Allen W. Cowley - US grants

vasopressins; meeting /conference /symposium; travel;

The purpose of this U.S.-Hungary cooperative research project between Dr. Allen Cowley of the Medical College of Wisconsin and Dr. Emil Monos of the Semmelweis Medical University is to take advantage of the complementary expertise of the two groups to investigate the contractile mechanisms of blood vessels. In particular, combination of the Wisconsin group's expertise on the ionic basis for depolarization of vascular and muscle cells, combined with the Hungarians'expertise on the biomechanics of the blood vessel wall, is expected to lead to improved understanding of venous myogenicity (the extent to which venous pressure causes contraction of the vein wall), the scientific basis of which is virtually unknown. Improved understanding of this phenomenon is crucial for defining the basic mechanisms which regulate vascular compliance in the cardiovascular system.

The overall objective of this Program is to evaluate broadly the mechaniss of arterial pressure regulation in normal and hypertensive states. Two major areas of study are emphasized: first, the regulation of body fluid volumes and electrolytes; second, the regulation of vascular smooth muscle tone and systemic vascular resistance. The Program integrates many levels of biological function ranging from cellular events to integrative mechanisms of the whole organism that ultimately determine arterial pressure. The conceptual framework of the Program has evolved from utilization of mathematical analysis (computer systems analysis) to evaluate the complex interactions of cardiovascular function.

arginine vasopressin; inhibitor /antagonist

The goal of this SCOR is to identify genetic loci and specific genes whose expression results in hypertension and in important phenotypic changes associated with hypertension. We have proposed 4 closely inter- related scientific projects, two human, one animal, and one theoretical development of mathematical tools for analysis, which brings together a team of uniquely qualified geneticists, clinical investigators, physiologists, and biomathematicians. Project 1 will conduct a linkage analysis, using a total genomic search strategy, in 400 African American sib pairs from Milwaukee/Chicago in which each sibling has hypertension and hypercholesterolemia. Hypertension associated phenotypes (renal function, renal and peripheral vascular sensitivity, stress responses) will be studied in 200 of these sib pairs. An unrelated African American control group will be used in association studies to determine which loci and risk factors contribute most to hypertension in this population. Project 2 will conduct a genome wide search of 200 similarly affected sib pairs obtained from a unique population of genetic isolates residing in the Chicoutimi Provence in Canada. The general linkage analysis will be refined by determining mutations that exist in linkage disequilibrium. Project 3 will conduct a genome wide search of an F2 population of inbred rats (Dahl S/jr x Brown Norway) to define hypertension susceptibility loci and loci influencing renal and vascular function. Additionally, congenic rat strains will be constructed that differ only in the region of individual loci that are linked to hypertension in order to study the physiological mechanisms by which the expression of this locus influences blood pressure. Homologous genetic loci which are found to influence blood pressure and/or important hypertension associated phenotypes in the F2 rat cross will be used as candidate genes in the human populations. Project 4 will design and apply analytic tools to be used in the gene mapping of studies of Projects 1-3 and develop novel extensions of traditional gene mapping methods for complex traits, accounting for heterogeneity and effects of concomitant variables such as age, sex, and shared environment. Novel techniques to analyze the dynamic nature of blood pressure will be undertaken in order to detect additional hypertension associated phenotypes. The four Cores which will provide support for the entire SCOR include: A) Administrative Core (Milwaukee); B) Informatics and Computational Resources (Milwaukee); C) Genotyping Core (Boston); D) Biochemical Core (Milwaukee)

The goal of Project 3 is to locate "hypertensive" loci using a large (>300) F2 cross derived from normotensive Brown Norway and Dahl salt- sensitive hypertensive rats. The F2 progeny will be extensively phenotyped after exposure to a high salt diet. In addition to salt- sensitivity, the Dahl S model of hypertension shares many similarities with phenotypic traits associated with hypertension in African Americans including insulin resistance, hyperlipidemia, and rapid development of severe progressive-hypertensive glomerulosclerosis that leads to end- stage renal disease. The F2 progeny will be genotyped using a total genomic search strategy and a candidate gene approach for determination of loci that cosegregate with arterial pressure and hypertension related phenotypes which correspond to those studied in the human populations on Projects 1 and 2 (e.g. vascular reactivity, renal function, and hyperlipidemia). QTLs which cosegregate with blood pressure or hypertension related phenotypes will be converted to homologous regions in the human and then be used to screen our patient populations. Once a region or regions are identified which cosegregate with salt-sensitive hypertension, we propose to enhance the localization of the blood pressure regulatory genes by creating new strains of rats that are identical except for selected regions of a single chromosome (congenic lines). These rats will then enable us to study the physiological mechanisms by which these loci influence blood pressure.

Project 1 examines the role of nitric oxide (NO) in the regulation of blood flow to the renal medulla. Although the medulla receives only about 5-10% of total renal blood flow, we have shown that perfusion of this region plays an important role in the regulation of sodium excretion and in the long-term control of arterial pressure. The mechanisms which regulate medullary blood flow remain poorly understood, but NO appears to be importantly involved. We have shown that nitric oxide synthase (NOS) activity is substantially greater in the medulla than in the cortex. Furthermore, chronic inhibition of medullary NOS activity greatly reduces medullary blood flow and results in sodium retention and hypertension. The goal of this project is to determine in medullary [NO] plays an important role in the normal homeostatic regulation of medullary flow by moderating the effects of vasoconstrictor hormones. such as angiotensin II (ANGII) and norepinephrine (NE), which may be trapped and concentrated by the counter-current vasa recta circulation. We hypothesize that these compounds stimulate the release of NO which in turn buffers reductions of medullary flow, tissue P0/2, and prevents hypertension. The application of techniques ranging from the molecular to the whole animal will enable examination of this hypothesis. We have developed a method for the direct tissue measurement of NO concentrations within the renal medulla in both anesthetized and conscious instrumented rats (microdialysis oxyHb-NO trapping). Implanted optical fibers and laser-Doppler flowmetry will also be used to measure regional blood flow changes in the renal medulla and cortex in unanesthetized rats. Techniques have also been developed to quantify regional changes of NOS gene expression, protein expression, enzyme activity and L-arginine concentrations in whole tissue and in isolated medullary microvessels and tubules of the renal medulla. Studies will determine if circulating angiotensin II (ANGII) and norepinephrine (NE) stimulate medullary NO production which buffers against acute reductions of regional blood flow. We will determine whether medullary NO serves to protect flow to the renal medulla and prevention hypertension in the face of chronic elevations of ANGII or NE. Towards this end, we will compare the influence of ANGII and NE on medullary blood flow in rats in which medullary NOS production is blunted pharmacologically with L-NAME and in the Dahl S rat, a non-pharmacological model which we find has a reduced capacity to release NO. Localization and quantification of acute and chronic effects of ANGII and NE on medullary microvascular and tubular NOS mRNA (nNOS, iNOS, eNOS), NOS enzyme activity and protein expression will be determined in Sprague Dawley rats. The proposed project builds upon the unique interdisciplinary and collaborative strengths of this PPG and will provide important new insights regarding the role of NO in the regulation of renal medullary blood flow and the long-term control to arterial pressure.

The goal of this SCOR is to identify genetic loci and specific genes whose expression results in hypertension and in important phenotypic changes associated with hypertension. We have proposed 4 closely inter- related scientific projects, two human, one animal, and one theoretical development of mathematical tools for analysis, which brings together a team of uniquely qualified geneticists, clinical investigators, physiologists, and biomathematicians. Project 1 will conduct a linkage analysis, using a total genomic search strategy, in 400 African American sib pairs from Milwaukee/Chicago in which each sibling has hypertension and hypercholesterolemia. Hypertension associated phenotypes (renal function, renal and peripheral vascular sensitivity, stress responses) will be studied in 200 of these sib pairs. An unrelated African American control group will be used in association studies to determine which loci and risk factors contribute most to hypertension in this population. Project 2 will conduct a genome wide search of 200 similarly affected sib pairs obtained from a unique population of genetic isolates residing in the Chicoutimi Provence in Canada. The general linkage analysis will be refined by determining mutations that exist in linkage disequilibrium. Project 3 will conduct a genome wide search of an F2 population of inbred rats (Dahl S/jr x Brown Norway) to define hypertension susceptibility loci and loci influencing renal and vascular function. Additionally, congenic rat strains will be constructed that differ only in the region of individual loci that are linked to hypertension in order to study the physiological mechanisms by which the expression of this locus influences blood pressure. Homologous genetic loci which are found to influence blood pressure and/or important hypertension associated phenotypes in the F2 rat cross will be used as candidate genes in the human populations. Project 4 will design and apply analytic tools to be used in the gene mapping of studies of Projects 1-3 and develop novel extensions of traditional gene mapping methods for complex traits, accounting for heterogeneity and effects of concomitant variables such as age, sex, and shared environment. Novel techniques to analyze the dynamic nature of blood pressure will be undertaken in order to detect additional hypertension associated phenotypes. The four Cores which will provide support for the entire SCOR include: A) Administrative Core (Milwaukee); B) Informatics and Computational Resources (Milwaukee); C) Genotyping Core (Boston); D) Biochemical Core (Milwaukee)

PROPOSED PROGRAM (Adapted from the applicant's abstract) The goal of this SCOR renewal is to build upon quantitative trait loci (QTLs) that they have identified to be in common in two human populations and in the rat to search for specific genes that importantly influence hypertension and related phenotypes. They propose four closely inter-related scientific projects (two human, one animal, and one theroretical) that bring together a team of geneticists, clinical investigators, physiologists, and biomathematicians. Building on the results of the QTL linkage analysis, Project 1 will study extended families from the largest "closed population" in North America (Chicoutimi-Saguenay-Lac St. Jean, Canada). Regions of three selected QTLs found to be in linkage disequilibrium will be mapped with a family based association haplotype analysis using single nucleotide polymorphisms (SNPs) as genetic markers coupled with a transmission disequilibrium test (TDT). In Project 2, haplotype regions narrowed in the Canadian families within the area of likage disequilibrium will be utilized in a case-control study to identify genes in African Americans of Milwaukee using SNPs in positional candidate genes. Both clinical projects will build upon broad based phenotyping data to determine if distinct clusters of traits can be identified to stratify hypertensive subjects for further genetic analysis. Project 3 will build upon the extensive rat linkage analysis just completed and utilize chromosomal substitution (consomics/congenics) and expression profiling to examine the functional relevance of several specific QTLs and to identify differentially expressed genes of relevance within these regions. Project 4 focuses on the development of analytical tools to assess linkage and association data and to evaluate the functional significance of multiple haplotypes in genomic regions that influence blood pressure and associated traits. The proposed studies exploit novel Bioinformatic tools to more adequately characterize regions of homology between the rat and human genomes. The four Cores that will provide support for the SCOR include: Core B) Bioinformatics; Core C) Genomic; Core D) Biochemical Core.

DESCRIPTION (Adapted from the applicant's abstract) The proposed studies examine the hypothesis that the phenotypes of blood pressure salt sensitivity, renal failure, and hyperlipidemia comprise a syndrome that is caused, in part, by closely linked genes, or genes exhibiting pleiotropy, localized to rat chromosomes 13 and 18. The goal of this project is to apply chromosomal substitution techniques (consomic and congenic inbred rat strains) to partition these traits and identify putative candidate genes based upon detailed physiological studies and expression profiling of discrete regions of these chromosomes. Aim 1 is to determine if hypertension salt- sensitivity, hyperlipidemia, and renal dysfunction are reduced by the introgression of either Chr 13 or Chr 18 from Brown Norway rats onto the genomic background of Dahl S salt sensitive rats (consomic inbred strains). Given the modification of the trait(s) of interest, in Aim 2 ten informative congenic sublines will be developed to partition each of these chromosomes into approximately 10 cM overlapping genomic regions. Each of these sublines will be screened for the traits of interest to select the congenic strains most affected by the introgression of BN alleles. These two congenic sublines will undergo detailed studies of renal physiology and cDNA expression profiling proposed in Aim 3. The goal of these studies is to quantify the influence of genes in these narrow regions upon the physiological pathways that determine the renal pressure-natriuresis relationship such as glomerular filtration, renal cortical and medullary blood flow, tubuloglomerular feedback, and other aspects of kidney function. Finally, in Aim 4 they propose to narrow the QTL regions containing genes that influence arterial pressure salt-sensitivity, renal dysfunction and hyperlipidemia by carrying out backcrosses of the congenic substrains studied in Aim 3 to the parental Dahl salt sensitive rats. The informative recombinant rats will be selected and phenotyped in order to reduce the QTL regions to between 0.5 and 1.0 cM.

DESCRIPTION (provided by applicant). The overall goal of this PPG since its inception in 1982 has been to achieve an understanding of the long-term regulation of arterial pressure and the consequences of high blood pressure. The studies are focused upon the regulation of body fluid volume by the kidneys and the regulation of systemic arterial vascular resistance This grant contains five interwoven scientific projects. Project 1 examines how excess production of reactive oxygen species (ROS) in inbred salt-sensitive Dahl rats (SS/Mcw) affects NO production within the renal medulla and the functional consequences of these interactions on the regulation of medullary blood flow, sodium homeostasis and arterial pressure. Project 12 will determine if a deficiency in L-arginine transport in the kidney of SS/Mcw rats contributes to the decreased availability of NO, elevation in ROS, and reduced excretory function. Project 3 will determine if pressure natriuresis is mediated through changes in renal interstitial hydrostatic pressure with an increased production of 20-HETE and/or EETs in the proximal tubule and 20-HETE in the thick ascending limb of Henle. Project 10 will determine whether the low ANG11 levels seen in Dahl salt-sensitive rats are responsible for the impaired response of cerebral arteries to vasodilator stimuli and if these impaired responses involve the endothelium or receptors in the vascular smooth muscle cells. Project 13 will study the role of locally produced ANG11 and the interaction between ANG11, NO, and 20-HETE in the regulation of microvessel density. Each of these projects will utilize unique genetic inbred strains (consomic and congenic lines) that provide the trait of interest and a genetically defined control strain. Three core units Administrative, Biochemistry/Analytical, and Research Services will support and facilitate the research in this program. The program reflects a long-standing experience of shared ideas and techniques that has provided a synergistic environment for advancing our understanding of arterial blood pressure regulation and hypertension.

DESCRIPTION (provided by applicant): This is a competitive renewal for a postdoctoral training grant (5 T32 HL07792) for M.D. and Ph.D. fellows studying the pathophysiotogical mechanisms leading to development of hypertension, renal and vascular disease. Training focuses on clinical and basic (mechanistic) aspects of abnormal vascular regulation in disease states with a major focus on hypertension. Skills which will be developed include: fundamental and advanced understanding of genetics, molecular biology, analytical chemistry, in-vivo animal models, in-vitro techniques for determination of mechanisms of vascular and cardiac muscle activation, cell biology, etectrophysiology (including patch-clamping of ion channels and organ and whole animal electrophysiological techniques), in-vivo blood flow techniques, radiographic techniques including digital x-ray and fMRI, and microscopic techniques such as confocal microscopy. Along with these basic techniques, specialty clinical techniques will be taught depending upon discipline and interest. The Medical College of Wisconsin provides state-of-the-art facilities for this training program, and all mentors have current NIH funding. The inter-departmental environment of the MCW Cardiovascular Research Center provides exceptional opportunities for scientific crossfertilization and interdepartmental contacts. During the initial four years of this grant four M.D. and four Ph.D. fellows were trained, and an additional M.D. fellow was funded by a minority supplement. During the last four years of this grant, five M.D. and ten Ph.D. fellows were trained or remain in the training program, along with one M.D., Ph.D. fellow. Most trainees remain in the program for two years. The quality of fellows and training within the program is exceptional as evidenced by the ability of the trainees to obtain individual training grants and to receive advanced academic degrees. The trainees also have a high level of productivity as evidenced by the more than twenty peer-reviewed publications. We have been fortunate in our ability to attract quality applicants, and we have had many more applicants than positions available. The proposed continuation of this program will continue to provide a unique and state-of-the-art training opportunity for our fellows. We will stress, even more strongly, translational and integrative research in which fellows will translate cellular and molecular data with respect to their physiological function at the organ, experimental and human level. We have added new mentors to the program who will facilitate these objectives. The continued funding of the MCW General Clinical Research Center will greatly augment the ability of our fellows to translate their basic research findings into clinically relevant scenarios. We are encouraged by the success of the training program thus far and the number of high quality applicants in our pools, so we are therefore requesting that the number of slots be increased from five to nine.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The goal of this PPG is to test specific hypotheses of how genes within four discrete regions of Chr 13 initiate the cascade of events determining blood presure salt-sensitivity and renal dysfunction in the Dahl S (SS) rat; and to identify specific genetic polymorphisms in two of these regions that determine blood pressure, salt-sensitivity, renal damage, and vascular angiogenesis. We have demonstrated that introgression of small regions of Chr 13 from the inbred Brown Norway (BN) strain of rat into the genomic background of the SS rat strain (consomic SS-13BN) substantially reduced salt-induced hypertension and restored angiogenic capacity to the SS rat. Four of 26 overlapping SS.BN congenic strains within Chr 13 containing BN substitutions (congenic strains 1,5,9, and 26) have been selected based on their protective actions on salt-induced hypertension and vascular angiogenesis effects. We propose three closely inter-related scientific projects that bring together a team of uniquely qualified geneticists and physiologists. Project by Cowley will determine the sequential physiological and gene expression changes in response to salt intake upon the kidney, adrenal gland, and vasculature. Since sex differences affected the degree of protection from salt-induced hypertension in several of the congenic strains, one of these strains (strain 9) has been selected to determine variations of genomic and physiological pathways that may explain these differences. Project by Roman focuses on a region of Chr 13 congenic strain 5 to identify the specific gene that "protects" from salt-induced hypertension in both male and female rats. Project by Greene focuses on Chr 13 congenic strain 9, to characterize the mutation that regulates the renin gene and thereby impacts upon angiotensin II formation and angiogenesis. Validation of differentially expressed and/or candidate genes will be tested using rat transgenic apporaches for Projects by Roman and Greene. The four Cores that will provide support for the PPG include: A) Administrative Core; B) Genomics Core; C) Transgenics Core; D) Research Services Core. [unreadable]

The Administrative Core of this Program Project Grant provides the vital support for all programmatic activities, project investigators and staff in general. This Core bridges the activities of the Program with the National Institutes of Health, the Medical College of Wisconsin administrative departments, the Department of Physiology, and the investigators of the Program Project Grant. The staff of this Core is responsible for matters of personnel, purchasing, centralization of service contracts, centralization of biostatistical services, and fiscal management. Core A coordinates annual reports to the National Institutes of Health, programmatic travel by Program Investigators, and visits by invited speakers and/or consultants. Core A also coordinates dissemination of vital information within the Program, organizes scientific meetings and seminars related to the Program, and coordinates the activities of the internal and external advisory committees.

The goal of Project by Cowley is to use physiological and gene microarray expression data to determine how genes[unreadable] within four discrete regions of Chromosome 13 (Chr 13) initiate and/or maintain the cascade of events[unreadable] determining blood pressure salt-sensitivity and renal dysfunction in the Dahl S (SS) rat. As shown previously,[unreadable] introgression of the entire Chr 13 from the inbred Brown Norway (BN) strain of rat into the genomic background[unreadable] of the SS rat strain (consomic SS-13BN) substantially reduces salt-induced hypertension and proteinuria. We[unreadable] have now completed development of and have phenotyped 23 congenic inbred strains with overlapping BN[unreadable] Chr 13 chromosomal segments introgressed into SS. Four discrete congenic regions of these BN substitutions[unreadable] within the SS genomic background (congenic strains 1, 5, 9 and 26), that range in size from 4.5 to 16 Mbp[unreadable] resulted in significant protection from salt-induced hypertension in female rats. Reductions of the levels of[unreadable] hypertension ranged from 22 to 32 mmHg among these congenic strains. In addition, since sex differences[unreadable] affected the degree of protection from salt-induced hypertension in several of the .congenic strains, one of[unreadable] these strains (congenic strain 9) was selected to determine variations of genomic and physiological pathways[unreadable] that may explain these male and-female differences. We hypothesize that genes within these four congenic[unreadable] regions of Chr 13 collectively contribute to genome-wide responses and operate through shared functional[unreadable] pathways to improve the sodium excretory function of the kidney and thereby protect the organism from salt-induced[unreadable] hypertension. Three systems important in the regulation of sodium homeostasis and arterial pressure[unreadable] regulation will be assessed, the kidneys (cortex and medulla), the adrenals (reflecting autonomic and[unreadable] endocrine function) and the vasculature. Studies in Aim I will determine the sequential physiological changes[unreadable] in pathways under conditions of 0.4% salt diet and at 16 hr, 3 and 12 days after switching to a 4.0% salt diet.[unreadable] Aim 2 will utilize gene microarrays as a powerful assay system to identify pathways and networks that are[unreadable] linked to whole system physiology. Molecular profiles reflected by mRNA expression will be combined with[unreadable] physiological profiles obtained in Aim 1 in an integrative'analysis to identify the molecular patterns and[unreadable] pathways that underlie common physiological systems responsible for the phenotypic differences .between the[unreadable] SS rat and'the reduced salt-sensitivity of the congenic strains. >Even if little is currently known of the function of[unreadable] a differentially expressed gene, gene function can be reverse engineered by placing .them into the context of[unreadable] an overall functional pathway to predict gene-function relationships. The use of congenic strains within Chr 13[unreadable] with well-defined blood pressure phenotypes provides a unique opportunity to produce an integrated picture of[unreadable] how genes within four discrete regions of Chr 13 modify salt-induced hypertension. This may provide valuable[unreadable] clues to define these critical pathways responsible for salt-induced forms of hypertension in human subjects,[unreadable] especially in high risk populations such as African-Americans.

DESCRIPTION (provided by applicant): It is recognized that hypertension can be a consequence of various types of renal dysfunction but the impact of elevated blood pressure upon the development of renal injury remains poorly understood. We hypothesize that elevations of renal perfusion pressure can be an important cause of renal injury, starting first in the renal outer medulla. The proposed studies will explore the consequences of the direct effects of elevated arterial pressure upon renal injury in Sprague Dawley rats and focus on three important questions: 1) What are the direct contributions of chronic elevations of renal perfusion pressure to tissue injury in the renal cortex and outer medulla;2) To what extent is the renal vulnerability to pressure-induced injury correlated to systemic factors (such as angiotensin II and norepinephrine) and the local balance between reactive oxygen species and nitric oxide in the kidney;3) What is the role of hydrogen peroxide (H2O2) as a key mediator of the pressure-induced injury? Novel servo-control techniques will be used that enable pressure to the left kidney to be maintained at a normal level during the development of hypertension while the contralateral right kidney is exposed chronically to elevated pressure. Different states of vulnerability to pressure-induced injury will be produced using three models of hypertension (Specific Aims 1-3). Aim 4 determines the role of H2O2 in pressure-induced injury to the renal medulla by infusing catalase into the medulla of the left kidney during the development of angiotensin lI+L-NAME-induced hypertension and conversely by infusing H2O2 into the left kidney of normal rats. Renal injury in each of the Aims will be assessed at three time points during the development of hypertension (days 3, 7 and 14) using techniques of microdialysis to collect interstitial fluid for the determination of H2O2 and nitric oxide concentrations. Renal tissue will be collected and protein expression and enzyme activities related to the pathways of synthesis and degradation of these reactive oxygen species measured. Histological techniques and immunostaining will be utilized to quantify the degree of tissue injury. The results of the proposed studies should significantly advance our understanding of the impact of blood pressure upon the development of renal interstitial fibrosis and tubuloglomerular injury in hypertension and the role of oxidative stress in this process. An understanding of these mechanisms could lead to new therapeutic approaches for the prevention of kidney disease in hypertension.

The goal of Project by Cowley is to use physiological and gene microarray expression data to determine how genes within four discrete regions of Chromosome 13 (Chr 13) initiate and/or maintain the cascade of events determining blood pressure salt-sensitivity and renal dysfunction in the Dahl S (SS) rat. As shown previously, introgression of the entire Chr 13 from the inbred Brown Norway (BN) strain of rat into the genomic background of the SS rat strain (consomic SS-13BN) substantially reduces salt-induced hypertension and proteinuria. We have now completed development of and have phenotyped 23 congenic inbred strains with overlapping BN Chr 13 chromosomal segments introgressed into SS. Four discrete congenic regions of these BN substitutions within the SS genomic background (congenic strains 1, 5, 9 and 26), that range in size from 4.5 to 16 Mbp resulted in significant protection from salt-induced hypertension in female rats. Reductions of the levels of hypertension ranged from 22 to 32 mmHg among these congenic strains. In addition, since sex differences affected the degree of protection from salt-induced hypertension in several of the .congenic strains, one of these strains (congenic strain 9) was selected to determine variations of genomic and physiological pathways that may explain these male and-female differences. We hypothesize that genes within these four congenic regions of Chr 13 collectively contribute to genome-wide responses and operate through shared functional pathways to improve the sodium excretory function of the kidney and thereby protect the organism from salt-induced hypertension. Three systems important in the regulation of sodium homeostasis and arterial pressure regulation will be assessed, the kidneys (cortex and medulla), the adrenals (reflecting autonomic and endocrine function) and the vasculature. Studies in Aim I will determine the sequential physiological changes in pathways under conditions of 0.4% salt diet and at 16 hr, 3 and 12 days after switching to a 4.0% salt diet. Aim 2 will utilize gene microarrays as a powerful assay system to identify pathways and networks that are linked to whole system physiology. Molecular profiles reflected by mRNA expression will be combined with physiological profiles obtained in Aim 1 in an integrative'analysis to identify the molecular patterns and pathways that underlie common physiological systems responsible for the phenotypic differences .between the SS rat and'the reduced salt-sensitivity of the congenic strains. >Even if little is currently known of the function of a differentially expressed gene, gene function can be reverse engineered by placing .them into the context of an overall functional pathway to predict gene-function relationships. The use of congenic strains within Chr 13 with well-defined blood pressure phenotypes provides a unique opportunity to produce an integrated picture of how genes within four discrete regions of Chr 13 modify salt-induced hypertension. This may provide valuable clues to define these critical pathways responsible for salt-induced forms of hypertension in human subjects, especially in high risk populations such as African-Americans.

The overall goal of this project is to advance our understanding of the role that reactive oxygen species (ROS) and reactive nitrogen species (RNS) play in determining renal function, especially that of the renal medulla, a region that is vulnerable to excess O2- production. We have previously established that changes in renal medullary blood flow play an important role in sodium homeostasis and the long-term regulation of arterial pressure and shown that nitric oxide (NO) production plays a key role in the regulation of blood flow to this region. We hypothesize that excess production of ROS in the renal medulla reduces NO bioavailability, lowers medullary blood flow, increases tubular sodium reabsorption and results in a salt-sensitive form of hypertension. This project, as all others in this PPG, utilizes Dahl salt-sensitive rats that are genetically defined (SS/Mcw) and a newly developed consomic control strain derived from the inbred SS/Mcw strain in which chromosome 13 from the salt-insensitive BN/Mcw strain has been introgressed into the genomic background of the SS/Mcw rat (SS.BN13 consomic strain). This consomic SS.BN13 strain is 98% identical to the SS/Mcw strain but is relatively salt-insensitive. Aim 1 will determine if the renal medulla of Dahl salt-sensitive rats (SS/Mcw) produces excess ROS (O2, H2O2, ONOO) that feeds back to uncouple nitric oxide syntheses enzymes (NOS) and reduce the redo ratio of tetrahydrobiopterin (BH4)/dihydrobiopterin (BH2) (i.e., a vicious cycle of O2. production). Newly developed fluorescent microdialysis and HPLC analytical techniques will enable comparison of ROS and NO pathways in salt-sensitive (SS/Mcw) and in salt-insensitive consomic SS.BN13 rats. Our preliminary data indicate that this control strain exhibits significantly lower levels of medullary ROS. Studies will also determine whether lowering of ROS in the medulla of SS/Mcw rats by chronic medullary infusion of antioxidants increases medullary blood flow and reduces salt-induced hypertension. Aim 2 will determine the effects of induced medullary elevations of ROS in salt-insensitive SS.BN13 consomic rats. Medullary blood flow, renal interstitial hydrostatic pressure (RIHP), arterial pressure (AP) and the pressure-natriuresis relationships will be determined in response to chronic elevations of O2-, H2O2, and ONOO-. The concept of NOS uncoupling will be examined by measurement of medullary tissue BH4/BH2 ratios determined by HPLC, and NO, O2 and H2O2 values obtained by microdialysis techniques. Aim 3 will determine the dynamic inter-relationships of O2 and NO within and between vascular and tubular segments of the renal medulla using thin, superfused medullary tissue stripes. NO responses to agents that stimulate and inhibit ROS will be compared in control SS.BN13 and salt-sensitive SS/Mcw rats. Novel fluorescent dyes, coupled with high speed capture of microscopic images will enable the measurement of intracellular NO independent from NO in the interstitial space thereby determining whether NO released from tubules could act as a paracrine substance to control medullary blood flow. Taken together, these studies will provide novel and clinically relevant data defining the role that ROS play in the regulation of renal medullary function and the development of salt-sensitive hypertension, information that would be expected to lead to new therapeutic modalities in the treatment of human hypertension.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The goal of this PPG is to test specific hypotheses of how genes within four discrete regions of Chr 13 initiate the cascade of events determining blood presure salt-sensitivity and renal dysfunction in the Dahl S (SS) rat; and to identify specific genetic polymorphisms in two of these regions that determine blood pressure, salt-sensitivity, renal damage, and vascular angiogenesis. We have demonstrated that introgression of small regions of Chr 13 from the inbred Brown Norway (BN) strain of rat into the genomic background of the SS rat strain (consomic SS-13BN) substantially reduced salt-induced hypertension and restored angiogenic capacity to the SS rat. Four of 26 overlapping SS.BN congenic strains within Chr 13 containing BN substitutions (congenic strains 1,5,9, and 26) have been selected based on their protective actions on salt-induced hypertension and vascular angiogenesis effects. We propose three closely inter-related scientific projects that bring together a team of uniquely qualified geneticists and physiologists. Project by Cowley will determine the sequential physiological and gene expression changes in response to salt intake upon the kidney, adrenal gland, and vasculature. Since sex differences affected the degree of protection from salt-induced hypertension in several of the congenic strains, one of these strains (strain 9) has been selected to determine variations of genomic and physiological pathways that may explain these differences. Project by Roman focuses on a region of Chr 13 congenic strain 5 to identify the specific gene that "protects" from salt-induced hypertension in both male and female rats. Project by Greene focuses on Chr 13 congenic strain 9, to characterize the mutation that regulates the renin gene and thereby impacts upon angiotensin II formation and angiogenesis. Validation of differentially expressed and/or candidate genes will be tested using rat transgenic apporaches for Projects by Roman and Greene. The four Cores that will provide support for the PPG include: A) Administrative Core; B) Genomics Core; C) Transgenics Core; D) Research Services Core. [unreadable]

DESCRIPTION (provided by applicant): Since the inception of this PPG in 1982, the overall goal of this program has been to achieve an understanding of the long-term regulation of arterial pressure and the consequences of high blood pressure. The unifying hypothesis of the present grant centers on the concept that the kidney controls the long-term level of arterial pressure and when genetically predisposed salt intake can importantly influence kidney function and the structure and function of the systemic vasculature. Project 1 will utilize Dahl salt-sensitive rats (SS) to study how a high salt diet may stimulate excess production of reactive oxygen species (ROS) in the renal medullary thick ascending limb (mTAL) leading to reduction of blood flow to the renal medulla, reduction in sodium excretion and hypertension. Project 2 hypothesizes that during the early phase of saltinduced hypertension an inflammatory process is initiated by infiltrating macrophages that produces angiotensin II (ANGII) that stimulates the production of ROS thereby increasing the severity of hypertension and renal injury. Project 3 will search for a mutation in one of the cytochrome P4504A genes (CYP4A) that is the underlying genetic defect in the SS rat that plays an important causal role in the impaired pressurenatriuresis and the development of hypertension. Project 4 examines the permissive role that ANGII plays in maintaining normal vascular reactivity and how defects in the SS renin allele lead to increased oxidative stress and impaired vascular relaxation. Project 5 focuses on the microcirculation and the mechanisms that control and alter organ and tissue perfusion in hypertension and the impact that reductions in ANGllstimulated O2 inhibit the VEGF signaling pathway leading to microvessel rarefaction and inhibition of angiogenesis that is found in the SS rat. Each of these projects utilize unique genetic rat strains (consomic, congenic, and transgenic) that provide the trait of interest and a genetically defined control strain. Hypertension affects more than 50 million Americans and remains largely uncontrolled in 75% of patients in North America leading to an increased incidence of stroke, heart, and renal disease, that contribute to escalating health care costs. The program reflects a long-standing experience of shared ideas in a synergistic environment aimed toward advancing our understanding of hypertension and the identification of novel targets for drug design that may better control this disease.

Project 1 hypothesizes that Dahl salt-sensitive (SS) rats when fed a high salt diet will reabsorb greater than normal amounts of NaCI in the renal medullary thick ascending limb (mTAL) that in turn will stimulate excess production of reactive oxygen species (ROS) and thereby reduce medullary blood flow (MBF). The mTAL of SS rats express greater amounts of Na/K/2CI co-transporter than salt-insensitive control SS.13 rats and it will be determined if they also express greater amounts of isoforms of the Na/H exchanger (NHE) that could account for excess superoxide (O*2~) production. We propose that increased luminal delivery of NaCI together with increases of outer medullary interstitial NaCI concentration may stimulate NAD(P)H oxidase and enhance production of O*2 and H2O2. It will be determined if greater diffusion of O'2~ and/or H2O2 occurs from mTAL to vasa recta pericytes in the presence of reduced NO bioavailability and whether this leads to a reduction of MBF. These events will be studied using time resolved fluorescence videomicroscopy of medullary tissue strips. Physiological relevance of these observations will be assessed by studies in conscious instrumented rats. Sequential measurements of medullary interstitial O'2~, H202, NO and medullary interstitial [NaCI] will be obtained using implanted microdialysis fibers. Changes of medullary blood flow and arterial pressure will be measured daily in these conscious rats using implanted optical fibers and laser-Doppler techniques together with indwelling arterial catheters before and following an increase in salt (NaCI) intake and the development of hypertension. The project is defined by three specific aims: 1) To determine if exposure of mTAL to increased NaCI by either tubular microperfusion and/or bath superfusion results in greater mTAL production of O*2 or H2O2 and subsequent constriction of surrounding vasa recta vessels in SS compared to the control SS.13BN rats; 2) To explore the cellular mechanisms whereby increased luminal delivery of NaCI to mTAL and increases of interstitial NaCI concentrations enhance 0*2 production in SS rats; 3) To characterize the temporal responses of renal medullary production of O*2, H2O2, and NO in conscious SS rats to determine if increases in ROS initiate reductions of medullary blood flow following increased NaCI intake.

DESCRIPTION (provided by applicant): The Cardiovascular Center (CVC) at the Medical College of Wisconsin (MCW) is a Biomedical Core Center with ongoing research related to the prevention, detection, treatment and cure of the complex set of cardiovascular diseases related to heart, lung, stroke, high blood pressure and renal injury, diabetes, and obesity. The most recent Strategic Plan designated the expansion of the cardiovascular research activities at MCW to be one of the highest priorities. Recent allocation of expanded research space and resources to the CVC to recruit new faculty demonstrates the institutional support of this expansion. We have identified an exciting young scientist, Dr. Aron Geurts, who would advance our goals to develop an innovative research program of excellence in stem cell and regenerative cardiovascular biology. Dr. Geurts with ten years of experience in cell culture and technology development, has been a postdoctoral fellow for the past three years in the Human and Molecular Genetics Center (HMGC) working in the laboratory of Dr. Howard Jacob. He has led the development and application in rats of gene-trap transposon mutagenesis for our NHLBl-PGA program;the creation of the first targeted knockout rats using zinc-finger nucleases;and the reprogramming of rat fibroblasts into induced pluripotent stem cells. This PSO grant would enable the immediate opportunity to provide start-up funds and protected time for Dr. Geurts as a new tenure-track faculty member to develop his own research initiatives to become an independently-funded, NIH-supported investigator. Not only his specific scientific skills and prowess but also his remarkable collaborative abilities make Dr. Geurts a strong candidate for recruitment to enhance research resources in the area of stem cell and regenerative cardiovascular biology developed by the CVC together with the Departments of Physiology, and Cell Biology, Neurobiology and Anatomy, the HMGC, and the Bioengineering and Biotechnology Center. The extensive resources on this campus would provide a pathway of independence for Dr. Geurts while at the same time expanding the institution's community of multidisciplinary researchers focusing on the specific interests of the NHLBI. The CVC has identified an exciting young scientist. Dr. Aron Geurts, who would advance our goals to develop an innovative research program of excellence in stem cell and regenerative cardiovascular biology. The recruitment of Dr. Geurts would enhance research resources in the area of stem cell and regenerative cardiovascular biology for the CVC at MCW.

DESCRIPTION (provided by applicant): The goal of this PPG is to advance our understanding ofthe complex regulation and interplay of a set of genes residing in two different regions of chr 13 but which together are responsible, in large measure, for salt-induced hypertension, renal injury, and vascularity/angiogenesis of the microcirculation in the saltsensitive (SS) rat. The studies are collectively designed to explore how genetic polymorphisms are translated into integrated cellular, tissue, organ and whole animal function. Project 1 hypothesizes that sequence variants of one or more ofthe genes within a congenic region of chr 13 alter molecular regulatory networks affecting function of the medullary thick ascending limb (mTAL) of SS rats and contribute to the development of salt-sensitive hypertension and renal injury. This will tested by: 1) generating finished-level genomic sequence with complete annotation of this congenic region;2) by constructing a molecular and physiological regulatory network of the mTAL epithelial cell and identifying pathways and genes that may contribute to hypertension and renal injury;and 3) by studying the impact of removing or overexpressing an important gene in the transcriptome/proteome/metabolome associated network. Project 2 will examine a novel hypothesis that non-protein-coding genes may play important roles in hypertension and related tissue injury. We hypothesize that miR-214 contributes to the development of salt-sensitive hypertension and renal injury and examine: 1) the functional contribution of the microRNA within the kidney;2) examine downstream mechanisms mediating its effects, examine upstream trans and cis mechanisms;and 3) carry out a pilot study of miR-214 in human salt-sensitive hypertension and renal injury. Project 3 hypothesizes that a mutation(s) in the SS rat is responsible forthe impaired angiogenesis in this model. We will: 1) identify sequence variants;2) demonstrate that these variants impact renin regulation in vitro;and 3) using a transgenic approach, demonstrate that the SS allele is capable of eliminating normal renin regulation and the angiogenic phenotype in vivo. The collaborative research of this PPG will be supported by Administrative Core A, Genomic and Transgenic Core B, and the Research Services Core C.

INTRODUCTION: The Administrative Core provides the vital support for all programmatic activities, project investigators and staff in general. This Core bridges the activities of the Program with the National Institutes of Health, the Medical College of Wisconsin administrative departments, the Department of Physiology, and the investigators of the Program Project Grant. The staff of this Core is responsible for matters of personnel, purchasing and fiscal management. Core A coordinates annual reports to the National Institutes of Health, programmatic travel by Program Investigators, and visits by invited speakers and/or consultants. Core A also coordinates dissemination of vital information within the Program, organizes scientific meetings and seminars related to the Program, and coordinates the activities of the internal and external advisory committees.

DESCRIPTION (provided by applicant): Since the inception of this PPG in 1982, the overall goal of this program has been to achieve an understanding of the long-term regulation of arterial pressure and the consequences of high blood pressure. The unifying hypothesis of the present grant centers on the concept that the kidney controls the long-term level of arterial pressure and when genetically predisposed salt intake can importantly influence kidney function and the structure and function of the systemic vasculature. Project 1 will utilize Dahl salt-sensitive rats (SS) to study how a high salt diet may stimulate excess production of reactive oxygen species (ROS) in the renal medullary thick ascending limb (mTAL) leading to reduction of blood flow to the renal medulla, reduction in sodium excretion and hypertension. Project 2 hypothesizes that during the early phase of saltinduced hypertension an inflammatory process is initiated by infiltrating macrophages that produces angiotensin II (ANGII) that stimulates the production of ROS thereby increasing the severity of hypertension and renal injury. Project 3 will search for a mutation in one of the cytochrome P4504A genes (CYP4A) that is the underlying genetic defect in the SS rat that plays an important causal role in the impaired pressurenatriuresis and the development of hypertension. Project 4 examines the permissive role that ANGII plays in maintaining normal vascular reactivity and how defects in the SS renin allele lead to increased oxidative stress and impaired vascular relaxation. Project 5 focuses on the microcirculation and the mechanisms that control and alter organ and tissue perfusion in hypertension and the impact that reductions in ANGllstimulated O2 inhibit the VEGF signaling pathway leading to microvessel rarefaction and inhibition of angiogenesis that is found in the SS rat. Each of these projects utilize unique genetic rat strains (consomic, congenic, and transgenic) that provide the trait of interest and a genetically defined control strain. Hypertension affects more than 50 million Americans and remains largely uncontrolled in 75% of patients in North America leading to an increased incidence of stroke, heart, and renal disease, that contribute to escalating health care costs. The program reflects a long-standing experience of shared ideas in a synergistic environment aimed toward advancing our understanding of hypertension and the identification of novel targets for drug design that may better control this disease.

DESCRIPTION (provided by applicant): The goal of this PPG is to advance our understanding ofthe complex regulation and interplay of a set of genes residing in two different regions of chr 13 but which together are responsible, in large measure, for salt-induced hypertension, renal injury, and vascularity/angiogenesis of the microcirculation in the saltsensitive (SS) rat. The studies are collectively designed to explore how genetic polymorphisms are translated into integrated cellular, tissue, organ and whole animal function. Project 1 hypothesizes that sequence variants of one or more ofthe genes within a congenic region of chr 13 alter molecular regulatory networks affecting function of the medullary thick ascending limb (mTAL) of SS rats and contribute to the development of salt-sensitive hypertension and renal injury. This will tested by: 1) generating finished-level genomic sequence with complete annotation of this congenic region; 2) by constructing a molecular and physiological regulatory network of the mTAL epithelial cell and identifying pathways and genes that may contribute to hypertension and renal injury; and 3) by studying the impact of removing or overexpressing an important gene in the transcriptome/proteome/metabolome associated network. Project 2 will examine a novel hypothesis that non-protein-coding genes may play important roles in hypertension and related tissue injury. We hypothesize that miR-214 contributes to the development of salt-sensitive hypertension and renal injury and examine: 1) the functional contribution of the microRNA within the kidney; 2) examine downstream mechanisms mediating its effects, examine upstream trans and cis mechanisms; and 3) carry out a pilot study of miR-214 in human salt-sensitive hypertension and renal injury. Project 3 hypothesizes that a mutation(s) in the SS rat is responsible forthe impaired angiogenesis in this model. We will: 1) identify sequence variants; 2) demonstrate that these variants impact renin regulation in vitro; and 3) using a transgenic approach, demonstrate that the SS allele is capable of eliminating normal renin regulation and the angiogenic phenotype in vivo. The collaborative research of this PPG will be supported by Administrative Core A, Genomic and Transgenic Core B, and the Research Services Core C.

ABSTRACT Essential hypertension affects more than 50 million Americans and increased blood pressure salt-sensitivity is a prominent feature in certain populations of hypertensive patients, especially African Americans. Although it is evident that the common forms of hypertension are multifactorial (polygenic and environmental), the genetic basis of the frequent forms of this disease remain elusive. The Dahl salt-sensitive (SS) rat is a widely used animal model that recapitulates many aspects of human salt-sensitive hypertension and associated renal injury. The goal of Project 1 is to study the interplay of gene and protein expression using integrative systems approaches to determine the functionality of a single cell type of the kidney (renal medullary thick ascending limb of Henle = mTAL). The mTAL is known to play an important role in renal medullary function, sodium excretion, and hypertension in the SS rat and in human hypertension. We hypothesize that gene(s) sequence variants within a congenic region of chromosome 13 (SS. 13[BN26] ; BN alleles substituted for SS) alter molecular regulatory networks affecting function of the mTAL thereby contributing to salt-sensitive hypertension and renal injury. In Aim 1, the congenic region (SS.13[BN26],13.2 Mb) will be sequenced to obtain a finished high-quality assembly of this region and annotated to search for gene sequence variants. Subcongenic strains will be developed to identify a narrow region that attenuates salt-sensitivity to ~ 5 Mb and a sequence capture chip will then be utilized for follow-up sequencing of this region in 14 additional strains that have a different evolutionary history from the SS and BN. The sequence differences (SNPs and other genomic variants) will be filtered using the criteria that a casual variant in the SS should only be in common with other salt-sensitive strains. Aim 2 will construct a molecular and physiological regulatory network (BayeN) of the mTAL epithelial cell and use that model to identify pathways and genes that may contribute to salt-sensitive hypertension and renal injury in SS rats. Aim 3 will select a candidate gene based on results from Aims 1 and 2 and either knock the gene out using zinc finger nucleases (ZFN) and/or over-express the gene using transgenic approaches to study the impact of this gene upon the transcriptome/proteome/metabolome and associated functional network. Several technological leaps and conceptual approaches are unique to this Project. These include 1) Next generation sequencing to provide very high resolution sequencing of a 13.2 Mb region of SS and congenic SS.13[BN26] strains coupled with a NimbleGen sequence capture chip for sequencing of additional rat strains; 2) Transcriptome analysis (Affymetrix) and a mass spectrometry sub-proteome and metabolome analysis using isolated and purified mTAL cells comparing the SS and salt-resistant congenic rats; 3) The use of a large-scale Bayesian analysis which is knowledge-constrained to seek yet unknown pathways hidden using the transcriptome and functional data; 4) A novel technology (zinc finger nucleases) to efficiently knock out a gene found to contribute importantly to the mTAL regulatory network, providing a definitive way to validate and characterize functional relevance. Project 1 addresses a critical challenge facing the field of hypertension: to identify the complex components (genes, proteins and pathways) that are responsible for alterations in kidney function leading to salt-sensitive hypertension. It is highly synergistic with Project 2 which tests the innovative concept that non-protein-coding genes (microRNA) may play an important role in hypertension and renal injury and Project 3 which aims to Identify the mechanisms by which mutations distant from the renin gene on chr 13 regulate the activity of renin and angiogenesis in the SS rat. All projects benefit greatly from genomic, genetic, proteomic and computational technological advances provided by Cores 8 and C.

The integrating theme and unifying hypothesis of this PPG centers on the concept that H202 production in the renal outer medulla (CM) plays a dominant role in the development of salt-sensitive hypertension. Studies in this PPG will use Dahl salt-sensitive (SS) rats to examine physiological mechanisms and molecular pathways underlying salt-sensitive hypertension; it does not focus on the generic aspects of this disease. This rat model recapitulates many aspects of human salt-sensitive hypertension and demonstrates disease phenotypes which are very similar to those observed in African Americans. Project 1 explores the role of the medullary thick ascending limb (mTAL) and tests the hypothesis that increased salt intake leads to greater mTAL NaCI delivery resulting in excess production of H202 in SS rats as amplified by a greater expression of the p67phox subunit of NADPH oxidase. It is proposed that this results in greater H202 diffusion into the interstitial space thereby constricting vasa recta and reducing medullary perfusion. Project 2 hypothesizes that the initials rise of arterial pressure following an increase in salt intake leads to the infiltration of T-cells in the kidney which exaggerates salt-sensitive hypertension and renal disease by increasing H202 and cytokines. The resulting T-lymphocyte infiltration into the kidney, we propose, is importanfiy influenced by Sh2b3, a gene recently identified by GWAS to be associated with human hypertension. Project 3 hypothesizes that a newly discovered pathway of H202 production related to cellular metabolism contributes importantly to the development and maintenance of hypertension in SS rats. Specifically, fumarase insufficiency in SS rats results in an increase of fumaric acid and glycolytic activity which stimulates H202 production and contributes to the salt-induced hypertension. These conceptually unique hypotheses combined with novel technological tools will advance our understanding of the molecular and physiological mechanisms underlying salt-sensitive hypertension that remain largely unclear. This highly integrated and collaborative program of three projects is supported by an Administrative Core A, the Biochemistry/Microscopy Core B, and Genetic Model Tracking and Monitoring Core C.

Superoxide and nitric oxide production within the outer medulla (OM) of the kidney are known to play an important role in sodium homeostasis and in salt-sensitive hypertension and renal injury. Little is yet known about the highly reactive H2O2 molecule which is involved in these processes and may account for as much as 50% of the hypertension and renal injury observed in the Dahl salt-sensitive (SS) rat. We hypothesize that increased NaCI delivery to the medullary thick ascending limb (mTAL) of the OM, as occurs following an increase in NaCI intake, stimulates mitochondrial production of H2O2 in the mTAL epithelial cells that diffuses to the cell membrane and enhances the activity of NADPH oxidase leading to an overall increase of cellular levels of H2O2 (Aim 1). We propose that this H2O2 response is significantly amplified in SS rats by greater expression of the p67phox cytosolic subunit of NADPH oxidase compared to a salt-resistant control strain as explored in Aim 2. The contribution of p67phox to these events will be determined ufilizing SS rats with a ubiquitous null mutation in the p67phox gene and salt-resistant SS.13BN26 rats with mTAL-specific transgenic overexpression of p67phox. In Aim 3, studies will determine if the greater production of H2O2 in the mTAL of SS rats results in diffusion of H2O2 into the interstitial space of the surrounding vasa recta which results in pericyte-mediated vasoconstriction and reduction of medullary blood flow leading to the initial moderate rise of arterial pressure. Aim 4 will determine if the rise of blood pressure with salt intake provokes renal T-lymphocyte infiltration, excess p67phox and fumaric acid in the mTAL leading to greater H2O2 production and a progression from a mild to severe form of hypertension and renal injury. This is a highly collaborative protocol between Projects 1, 2 and 3 that will utilize a computer-controlled system to examine the consequences of the elevated renal perfusion pressure by protecfing one kidney from the hypertension while the other is exposed to the elevated blood pressure. Since technical limitations have impeded a thorough mechanistic understanding of the role of H2O2 in renal function and hypertension, a number of new fluorescent imaging approaches, a novel fluorescent probe that specifically detects mitochondrial changes of H2O2, and a novel inhibitor of mitochondrial H2O2 will be utilized to advance our understanding of this field. Several novel genetically engineered rat model systems have been developed to test several of the key hypotheses including an SS rat with a ubiquitous null mutation in the p67phox gene and a salt-insensitive SS.13BN26 in which p67phox is transgenically overexpressed only in the thick ascending limb of Henle. H2O2 appears to be an important signaling molecule in the OM of the kidney. If more effective antioxidant therapies are to be developed it will require knowledge of the expression and regulation of the key pathways and enzymes responsible for H2O2 formation and their functional relevance at the level of the tissue and whole organism. By identifying the two novel controllers of H2O2 production, one related to the mitochondria and the other to the p67phox gene, we anticipate identifying new therapeutic targets for hypertension.

? DESCRIPTION (provided by applicant): Sodium and water regulation by the kidney plays a key role in hypertension and can be significantly compromised by pathways of oxidative stress. Two tubular elements are of major importance in establishing Na+ homeostasis and both are known to participate in salt-sensitive forms of hypertension, the medullary thick ascending limb of Henle (mTAL) and the aldosterone sensitive distal nephron (ASDN). The mTAL of SS rats produces excess ROS and the chronic intramedullary infusion of catalase, a scavenger of H2O2, reduces salt- induced hypertension nearly 50% in SS rats. Conversely, medullary infusion of H2O2 to normal rats reduces MBF and Na+ excretion resulting in a salt-sensitive form of hypertension. SS rats fed a high salt diet also exhibit greater expression and activity of ENaC in the ASDN segments leading to greater reabsorption of Na+ and enhancement of salt-induced hypertension. The major source of ROS and H2O2 in the kidney is NADPH oxidase but the roles of specific Nox isoforms such as Noxs 1, 2 and 4 and the mechanisms whereby they affect renal function have not been well elucidated. The most abundant isoform in the kidney is Nox4 which is unique in that it releases predominantly H2O2. Yet no studies have been carried out to determine the role of Nox4 in Na+ homeostasis and hypertension. We hypothesize that Nox4 plays a dominant role in determining blood pressure salt-sensitivity in the SS rat in two ways: 1) By excess production of H2O2 in the renal outer medullary thick ascending limbs of Henle (mTAL) which diffuses to surrounding vasa recta (VR) pericytes causing constriction and reduction of MBF; 2) Through H2O2-mediated increases of ENaC activity in the ASDN. To explore the role of Nox4, we have created a novel rat model with a null mutation of Nox4 in the SS rat. We will compare the responses of this mutant rat, SSNox4-/-, to those of the SS rat in four Specific Aims: 1- Determine physiological consequences of a null mutation of Nox4 in SS rats (SSNox4-/-) upon whole kidney function (MBF and GFR), renal oxidative stress, pressure-natriuresis, salt-induced hypertension and renal injury. 2-(New Aim) Determine the extent to which the reduced renal injury in SSNox4-/- rats is a consequence of a lower renal perfusion pressure versus an inherent intrarenal reduction of ROS production (servo-control of renal perfusion pressure studies). 3-Determine if Nox4 is importantly involved in H2O2 production in mTAL in response to increased luminal Na+ delivery and whether H2O2 can diffuse from mTAL to constrict surrounding VR. 4-Determine if production of H2O2 and ENaC expression/activity in ASDN of SS rats is Nox4-dependent. Studies are multiscale in design ranging from intracellular to those utilizing chronically instrumented rats which monitor changes in MBF and GFR over several weeks. The results are expected to greatly enhance our understanding of the role of Nox4 in renal function and lead to novel ways to target pathways of oxidative stress in the treatment of hypertension and renal disease.

PROJECT SUMMARY Salt-sensitive hypertension is a significant health problem; there is a need to understand the underlying mechanisms to enable more effective treatments. The proposed studies are based on a strong scientific foundation with experiments performed in our laboratories in Dahl salt-sensitive (SS) rats demonstrating that both renal H2O2 production and inflammation play key roles in the initiation and progression of salt-sensitive hypertension, but the connection between these two has thus far not been elucidated. Based on prior work and exciting preliminary data, we hypothesize that renal inflammation in SS rats fed high salt is driven by an initial rise of arterial pressure with elevated Nox4-derived H2O2 production. This, in turn, results in leukocyte adhesion and infiltration into the kidney via activation of matrix metalloproteinase (MMP)-2 and -9 and the NLRP3 inflammasome. The infiltrated immune cells produce additional MMP-2 and H2O2 (and superoxide [O2-]) from Nox2 which facilitates the transition from a pre-hypertensive state to accelerated malignant hypertension and end-stage renal injury. Several unique methodologies enable us to address this overall hypothesis. First, we have developed an SS rat containing a null mutation in Nox4 (SSNox4-/-), a unique model of reduced H2O2 production with reduced salt-sensitive hypertension. Second, we have also developed novel SS rat strains with null mutations in MMP-2, MMP-9, and NLRP3 (SSMMP2-/-, SSMMP9-/-, and SSNLRP3-/-) enabling us to determine their involvement in salt-sensitive hypertension. Third, a custom-designed servo-control system which is unique to our laboratory enables the precise chronic control of renal perfusion pressure (RPP) to the left kidney of SS rats thereby allowing us to separate the effects of renal H2O2 production from those of differing RPPs. Moreover, given the profound effect of RPP on renal immune cell infiltration, this system enables us to perform pressure-matching studies to isolate the effects of genetic mutation of NLRP3, MMP-2 and MMP-9 on renal inflammation from the confounding effects of different RPP in the models. Finally, sophisticated bone marrow transfer studies, which utilize our genetically modified SS strains (SSp67phox-/- and SSMMP2-/-), will enable us to determine the relative contributions of parenchymal- and hematopoietic-derived H2O2/reactive oxygen species and MMP-2 to salt-sensitive hypertension and renal injury. This proposal has three Specific Aims: 1) will test the hypothesis that an initial rise of BP accompanied by elevated Nox4-derived H2O2 mediates immune cell infiltration into the kidney of hypertensive rats fed high salt; 2) will test the hypothesis that upregulation of MMP-2, MMP-9 and the NLRP3 inflammasome mediates renal immune cell infiltration in SS rats and participates in the progression of salt-induced hypertension; and 3) will test the hypothesis that T-cells that infiltrate into the kidney enhance H2O2 production via Nox2 and serve as a source of MMP-2 which further amplify immune cell infiltration and hypertension (i.e. creates a vicious cycle). Our unique ability to perform these studies will provide important insight into our fundamental understanding of salt-sensitive hypertension.

PROJECT 1 PROJECT SUMMARY The scientific premise of this project is based upon the significance of salt-sensitive hypertension as a health problem and the need to understand the underlying mechanisms to enable development of more effective treatments. The studies proposed pursue mTOR signaling, which has long been known to be important in the regulation of cell cycle events and in human cancers, but has remained unexplored in hypertension. It is also recognized that mTOR complexes can participate in autoimmune responses and cardiovascular diseases and studies find they are important for renal podocyte homeostasis and tubular epithelial Na+ and K+ transport. Despite evidence that many aspects of kidney physiology known to be altered in hypertension are negatively affected by stimulation of mTOR complexes, the therapeutic benefit of inhibition of these pathways has not been studied. Indeed, remarkably little is known about the contributions of mTOR to the pathophysiology of hypertension. Our preliminary studies find that inhibition of the mTOR complexes virtually abolished salt-induced hypertension in a rat model of salt-sensitive hypertension (Dahl SS rats). Given that a high salt diet stimulates H2O2 production in the kidney of SS rats, and based on our in vitro evidence, we hypothesize that in the SS rat model of salt-sensitive hypertension enhanced intrarenal production of H2O2, largely from Nox4, enhances mTOR signaling. We propose that activated mTORC2 phosphorylates and activates SGK1; thereby increasing tubular Na+ reabsorption which results in Na+ retention and hypertension (and as studied in Project 3, enhanced inflammation). Project 1 has three Specific Aims: Aim 1 will test the hypothesis that therapeutic suppression of the mTORC2 pathway will reduce blood pressure salt-sensitivity and renal injury in SS rats. Aim 2 will test the hypothesis that mTORC2/SGK1 contributes to enhanced tubular Na+ reabsorption and salt-sensitivity of SS rats. Aim 3 will test the hypothesis that elevations of intrarenal H2O2 in SS rats fed a high salt diet contribute importantly to the activation of the mTOR signaling pathway. These mechanisms are not just of academic interest since understanding of these pathways could yield novel therapeutic targets.

PROJECT SUMMARY Salt-sensitive hypertension is a significant health problem worldwide and there is a need to understand the underlying molecular mechanisms to enable more effective treatments. The proposed studies are based on a strong scientific foundation with experiments performed in our laboratories in Dahl salt-sensitive (SS) rats which mimic the human condition of the disease. We have demonstrated that this form of hypertension is associated with excess renal and vascular reactive oxygen species (ROS) production and reduced ability to excrete Na+. Excess reabsorption occurs in the renal medullary thick ascending limb (mTAL) leading to greater reabsorption of filtered Na+. Most relevant to this grant, SS rats exhibit a reduced ability to generate ATP through mitochondrial respiration in the mTAL, the tubular segment that is responsible for reabsorption of nearly 25% of the filtered Na+ of the kidney. In this region of the kidney, there exists high levels of oxidative stress (excess ROS production) emanating from both the mitochondria and cell membrane NADPH oxidases (NOX2 and NOX4). Two of the major gaps that remain in this field are first a lack of mechanistic studies of cellular/mitochondrial metabolism, and second, an absence of approaches to quantitatively evaluate the interdependence of the complex cellular processes. We hypothesize that a high salt diet which increases the delivery of Na+ to the mTAL of SS rats results in excess Na+ reabsorption and an increase of mTAL cytosolic [Na+] which stimulates mitochondrial ATP synthesis and ROS production which in turn stimulates membrane NOXs (ROS-ROS crosstalk and vicious cycle) leading to uncoupling of mitochondrial oxidative phosphorylation (OxPhos) and tissue injury. Aim 1 will utilize intact microdissected mTAL to test the hypothesis in SS rats that high salt diet increases cytosolic [Na+] thereby stimulating mitochondrial ROS production which in turn enhances greater uptake of Na+ into the cell and though ROS-ROS crosstalk of mitochondria and membrane NOX2 and NOX4 which amplifies total intracellular ROS production leading to OxPhos uncoupling. Contribution of membrane NOXs and mitochondrial ROS interactions will be determined using novel genetically engineered knockout strains SSNox4KO and SSp67/Nox4DKO rats. Aim 2 will determine the progression of the postulated bioenergetic events in isolated mitochondria of the kidney (both outer medulla and cortex) of high salt fed SS rats. Progressive alterations of mitochondrial bioenergetics and ROS production will be determined at four time points during the three weeks of high salt feeding. Aim 3 will utilize the measured data-driven computational modeling to provide a quantitative, integrated, and mechanistic framework that can predict the complex relationships existing between cellular oxygen utilization, energy production, and oxidative stress in the kidney during the development of salt-sensitive hypertension.