Eric Delpire - US grants

DESCRIPTION: (Applicant's Abstract) Electroneutral Na-Cl, Na-K-2Cl, and K-Cl cotransporters participate in the movement of salt and fluid across epithelia and play a major role in the maintenance and regulation of the volume of a wide variety of cells. While these transporters have been studied extensively during the past 15 years, it is only recently that their molecular identity has been revealed. We have recently shown that two of these cotransporters, the Na-K-2Cl cotransporter and the K-Cl cotransporter, are expressed highly in the brain. Their role in the nervous system is incompletely understood. Growing evidence suggests that besides playing an important role in regulating the composition of the CSF, they may also play a major role in neuronal function. In fact, Na-K-2CI cotransporter expression in neurons has been suggested to determine the effect of GABA on membrane potential, which in turn may be important during neuronal maturation and response to neuronal injury. This proposal is aimed at understanding the physiology of these cotransporters in neuronal cells. Through detailed quantitative studies we will investigate 1) the role of the secretory Na-K-2Cl cotransporter (BSC2), an inward transport mechanism, and the neuronal-specific K-Cl cotransporter (KCC2), an outward transport mechanism, in establishing the steady-state intracellular Cl- concentration in neurons, and 2) the developmental regulation of these two cotransporter mechanisms. These studies, by involving functional and molecular approaches, will lead to a better understanding of the role of these electroneutral cation-chloride cotransporters in neuronal function. Regulation of intracellular and extracellular ion composition has important implications for the physiology and pathophysiology of the brain. For instance, regulation of intracellular Cl- concentration is crucial for controlling the function of GABA in the CNS. The GABA response in turn is an important feature of the normal function of the neuron, e.g. for the control of slow wave sleep in the thalamus, or the control of diurnal rhythmicity in the hypothalamus, a critical feature of neuronal plasticity during development, and a critical feature in various pathologies such as cerebral trauma, cerebral ischemia, where neuronal damage involves changes in GABA-induced excitability properties, potentially due to significant changes in intracellular Cl- concentration.

DESCRIPTION: (Applicant's Abstract) Electroneutral Na-Cl, Na-K-2Cl, and K-Cl cotransporters participate in the movement of salt and fluid across epithelia and play a major role in the maintenance and regulation of the volume of a wide variety of cells. While these transporters have been studied extensively during the past 15 years, it is only recently that their molecular identity has been revealed. We have recently shown that two of these cotransporters, the Na-K-2Cl cotransporter and the K-Cl cotransporter, are expressed highly in the brain. Their role in the nervous system is incompletely understood. Growing evidence suggests that besides playing an important role in regulating the composition of the CSF, they may also play a major role in neuronal function. In fact, Na-K-2CI cotransporter expression in neurons has been suggested to determine the effect of GABA on membrane potential, which in turn may be important during neuronal maturation and response to neuronal injury. This proposal is aimed at understanding the physiology of these cotransporters in neuronal cells. Through detailed quantitative studies we will investigate 1) the role of the secretory Na-K-2Cl cotransporter (BSC2), an inward transport mechanism, and the neuronal-specific K-Cl cotransporter (KCC2), an outward transport mechanism, in establishing the steady-state intracellular Cl- concentration in neurons, and 2) the developmental regulation of these two cotransporter mechanisms. These studies, by involving functional and molecular approaches, will lead to a better understanding of the role of these electroneutral cation-chloride cotransporters in neuronal function. Regulation of intracellular and extracellular ion composition has important implications for the physiology and pathophysiology of the brain. For instance, regulation of intracellular Cl- concentration is crucial for controlling the function of GABA in the CNS. The GABA response in turn is an important feature of the normal function of the neuron, e.g. for the control of slow wave sleep in the thalamus, or the control of diurnal rhythmicity in the hypothalamus, a critical feature of neuronal plasticity during development, and a critical feature in various pathologies such as cerebral trauma, cerebral ischemia, where neuronal damage involves changes in GABA-induced excitability properties, potentially due to significant changes in intracellular Cl- concentration.

DESCRIPTION (provided by applicant): The overall goal of the INIA-stress consortium is to integrate efforts to define the allostatic state of the cortical-limbic-HPA axis produced by excessive alcohol consumption as a platform for studying the relationship between stress, the subjective state of anxiety, and excessive drinking. The objective of the Gene-Targeted Mouse Core is to create novel animal models that allow spatial and/or temporal disruption of specific brain targets. These targets are receptors or their ligands that play key roles in modulating biological attributes such as anxiety, reward, and emotional behavior. Circuitries underlying these attributes are all affected by alcohol abuse. The use of targeted gene disruption will allow the dissection of individual pathways in relationship to alcohol consumption and alcoholism. For this renewal, the Core has been tasked to create seven new mouse models while taking advantage of available gene-targeted embryonic stem cell lines generated by the European Knockout Consortium and the Knockout Mouse Project (KOMP). When available, the ES cell lines will be obtained and injected into C57 albino blastocysts. Homozygous floxed mice will be mated to mice carrying inducible, forebrain-specific recombinase transgenes. For targets with unavailable ES cell lines, the Core will engineer a targeting construct using BAC recombineering techniques and target ES cells for recombination. Once completed, inducible knockout mice will be shipped to the Neuroanatomy Phenotyping Core for anatomical analyses and a larger number of breeders will be shipped to the Mouse Chronic Intermittent Ethanol (CIE) Core for alcohol phenotyping. The Core will also breed, maintain, distribute to INIA-stress investigators, and cryopreserve all mouse lines. Finally, the Core will provide genotyping services to INA-stress investigators.

DESCRIPTION (provided by applicant): g-Aminobutyric Acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain, and plays a prominent inhibitory role in the brainstem and spinal cord as well. One mechanism through which GABA produces its inhibitory action is via GABAA receptors which produce fast synaptic inhibition of neurons by activation of intrinsic CI- channels. GABA opening of CI- channels produces an inward movement of CI-, driven by a low intracellular CI- concentration which is maintained by an active CI- extrusion mechanism: presumed to be the neuronal-specific isoform of the K-CI cotransporter, KCC2. We have disrupted Sic12a5, the gene encoding this isoform of the K-CI cotransporter, and the homozygous mutant mice die shortly after birth of repeated seizures. Epileptic seizure activity in the KCC2 knockout brain suggests hyper-excitability, in agreement with the putative role of KCC2 in controlling hyperpolarizing GABA responses. This proposal is aimed at understanding the role of KCC2 in controlling CNS excitability and epilepsy. We will 1) investigate the developmental role of KCC2 in regulating intracellular CI- and controlling the maturation of GABA hyperpolarization, 2) investigate the role of KCC2 in preventing hyper-excitability and the participation of the cotransporter in depolarizing GABA responses during high frequency synaptic activation. This will be achieved through detailed electrophysiological measurements in hippocampal slices and isolated cortical neurons, 3) develop brain-region-specific and inducible knockout of KCC2 to study the knockout phenotype in the adult and examine the effect of graded reduction in KCC2 expression, and 4) examine the determinants of KCC2 function and regulation, by focusing mainly on phosphorylation/dephosphorylation of the protein. The epileptic seizure phenotype of the KCC2 knockout mouse demonstrates the importance of KCC2 in preventing hyper-excitability and controlling CNS function. Results of these molecular, physiological, and behavioral studies will lead to a better understanding of the relationship between cation-chloride cotransporters, ion homeostasis, synaptic transmission and brain excitability.

DESCRIPTION (provided by applicant): Coupled movement of Na+, K+, and Cl-through cell membranes occurs primarily through Na-K 2CI and K-CI co-transporters, which belong to the Slc12 family of membrane transporters. Until the identification of the multiple genes that constitute this family, the physiology of cation-chloride co-transporters was dominated by the critical role that some of these co-transporters play in water and ion homeostasis in the kidney. In fact, the co-transporters are best known through their inhibitors: the loop and thiazide diuretics which are widely used in clinical medicine to volume deplete patients. The cloning of the human and mouse genomes identified 9-10 members of the Slc12 family of solute carriers. Among them, 7 are functionally well characterized: there are 1 Na-CI co-transporters, 2 Na-K-2CI co-transporters, and 4 K-CI co-transporters. These transporters participate in a wide variety of function which range from fluid secretion/absorption in multiple epithelia, modulation of synaptic transmission, cell volume control and regulation, cell proliferation. Mutations in cation-chloride co-transporters are responsible for diseases such as salt wasting disorders and peripheral nerve degeneration. They are also possibly involved in hypertension, age-related loss of hearing, neurological and psychiatric disorders. Whereas loop diuretics such as furosemide and bumetanide inhibit most cation-chloride co-transporters, these drugs are rather unspecific and not very potent. Thus, there is a critical need for the development of new compounds targeting cation-chloride co-transporter function. The development of novel fluorescent-based reagents, sensitive to anions and cations, now permits the development of better methods for High Throughput Screening (HTS). We propose to 1) Develop a fluorescence-based assay to monitor the activity of cation-chloride co-transporters. This will be achieved through heterologous expression of NKCC1 and KCC2 together with anion-sensitive fluorescent protein in HEK-293 cells. Based on studies showing that K+ channels can carry thallium, we will also examine the feasibility of use of a thallium-sensitive indicator dyes. 2) Provide a validation the assay for High Throughput Screening. This will be achieved through a detailed analysis of effects of a known inhibitor (e.g. bumetanide) on fluorescent signals generated by the movement of the surrogate anion or cation through the cell membrane and a through a test screening of a library consisting of 10,000 compounds. The development of a High Throughput fluorescent method to measure cation-chloride co-transporter function will allow us to transit to actual HT screens to identify novel compounds targeting the function of cation-chloride co-transporters.

[unreadable] DESCRIPTION (provided by applicant): Cation-chloride cotransporters, e.g. Na-K-21 and K-CI cotransporters, play fundamental roles in a variety of cells and tissues. In the nervous system, they modulate inhibitory synaptic transmission and participate to the movement of salt and fluid across epithelia. The Na-K-2CI cotransporter, NKCC1, accumulates Cl- in sensory neurons (DRG, olfactory), thus promoting GABA depolarizing responses. The cotransporter also participates in the production of the inner ear fluid and the reabsorption of K+ from the CSF. Knockout of NKCC1 leads to multiple phenotypes including increased sensitivity to pain and sensorineural deafness. Cation-chloride cotransporters are regulated by a variety of stimuli, most of them (if not all of them) converging to phosphorylation/dephosphorylation of the transporters, but not much is known about the kinases affecting them. We have recently identified, as interactors of cation-chloride cotransporters, stress kinases related to the yeast Ste20 kinase family. Together with WNK4, a kinase that is associated with human hypertension, the stress kinases modulate the activity of the cotransporters. We have also identified a SPAK-interacting tyrosine kinase which negatively regulates NKCC1 activity. To understand the physiological significance of the interaction between these kinases and the cotransporters, we propose to 1) Define the role of the stress kinases and WNK4 in modulating the activity of NKCC1 and 2) Define the role of the tyrosine kinase AATYK in the regulation of NKCC1 activity. This will be achieved through functional studies of cotransporter and kinase mutants in Xenopus laevis oocytes and through in vitro phosphorylation experiments. We will also address the role of PP1 and its putative binding to a site that overlaps with a SPAK binding domain. 3) Utilize a cell line which expresses NKCC1, SPAK, OSR1, WNK4, and AATYK to address through silencing experiments the specific role of each kinase in the regulation of the cotransporter. These studies will elucidate novel aspects of cation-chloride cotransporter function and regulation and provide a better understanding of cation-chloride cotransporter links to CNS-related disorders. Mechanisms that transport inorganic ions across cell membranes are involved in a variety of disorders: salt wasting disorders, hypertension, nerve degeneration and brain hyperexcitability. The transporters do not function in isolation but are tightly regulated by a variety of other proteins that also have the potential to participate in these diseases. Detailed studies of the interaction between regulatory proteins and the transporters are of critical importance for understanding these human disorders. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Mutations in K-Cl cotransporter 3 (KCC3) are responsible for one of the most severe, early onset peripheral neuropathy disorders called ACCPN or "Agenesis of Corpus Callosum associated with Peripheral Neuropathy". We have disrupted the KCC3 gene by homologous recombination in mouse embryonic stem cells and the mouse peripheral nerve phenotype is similar to that observed in ACCPN patients. Due to the difficulty in studying K-Cl cotransport function in peripheral nerves, we propose to further examine the role of KCC3 in ACCPN by creating additional mouse models. First, as all but one human mutation results in truncated KCC3 proteins with shorter carboxyl- termini, we propose to create a knock-in mouse with a single missense amino acid substitution and assess whether absence of cotransporter function or absence of protein-protein interaction is causing the disorder. Second, as the cotransporter is expressed in both sensory neurons and Schwann cells, we propose to create mice with tissue-specific deletions of the cotransporter. To significantly reduce the time necessary to assemble the constructs and increase efficiency in obtaining these new mouse models, we have designed a common modular targeting construct. As we have identified a single residue substitution that renders the cotransporter non-functional without affecting its membrane expression, we will utilize the exon encoding this residue as our targeted region to construct the three mouse lines. In this way, both constructs will contain the same arms of recombination and allow for a common ES cell screening strategy to identify recombination events. Mouse phenotype will be analyzed through locomotor activity and peripheral nerve pathology. These two new mouse models will allow us to further understand the developmental versus degenerative nature of the neuropathy, as well as the molecular basis and cell-type origin of the disorder. PUBLIC HEALTH RELEVANCE The goal of this proposal is the creation of three novel KCC3 mouse models that will advance our understanding of the peripheral neuropathy disorder associated with disruption of the KCC3 gene in humans.

DESCRIPTION (provided by applicant): Inherited mutations in SLC12A1 and SLC12A2, the genes encoding the renal electroneutral Na-Cl and Na-K-2Cl co-transporters, result in salt wasting disorders associated with changes in blood pressure. The syndromes, called Gittleman and Bartter, are due to decreased Na+-re-absorption in distal convoluted tubule and thick ascending limb of Henle, respectively. Two Sterile20 kinase, SPAK, and OSR1, are involved in regulating the co-transporters. The kinase directly binds to the N-terminal tails of the co-transporter and phosphorylates them. A whole-genome study found an association between SPAK and increased blood pressure, and SPAK knockout mice exhibit a Gittleman-like phenotype. Surprisingly, NKCC2 phosphorylation is increased in SPAK knockout animals, but is blunted in phosphorylation-deficient knock-in SPAK mice, suggesting a possible interaction between SPAK and OSR1 in the thick ascending limb. The purpose of this 2 PD/PI application is to understand the molecular details leading to increased Na-K-2Cl co-transporter phosphorylation in the SPAK knockout and to define the role of each kinase in modulating Na+-re-absorption in the two tubule segments. In this application, we propose to 1) examine the mechanisms of SPAK/OSR1 activation, determine the role of dimerization, and establish the existence of SPAK-OSR1 interactions; 2) examine the function of putative inhibitory isoforms and of Cab39, a scaffold calcium binding protein; and 4) establish the role of SPAK and OSR1 in regulating renal salt transport and maintaining blood pressure. The first two aims will involve functional studies using heterologous expression in both Xenopus laevis oocytes and mammalian kidney cells, as well as molecular studies involving protein-protein interaction both in vitro and in vivo. The third aim will consist of detailed phenotypic analysis of genetically-modified mice under different diet regiments, and the development and study of a novel constitutively active OSR1 knock-in mouse. This application is a logical extension of extensive studies of cation-chloride co-transporters and their regulation by the submitting PI and a natural extension of a successful ongoing AARA-funded Challenge grant collaboration between the two PD/PIs. Upon completion, the proposed studies will clarify the role of two kinase and demonstrate that they are integrated into precise signaling networks in the thick ascending limb of Henle and the distal convoluted tubule.

DESCRIPTION (provided by applicant): The training program, open to physicians (MD or MD/PhD)during their residency training or as a post- graduate research fellowship and to basic scientists (PhD) early in their research career, is designed to provide 2 years of research training in perioperative science. The major goal of this training program is to provide the highest caliber research training in four Specific Themes originating from existing strengths of our faculty at Vanderbilt University Medical Center. The Research Themes include: 1) Mechanisms and Management of Pain; 2) Perioperative Stress Biology and Outcomes; 3) Perioperative Health Services and Translational Research; and 4) Personalized Medicine and Pharmacogenomics. Each of these themes directly relates to the overarching aim of the program - to train the next generation of scientists to create new knowledge and translate it into best evidence for personalized perioperative care and pain management at a population level. The training faculty will consist of an exceptionally strong collection of physician-scientists and basic scientists who offer superb interdisciplinary research training opportunities in 7 different academic departments. The training program will accept three new trainees per year (maximum 6 participants per year) with ratios depending on availability: 4 clinicians and 2 PhDs or 3 clinicians and 3 Ph.Ds. Clinicians who show exceptional aptitude for successfully pursuing an academic research career and Ph.D. scientists who demonstrate the best aptitude to develop towards independence will be considered for participation. Each participant will commit to a 2 year basic science, clinical and/or translational research project with 75% effort for Clinicians and 100% effort for PhDs. In addition to their research project training, trainees will receive coursework instruction in research related processes such grant writing, publications, ethics, and responsible conduct of research. This training will prepare them to utilize the skills they acquire in the pursuit of future academic research careers.

? DESCRIPTION (provided by applicant): Living organisms necessitate the uptake of energy and of building blocks, and the elimination of waste. Therefore, the barrier that separates them from the environment cannot be completely tight to the movement of water, ions, and small organic molecules. As a consequence, cells from their beginning created basic mechanisms to maintain and regulate their water content and volume. When disrupted, these basic functions can have severe consequences for an organism, and diseases of salt and water transport are involved in both acute and chronic conditions that impact over 50% of the population, and have been documented to cost the health care system billions of dollars annually. The goal of the proposed symposium Cell Biology of Volume Regulation and Fluid Homeostasis, an 11th International Symposium on Cell Volume Regulation is to cover both the basic mechanisms of cell volume regulation and their implications in several major diseases including hypertension, brain disorders and lung and kidney diseases. Specifically, we propose 10 scientific sessions: The first part of the conference Cell Biology of Volume Regulation and Fluid Homeostasis, is divided into 5 sessions: I.1 Molecular Mechanisms of Cell Volume Regulation: Transporters and Ion Channels; I.2 Salt-sensitive Mechanisms in Regulation of Apoptosis and Autophagy; I.3 Cell Volume Regulation in Cell Proliferation and Migration; I.4 Lipid Regulation of Osmotic- and Mechano-sensitive Ion Transport Mechanisms; and I.5 Hydration and water transport through the membrane. The second part of the conference Diseases of Volume Regulation and Fluid Homeostasis, is divided into 4 sessions: II.1 Role of Salt Transport in Hypertension; II.2 Fluid-Electrolyte Contributions to Disease Progression in Polycystic Kidney Disease; II.3 Osmoregulation and Hydration in Cystic Fibrosis; and II.4 Osmoregulation in Neurological and Brain Disorders. A 10th scientific session will be dedicated to Young Investigators. In addition, we propose a new educational outreach initiative that will include high school and community college students. This will be achieved via a Lunch and Learn with a Professor sessions that will focus on salt and water in the diet and on small groups discussions about science and scientists personal experiences. It is the first time in the last decade that an International Symposium on Cell Volume Regulation is organized in the United States, which we believe is critically important to facilitate the interaction of the American scientists to the international community and foster international collaborations in this area. Narrative: The major goal of this international scientific meeting on Cell Volume Regulation and Fluid Homeostasis is to cover both the basic mechanisms of cell volume regulation and the roles of these mechanisms in several major diseases including hypertension, brain disorders and lung and kidney diseases. In addition, the meeting will include an educational outreach program that will include high school students and community college students to foster the interest of the students to science.

?DESCRIPTION (provided by applicant): NKCC1 is an electroneutral cation?chloride cotransporter which belongs to SLC12A, an evolutionary ancient gene family. The transporter is found from bacteria to humans and thus has evolved to fulfill a multitude of cellula functions. NKCC1 is expressed on the basolateral membrane of Cl? secreting epithelia such as airway, intestine, salivary gland, sweat gland, etc. Or K+ secreting epithelia, e.. stria vascularis (inner ear), where it participates to the transepithelial movement of Cl? or K+, respectively. In neurons, NKCC1 is involved in modulating intracellular Cl? thereby affecting GABAergic and glycinergic neurotransmission. The cotransporter is also a key factor for cell hydration as it is activated by loss of cell water and consequently participates in the maintenance and regulation of cell volume. Recently, a de novo deletion of 11 bases was found in exon 22 of SLC12A2, the gene encoding NKCC1, in a 12?year old patient. The deletion truncates 40% of the carboxyl?terminal tail of the cotransporter. We hypothesze that the mutant cotransporter is expressed and exerts dominant?negative effects on wild?type cotransporter, or exerts toxic effects on cell metabolism. In this application, we propose to 1) examine function of the truncated cotransporter in heterologous expression systems; and 2) examine expression and function of the mutant cotransporter in cells that are isolated and cultured from the patient; and create a mouse model recapitulating this mutation. The first aim will involve studies of expression, trafficking, function, interacton, of both truncated and wild?type cotransporter in Xenopus laevis oocytes and HEK293 cells. This study will allow us to characterize in details the mutant cotransporter and learn about the molecular role of the carboxyl?terminal tail of the cotransporter. Attempts wil be made to assess whether it is possible to rescue the truncation cotransporter functin. The second aim will examine the impact of the mutant transporter on ions and volume homeostasis, as well as basic metabolic properties of cells isolated from the patient (e.g. fibroblasts and/or transformed lymphocytes). Using CRISPR technology, we will also create and study a mouse model recapitulating the human mutation. These studies will allow us to gain insights into NKCC1 mutant?mediated cellular dysfunction and explain the clinical presentations of the patient.

7. PROJECT SUMMARY The distal convoluted tubule (DCT) of the kidney expresses the thiazide-sensitive Na-Cl cotransporter, NCC, a mechanism essential to Na+ reabsorption, control of blood pressure, and K+ secretion. NCC is activated by phosphorylation, which is mediated by a pair of protein kinases: SPAK, a Ste20p-like kinase which directly binds and phosphorylates the cotransporter, and WNK4, an upstream kinase, which phosphorylates and activates SPAK. Increasing consensus is developing that the function of the DCT is to sense and regulate plasma K+ through the modulation of K+ secretion in the aldosterone-sensitive distal nephron (ASDN). The precise mechanism(s) by which the DCT fulfills this function is still not well-understood. Preliminary data collected in the previous funding cycle indicated that even in the presence of a constitutively-active SPAK kinase, increase in plasma K+ still decreases phosphorylation of NCC leading to a first hypothesis that the K+ activated phosphatase, calcineurin, mediates inhibition of the cotransporter (Aim 1). Based on preliminary work that highlights the role of Cab39 adaptor proteins (Cab39 and Cab39l) in facilitating SPAK and WNK function we hypothesize that these proteins play a critical role in the pathway regulating NCC phosphorylation. In Aim 2, the molecular interaction between Cab39 and SPAK/WNK, the relationship with the Cl- sensitivity of WNK4, and the relevance of the adaptor proteins at the animal level will all be addressed. To account for the relationship between Na+ reabsorption in DCT and K+ secretion in the ASDN, we hypothesize that on top of the well-accepted view that Na+ delivery inversely modulates epithelial Na+ channel and ROMK, there is paracrine communication between the two nephron segments. This novel idea will be addressed in Aim 3. Finally, longer term coupling between the DCT and the ASDN involves significant remodeling for which we now have a precise signaling pathway to study. Through lineage mapping studies and Jagged-1 knockout studies we will test the hypothesis that NOTCH signaling increases the number of principal cells in the connecting segment (Aim 4). These new studies will provide a greater integrated view of the role of Na+ transport in the DCT in renal function.                                                                  

Thiazides are one of the most cost-effective and medically beneficial first line antihypertensive agents. However, they don?t work for everyone, and in some patients they may lower blood pressure for a while but then wear off. The mechanisms responsible for thiazide resistance have been mysterious, until recently. Our recent systems-biology investigations revealed a salt-transport process is activated by a novel mechanism to limit urinary salt wasting when NCC, the thiazide target, is inhibited or hypokalemic intravascular volume depletion occurs. We discovered that a salt reabsorption pathway is created by the coordinate induction of a multi-gene transport system in non-? intercalated cells, highlighting the Cl/HCO3- exchanger, pendrin, alpha-ketoglutarate (?-KG) and the ?-KG G-Protein Coupled Receptor, OXGR1. Our recently published and preliminary data strongly suggest that paracrine delivery of ?-KG stimulates OXGR1 in non-? cells and this activates pendrin, stimulates salt reabsorption, and potentially lowers the diuretic response. Here, we have assembled a highly collaborative, multidisciplinary team to rigorously test the central tenants of the ?- KG /OXGR1/pendrin hypothesis (Aim 1), explore the underlying molecular mechanism(s) linking OXGR1 to pendrin activation (Aim 2), elucidate the physiological stimuli and consequences of the Renal ?-KG/OxGR1 Paracrine system (Aim 3), building on our recent discoveries. We expect these investigations will have a major impact on understanding how the kidney controls salt balance in health and disease, in ways that illuminate the central underpinnings of the variable diuretic response. Ultimately, these studies will provide new information and diagnostic tools that lead to the better treatment of hypertension.