John Stokes - US grants

The proposed work will investigate the effects of endogenous and exogenous prostaglandins on Na and water flow across the isolated perfused rabbit cortical collecting tubule. Arachidonic acid and meclofenamate will be used to stimulate and inhibit respectively prostaglandin production in cells during perfusion. The effects of prostaglandin E2 and F2 alpha on Na and C1 transport across the medullary thick ascending limb of Henle's loop will also be evaluated.

The longterm objectives of this research proposal are to understand in greater detail the mechanisms of Na+ and C1- absorption and K+ secretion by the urinary bladder of the winter flounder. This epithelium is unique in several aspects. It absorbs Na+ and C1- by a process which can be conducted in the absence of a transepithelial voltage or short-circuit current. Thus, the absorption is an electrically silent process. The bladder is a high resistance epithelia which makes the determination of transcellular fluxes more accurate than is the case for epithelia possessing a lower paracellular resistance. Another interesting feature is that in some bladders there does exist a short-circuit current which is completely attributable to K+ secretion into the lumen. Thus, one is able to determine independently NaC1 absorption and K+ secretion. An additional unique feature of this epithelia is its lack of sensitivity to furosemide and its sensitivity to thiazide diuretics. Thus, this epithelia is the only in vitro model for the mechanism of action of the thiazide diuretics. It appears that these diuretics inhibit a neutral NaC1 cotransporter which, in contrast to the furosemide-sensitive cotransporter, does not require K+ for its operation. The specific aims for the project are: 1) to determine the dose-response relationships for a variety of thiazide-type diuretics using tracer fluxes and electrophysiologic techniques; 2) to determine the mechanism of action of papaverine (which also inhibits NaC1 absorption) using electrophysiologic approaches; 30 to determine the kinetics of NaC1 absorption including the interrelationships between the mucosal concentration of Na+ and C1- and the stoichiometry of the cotransporter; 4) to examine the factors regulating basolateral membrane ion permeation; and 5) to conduct a detailed morphological examination of the bladder, its response to inhibitors of transport, and the pathways of lanthanum permeation. The understanding of this transport system will likely assist us in understanding the operation of the distal convoluted tubule of the kidney, the mechanism of action of thiazide diuretics. The results may lead to important opportunities for localizing the NaC1 cotransporter and determining its biochemical nature.

The renal cortical collecting tubule (CCT) absorbs Na and secretes K by processes which are interrelated. There is considerable evidence that the stoichiometry is not fixed but that under some conditions the two flows can be dissociated. The isolated, perfused rabbit CCT is an ideal model for the study of K secretion because of the relatively large fluxes which can be measured. In addition, a large portion of K permeation is via a transcellular route which has a conductive component. The magnitude of the cellular K permeation appears to be increased by mineralocorticoid hormone and decreased by inhibitors of K conductive pathways. The proposed experiments will examine some of the basic properties of cellular and paracellular K permeation using four separate approaches: a) tracer permeation and transepithelial conductance measurements for conductive pathways, b) net Na, K, and Cl transport using electron probe analysis, c) intracellular measurements of membrane voltages and fractional resistances of the apical and basolateral membrane, and d) analysis of single ion channels using the patch clamp technique. In addition, cell culture techniques will be used to make unique preparations of the CCT cells. Some aspects of a model of K secretion will be examined. Experiments will probe for a KCl cotransport system on the basolateral membrane. Other experiments will examine the mechanism of inhibition of K permeation following inhibition of the Na-K pump. Another group of experiments will characterize the nature of the altered K transport following in vivo manipulations designed to greatly increase or greatly decrease K secretory rates. These results will add to our general understanding of how K excretion is regulated by the kidney. The information will be useful in understanding various states of electrolyte imbalance.

The long term objectives of this proposal are to understand in greater detail the pathways and regulatory mechanisms of ion transport across cell membranes. The research focusses on the renal collecting duct because this structure has developed specialized transport systems to regulate ion excretion. The regulation of Na and Cl excretion, for example, plays a central role in the regulation of blood pressure. Precise regulation of H+ and K excretion in order to match intake is critical for prevention of acidosis (or alkalosis) and hyperkalemia (or hypokalemia), respectively. The collecting duct has developed systems to regulate the absorption or secretion of each ion species. Specific experimental approaches outlined in the application will examine the extent to which hormones and autocoids produce effects on one or more transport systems. Collecting duct ion transport can be divided into two general categories: acid-base and Cl transport is effected by intercalated cells, and Na and K transport is effected by principal cells. The specific aims of the application are to: a) investigate the mechanism(s) whereby cAMP and primary messengers stimulate H+ and HCO3 secretion by intercalated cells; b) determine the mechanisms where by hormones and autocoids alter Na and K transport by principal cells; and c) determine the linkage between oxidative phosphorylation and turnover of the Na-K pump in the K-K exchange mode. The methods to be used in the evaluation of these mechanisms include intracellular pH measurements, intracellular voltage measurements with equivalent circuit and cable analysis, tracer flux measurements, net transport measurements of Na, K, C1, and HCO3, and biochemical measurements in single nephron segments. The results will be relevant to our integrated understanding of salt and electrolyte balance. In addition, they will be important to the understanding of mechanisms of hormone action. Finally, the experiments may provide new insights regarding the ways the Na-K pump can be regulated both by hormones and by metabolism.

Recently, two forms or genetic hyperrension in humans have been described, both of which display salt sensitivity. These genetic defects involve a) overproduction of aldosterone and b) an abnormal epithelial Na channel. The complete understanding of how and why these genetic abnormalities produce hypertension is just beginning. It is likely that animal models will assist in the unraveling of the genetic determinants of salt sensitive hypertension. The most widely studied animal model is the Dahl SS/Jr rat; its relative, the Dahl SR/Jr rat does not develop hypertension eating a high salt diet. We have obtained preliminary data indicating that the inner medullary collecting duct cells of prehypertensive Dahl S rats, when cultured on filters, absorb twice as much Na as inner medullary collecting duct cells cultured from Dahl R rats. Aldosterone stimulates Na transport by both the S and R monolayers, but the stimulation is greater in S monolayers. This observation is consistent with the idea that an (inappropriately) elevated rate of Na absorption by S kidneys plays a crucial role in the development of hypertension. The analysis of the ion transport systems of these cells indicates that the higher rate of Na transport by S monolayers is largely due to the greater rate of Na entry into the cell across the luminal (apical) membrane. The molecular pathway involved is a Na channel, probably the same as has been discovered to be defective in one of the genetic forms of human hypertension (Liddle~s Syndrome). This Na channel can be regulated by aidosterone and other adrenocortical steroids. The proposed work will examine the hypothesis that genetic differences between the Dahl S and R rats are responsible for the differences in Na channel function. Three specific aspects of this general hypothesis will be addressed. First, we will determine if any of the 3 genes encoding the subunits of the Na channel cosegregate with hypertension in F2 populations. Second, we will determine the extent to which the mRNA for the 3 subunits is regulated by adrenal steroids. At the same time we will determine to what extent steroid - hormone regulation of these subunits differs between the S and R strains. Third, we will study certain aspects of Na transport in a newly developed congenic rat strain. This strain has the 11 beta-hydroxylase gene from the R rat superimposed on the S rat genetic background. Using this model, we will be able to address important aspects of the mechanism whereby (genetically) abnormal steroid production contributes to enhanced Na transport and to the pathogenesis of hypertension. The results of these experiments will provide important information regarding the genetic mechanisms contributing to elevated rates of Na transport by the kidney and insights as to the mechanisms whereby these abnormalities contribute to hypertension.

The discovery of the molecular nature of the epithelial Na channel (ENaC) has rapidly lead to the understanding that defects in this molecular complex, or in the factors responsible for its regulation, contribute strongly to abnormal regulation of blood pressure. the most convincing evidence that abnormal function of t his channel complex can produce hypertension comes from patients with Liddle~s Syndrome, a rare genetic disorder that produces an activating mutation in one of the channel subunits. This O~Brien Center Program will examine the important factors determining the structure and regulation of this molecular complex in order to gain insight into how abnormalities in its regulation might contribute to NaCI-sensitive hypertension. the first project will address the role of ENaC in the collecting duct and uropithelium in regulating NA exception. the experiments will test the novel hypotheses that overactivity of ENaC in the inner medullary collecting duct mechanosesation to afferent renal nerve activity. The second project will focus on the developmental regulation of ENaC in the normal rat and in rat models of salt-sensitive and -resistant hypertension. In addition, using a mouse model incapable of producing endogenous glucocorticoids, experiments will examine specific hypotheses on the role of glucocorticoid hormone on ENaC expression during development. the third project will focus on the regulation of the human ENaC gamma subunit gene. The preliminary data indicate that the 5' flanking region contains promoter activity and that steroid sensitivity might e conferred by a non-traditional response mechanism. Information from this project could provide critical information on regions of the gene leading to abnormal regulation of this subunit in hypertensive populations. The fourth project will test the hypothesis that specific mutations known to cause overactivity of ENaC in Liddle~s Syndrome do so by altering the insertion and retrieval of the channel complex in the plasma membrane. The fifth project will examine the integrated effects of over- and under-expressing ENaC in intact mice using genetic manipulation. The experiments will test several predictions derived from experimental results in cell and organ systems. The experiments will also test some important hypotheses regarding the role of ENaC in high renin hypertension. The separate projects are tightly interwoven to produce an interdisciplinary focus on a critically important molecular complex that, when abnormal, is capable of producing significant human disease.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] This application proposes continued funding for an interdisciplinary research training program in kidney diseases, hypertension, and cell biology. The purpose of this program is to provide training for young clinicians and scientists interested in pursuing research careers involved in diseases of the kidney, regulation of the blood pressure, and basic aspects of cell biology. Trainees will be attracted from programs in urology, pediatric nephrology, nephrology, and the basic sciences. The major theme of the program is to provide the means whereby highly motivated young physicians and scientists can spend an intensive and sustained period of research training under the guidance of an established and productive faculty mentor. A newly instituted part of the program includes a mentoring committee for all candidates. Specific training opportunities include the areas of basic and applied cell biology, abnormalities of the cardiovascular and renal systems in hypertension, developmental biology of the kidney, the biology of urogenital cancer, immunological disorders of the kidney, and clinical investigation in disease of the kidney and urinary tract. Additional opportunities will be available in related areas of physiology, biochemistry, pharmacology, and immunology. In addition, applicants will have the opportunity to work with investigators using epidemiologic and statistical methods. Over 30 faculty have indicated a willingness to participate in the training of such postdoctoral candidates. A broad range of highly specialized expertise permits such training in almost any area related to kidney diseases. [unreadable] The recent increase in the number and quality of applicants to our programs indicates that we are in a strong position to utilize these trainee positions. One of the major characteristics of research and training at the University of Iowa is the interdisciplinary nature of many of the research programs. Faculty members of this Training Program are unanimously eager to contribute to the education of potential young scientists and physicians. At Iowa, research training is an important component of our overall mission. A strong faculty, strong institutional resources, and a strong applicant pool are the hallmarks of this application. [unreadable] [unreadable]

The general goal of this project is to further understand the regulation and function of the gene products that form the recently discovered epithelial Na channel. the context in which these studies are undertaken relates to the pathogenesis of salt-sensitive hypertension. There are 2 subprojects. The first subproject capitalizes on new information indicating that primary cultures of the renal inner medullary collecting duct from salt-sensitive rates transport substantially more Nathan do these cells cultured from salt-resistant rats. The hypothesis derived from this data is that an abnormality in the epithelial Na channel complex, or in associated regulatory molecules, contributes to the pathogenesis of salt- sensitive hypertension in humans. the experiments are directed at 3 specific aims. The first aim is to determine the effect of physiologic maneuvers known to alter the activity of collecting duct Na transport on the messenger RNA and protein of each of the 3 major subunits. The focus will be on the kidney and uroepithelium, but responses in lung and colon will be measured in order to examine tissue heterogeneity. The second aim is to determine the biophysical properties of the epithelial Na channel as it exists in the apical membrane of inner medullary collecting duct cells from salt- sensitive and salt-resistant rats. the third aim is to determine the effect of overexpressing one or more of the epithelial Nachannel subunits in the inner medullary collecting duct on Na transport. Using transgenic mice, the rate-limiting subunit will be selectively overexpressed and the animals examined for their salt-sensitivity. These experiments will test the hypothesis that an overactive Na channel in the inner medullary collecting duct will contribute to salt-sensitive hypertension. The second subproject is based on the new observation that the uroepithelium contains epithelial Nachannel subunits. The experiments proposed in this portion will test the hypothesis that these subunits serve a different function that they do in the collecting duct. Rather than participate in Na reabsorption, the hypothesis is that they participate in activation of mechanosensitive nerve fibers. In the renal pelvis, afferent nerve activity is markedly activated by an increase in renal pelvic pressure. Preliminary data suggest that the epithelial Nachannel subunits participate in this activity. the specific aims are to determine the mechanisms by which ameliorate modulates the activation of afferent renal nerve activity. to determine the extent to which ameliorate inhibits the release of putative mediators of mechanoreceptor activation, and to determine the effect of elimination of one of the subunits in genetically altered mice on the activation of afferent renal nerve activity. These experiments will test a novel hypothesis which may have important implications for an interaction of Na channel function and the central nervous system.

This study will determine if human urine contains substances that can regulate Na ion channels that exist in the luminal membrane of distal nephrons.

The major goal of this laboratory is to identify the molecular mechanisms responsible for producing for producing salt-sensitive hypertension. The models used for the proposed studies are the Dahl salt-sensitive (S) and salt resistant (R) strains. These strains have renal mechanisms of Na handling that participate in the pathogenesis of elevated blood pressure. The proposed studies take advantage of previous observations that the inner medullary collecting duct cells of Dahl S rats, when cultured on filters, transport twice as much Na as those of the Dahl R rat. The proposed experiments will combine this preparation with DNA microarray analysis and expression screening to address the following aims. First, differentially expressed genes in the cultured inner medullary collecting ducts will be identified and localized on the rat chromosome. Second, differentially expressed genes will be screened using the oocyte expression system to determine if they alter Na transport will be further characterized and their mechanism of action channel. Those that do alter Na transport will be further characterized and their mechanism of action ascertained. Finally, genes that are differentially expressed and affect Na currents in oocytes will be over- or under-expressed in mammalian cell models. The experiments will be design to take advantage of other components of this SCOR program to integrate candidate genes into models where the mechanisms of Na channel and blood pressure regulation can be better understood. This information should greatly accelerate the progress toward identifying the molecular causes of salt- sensitive hypertension and thus lead to improved strategies for identification, treatment, and prevention.

DESCRIPTION OF OVERALL CENTER (provided by applicant) The major objective of this O'Brien Kidney Research Center is to identify the molecular mechanisms involved in the regulation of the epithelial Na channel (ENaC). The rationale for this approach is to broaden the scientific framework from which the mechanisms of salt-sensitive hypertension can be explored. This application includes three regular projects, and two pilot and feasibility studies. The themes of the regular projects converge on aspects of ENaC regulation. Project 1 will examine the mechanism for the increase in ENaC activity recently shown to be mediated via the carboxy terminus of the alpha subunit. The role of endogenous kinase activity and the relationship of four identified candidate proteins to this regulation will be explored. Project 2 will examine the mechanisms whereby the Nedd4 family of proteins alters the activity of ENaC. Specific experiments will be directed at the interactions between WW domains within Nedd4 proteins and their targets, the PY motif present in each of the ENaC subunits. Project 3 will use gene targeting to develop a mouse that can express Cre recombinase under the control of tamoxifen in the collecting duct principal cell. This mouse, which will be of potential use to many investigators, will be used to develop a mouse that lacks expression of the Nedd4-2 gene product specifically in those cells in a time dependent fashion. The phenotype will be examined under a variety of conditions predicted to produce derangements in blood pressure and electrolyte balance. A second objective of this O'Brien Center is to foster the development of promising young scientists who will make contributions to the understanding and treatment of kidney diseases. The Pilot and Feasibility Studies of two such investigators are included. One proposes to examine some specific aspects of a newly discovered protein expressed in the kidney that may be a Mg ion channel. The second proposes a novel approach to the treatment of renal cell carcinoma using adenoviral expression of TRAIL, a protein causing apoptosis of tumor cells. The O'Brien Center will interface with the program on the biology of ENaC/degenerins lead by Dr. Welsh and with the SCOR in the Molecular Genetics of Hypertension lead by Dr. Sigmund.

DESCRIPTION (provided by applicant): The long term objective of this research is to improve the health of patients with End Stage Renal Disease who require hemodialysis. This proposed study is ancillary to the Frequent Hemodialysis Network (FHN) trial, a trial designed to determine if daily dialysis in a center, or nocturnal dialysis at home every night will substantially improve those factors that are responsible for the high mortality of dialysis patients. The proposed studies stem from two lines of reasoning. First, the FHN trial offers an unparalleled opportunity to determine if frequent dialysis will improve patients'cognitive function. The majority of the literature indicates that large numbers of dialysis patients have cognitive defects, but whether today's dialysis patients will improve their cognitive function with frequent dialysis is not so clear. We submit that the tests that will be used to measure cognitive function in the FHN trial as presently designed are not sensitive or specific enough to make clear conclusions. Second, the practical effects of improvements in cognitive function (as measured by modern tests) on real-life function is not clear. We propose to conduct a series of sensitive cognitive function tests on a subset of patients who will be enrolled in the FHN trial to determine if cognitive function is improved, and if so, which aspects are improved to the greatest extent. We will make these measurements on 260 patients located in 11 sites where patients will be randomized to conventional dialysis or to nocturnal dialysis, and another 5 sites where patients will be randomized to conventional dialysis or daily dialysis. In a second part of the study, we will begin an assessment of the relationship between cognitive impairment exhibited by dialysis patients and their ability to perform driving tasks in a driving simulator. With these measurements we will be able to determine which cognitive functions improve with frequent dialysis and which cognitive functions predict impaired driving skills in dialysis patients. The importance of these studies to the public health relates to improved health and function of patients who have kidney failure and who require dialysis. Knowing which impaired cognitive functions predict impaired driving skills will help develop strategies to improve cognition and thus enhance safe driving. Such strategies have the potential to make the highways safer for everyone.