate the molecular mechanisms underlying the pathophysiology of kidney diseases;and provide human genetics and clinical research services to apply genetic and genomic technologies to the study of human kidney diseases. We have identified a research user base of 51 investigators, including 18 at outside institutions, who have expressed specific interest in using core services of the Yale Kidney Center. A Pilot and Feasibility Program will be established to provide initial project funding for young investigators, to attract new investigators into the field of kidney disease research, and to foster translational and clinical studies directly related to kidney diseases. In addition, an Enrichment Program will be established to promote interdisciplinary interactions and collaborations among renal investigators participating in the Center;facilitate the application of new technologies to kidney disease research;and provide an online index of resources developed in the Center (e.g. antibodies, transgenic mouse lines, cell lines) so that they can be made available to renal investigators both at Yale and other institutions. The Center will also include a Research Training Program to enhance the training of graduate students, medical students, and postdoctoral fellows in kidney disease research in general, and in the specialized methods provided by the Center Cores in particular.

Project Summary/Abstract-Adminstrative Core The overall objective of the Administrative Core of the George M. O'Brien Kidney Center at Yale is to ensure that Center functions and activities are optimized to fulfill the overarching goal of facilitating basic, translational and clinical research that will advance the prevention and treatment of kidney diseases. To achieve this objective, the Administrative Core will pursue the following specific aims: 1. Provide central oversight and financial management of the Center 2. Organize and coordinate activities of all administrative committees 3. Monitor core use and services, and allocate resources among Center cores 4. Facilitate communication with kidney research community including maintenance of Center website 5. Manage Pilot and Feasibility Grant Program 6. Monitor and allocate resources to Enrichment Program

PROJECT SUMMARY (See instructions): This is an application for renewal of the George M. O'Brien Kidney Center at Yale. This center was established with the overarching goal to facilitate basic, translational and clinical research that will advance the prevention and treatment of kidney diseases. Major research areas of emphasis are renal epithelial cell biology and physiology; inherited kidney disease and kidney development; acute kidney injury (AKI) and chronic kidney disease (CKD); and vascular biology, inflammation and glomerular disease. A critically important benefit of the Center is to provide renal investigators both at Yale and across the country with access to highly specialized services not otherwise routinely available to support their research. To this end, the Center includes three cores to provide small animal physiology and phenotyping services to enable detailed characterization of renal function at the level of the tubule, the kidney, and the intact organism in normal animals and in animal models of renal injury and kidney disease; provide mouse genetics and cell line services to develop novel animal models and kidney cell lines to elucidate the molecular mechanisms underlying normal renal function and the pathophysiology of kidney diseases; and provide human genetics and clinical research services to enhance the ability of renal investigators to apply advanced genetic and genomic technologies to human investigation. Our cores currently have a combined user base of approximately 90 investigators, including over 40 at outside institutions. A Pilot and Feasibility Program has the goals of providing initial project funding for young investigators, attracting new investigators into the field of kidney disease research, and fostering translational and clinical studies directly related to kidney diseases. In addition, an Enrichment Program enhances kidney disease research by maximizing interaction and information sharing among renal investigators and trainees, and by providing activities to enhance recruitment and education of all levels of trainees from undergraduate students to postdoctoral fellows.

? DESCRIPTION (provided by applicant): The Yale University School of Medicine has a long tradition and record of accomplishments in the training of medical students for careers in academic medicine and research. This application is a new grant requesting NIDDK support for medical students (Short-Term Training: Students in Health Professional Schools) to carry out research primarily with NIDDK funded investigators The purpose of the renewal is to provide intensive short-term training in research for selected pre-doctoral medical students in the most outstanding laboratories and training sites in the Yale University School of Medicine. The specific laboratory experiences will range from fundamental molecular biology, cellular and organ physiology to applied clinical research in cardiovascular and pulmonary disease. The program is designed to attract the most highly qualified Yale medical students into careers as physician-scientists in the biomedical sciences. An extensive follow-up documents a high level of subsequent research training, research productivity, and faculty appointments (40.2%) among previously supported students. Trainees will be chosen upon application of pre-doctoral medical students who have completed in good standing one year of the curriculum of the Yale University School of Medicine. 30 students per year will be selected competitively for this short-term training support on the basis of the quality of a formal written proposal of the planned research and the quality of the mentor and the training environment. The participating departments and sections will include: cell biology, cellular and molecular physiology, urology, internal medicine, including endocrinology, nephrology, digestive diseases, hepatology, hematology, emergency medicine, epidemiology and public health, genetics, immunobiology, laboratory medicine, molecular biophysics and biochemistry, molecular, cellular & developmental biology, pediatrics (endocrinology and nephrology, perinatal medicine), pharmacology, urology and related interdisciplinary centers.

Pilot and Feasibility Project Summary/Abstract The overall goals of the Pilot and Feasibility Program are to provide funding for young investigators who have received no prior NIH project grant support, to attract new investigators into the field of kidney disease research, and to foster translational and clinical studies directly related to kidney diseases. To achieve these objectives, the Pilot and Feasibility Program will pursue the following specific aims: 1. Provide support for Pilot and Feasibility Grants of $40,000 per year in direct costs up to 2 years 2. Disseminate announcements of the availability of Pilot and Feasibility Grants in advance of each application cycle 3. Provide a review process to evaluate the eligibility of applicants, scientific merit of the proposed projects, and budget justification 4. Monitor progress of funded projects 5. Provide mentoring to grant recipients who are young investigators

Pilot and Feasibility Project Summary/Abstract The overall goals of the Pilot and Feasibility Program are to provide funding for young investigators who have received no prior NIH project grant support, to attract new investigators into the field of kidney disease research, and to foster translational and clinical studies directly related to kidney diseases. To achieve these objectives, the Pilot and Feasibility Program will pursue the following specific aims: 1. Provide support for Pilot and Feasibility Grants of $40,000 per year in direct costs up to 2 years 2. Disseminate announcements of the availability of Pilot and Feasibility Grants in advance of each application cycle 3. Provide a review process to evaluate the eligibility of applicants, scientific merit of the proposed projects, and budget justification 4. Monitor progress of funded projects 5. Provide mentoring to grant recipients who are young investigators

Pilot and Feasibility Project Summary/Abstract The overall goals of the Pilot and Feasibility Program are to provide funding for young investigators who have received no prior NIH project grant support, to attract new investigators into the field of kidney disease research, and to foster translational and clinical studies directly related to kidney diseases. To achieve these objectives, the Pilot and Feasibility Program will pursue the following specific aims: 1. Provide support for Pilot and Feasibility Grants of $40,000 per year in direct costs up to 2 years 2. Disseminate announcements of the availability of Pilot and Feasibility Grants in advance of each application cycle 3. Provide a review process to evaluate the eligibility of applicants, scientific merit of the proposed projects, and budget justification 4. Monitor progress of funded projects 5. Provide mentoring to grant recipients who are young investigators

Project Summary The present proposal is for continuation of a longstanding research program that had originally been directed at studying the ion exchangers mediating acid-base and NaCl transport in the proximal tubule. In the course of this work the applicants cloned and characterized a novel transporter expressed on the apical membrane of proximal tubule cells, SLC26A6. They showed that SLC26A6 is capable of mediating Cl- formate and Cl-oxalate exchange, and generated Slc26a6-null mice to evaluate its role in proximal tubule NaCl transport. However, they unexpectedly observed a striking phenotype of calcium oxalate urolithiasis due to hyperoxaluria. They found that the cause of the hyperoxaluria is increased net absorption of dietary oxalate due to a defect in SLC26A6-mediated oxalate secretion in the intestine. In addition, work by others indicated that a defect in SLC26A1-mediated oxalate transport could also result in calcium oxalate urolithiasis. The research program was therefore re-directed to focus on the roles of SLC26A6 and SLC26A1 in oxalate homeostasis and hyperoxaluria. The apical transporter SLC26A6 and basolateral transporter SLC26A1 are each expressed in the epithelial tissues participating in oxalate homeostasis: intestine, liver and kidney. Moreover, the applicants have recently identified SLC26A6-mediated oxalate transport in macrophages. The overall goal of this project is to unravel the tissue-specific roles of SLC26A6 and SLC26A1 in oxalate homeostasis and kidney disease. During the next project period, the following specific aims will be pursued: 1. Determine tissue-specific roles of SLC26A6 and SLC26A1 in oxalate homeostasis. Generate mouse lines with tissue-specific disruption of the genes encoding SLC26A6 and SLC26A1 in intestine, liver and kidney, and characterize the tissue-specific roles of these transporters in defending against hyperoxalemia and hyperoxaluria resulting from ingested or endogenously produced oxalate loads. 2. Determine tissue-specific roles of SLC26A6 and SLC26A1 in defending against hyperoxalemia in CKD. Characterize tissue-specific roles of SLC26A6 and SLC26A1 in defending against hyperoxalemia in a model of CKD induced with aristolochic acid, and evaluate whether exaggerated hyperoxalemia resulting from tissue-specific deletion of SLC26A6 or SLC26A1 leads to accelerated progression of CKD. 3. Determine tissue-specific roles of SLC26A6 and SLC26A1 in the pathogenesis of oxalate-induced nephropathy. Characterize the role of SLC26A6 in mediating oxalate transport in inflammatory cells, assess the impact of deleting SLC26A6 specifically in inflammatory cells on oxalate-induced nephropathy, and assess the impact of kidney-specific deletion of SLC26A6 and SLC26A1 on oxalate nephropathy. The proposed studies will provide important new insights into the roles of oxalate transporters in governing urinary oxalate excretion in response to oxalate loading, in defending against hyperoxalemia and progressive loss of GFR in CKD, and in modifying oxalate-induced nephropathy.