Chou-Long Huang - US grants

DESCRIPTION (provided by applicant): ROMK K+ channels play an essential role in K+ secretion in cortical collecting ducts (CCDs). The activity of the ROMK channels are regulated by multiple signaling pathways including protein kinase A (PKA), protein kinase C (PKC) and intracellular pH (pHi). Recently, we reported a novel mechanism for regulation of ROMK via direct interaction with membrane phospholipid, phosphatidylinositol-4 ,5-bisphosphate (PIP2). The interaction occurs between positively charged amino acids in the proximal C-terminal region of ROMK1 and PIP2. We hypothesize that the direct interaction between the anionic PIP2 in the inner leaflet of the plasma membrane and the cationic amino acids in this proximal C-terminal region of ROMK1 stabilizes the channels in the open state. The long-term objectives of PI's research are to understand the molecular mechanisms for PIP2 regulation of ROMK and the physiological importance of this regulation. First, we will test the hypothesis that PIP2 regulates ROMK1 opening by anchoring the proximal C-terminal region of the channel to the plasma membrane. Binding of green fluorescent protein (GFP) fusion proteins of the C-terminus of ROMK to plasma membrane PIP2 in living cells will be examined using laser scanning confocal imaging system. Effects of anchoring the proximal C-terminus of ROMK1 to the plasma membrane on channel activity will be studied using palmitoylation as an alternative membrane anchor. Second, we will examine the molecular mechanism for regulation of the K+ channels in CCDs by PLC-activating hormones. Activation of PKC by phorbol ester inhibits K+ channels in rat CCDs. This effect of PKC is likely the mechanism for regulation of K+ secretion by the phospholipase C (PLC)-activating hormones such as bradykinin and epidermal growth factor. Our preliminary results showed that PKC inhibits K+ channels by reducing membrane PIP2 content. The effects of PKC on PIP2 content and on phosphorylation of ROMK in rat CCDs as well as in heterologous expression systems (such as cultured cells and Xenopus oocytes) will be studied. The biochemical studies will be correlated with electrophysiological recording of channel activity.

DESCRIPTION (provided by applicant): ROMK potassium (K+) channels are present in the apical membrane of principal cells of the kidney cortical collecting ducts (CCD) and are responsible for luminal K+ secretion in this nephron segment. K+ secretion in CCD is regulated by dietary K+ intake and the hormone vasopressin. It has been shown that dietary K+ intake and vasopressin alters K+ secretion, at least partly, by altering the number of active K+ channels in CCD. How this occurs is not fully understood, but may involve alteration of membrane trafficking of the channels and/or activation of silent channels. Our long-term objectives are to understand the molecular mechanism for membrane trafficking of renal K+ channels and its importance in physiological regulation of the channels. There are multiple pathways for endocytosis. Our preliminary results indicate that endocytosis of ROMK is mediated via clathrin-coated vesicles (CCVs). The specific aims of the first part of the application are to examine the structural elements of ROMK and CCVs involved in the endocytosis of the channels and to examine whether low dietary K+ intake decreases K+ channels by increasing endocytosis and subsequent degradation of ROMK. Association of ROMK with CCVs will be studied by immunofluorescent colocalization, biochemical purification, and immunocoprecipitation. Functional significance of protein interaction will be examined using two-electrode voltage clamp and patch-clamp recording in Xenopus oocytes as well as CCD. We also found that some of the ROMK channels in the apical membrane of CCD are inhibited by syntaxin-lA. The second part of the application is to test the hypothesis that vasopressin increases density of active channels by activating pre-existing silent channels. Biochemical binding assay and patch-clamp recording will be performed to examine this hypothesis.

The purpose of this core is to provide support for studies on epithelial Ca 2+ channels (ECaC) and the electrogenic sodium/dicarboxylate co-transporter (NaDC-1) in components I, III, and IV. ECaC channels are present in the apical membrane of intestine and the distal convoluted tubule (DCT) of kidney and serve as gatekeepers for transcellular Ca 2+ transport in these sites. ECaC is a major target for many hormones that regulate gastrointestinal and kidney Ca 2+ absorption, such as parathyroid hormone, vitamin D, etc (1). cDNAs for ECaC and its isoforms have recently been isolated by Dr. Bindels' (2) and Dr. Hediger's groups (3, 4) independently. We have also isolated cDNA for rabbit ECaC1 and obtained cDNA for mouse ECaC2 from Dr. Bindels (consultant for this core) NaDC-1 in the proximal tubules reabsorbs filtered Krebs cycle intermediates and plays an important role in the regulation of urinary citrate concentrations (5). Low urinary citrate is an important risk factor for formation of kidney stone. In component I, we will test the hypothesis that protein product of an absorptive hypercalciuria (AH)-related gene, AH-related adenylate cyclase (AHRAC), regulates ECaC channel activity. In component III project 1, we will investigate the role of intracellular vs extracellular pH in the regulation of the electrogenic NaDC-1 (6). In component III project 2, we will test the hypothesis that acid inhibition of ECaCl-mediated Ca 2+ reabsorption in DCT contributes to the hypercalciuria induced by a high dietary protein intake and the hypothesis that membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2) regulates ECaC1 channel activity. In component IV, we will test the hypothesis that down-regulation of calbindin-D28k by estrogen lack (in addition to the downregulation of ECaC) is important for renal calcium. leak and examine the molecular mechanism by which calbindin- D28k regulates Ca 2+ reabsorption through ECaC.

The goal of this project is investigate pathophysiological basis of high protein intake-induced hypercalciuria. Epithelial Ca 2+ channels (ECaC1) are present in the apical membrane of the distal convoluted tubule (DCT) of kidney and play an important role in the maintenance of overall calcium homeostasis. Our preliminary results indicate that hypercalciuria associated with a high protein intake is, at least partly, caused by acid inhibition of ECaC1-mediated Ca 2+ reabsorption in DCT. In Specific Aim 1, we will examine the molecular mechanism of acid inhibition of ECaC1. Likely candidates of "pH sensor" for acid regulation of ECaC1 will be mutated by site-directed mutagenesis. The activity of wild type and mutant channels will be examined by whole-cell patch-clamp recording. Membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2) has recently emerged as a unique regulator of channel function. In Specific Aim 2, we will examine the hypothesis that PIP2 regulation of ECaC1 channel underlines the mechanism by which prostaglandin E2 worsens the high protein intake-induced hypercalciuria. In Specific Aim 3, we will confirm that luminal acidification inhibits Ca 2+ reabsorption in the distal convoluted tubules (DCT). In vivo microperfusion of rat DCT will be performed to examine the effect of luminal pH on Ca 2+ reabsorption. These studies will help understand the mechanism of hypercalciuria, not only during high dietary protein intake but also under conditions of chronic metabolic acidosis.

DESCRIPTION (provided by applicant): The kidney is critical for maintaining calcium homeostasis. Most of the calcium ion (Ca2+) filtered at the glomerulus must be reabsorbed by tubules through both paracellular and transcellular pathways. The transcellular Ca2+ reabsorption occurring in the distal renal tubules accounts for ~10-20% of total reabsorption and is believed to be the primary target for regulation of calcium homeostasis by hormones (such as parathyroid hormone) and acid-base status. Transient receptor potential type V5 (TRPV5) channel localized to the apical membrane of distal renal tubules is a gatekeeper for transcellular Ca2+ reabsorption in the kidney. The overall long- term goal of our research is to understand the molecular mechanisms of regulation of TRPV5 in physiological and diseased states associated with disturbances of renal calcium transport. To this end, we will investigate the following 3 aims in the current proposal. Aim 1 will examine the mechanism and interrelationship between pH and Mg2+ regulation of TRPV5. Aim 2 will examine the mechanism of regulation of TRPV5 by parathyroid hormone (PTH). Aim 3 will examine the molecular mechanism of Klotho, an anti-aging hormone, in the regulation of TRPV5. We will use a combination of complementary biochemical, electrophysiological, and animal approaches. These studies are directly relevant to nephrolithiasis, since Mg2+ and alkali have been used in the treatment of kidney stone disease, and alterations in urinary Mg2+ and urinary pH influence urinary Ca2+. PTH is a principal calcitropic hormone but the mechanism by which PTH regulates renal Ca2+ reabsorption remains largely elusive. The study of Klotho will shed lights on our understanding of how Klotho can lower serum phosphorus (an important factor for longevity of life) without causing bone problems. Urinary Ca2+ concontration is critical determinant of kidney stone diseases. These studies will greatly advance understanding of TRPV5 biology and molecular regulation. Results of this proposal may provide improved undestanding of the process of stone formation and of treatment.

DESCRIPTION (provided by applicant): The kidney adjusts K+ secretion in the distal nephron in response to variations in dietary intake to maintain K+ homeostasis. The distal K+ secretion involves K+ efflux from the cell into the lumen through apical K+ channels such as ROMK. Na+ reabsorption via the epithelial Na+ channel ENaC provides the electrical driving force for K+ secretion. An increase in the activity of the Na-Cl cotransporter NCC may decrease Na+ delivery to ENaC thus diminishing K+ secretion. In support for the role of NCC as well as ROMK in maintaining K+ homeostasis during dietary variations of K+ intake, high K+ loading in rodents increases the density of ROMK while decreasing the density of NCC in the distal nephron. The upstream regulation of ROMK and NCC remains incompletely understood. WNK1 is a protein kinase of which gene mutations resulting in increased expression cause pseudohypoaldosteronism type II (PHA2), an autosomal-dominant disease characterized by hypertension and hyperkalemia. WNK1 has several alternatively spliced variants including a ubiquitous full- length long WNK1 (L-WNK1) and a shorter kidney-specific WNK1 (KS-WNK1). Cell-based expression studies have shown that L-WNK1 activates NCC and inhibits ROMK. KS-WNK1, by itself, does not regulate NCC and ROMK but reverses L-WNK1-mediated activation and inhibition of NCC and ROMK, respectively. The long- term goal of our research is to understand K+ homeostasis in the normal physiology and in diseased states. Here, we will examine three specific aims. Aim 1 is to examine the physiological role and mechanism of L- WNK1 in the regulation of renal Na+ and K+ transport. Aim 2 is to examine the hypothesis that KS-WNK1 antagonizes L-WNK1 regulation of renal Na+ and K+ transport and changes in dietary K+ affect the ratio of L- over KS-WNK1 to regulate NCC and ROMK. Aim 3 is to examine the interplay between aldosterone and L- and KS-WNK1 pathway in the regulation of renal Na+ and K+ transport and the response to variations in dietary K+. We have generated mouse models with genetically altered expression of L-and/or KS-WNK1. Renal Na+ and K+ transport in these mice will be studied by measuring blood and urinary electrolytes and expression of Na+ and K+ transporters in response to dietary Na+ or K+ perturbations and to diuretics. K+ secretion will also be studied by in vitro microperfusion of isolated CCD tubules. Surgical adrenalectomy will be performed to examine interactions between aldosterone and WNK1 pathway. These studies will provide important insights to the in vivo role of WNK1 in renal Na+ and K+ transport in the normal physiology and in diseased states.

Genetically modified mice are important tools for studying kidney physiology and diseases. However, expertise and equipment for physiological analysis of kidney function in mice are not readily available to many investigators in the renal research community. Furthermore, production of mutant mice has allowed many investigators outside the renal community to uncover unexpected renal phenotypes and enter into renal research. The objective of the Physiology Core is to assist center investigators with a wide range of physiological techniques to study kidney physiology and diseases at the cellular, organ, and whole animal level. To achieve these objectives, the Core will provide the following specific services: (1) Whole animal clearance studies; (2) Measurements of serum and urine electrolytes and chemistry; (3) Measurement of blood pressure; (4) Measurement of electrolytes in nl volume using ion-selective electrode (5) Isolation of individual tubules for real-time PCR and immuno-fluorescent staining; (6) In vitro microperfusion of individually isolated tubules; (7) Electrophysiological studies. Procedures will be in place to evaluate efficiency and to maintain appropriate quality control of the services that are provided. The Core will also provide education and training to the staffs of center investigators. The core directors have a track record of providing assistance and training to visiting scientists locally and nationally. The Core director. Dr. Chou- Long Huang, received his PhD in Physiology from UCSF working with Dr. Floyd Rector on renal physiology and completed postdoctoral training with Dr. Lily Jan at UCSF working on K+ channels. He is well recognized for his studies of renal ion channels. His laboratory actively employs electrophysiological and other physiological approaches and mouse models. The co-director. Dr. Michel Baum, also trained in Dr. Floyd Rector's lab in UCSF. Throughout his independent research career, he has studied renal ion transport using in vitro microperfusion and microanalytic and imaging techniques on individually isolated tubules. The Physiology Core will be housed in the laboratories of the core directors in the Departments of Internal Medicine and Pediatrics at UTSW.

Project Summary Chronic kidney disease (CKD) affects approximately 10% of the general population. The prevalence of cardiac hypertrophy is markedly increased in CKD patients, reaching as high as 90% in advanced stages of CKD. Cardiovascular disease is the main cause of death for CKD patients; among which cardiac hypertrophy is an important underlying cause. Risk factors for cardiac hypertrophy in CKD include CKD-specific risk factors as well as conventional risk factors (hypertension and volume expansion, etc). Several CKD-specific risk factors have been proposed but their roles remain inconclusive. Klotho is a membrane protein predominantly produced in the kidney. The extracellular domain of Klotho (soluble klotho; sKL) is released into the systemic circulation and functions as a soluble endocrine hormone. Serum levels of soluble Klotho are decreased in human CKD patients and in mouse models of CKD. We recently reported that sKL protects the heart by inhibiting TRPC6-mediated abnormal Ca2+ signaling and that membrane lipid rafts are receptors for sKL. Our over-arching hypothesis is that sKL binds lipid rafts to exert cardiac protection and that sKL deficiency is a cause of uremic cardiac hypertrophy. To support this hypothesis along with the long-term goal of developing potential treatment, we propose two aims. Aim-1 will identify and develop potential sKL-mimetic that exerts organ protection by binding and targeting sialogangliosides and lipid rafts. We will produce recombinant sKL and sKL-mimetic proteins and examine their effects to bind sialoganglioside moiety in vitro and to protect organ in vivo. Aim-2 will further elucidate the molecular mechanism for sKL regulation of TRPC6-mediated abnormal Ca2+ signaling. Supported by the preliminary data, we will test the hypothesis that TRPC6-containing vesicles are pre-docked to lipid rafts and that binding of cationic amino acids in the C-terminal region of TRPC6 to PIP3 (stimulated by PI3K) in the inner leaflet of raft membrane is important. Furthermore, we will examine molecular mechanism by which DAG stimulates TRPC6 vesicle exocytosis, thereby sKL inhibits TRPC6 function. We will use combined biochemical, electrophysiological, and imaging approaches. Our proposed studies in mice will provide important pre-clinical information that may lead to treatment of CKD-induced cardiomyopathy. Furthermore, upregulation of TRPC6 and abnormal Ca2+-calcineurin-NFAT signaling is critical for sustaining and amplifying pathological cardiac hypertrophy and remodeling from diverse causes. Klotho-based therapeutic strategies may be applicable to diverse cardiac diseases. .

Project Summary TRPV5-mediated renal Ca2+ reabsorption is critical for maintaining Ca2+ homeostasis. Klotho is a type-1 transmembrane protein predominantly produced in the kidney. Klotho exists either in the membranous klotho form or the soluble ectodomain that is shedded into urine or systemic circulation to function as a paracrine or endocrine factor. Previously, we have found that soluble klotho increases surface abundance of TRPV5 by removing terminal sialic acids from the N-linked glycan chains of the channel. Removal of sialic acids exposes underlying disaccharide N-acetyl-lactosamine, a ligand for galectin-1 that is ubiquitously present on the external surface of cells. Binding to galectin-1 at the extracellular surface leads to accumulation of functional TRPV5 on the plasma membrane. To support this novel hypothesis for mechanism of action for klotho, we propose the following studies to extend our in vitro findings into in vivo and to investigate pathophysiological relevance of our findings. Aim-1 will test the hypothesis that soluble klotho regulates renal Ca2+ reabsorption via TRPV5 in vivo and that it does so through the putative sialidase activity of klotho. To examine the role of soluble klotho in renal Ca2+ reabsorption in vivo, we will generate transgenic mice that express soluble klotho in the background of klotho-deficient mice (rescued from death by dietary phosphate and vitamin D restriction) and examine urinary calcium excretion, TRPV5 expression, several gene expression parameters related to TRPV5-mediated Ca2+ reabsorption, and patch-clamp recording of TRPV5 channel activity in the native renal tubules. We will also define domains of soluble klotho involved in klotho's putative sialidase activity in vitro and test the effect of mutant soluble Klotho carrying sialidase activity-inactivating mutations in vivo. Chronic kidney disease (CKD) is a klotho-deficient state. Aim 2 will test the hypothesis that defect in TRPV5-mediated renal Ca2+ reabsorption from soluble klotho deficiency contributes to secondary hyperparathyroidism in chronic kidney disease. Elevation of parathyroid hormone (PTH) occurs in patients with CKD as a response to combat hyperphosphatemia and hypocalcemia. Hyperparathyroidism yet causes multiple adverse consequences in CKD. We will test the hypothesis that decrease in soluble klotho and downregulation of TRPV5 plays a role in hyperparathyroidism of CKD by using two mouse models of CKD. The effect of transgenic delivery of wild-type or inactive mutant soluble klotho on serum PTH and Ca2+ levels, urinary Ca2+ excretion, and other mineral metabolites in CKD will be examined. These studies on the mechanism by which klotho stimulates renal calcium reabsorption and its role in mineral metabolism of CKD will be important for our understanding the pathophysiology of mineral disorder of CKD and may help management or deign future therapy of mineral disorder in CKD patients.

Project Summary The kidney plays a critical role in regulating potassium homeostasis by adjusting secretion in the distal nephron to match daily dietary intake. Distal K+ secretion involves K+ efflux through apical ROMK K+ channels, a process that requires Na+ reabsorption via ENaC to provide the electrical driving force for K+ secretion. The activity of the Na+- Cl- cotransporter, NCC, impacts distal K+ secretion by altering Na+ delivery to ENaC. Thus, besides upregulating ROMK, high dietary K+ intake enhances distal K+ secretion by downregulating NCC. WNK1 and WNK4 are protein kinases in which gain-of-function mutations cause an autosomal-dominant hypertension and hyperkalemia syndrome, pseudohypoaldosteronism type II, which occurs at least in part by increasing Na+ reabsorption via NCC and decreasing K+ secretion via ROMK. How dietary K+ intake regulates NCC and how WNK1 and 4 activate NCC and inhibit ROMK remain unresolved. As a part of long-term goal to understand the mechanism of renal Na+ and K+ transport in health and disease, we propose the following studies. Aim-1 will test the hypothesis that WNK1 and WNK4 both activate NCC in vivo and that it occurs via the OSR1/SPAK kinase cascade. We will examine the role of WNK1 and 4 on NCC in vivo using kidney-specific conditional Wnk1-knockout mice (to circumvent embryonic lethality of global Wnk1-deleted mice) and global Wnk4-deleted mice, respectively. Double knockout mice will be studied to investigate whether WNK1 and 4 are additive or antagonistic. The role of OSR1/SPAK in mediating WNK1/4 regulation of NCC will be investigated based on the ability of expression of catalytically constitutive-active OSR1/SPAK to rescue loss-of-function of NCC in mice lacking Wnk1 and/or Wnk4. The activity and expression of NCC in kidney will be assessed by analyzing the increase of urinary Na+ excretion in response to hydrochlorothiazide and western blot analysis of the total and phosphorylated NCC. Aim-2 will examine the hypothesis that WNK kinase cascade mediates dietary K+ intake-induced regulation of NCC. Abundant in vitro and cell-based evidence implicate that dietary K+ intake regulates NCC by altering intracellular chloride concentration to affect WNK kinase activity. We will test the hypothesis in vivo using mice carry Wnk4-null alleles and kidney- specific conditional Wnk1-knockout mice. Mice will be fed a normal K+ or low K+ diet and studied for NCC activity. Aim-3 will test hypothesis that WNK kinases can directly inhibit renal K+ secretion independently of the effect via increasing NCC-mediated Na+ reabsorption using whole animal clearance studies and in vitro microperfusion of cortical collecting ducts isolated from wild-type mice versus mice whose WNKs are deleted but NCC activity is normalized by constitutive-active SPAK/OSR1. These studies using state-of-the-art animal models and physiological approaches will provide important in vivo information for our understanding of the mechanism of renal Na+ and K+ handling in physiological and diseased states.

Abstract This application proposes continued funding for an interdisciplinary Iowa Training Program in Kidney and Hypertension research. The program provides research training for adult and pediatric nephrology fellows as well as PhD scientists interested in kidney and hypertension research in the University of Iowa, Carver College of Medicine. The primary purpose of the program is to provide intensive rigorous training for clinicians and scientists in broad ranges of areas related to kidney biology and disease and hypertension to transform this knowledge into improvements in patient care. The training program has been continuously funded for the past 25 years. The applicant pool is robust, and the program has filled consistently. The graduates from the program have published important contributions to biomedical research, and many have obtained faculty positions and received external funding for their research. The program faculty comprises 41 physician- scientists and basic scientists from 11 Departments with expertise in a broad range of scientific disciplines that are relevant to the kidney and hypertension. The training program research portfolio is organized into five themes based on and integrated with the existing strengths of research on campus: ion transport physiology and cell biology, renal genetics, hypertension, diabetes and renal metabolic diseases, and clinical and translation research in kidney diseases. The training curriculum includes mentored research training, didactic courses that include optional elective graduate-level courses, grant-writing workshop, and required courses in ethics and the responsible conduct of research, and attendance to journal clubs, works-in-progress and research seminar series. Specific training opportunities for patient-oriented research consist of a certification program in patient-oriented research and a Master or PhD degree in Translational Medicine.