Mingyu Liang - US grants

DESCRIPTION (provided by applicant): The overall goal of this grant proposal is to advance the understanding of the pathophysiology of salt-sensitive hypertension by investigating the role of renal 11beta-hydroxysteroid dehydrogenases (11beta-HSDs). 11beta-HSDs are critical controllers of local levels of active glucocorticoids. Alterations of 11beta-HSDs that cause local excess of glucocorticoids in the kidney or other tissues (e.g., transgenic over-expression of the glucocorticoid-regenerating isoform 11beta-HSDI) result in hypertension. However, the importance of 11beta- HSDs in common forms of hypertension remains unclear. The Dahl salt-sensitive (SS) rat is a widely used model of human salt-sensitive hypertension. The consomic SS-13BN rat is genetically highly similar to SS, but has substantially reduced blood pressure salt-sensitivity. Previous studies from this laboratory found that 11beta-HSDI was over-expressed in the renal medulla (a kidney region critical for long-term blood pressure regulation) of SS compared to SS-13BN when rats were exposed to a high-salt diet. The proposed studies will test the hypothesis that dysregulation of renal medullary 11beta-HSD1 contributes to the development of salt-sensitive hypertension in SS rats. Studies are designed to: 1) characterize the expression and localization of 11beta-HSDI and related genes in SS and SS-13BN rats; 2) utilize in vivo small interfering RNA techniques to selectively suppress the expression of renal medullary 11beta-HSDI, which is not achievable with common pharmacological inhibitors, and examine the effect of such suppression on salt-induced hypertension in chronically instrumented SS rats; 3) begin to examine the functional mechanism for the role of 11beta-HSDI in salt-sensitive hypertension by investigating the effect of 11beta-HSDI suppression on renal tubular segmental fluid reabsorption in SS rats using micropuncture. Preliminary data has demonstrated the feasibility of the proposed studies and appeared to support the hypothesis. These studies will combine innovative molecular analysis/manipulation and functional assessment in genetically defined or modified animal models. The results are expected to elucidate a novel aspect of the pathophysiology of salt- sensitive hypertension and to provide potential new targets for prevention and treatment of hypertension.

DESCRIPTION (provided by applicant): The overall goal of this grant proposal is to advance the understanding of the pathophysiology of salt-sensitive hypertension by investigating the role of renal 11beta-hydroxysteroid dehydrogenases (11beta-HSDs). 11beta-HSDs are critical controllers of local levels of active glucocorticoids. Alterations of 11beta-HSDs that cause local excess of glucocorticoids in the kidney or other tissues (e.g., transgenic over-expression of the glucocorticoid-regenerating isoform 11beta-HSDI) result in hypertension. However, the importance of 11beta- HSDs in common forms of hypertension remains unclear. The Dahl salt-sensitive (SS) rat is a widely used model of human salt-sensitive hypertension. The consomic SS-13BN rat is genetically highly similar to SS, but has substantially reduced blood pressure salt-sensitivity. Previous studies from this laboratory found that 11beta-HSDI was over-expressed in the renal medulla (a kidney region critical for long-term blood pressure regulation) of SS compared to SS-13BN when rats were exposed to a high-salt diet. The proposed studies will test the hypothesis that dysregulation of renal medullary 11beta-HSD1 contributes to the development of salt-sensitive hypertension in SS rats. Studies are designed to: 1) characterize the expression and localization of 11beta-HSDI and related genes in SS and SS-13BN rats; 2) utilize in vivo small interfering RNA techniques to selectively suppress the expression of renal medullary 11beta-HSDI, which is not achievable with common pharmacological inhibitors, and examine the effect of such suppression on salt-induced hypertension in chronically instrumented SS rats; 3) begin to examine the functional mechanism for the role of 11beta-HSDI in salt-sensitive hypertension by investigating the effect of 11beta-HSDI suppression on renal tubular segmental fluid reabsorption in SS rats using micropuncture. Preliminary data has demonstrated the feasibility of the proposed studies and appeared to support the hypothesis. These studies will combine innovative molecular analysis/manipulation and functional assessment in genetically defined or modified animal models. The results are expected to elucidate a novel aspect of the pathophysiology of salt- sensitive hypertension and to provide potential new targets for prevention and treatment of hypertension.

DESCRIPTION (provided by applicant): Project Summary The goal of the proposed study is to identify renal microRNAs and their target genes that could potentially contribute to the development of salt-induced hypertension and renal injury in the Dahl salt-sensitive (SS) rat. The SS rat is a widely used animal model for human salt-sensitive forms of hypertension and renal injury, the molecular mechanisms of which are poorly understood. MicroRNAs are a class of small regulatory RNA, the discovery of which has been hailed as one of the most important breakthroughs in biology in recent years. MicroRNAs are encoded by specific genes in plant and animal genomes, and have been predicted to regulate the protein expression of thousands of mammalian genes, primarily through translational repression. Intensive research in the last two years has assigned important and diverse functions, ranging from the regulation of cell differentiation to insulin secretion, to several dozen mammalian microRNAs. The relevance of microRNA to complex mammalian physiology such as blood pressure regulation is unknown. We hypothesize that specific microRNAs, through the suppression of their target genes, are involved in the development of Dahl salt- sensitive hypertension and renal injury. The current proposal outlines the exploratory/developmental phase of the study that will achieve two goals: discovering renal microRNAs potentially relevant to the SS model, and identifying their target genes. In Aim 1, a custom-made microRNA microarray platform combined with a modified real-time PCR assay will be used to examine renal microRNA expression profiles in the SS rat. SS- 13BN, a consomic strain of rat with substantially attenuated hypertension and renal injury, will be used as the control. In Aim 2, target genes for differentially expressed microRNAs found in Aim 1 will be identified according to four criteria including 1) the presence of certain sequence and thermodynamic characteristics, 2) an inverse correlation between the levels of the microRNA and the target protein in SS and SS-13BN rats, 3) differences in the level of the target protein unexplainable by mRNA, and 4) up- and down-regulation of the target protein occurring when the microRNA is inhibited and overexpressed, respectively, in cultured rat cells. Preliminary studies indicate that 1) microRNA expression can be reliably analyzed using custom-made microarrays;2) differential expression of microRNA exists between SS and SS-13BN rats;3) many proteins differentially expressed between SS and SS-13BN are not explained by mRNA levels and are predicted targets for microRNAs;and 4) several genomic regions associated with SS phenotypes contain microRNA genes. The results of the proposed, exploratory study will become the foundation for future studies determining the in vivo pathophysiological significance of the identified microRNAs. Project Narrative Relevance Identification of specific microRNAs that are functionally important in the SS model will open new directions of research exploring the significance of non-protein-coding genes in complex mammalian physiology, and improve our understanding of the pathophysiology of hypertension and renal injury.

DESCRIPTION (provided by applicant): mRNAs through imperfect complementarities and reduce the level of target protein expression. Numerous studies have begun to reveal a critical and wide-spread role for microRNA in mammalian physiology and disease. The arguably most important yet challenging task in the field of microRNA research is experimental identification of specific targets of microRNA. It is relatively straightforward to identify microRNAs that are altered in a disease or experimental condition. But then investigators have to "guess" from hundreds or more computationally predicted targets of each microRNA what the real targets might be. The very small number of verified microRNA-target pairs available and the inability to nominate likely microRNA-target pairs severely limits the entire field of microRNA research. The 3'-UTR reporter analysis provides the most specific evidence for a microRNA-target interaction. However, without high-probability candidates, performing the 3'-UTR reporter analysis for a large number of predicted interactions would be prohibitively costly with limited benefits. Our laboratory has developed a new approach to nominate high-probability, physiologically relevant microRNA-target pairs. It combines proteomic, microRNA expression, and bioinformatic analyses to identify microRNA-target pairs supported not only by computational prediction but also by reciprocal expression. The approach has been applied to identify 98 such pairs in kidney regions or a cell model of epithelial mesenchymal transition (EMT). The findings provide a unique basis that makes it practical to verify a large number of microRNA-target pairs using the 3'-UTR analysis. We propose to develop a high-throughput 3'- UTR assay to examine the 98 microRNA-target interactions as well as approximately 1,350 additional predicted interactions (Aim 1). Preliminary study has verified a microRNA-target interaction using the assay. We will perform a proof-of-concept study (Aim 2) to examine the functional significance of selected microRNA target pairs verified in Aim 1. Preliminary study has identified one such microRNA, the inhibition of which significantly attenuated transforming growth factor [unreadable]1-induced EMT. The proposed study will directly address a critical barrier in a rapidly emerging field and could have far-reaching implications. 1) The large number, possibly hundreds, of verified microRNA-target pairs will provide an extensive, unique, and much-needed resource for microRNA research in any subject area, particularly in renal physiology and disease. 2) The study will allow us to evaluate and possibly improve various approaches for nominating microRNA-target pairs, providing methodological advances critical to the field of microRNA research. 3) The study may reveal novel mechanistic insights into the role of specific microRNAs in EMT and renal fibrosis that may be further investigated in future studies. PUBLIC HEALTH RELEVANCE: The result of the study in this R21 proposal may greatly facilitate future studies of microRNA in human health and disease and provide novel insights into the mechanisms underlying the development of chronic renal injury.

DESCRIPTION (provided by applicant): The annual meeting of the American Society of Nephrology (ASN) is the largest gathering in the world of clinical and basic scientists working in the renal field. Prior to each annual meeting the ASN sponsors an intensive basic science symposium whose topic, selected by the ASN's Basic Science Committee, reflects an area of investigation with major advances that impact the renal field. The conference is intended to foster new collaborations and promote applications of new technologies for studying the kidney. The 2011 Advances in Research Conference to be held in Philadelphia on November 8-9, 2011 is entitled Micro-RNA. The conference will feature 24 established investigators, most of them from outside the field of nephrology, who will speak on diverse aspects of micro-RNA research. The specific scientific themes of the meeting to be addressed by the Speakers include diverse aspects of micro-RNA research, including the basic biology of micro-RNAs (expression, processing, cellular action, and interacting proteins), significance of micro-RNAs in cardiovascular disease, cancer, and development, and state-of-the-art in vitro and in vivo methods for micro-RNA research. Studies of micro-RNAs in the kidney and hypertension began only very recently, yet the significance of this area is rapidly becoming clear. For example, micro-RNAs have been shown to be important for the development of glomerular podocytes, the development of renin-producing juxtaglomerular cells, the development of acute renal injury, diabetic kidney disease, hypertension, and renal interstitial fibrosis, and the regulation of normal renal physiology. At this conference, ASN members and other interested participants will have the chance to interact with world leaders in the field and will be exposed to state-of-the-art research. The purpose of this grant application is to request fund for junior faculty, postdoctoral fellow, and graduate student travel awards so that members can attend this conference prior to the ASN annual meeting in Philadelphia. PUBLIC HEALTH RELEVANCE: The goal of this annual conference is to update Renal Community researchers in a field that is instrumental to advancing our understanding of kidney function and/or disease. The focus of this year's conference is micro-RNAs. As usual, the majority of the speakers for the two day conference are top scientists from outside the Renal Community, who were selected for their scientific contributions in their respective fields. In the past, conference participants have developed collaborative relationships with some of the speakers, which greatly facilitates advancing kidney research and the potential to develop therapies that can improve the health of the population.

DESCRIPTION (provided by applicant): The goal of the proposed project is to understand the protein composition of ribonucleoprotein complexes containing microRNA miR-29b. MicroRNAs are endogenous, small regulatory RNA molecules. Numerous recent studies have demonstrated that microRNAs are important regulators of gene expression and play crucial roles in a wide range of physiological processes and diseases including cardiovascular and renal diseases. The effect of microRNAs on target genes is mediated by interactions of microRNAs with proteins that, together, form microRNA-containing ribonucleoprotein complexes (miRNPs). Despite the crucial and wide-spread functional role of microRNAs, the number of protein components of miRNPs that have been characterized is surprisingly small. The understanding of proteins interacting with specific microRNAs is particularly lacking, despite the recognition that different microRNAs may act differently. The shortage of knowledge in this critical area significantly hampers in-depth understanding of how microRNAs act, how the action of microRNAs is regulated, and how microRNAs contribute to the regulation of physiology and disease. We propose to develop a new approach for identifying protein components of miRNPs specifically containing miR-29b, a microRNA that we have shown to be functionally important in the protection against hypertensive renal injury. Aim 1 will develop the approach, identify the protein components, and provide initial characterization of the newly identified proteins. Aim 2 will examine the functional significance of the newly identified proteins. PUBLIC HEALTH RELEVANCE: The result of the proposed study could significantly improve our understanding of how the action of microRNAs is mediated and enable in-depth studies of how microRNAs contribute to physiological regulation and the development of disease.

The goal of Proiect 2 is to examine a novel role of microRNA miR-214 in the development of salt-sensitive hypertension and renal injury. Increased blood pressure salt-sensitivity is a prominent feature in certain populations of hypertensive patients, especially hypertensive African Americans. The molecular mechanism underlying salt-sensitive hypertension and renal injury remains largely unclear. The Dahl salt-sensitive (SS) rat is a widely used animal model of human salt-sensitive forms of hypertension and renal injury. The consomic SS.13[BN] rat and the congenic SS.13[BN26] rat have chromosome 13 (chr 13) or a segment of chr 13 from the Brown Nonway (BN) rat substituted into the SS genome. These rat strains exhibit significantly attenuated saltinduced hypertension and renal injury, and have been highly valuable for studying the disease phenotypes in the SS rat. MicroRNAs are endogenous, regulatory RNAs. Numerous recent studies have demonstrated that microRNAs represent one of the most important mechanisms for the regulation of gene expression and play crucial roles in a wide range of physiological processes and diseases including cardiovascular and renal functions. However, it remains largely unknown if or how microRNAs contribute to the regulation of arterial blood pressure or related tissue injury. We have found that microRNA miR-214, which is located in the SS.13[BN26] congenic region, was up-regulated in the kidneys of the SS rat compared to SS.13[BN26] and SS.13[BN26] rats. Several lines of exciting preliminary evidence suggest that miR-214 might play a significant and novel role in salt-sensitive hypertension and renal injury both in SS rats and in patients. We hypothesize that miR-214 contributes to the development of salt-sensitive hypertension and renal injury. We propose to test the hypothesis in four specific aims. Aim 1 will examine the functional contribution of miR-214 to salt-sensitive hypertension and renal injury in the SS rat. Aim 2 will investigate possible downstream mechanisms mediating the effect of miR-214. Aim 3 will examine upstream trans and cis regulatory mechanisms underiying the up-regulation of miR-214 in the SS rat. Aim 4 will complete a pilot study of the clinical relevance of miR-214 in human salt-sensitive hypertension and renal injury. Project 2 will use several state-of-the-art approaches and techniques to examine the innovative concept that non-protein-coding genes may play important roles in hypertension and related tissue injury. Project 2 will unitilize data from Project 1, feed new data into the network construction in Project 1, share technological advances in proteomics and promoter analysis with Project 3, and contribute to the overall goal of the PPG, which is achieving a significantly deeper understanding of the molecular pathophysiology of hypertension, renal injury, and angiogenesis. Project 2 will benefit greatly from genomic, genetic, proteomic, computational and animal research technological advances provided in Cores B and C.

PROJECT 2 PROJECT SUMMARY The goal of Project 2 in this PPG is to understand renal cellular metabolic mechanisms of salt-sensitive hypertension. Recent work, including work in this project in the current cycle of PPG, has discovered a novel role of fumarase (Fh1) and fumarate metabolism in hypertension in the Dahl salt-sensitive (SS) rat. Fumarase primarily catalyzes the conversion of fumarate to malate in the tricarboxylic acid (TCA) cycle in mitochondria. Fumarase enzyme activity is lower in the kidneys of SS rats than SS.13BN or Sprague-Dawley (SD) rats, and fumarate is higher and malate is lower in the kidneys of SS rats. Transgenic over-expression of fumarase on the background of SS rat (SSTgFh1) attenuates hypertension. Intravenous infusion of a fumarate precursor in SS.13BN rats exacerbates hypertension. It remains unknown 1) what mechanisms mediate the effect of fumarase-related metabolites on salt-sensitive hypertension, and 2) whether blood pressure salt-sensitivity in humans is associated with abnormalities in intermediary metabolism. Aim 1 of the proposed study will test a novel hypothesis that fumarase insufficiency contributes to salt-sensitive hypertension in SS rats in part by reducing arginine regeneration and nitric oxide (NO) levels in the kidney. Aim 2 studies are designed to identify metabolites associated with blood pressure in humans on low- or high-sodium intake. The proposed study could provide novel insights into the mechanisms by which fundamental metabolism contributes to hypertension. In addition, it could identify specific metabolic intermediaries that might be important for mediating the effect of dietary salt intake on blood pressure in humans.

DESCRIPTION (provided by applicant): The goal of the current project is to investigate the role of microRNA miR-29 in the development of hypertension and renal injury and understand the mechanisms involved. MicroRNAs are endogenous, regulatory RNAs that primarily reduce protein expression by binding to the 3'-untranslated region (UTR) of target mRNAs. Numerous studies have demonstrated that microRNAs are powerful regulators of gene expression and play crucial roles in a wide range of disease processes including several cardiovascular and renal diseases. However, the functional role of most microRNAs in the development of systemic hypertension and hypertensive tissue injury remains unknown. The Dahl salt-sensitive (SS) rat is a widely used animal model of human hypertension and related renal injury especially in African Americans. The consomic SS.13BN rat exhibits significantly attenuated hypertension and renal injury and has been used as a control for studying disease mechanisms in the SS rat. We have reported that microRNA miR-29b is down-regulated in the kidneys of SS rats on a high-salt diet compared to SS.13BN rats. miR-29 directly suppresses at least 16 genes related to extracellular matrix (ECM) and helps to prevent interstitial fibrosis in SS.13BN kidneys. Moreover, we found in ongoing human studies that miR-29b was dysregulated in the kidneys of patients with hypertensive renal injury. However, it remains unknown whether the insufficiency of renal miR-29 contributes to the development of hypertension and renal injury in the SS rat, whether renal miR-29 protects SS.13BN rats from developing hypertension, what mechanisms mediate any effect of miR-29 on hypertension, and how the expression of miR-29 is regulated in the kidneys of SS rats. We hypothesize that miR-29 insufficiencies, specifically in the kidney, contribute to the development of salt-induced hypertension and renal injury. In Aim 1, we will examine the functional role of miR-29 in the development of salt-induced hypertension and renal injury in the SS rat using tissue-specific miR-29 transgenic rats that we just developed and local knockdown of miR-29. In Aim 2, we will examine mechanisms underlying the regulation of miR-29 and its protective effect using several approaches including a molecular complex pull-out and deep sequencing method that we just developed. Exciting preliminary data support the feasibility of the proposed project.

? DESCRIPTION (provided by applicant): Microvascular endothelial dysfunction precedes the development of vascular disease and portends future adverse vascular events. Endothelial dysfunction is readily identified by a characteristic loss of nitric oxide (NO) bioavailability, whch results from the dysregulation of a wide variety of proteins and molecular pathways. However, we lack important knowledge of how these proteins and molecules are coordinately regulated. MicroRNAs have been demonstrated to act as master regulators of physiological or disease processes by coordinately targeting multiple genes involved in the process. However, the role of microRNAs in endothelial dysfunction, especially in the context of diabetes, remains largely unexplored. We have obtained preliminary data suggesting a homeostatic level of vascular miR-29b is critical for the maintenance of normal microvascular NO bioavailability and endothelium-dependent vasodilation in humans and animal models. Conversely, in type 2 diabetes mellitus, a disease typified by reduced microvascular NO bioavailability and microvascular morbidity, miR-29b could restore NO bioavailability and endothelium- dependent vasodilation. MiR-29b's impact on NO bioavailability appears to arise from its coordinated effects on several genes that alter NO bioavailability at multiple levels of regulation. We propose in this application to investigate the novel role of miR-29b in microvascular endothelium- dependent vasodilation and NO bioavailability in health and in type 2 diabetes and identify the mechanisms mediating these effects of miR-29b. The proposed project will employ a highly translational approach that combines functional studies of a newly developed knockout rat model with studies of intact human vessels obtained from well-phenotyped volunteers. We will also employ newly developed methods for identifying target genes and molecular pathways involved in the effect of miR-29b. Specifically, we will test the hypothesis that endogenous miR-29b is critical to maintaining normal endothelium-dependent vasodilation and NO bioavailability in resistance vessels in healthy humans and animals in Aim 1. Aim 2 studies will test the hypothesis that miR-29b can restore endothelium-dependent vasodilation and NO bioavailability in resistance vessels from T2DM patients and db/db mice. The molecular mechanisms underlying the role of miR-29b in endothelium-dependent vasodilation and NO bioavailability will be examined in Aim 3. The study will be carried out by a team of experienced researchers led by a physician scientist and a basic scientist who possess complementary expertise ideally suited for the proposed project. Successful completion of the project will reveal novel mechanisms regulating endothelial function and demonstrate their clinical relevance.

PROJECT SUMMARY Project II. MicroRNAs in Anesthetic Cardioprotection (PI: Mingyu Liang) The goal of Project II is to test the hypothesis that miR-21 contributes to cardioprotection conferred by anesthetics in animal models and in human cardiomyocytes. MicroRNAs are endogenous small RNA molecules that regulate a wide range of cellular functions primarily through reducing the abundance of target proteins. MicroRNAs have been shown to be powerful regulators of cardiac development, injury, and remodeling. However, the role of microRNAs in anesthetic cardioprotection and its impairment by diabetic conditions remains unknown. We and other investigators have reported that microRNA miR-21 contributes to ischemic preconditioning of the heart and the kidney and to xenon-induced protection of the kidney. Importantly, we have obtained very exciting preliminary data strongly supporting an important role of miR-21 in isoflurane-induced cardioprotection and its impairment by diabetic conditions in animal models and in human cardiomyocytes generated from non-diabetic individual-derived induced pluripotent stem cells (iPSCs) (N-CM) or from a type 2 diabetic patient-derived iPSCs (T2-CM). Based on these novel findings, we propose in Aim 1 to use gene knockout or transgenesis to further examine the role of miR-21 in anesthetic cardioprotection in animal models and in possible restoration of anesthetic cardioprotection in models of type 2 diabetes. In Aim 2, we will translate the findings to human using patient-specific cardiomyocytes including N-CM and T2-CM. Finally, in Aim 3, we will investigate the molecular mechanisms involved by examining miR-21 target genes and their downstream pathways linked to mitochondrial function and mitochondrial permeability transition pore dysregulation as well as additional mechanistic pathways. Project II shares the central theme of the PPG, which is to investigate cellular mechanisms underlying anesthetic cardioprotection and its impairment by diabetic conditions. Project II will interact extensively with Projects I and III by studying shared mechanistic pathways leading to mitochondrial dysfunction and cell death. Results of Project II will be used to test, validate, and extend the mathematical models developed in Project III.

CORE A PROJECT SUMMARY The Administrative Core (Core A) has two major functions: to provide an effective venue for efficient coordination of the projects and the scientific core to ensure the success of this PPG, and to provide administrative support to the Program Director and all three projects and the scientific core in this PPG to ensure effective and open communication, fiscal soundness, and regulatory compliance. Core A is a conduit for communication and coordination of this PPG with internal and external advisory boards and other stake- holders. The Administrative Core will be led by the Program Director who will be assisted by Core A staff including an experienced Program Coordinator. The Executive Committee, which comprises project and core leaders and essential co-investigators, is the primary vehicle that the Program Director / Administrative Core Leader uses to direct and manage this PPG.

PROJECT 1 PROJECT SUMMARY The vast majority of single nucleotide polymorphisms (SNPs) associated with complex human traits, including blood pressure (BP), are located in noncoding regions of DNA. These noncoding SNPs, especially the ones located in haplotype regions far from protein-coding genes, most likely influence BP by regulating gene expression. Expression quantitative trait locus (eQTL) studies are beginning to enable the identification of associations between noncoding SNPs and gene expression. However, very few studies have gone beyond associations to examine the functional effect of BP- associated noncoding SNPs on gene expression. Such studies would have to overcome substantial challenges. First, BP-associated noncoding SNPs could regulate the expression of distant protein-coding genes through chromatin folding or noncoding RNA, which means one cannot assume that the protein-coding genes that the SNPs regulate are the ones that are sequentially closest to the SNPs. Second, results from eQTL and other studies indicate the effect of noncoding SNPs on gene expression is often cell type-specific. In Project 1 of this PPG, we have developed several approaches and methods to overcome these challenges and to test the overall hypothesis that BP-associated noncoding SNPs regulate the expression of genes that are physiologically important to BP regulation. All three aims in Project 1 share the goal of identifying the effect of BP-associated noncoding SNPs on gene expression in BP-relevant cell types. Each aim will focus on a group of LD (linkage disequilibrium) regions located far from any protein-coding gene and a specific hypothesis. The mechanistic aspect of each hypothesis will be tested in Projects 2 and 3 of this PPG. The feasibility of Project 1 is supported by an extensive series of preparatory and preliminary studies. The overall goal of this PPG proposal is to identify genes regulated by human BP-associated noncoding SNPs, examining the underlying mechanisms, and investigating their influence on BP. Project 1 focuses on the first part of the overall goal and will contribute to the goal of the program through extensive integration with Projects 2 and 3. Project 1 will rely on Core B for extensive RNA-seq analysis.

OVERALL PROGRAM SUMMARY One of the most significant challenges for understanding genetic control of blood pressure (BP) is that the vast majority of BP-associated single nucleotide polymorphisms (SNPs) in humans are located in noncoding regions of DNA. Many of these noncoding SNPs are located in haplotype regions thousands of base pairs away from any protein-coding gene and their effects on BP cannot be explained by any currently known coding or other functional sequence variant, making it nearly impossible to link these noncoding SNPs to genes or physiological pathways that regulate BP based on genomic sequence. Understanding the effect of intergenic noncoding SNPs on gene expression and the underlying mechanisms is a major challenge not just for BP and hypertension research, but for research on nearly all complex traits and common diseases. The goal of this PPG proposal is to begin to address this major challenge and test the overall hypothesis that noncoding SNPs associated with human BP but located far from any protein-coding gene regulate gene expression in specific BP relevant cell types through epigenetic mechanisms and these mechanisms can influence BP. We have developed three projects that each address one aspect of this overall hypothesis. Project 1 will use precision genome editing to identify the effect of specific BP- associated noncoding SNPs on gene expression in BP-relevant human cell types. Project 2 will test the hypothesis that BP-associated noncoding SNPs influence the expression of BP-relevant genes through epigenetic mechanisms including chromatin looping, enhancer function and noncoding RNA in human cells and tissues. Project 3 will take this line of research to animal models in vivo to test directly the novel hypothesis that chromatin conformation plays a role in BP regulation. The three projects will interact with, and inform, each other extensively and, together, will achieve the overall goal of the program. All three projects will rely on Core A for administrative support and Core B for sequencing coordination and data analysis. We have published at least 16 papers in the last few years that provide direct support for key aspects of the conceptual validity and technical feasibility of this PPG. In addition, we have obtained a large amount of preliminary data to further support the feasibility of the wide range of sophisticated and new technologies that we will use and the validity of proposed novel hypotheses. This PPG represents a fundamentally new direction for hypertension research. It will establish several novel approaches and technologies, generate unique and extensive datasets, and provide new biological insights, all of which will help to advance genetic and epigenetic research in hypertension and other disease areas.

PROJECT SUMMARY More than 50% of hypertensive patients show an increased blood pressure sensitivity to salt intake. However, population-wide reduction of salt intake has proved to be difficult, making it ever more important to better understand the mechanism of salt-sensitive hypertension and provide a basis for developing new interventions. The kidney plays a key physiological role in the development of hypertension including salt- sensitive hypertension. Thousands of genes in the genome encode long non-coding RNAs (lncRNAs). lncRNAs can interact with and influence the function of other RNA or proteins. Few lncRNAs have been studied for their role in hypertension. Unlike most lncRNAs, MALAT1 (metastasis associated lung adenocarcinoma transcript 1; Malat1 in rodents) is conserved across many species and expressed at high abundance levels in several tissues including the kidney, which suggests MALAT1 might be important physiologically. However, MALAT1?s physiological and pathophysiological role remains largely unknown. We discovered recently that renal miR-214-3p targets and suppresses endothelial nitric oxide synthase (eNOS) directly, which contributes significantly to the development of salt-sensitive hypertension in rat models and possibly humans. This was supported by a systematic analysis of human sequence variants and all miRNA precursors, small RNA deep sequencing in human kidney biopsy specimens, kidney-specific inhibition of miR- 214-3p in Dahl SS rats, and a newly generated mutant rat strain. The Dahl SS rat is the model most widely used to study the molecular mechanism of human salt-sensitive hypertension. We have obtained a large series of preliminary data that suggest MALAT1 might be dysregulated in the kidneys of salt-sensitive humans and SS rats and might influence the development of salt-sensitive hypertension by regulating the renal miR-214-3p/eNOS pathway. We propose to investigate MALAT1?s role in the development of salt-sensitive hypertension (Aim 1), the role for the renal miR-214-3p/eNOS pathway in the effect of MALAT1 on hypertension (Aim 2), and the underlying molecular interactions (Aim 3). We will achieve these aims by using analysis of scarcely available human samples, combinatorial gene manipulation in animal models, and new methods including genome editing and RafTOP (rapid freezing with tagged oligonucleotide pullout). RafTOP is a method for identifying the native interactome for a specific RNA that we developed recently.