Anupam Agarwal - US grants

The major objective of this research program is to provide the applicant with new and intensive training in cell and molecular biology. To achieve this goal the applicant will train in the rich scientific environment of the University of Florida, under the close sponsorship of Dr. Harry Nick, in the Department of Biochemistry and Molecular Biology and the expert guidance of Dr. C. Craig Tisher, Chief, Division of Nephrology. The long-term goals of this proposal are to elucidate the cellular and molecular pathogenesis of renal injury, particularly those mechanisms that underlie the progression of renal disease. Renal tubular epithelial cells are exposed to an array of macromolecules including lipoproteins (LDL) that leak into the urinary space, consequent to proteinuria and disordered lipid metabolism, which occur in the nephrotic syndrome (primary and secondary). Oxidation of LDL (LDLox) is pathogenic in atherosclerosis and glomerulosclerosis. Preliminary studies demonstrate that oxidative processes are involved in LDLox-mediated renal tubular cytotoxicity and are associated with marked cellular expression of heme oxygenase, a redox sensitive gene, which is an adaptive response in cells exposed to, a wide variety of oxidant stress. The molecular mechanisms controlling the induction of heme oxygenase in a cell model of LDLox- mediated renal tubular injury will he examined, The proposed experiments will be the basis for training the applicant in molecular biology techniques such as nuclear run-on transcription assays, recombinant DNA, PCR, promoter deletion analysis, transfection studies and to study DNA-protein interactions by in vivo genomic footprinting. The observations in this cell model will be applicable to renal diseases such as diabetic nephropathy and nephrotic syndrome due to other causes. These studies would attempt to establish a pathogenic link between proteinuria and tubulointerstitial disease through the potential harmful effects of LDLox and may aid in developing new therapeutic interventions for modifying the progression of renal disease. Using this comprehensive training in later, independent research, the applicant will further investigate gene regulation in other animal models of renal injury as well. This phase of the applicant's scientific development will be greatly fostered by the ongoing sponsorship and guidance of Dr. Harry Nick and Dr. C. Craig Tisher.

This proposal is in response to PAR-98-087, "Small grant program for K08 recipients" and is linked to NIH K08 02446. The studies outlined in this proposal will evaluate the molecular mechanisms involved in the upregulation of heme oxygenase-1 (HO-1), a redox sensitive and cytoprotective gene, by oxidized low density lipoprotein (LDLox). LDLox is implicated in the pathogenesis of atherosclerosis and progression of renal disease. Previous studies have demonstrated that exposure to LDLox results in the induction of HO-1, in both renal epithelial and aortic endothelial cells. Induction of HO-1 is considered an adaptive response in cells and tissues exposed to oxidative stress of a diverse nature. LDLox is a complex structure consisting of several chemically distinct components including fatty acid hydroperoxides, modified apoB, oxidized sterols and phospholipids. These preliminary studies are the first to identify linoleyl hydroperoxide (LAox), an oxidized C:18 containing fatty acid, as the major component of LDLox responsible for HO-1 induction. Further, such induction is via increased HO-1 gene transcription through mechanisms different from known inducers of the gene. A approximately 4.5 kb HO-1 promoter which responds to heme and cadmium, known inducers of the gene, does not contain the cis-acting elements necessary for LAox-dependent gene induction. The goals of this proposal will be to delineate the region of the HO-1 gene that controls LDLox-mediated induction and will require further experiments using chromatin structure analysis and additional promoter transfections. Specific Aim I will identify specific locations of DNA-protein interactions that alter chromatin structure of the human HO-1 gene under basal and LAox-stimulated conditions. These studies will identify DNA sequences that could exist anywhere outside of the 4.5 kb HO-1 promoter that has already been analyzed. The results from chromatin structure analysis will aid in promoter deletion studies (Specific Aim II) using a larger (approximately 18 kb XhoI/XhoI) 5' upstream region of the HO-1 gene that has been characterized from a human P1 phage clone. These two additional specific aims are crucial preliminary data for completion of Specific Aim III of the original K08 grant application that will evaluate DNA-protein interactions by in vivo genomic footprinting to identify binding sites for cis-acting regulatory sequences on the human HO-1 gene. These studies have a potential application to both renal and cardiovascular diseases, wherein LDLox is an important mediator and may aid in developing new therapeutic interventions for modifying the progression of renal disease and atherosclerosis. This grant will also enhance research productivity, fiscal independence and career development of the applicant towards becoming an independent investigator.

DESCRIPTION (provided by applicant): The long-term objectives of this proposal are to understand the molecular mechanisms that regulate expression of the human heme oxygenase-1 (HO-1) gene in acute kidney injury. HO-1 is a key regulator of several important biological processes due to the anti-oxidant, anti-inflammatory and anti-apoptotic effects of its reaction products, namely carbon monoxide and biliverdin/bilirubin. Induction of HO-1 is an adaptive and protective response in acute kidney injury following ischemia-reperfusion, nephrotoxins, transplantation, acute glomerulonephritis and rhabdomyolysis as well as several non-renal settings of injury. During the current project period, we have characterized a novel intronic enhancer in the human HO-1 gene that, in conjunction with critical regulatory sites in the promoter, regulates gene expression by heme and nitric oxide, two major stimuli that are implicated in the pathophysiology of acute kidney injury. The transcription factors belonging to the Jun family and upstream stimulatory factor associate with the HO-1 promoter and mutations of the DNA-protein contact points in the HO-1 promoter significantly decrease enhancer function. The hypothesis of this renewal application is that heme oxygenase-1 induction in renal epithelial cells is a protective response in acute kidney injury and occurs through interaction of an intronic enhancer with promoter elements in the human HO-1 gene. In Aim 1, the location and level of HO-1 that is protective in acute kidney injury will be determined by both in vitro and in vivo approaches. In Aim 2, the interaction between the internal enhancer and regulatory sequences in the HO-1 promoter will be examined using the 3C (Chromosome Conformation Capture) and chromatin immunoprecipitation assays. In Aim 3, DNA-protein interactions will be tested in models of acute kidney injury in vivo in luciferase reporter mice driven by human HO-1 promoter/enhancer constructs. By understanding the molecular mechanism(s) that underlies the activation of the human HO-1 gene, we plan to develop specific molecular reagents to manipulate endogenous HO-1 gene expression and exploit its protective effects in renal pathophysiologic states. These studies will also be applicable to other disease settings such as atherosclerosis, sepsis and transplant rejection wherein HO-1 plays an important protective role. PUBLIC HEALTH RELEVANCE The goals of this proposal are to understand the molecular mechanisms that regulate expression of the human heme oxygenase-1 gene in acute kidney injury. The results of these studies will also be applicable to other disease settings such as atherosclerosis, sepsis and transplant rejection wherein heme oxygenase-1 plays an important protective role.

DESCRIPTION (provided by applicant): Induction of heme oxygenase-1 (HO-l) is an adaptive and cytoprotective response in cells and tissues exposed to oxidative stress of a diverse nature. Recent studies have demonstrated the importance of HO-l expression in atherogenesis. Oxidized LDL (oxLDL) is implicated in the pathogenesis of atherosclerosis, a major cause of morbidity and mortality, specifically in patients with predisposing factors such as diabetes mellitus, hypertension, hyperlipidemia, renal disease, and obesity. Our previous studies have demonstrated that exposure of human aortic endothelial cells to oxLDL results in the induction of HO-l. OxLDL is a complex structure consisting of several chemically distinct components. Our preliminary studies have identified linoleyl hydroperoxide (13-HPODE, LAox), an oxidized C: 18 containing fatty acid, as the major component of oxLDL responsible for HO- 1 induction. Most importantly, such induction occurs via increased HO-l gene transcription through molecular mechanisms different from known inducers of the gene. The studies in this proposal will evaluate the biological role of HO-l induction in response to oxLDL both in vitro and in vivo using HO-l knock out mice as well as delineate the regulatory elements that control oxLDL- mediated HO-1 gene expression in human aortic endothelial cells. Aim IA will evaluate the effects of an atherogenic diet in HO-l deficient mice in vivo. Aim IB will involve in vitro experiments in a model of oxLDL-induced injury in endothelial cells derived from the HO-l deficient mice. Aim IIA will evaluate specific regions of oxLDL-inducible altered chromatin structure of the human HO-l gene. Aim IIB will involve studies to characterize the oxLDL-responsive element using luciferase and human growth hormone reporter genes. The studies outlined in Aim IIIA will evaluate DNA-protein interactions at the single nucleotide resolution by in vivo footprinting and Aim IIIB will evaluate the functional significance of potential DNA-protein binding regions by site-directed mutagenesis. These studies have a potential application for the development of novel molecular approaches to manipulate expression of the human HO-l gene and thus exploit its cytoprotective effects in atherosclerotic cardiovascular diseases, wherein oxLDL is an important mediator.

DESCRIPTION (provided by applicant): The goals of this pilot proposal (PA-01-127, Pilot and Feasibility Program related to the Kidney) are to explore the ability of the different serotypes of recombinant adeno-associated viral (rAAV) vectors to enhance the efficacy of gene delivery to the kidney, particularly the intra-renal vasculature. While kidney transplantation remains the treatment of choice for end stage renal disease, chronic vascular injury secondary to transplant rejection, cyclosporine toxicity, hypertension and recurrence of disease all contribute to graft loss, necessitating return to dialysis or re-transplantation. Specific approaches that target "protective" genes into the kidney and more importantly the intra-renal vasculature would be highly beneficial in prolonging graft survival. However, most of the previous studies in this area of investigation have demonstrated limited success due to the transient nature of transgene expression and the development of adverse host immune responses. The hypothesis of this application is that the efficacy of gene delivery to the vasculature and the kidney is enhanced by the use of recombinant adeno-associated viral (rAA V) serotype 1 and 5 vectors. This hypothesis will be tested by (1) exploring the efficacy of gene delivery to the vasculature and the kidney using rAAV serotype 1 and 5 vectors in comparison to rAAV type 2, the prototypic serotype used in most gene therapy studies to date, and (2) evaluating the role of a protective gene (heme oxygenase-1, HO-1) in chronic transplant rejection using the ideal rAAV serotype. In preliminary studies, we have observed that in comparison to rAAV2, rAAV1 and rAAV5 demonstrate markedly higher transduction efficiencies at lower levels of multiplicity of infection in human vascular endothelial and smooth muscle cells. Initial studies will be performed using reporter genes, followed by transduction using rAAV vectors containing a protective gene, HO-1. The effects of rAAV-mediated HO-1 gene delivery will be evaluated in a rat aortic allograft model of chronic vascular rejection, followed by studies in a rat model of renal transplantation. These studies have important therapeutic implications in limiting vascular injury that occurs not only in renal transplantation but also in native kidney diseases secondary to diabetes and other renal diseases.

DESCRIPTION (provided by applicant): At present approximately 60,000 patients with chronic kidney disease are awaiting a kidney transplant but only 14,000 patients receive a graft each year due to a shortage of donor organs. Despite significant progress in the field of organ transplantation, loss of transplanted kidneys remains a major problem, largel. y due to accelerated vascular disease ("transplant arteriosclerosis") as the result of vascular endothelial and smooth muscle cell proliferation. This is propagated by multiple factors including endothelial injury due to immune and non-immune processes, drug nephrotoxicity and hypertension and leads to progressive narrowing of the vascular lumen, ischemia, fibrosis and loss of kidney function. Given the extent of the problem, a molecular approach to inhibit vascular inflammation and neointimal proliferation would effectively ameliorate chronic vascular rejection and prolong graft survival, limiting the need for subsequent retransplantation or the return to dialysis. Our preliminary studies demonstrate a pivotal role for interleukin-10 (IL-10) in inhibiting vascular neointimal proliferation and inflammation in a rat model of chronic vascular rejection. The vascular protective effects of IL-10 are mediated via induction of heme oxygenase-1 (HO-1), an enzyme with recently recognized anti-inflammatory, anti-apoptotic and immunomodulatory functions. The overall hypothesis of this proposal is that IL-10 prevents chronic vascular rejection by inducing HO-1 expression inhibiting thereby neointimal proliferation and inflammation. To address this central hypothesis, four aims are proposed. The studies in Aim 1 will examine the role of HO-1 in mediating the effects of IL-10 on vascular neointimal proliferation and inflammation in aortic and kidney transplantation using HO-1-/- and HO-1+/+ recipient mice. The experiments in Aim 2 will determine the relative contribution of HO-1 expression in infiltrating hematopoietic cells versus locally in the graft vasculature in mediating the protective effects of IL- 10. In Aim 3, the hematopoietic cell lineage required for the effects of IL-10 will be determined using adoptive transfer of specific cell populations from HO-1 and HO-1+/+ mice following aortic and kidney transplantation. Preclinical studies will be performed in Aim 4 to determine the link between IL-10 and HO-1 in an existing population of stable kidney allograft rhesus macaques that exhibit high levels of IL-10 and have no evidence of chronic vascular rejection. Lay Summary: Loss of kidney transplants due to progressive narrowing of blood vessels in the kidney is a significant clinical problem. The studies in this project will provide insights into potential novel therapies to prevent this condition and, hence, prolong survival of kidney transplants.

[unreadable] DESCRIPTION (provided by applicant): Transforming growth factor-B (TGF-B) is a regulatory cytokine that is implicated in a variety of kidney diseases where it promotes extracellular matrix deposition and fibrosis. Paradoxically, TGF-B has a critical role in normal homeostasis and stabilizes and attenuates tissue injury through mechanisms that are not clearly understood. We previously reported that, in renal tubular epithelial cells, TGF-B1 is a potent inducer of a cytoprotective enzyme, heme oxygenase-1 (HO-1). The overall hypothesis of this proposal is that HO-1 induction is a cytoprotective response to TGF-B1mediated cellular injury in the kidney. The mechanisms underlying the induction of HO-1 by TGF-B as well as the effects of HO-1 expression and its products on the renal epithelial cellular responses to TGF-B1 are not known. In preliminary studies, we have observed that kidneys from HO-1-/- mice (age 24 weeks) show significantly higher fibronectin expression than age matched HO-1 () mice. In contrast, HO-1 induction is associated with decreased levels of fibronectin. Furthermore, bilirubin, 1of the products of the HO-1 catalyzed reaction, significantly lowers TGF-B1-mediated increases in fibronectin levels in renal tubular epithelial cells. Our studies also demonstrate that the inhibitory Smad, Smad7, blocks HO-1 induction by TGF-B1 in renal epithelial cells. The in vivo relevance of the inverse relationship between Smad7 and HO-1 was also observed in kidneys from TGF-p overexpressing mice. TGF-B1-mediated HO-1 induction is transcriptionally regulated and requires a cis-acting region between-9.1 and-9.4kb of the human HO-1 promoter and may involve Sp1-like sequences. This proposal will evaluate the biological role of HO-1 induction in response to TGF-B1 in vitro, using HO-1 deficient cells (primary cells derived from HO-1 -/-, +/+ mice and HO-1 knockdown using siRNA in HK-2 cells) as well as HO-1 overexpressing HK-2 cells (Aim I). HO-1 -/- mice and their littermates will be used to study the role of HO-1 and HO reaction products in the pathogenesis of fibrosis in a model for obstructive uropathy and renal fibrosis, the unilateral ureteral obstruction (DUO) model (Aim II). Finally, the molecular regulation of TGF-B1 -mediated HO-1 gene expression will be characterized (Aim III) and integrated with Aim 1 by testing the effects of overexpression or dominant negative expression of the protein/transcription factor identified on the cellular responses to TGF-B1. Studies to understand the molecular mechanisms involved in the induction of HO-1 by TGF-B1 are timely and relevant for efforts to fine tune endogenous HO-1 activity in renal injury. [unreadable] [unreadable] [unreadable]

PROVIDED. The University of Alabama at Birmingham (UAB)-University of California at San Diego (UCSD) O'Brien Core Center for Acute Kidney Injury (AKI) will establish an interdisciplinary center of excellence in AKI related research. The main objective of this core center is to provide scientifically rigorous, state-of-the-art methodologies in a cost-effective manner to address experimental questions that will advance our understanding of the pathophysiology of AKI, enhance our diagnostic specificity and expand our therapeutic and preventive approaches for AKI, specifically in the intensive care unit and in the setting of kidney transplantation. This objective will be implemented in three specific aims: (i) Facilitate hypothesis-driven research through the support of shared core facilities and to leverage these core technologies into new projects, interactions and collaborations in AKI-related research, (ii) Foster meaningful interactions among 43 UAB-UCSD investigators from several different disciplines and extend these interactions to include a cadre of 55 investigators from multiple institutions at the regional, national and international levels - an extended research base and (iii) Provide, through the Biomedical Research Cores, a Pilot and Feasibility Program (PAF) and the Scientific Enrichment program, the intellectual resources and the research infrastructure to attract new and established investigators to AKI research. The core center investigators will benefit from access to a set of three complementary Biomedical Research Cores that will integrate existing intellectual and technological resources of UAB and UCSD and provide a defined set of services that will facilitate the research of investigators pursuing AKI-related basic and clinical research. The proposed Biomedical cores are: 1) Core A - Resource for Clinical Studies of AKI (clinical research, genomics and biorepository); 2) Core B - Resource for Pre-Clinical Studies of AKI (animal models, small animal imaging and physiology); 3) Core C - Bioanalytical Resource (proteomics/oxidative stress markers/molecular pathology). The Center includes a Biostatistical/Bioinformatics Resource that will provide support to the cores and pilot projects. The center also includes four well-designed PAF projects, each of which uses innovative approaches to address important mechanistic questions in AKI. An Educational Enrichment program will facilitate collaborative interactions among the research base.

DESCRIPTION (provided by applicant): This is a request for partial funding of the 6th International Conference on Heme Oxygenases "Heme Oxygenases in Biology and Medicine" scheduled for September 30th to October 4th, 2009 at Miami Beach, Florida. The goals of this conference are to attract and provide scientific direction to a wider group of young investigators and trainees to address both basic science and focused translational issues related to the heme oxygenase system;and to bring together investigators who will benefit from interdisciplinary cross-talk, thereby enabling scientific interaction and collaboration that would not have otherwise occurred. The conference addresses state-of-the-art topics related to heme oxygenases and will include new and cutting-edge advances in the field. The topics included in the program have not been covered by other conferences in the past two years. The program committee and invited speakers are broadly based between MD and PhD investigators and has diversity in gender and racial background. The incorporation of a "hot-topic" session is based on the best abstracts submitted to the meeting. The choice of Miami Beach offers a wonderful environment where established and young investigators and trainees can meet in an informal setting to exchange ideas. Also, by being readily accessed from Europe and Asia, the opportunity for a true international conference will be offered. Thus, facilitating attendance from those parts of the world, particularly young investigators, who otherwise may not be able to afford more distant and costly travel to attend such a conference. Since the 5th International Conference on Heme Oxygenases in 2007, there have been significant advances in the field including new discoveries, the availability of innovative tools and reagents, and results of pre-clinical and clinical trials becoming available. This meeting will therefore provide a forum for easy dissemination of knowledge and exchange to further advance the field.

DESCRIPTION (provided by applicant): The primary goal of this ongoing interdisciplinary training program, currently in the 25th year, is to bring together pre-clinical scientists and translational investigative researchers to catalyze the transformation of postdoctoral students from both basic science and clinical disciplines into independent academic faculty committed to kidney-relevant research. The training program builds on new initiatives in renal pathophysiology, cell and molecular biology, vascular biology, basic immunology, genetics, transplantation, acute and chronic kidney disease and in clinical and translational research. The UAB Clinical and Translational Science Award and the Office for Postdoctoral Education provide essential facilities that embrace trainee education as well as mentor training, also a major goal of this program. The Program accepts PhD and MD scientists from a large applicant pool that now includes physicians in the American Board of Internal Medicine Research pathway, which recruits highly meritorious candidates to pursue a career in academic medicine. Twenty-two (50%) of our 44 trainees are in academia, 20% are still pursuing post-graduate training and 7% have pursued non-academic positions, although still in science-based careers. Trainees have been successful in obtaining 20 highly competitive extramural grants, including 13 career development awards. During the past 10 years, 18 trainees have published 68 peer- reviewed publications including papers in high-impact journals. The program has benefitted from a multidisciplinary collaborative faculty from 9 Departments (Medicine, Cell Developmental and Integrative Biology, Epidemiology, Microbiology, Emergency Medicine, Genetics, Pathology, Pediatrics, Surgery) and robust institutional infrastructure and support. The strong commitment of the 34 preceptors, organized in four thematic areas - renal physiology and pathophysiology, epithelial biology, vascular biology related to kidney disease and clinical and translational research - who are actively involved in the training of young scientists in the use of basic and applied approaches are also strengths of this training grant. The collaborative environment of our institution, embodied in the University-Wide Interdisciplinary Research Centers Program, the Nephrology Research and Training Center, and the NIDDK-funded O'Brien Center, provides an ideal setting for the implementation of interdisciplinary kidney-related research and training. Based on the accomplishments during this cycle, continued support of four postdoctoral trainees is being requested in this competing renewal application.

DESCRIPTION (provided by applicant): The heme oxygenase-1 (HO-1) system consists of the enzyme heme oxygenase-1 and the products of the degradation of heme, carbon monoxide, biliverdin, and iron. Overexpression of HO-1 protects cells and tissues from immune-mediated injury as well as oxidative damage, indicating that HO-1 and its products are an important immunoregulatory mechanism. The mechanisms of this regulation are unknown. We have shown that the absence of HO-1 strongly affects differentiation of resident dendritic cell (DC) subsets in vivo and abrogates regulatory T cell mediated suppression of T cell activation by DC in vitro. These findings support the concept that modulation of the immune response, and therefore allograft rejection, is through the effect of HO- 1 on the differentiation and function of DCs. In support of our overall goal of identification of unique molecular targets for modulation of immune responses, we propose to determine how host HO-1 expression affects the differentiation of resident DC subpopulations and how expression of HO-1 by DCs modulates inflammatory responses in vivo in a renal allograft model of chronic rejection. This objective will be pursued using unique tools developed by the PIs, including i) a transgenic floxed HO-1 mouse strain permitting selective expression of HO-1 by cross-breeding with the appropriate cre recombinants; a CD11c-DC specific HO-1 overexpressing strain; ii) a GFP+ HO-1-/- mouse strain; and iii) an orthotopic murine renal transplantation model of chronic allograft nephropathy (CAN) with features that resemble human CAN. We will address the following specific aims: 1) to test the hypothesis that HO-1 regulates the distribution and differentiation of DC subpopulations in the peripheral lymphoid organs; 2) to test the hypothesis that HO-1 regulates the development of inflammation, fibrosis and vascular disease in CAN by modulation of DC activation and homing in renal allografts; 3) to determine the therapeutic potential of manipulation of HO-1 expression in DCs for kidney allografts by testing the hypothesis that HO-1 overexpressing DC will prolong graft survival, improve renal function and alleviate histological features of CAN. Current research and paradigms with respect to the control of the immune response to solid organ allografts are centered on control of T cell activation and clonal expansion. This approach has been successful in reducing acute allograft rejection, but has not made satisfactory inroads into long-term graft losses due to chronic rejection. The proposed studies are designed to address this need by examining the role of a unique molecular target, heme oxygenase-1 (HO-1), which is part of a group of stress proteins shown to provide cytoprotection. HO-1 is unique among these proteins because of its association with immunomodulation, and protection against fibrosis and vascular disease. Safe, effective therapeutic application of this molecule, effected by manipulation of HO-1 activity or the relative abundance of its substrates and products, requires a much better understanding of its role in immunity and long-term graft survival.

The University of Alabama at Birmingham (UAB)-University of California at San Diego (UCSD) O'Brien Core Center for Acute Kidney Injury (AKI) is designed to provide scientifically rigorous, state-of-the-art methodologies in a cost-effective manner to foster investigations that will (i) advance our understanding of the pathophysiology of AKI, (ii) enhance our diagnostic specificity and (iii) expand our therapeutic approaches for patients with AKI. The administrative core will coordinate and integrate the diverse activities of the UAB-UCSD O'Brien center, facilitate interactions and collaborations among the research base, ensure quality control of the core services and promote scientific development. The administrative core includes a Biostatistical Resource (BR), which will provide statistical support for the cores and research and pilot projects of the O'Brien Center. The administrative structure with clear lines of authority has been developed to ensure that the goals and objectives of the center are achieved. The Director and Associate Directors will be advised by an Internal Advisory Committee, who were carefully selected based on their experience in directing high quality large research programs and include the Directors of the Clinical and Translational Science Awards (CTSA) at both UAB and UCSD. In addition, the Center leadership will be advised by an External Advisory Committee, a Pilot and Feasibility (PAF) Program Committee and a Scientific Advisory Committee for each Core. The Administrative Core will continue to oversee the PAF Program and will be responsible for evaluating the Pilot Program's efficacy in promoting high quality AKI-related research that leads to extramural funding and publications. In addition, the Administrative Core will organize and support an enrichment program consisting of seminars with visiting speakers, journal clubs/work-in progress sessions, interface with CTSA enrichment programs. Summer Student's Training Program, and an Annual Comprehensive Research Symposium. The Administrative Core has also established a communication network through email, newsletters, live video conferencing and a website to inform Core Center investigators of the center activities and funding opportunities, AKI-related news, as well as the availability of new reagents and technologies through the Center. Therefore, the Administrative Core will enable optimal coordination of the various Core Center components through its committees, regularly scheduled meetings, seminar series, web-based communication mechanisms and video conferences between participating institutions.

DESCRIPTION (provided by applicant): Bromine (Br2) is a halogen used as a water disinfectant, for bleaching fibers, in the manufacture of antiepileptic drugs, dyestuffs, flame retardants, insecticides, drilling fluids, and gasoline additives When inhaled at higher concentrations, as may occur during transportation accidents, transfers among containers or acts of terrorisms, Br2 has caused death from respiratory failure. There are very few published studies evaluating acute and chronic sequelae of Br2 inhalation; treatment remains symptomatic and no effective countermeasures exist. Our exciting and highly novel preliminary data show that mice overexpressing the human form of the heme oxygenase (HO)-1 gene and protein (hHO-1 BAC) exhibit significantly lower mortality when returned to room air post Br2 exposure as compared to their wild-type littermate controls. In contrast, mice deficient in the HO-1 gene (HO-1-/-), display markedly increased mortality following Br2 exposure. We have also identified five compounds that are potent inducers of the human HO-1 gene using high-throughput screening. The goals of this application are: (1) to establish the role of HO-1 in protecting mice from Br2 induced injury and (2) test the efficacy of these compounds, administered in mice systemically, post Br2 exposure, to upregulate HO-1, and decrease mortality and lung injury. SA #1. We will expose hHO-1 BAC, HO-1-/- mice and their wild-type littermates to Br2 (600 ppm for 30 min) in environmental chambers, and return them to room air and measure mortality and lung injury for two weeks. SA #2: (i) We will expose confluent monolayers of human airway Clara-cell like cells (H441) to Br2 and return them to room air; we will then test the efficacy of the five compounds identified by high throughput screening, to increase HO-1 and decrease Br2 induced cellular necrosis and apoptosis. (ii) We will profile these compounds to determine those with the best pharmacokinetic properties related to intramuscular dosing. (iii) We will inject hHO-1 BAC and wild-type mice intramuscularly with the compound that exhibits the best cytoprotective effects in vitro and measure lung mRNA and hHO-1 activity at two, 24 and 72 post injection. (iv) We will expose hHO-1 BAC, HO- 1-/- mice and their wild-type littermates (all in C57BL/6 background) to Br2 (600 ppm for 30 min), return them to room air and inject them intramuscularly starting at 2 h post-exposure with this compound. We will measure mortality in all groups and indices of lung injury and lung hHO1 activity for 14 d as described in SA #1.

Arsenicals are an important category of chemical weapons due to their devastating effects on the skin as well as systemic effects damaging multiple organs including the kidney and lung. This project is based on our findings that cutaneous exposure to lewisite, an arsenical first synthesized during world war I, not only damages skin but is also rapidly absorbed and exerts toxic effects in the kidney leading to both acute and delayed kidney damage. Preliminary studies demonstrate that arsenicals cause epigenetic histone remodeling by hyperacetylation and recruitment of BRD4 to promoter regions of inducible genes associated with inflammation and tissue damage. BRD4 is a member of the bromo- and extra-terminal domain family of proteins. In addition, we observed marked upregulation of the cytoprotective protein, heme oxygenase-1 (HO- 1) in the kidney following topical exposure to lewisite. Arsenicals induce higher expression of BRD4 and inflammatory signaling genes in HO-1 knockout mice as compared to wild-type littermates, suggesting the importance of HO-1 in epigenetic regulation of inflammatory responses. Taken together, these studies underscore the significance of both acute and delayed kidney damage following a single cutaneous arsenical exposure and identify two potential inter-related molecular targets, BRD4 and HO-1 in renal injury. The overall goal of this project is to develop mechanism-based post-exposure countermeasures that can mitigate arsenical-induced kidney damage. Our hypothesis is that toxic doses of arsenicals cause acetylation of proteins (histones) and subsequent recruitment of bromodomain proteins resulting in activation of injury pathways and that blocking bromodomain signaling or its downstream effectors can mitigate kidney injury. In Aim 1, an arsenical mediated murine model of AKI will be characterized to determine the dose- and time-dependence of kidney damage. In Aim 2, we determine the mechanisms by which arsenicals cause AKI focusing on BRD4 and HO-1 for intervention in arsenicals-induced AKI. In Aim 3, we will develop targeted therapeutic intervention in arsenical-induced AKI to determine the optimal window for the beneficial effects by post-exposure treatment in animals exposed to arsenicals. Both FDA approved and novel small molecules will be assessed in this aim. Successful completion of our research as proposed here will not only provide an effective antidote for chemical injury but will also contribute to a broader understanding of how endogenous epigenetic responses can be exploited towards developing new therapeutic strategies for AKI.

Project Summary Kidney diseases in general and acute kidney injury (AKI) in particular impose substantial morbidity and mortality; these remain unabated in recent decades despite concerted efforts to reduce both. The overall mission of the University of Alabama at Birmingham (UAB)-University of California San Diego (UCSD) O'Brien Center for AKI Research is to improve the health of patients by fostering research specifically targeted to the prevention and treatment of AKI and its complications. To achieve this mission, this Center crosses institutional boundaries and research fields to harness the momentum and ingenuity of ongoing pre-clinical and clinical research in both institutions. The following aims are proposed: i) facilitate hypothesis-driven research through shared core facilities and leverage these technologies into new projects and collaborations, (ii) foster relevant interactions among UAB-UCSD investigators from different disciplines and extend these interactions to investigators from multiple institutions, (iii) provide, through the Biomedical Research Cores, a Pilot and Feasibility Grants (PAF) Program, and attract new and established investigators to AKI research by capitalizing on our research cores and infrastructure, (iv) build upon the progress during the last cycle by responding to the evolving needs of our investigators, (v) administer an Enrichment Program that delivers outstanding training and education across the continuum of research activity, and (vi) leverage substantial institutional commitments (>$1.5 million) to advance these aims. Three complementary Cores will continue to integrate existing intellectual and technological resources of UAB and UCSD and provide a defined set of services to facilitate investigator-initiated AKI- related research. The Cores include: 1) Resource for Clinical Studies of AKI (clinical research, registry and biorepository including human kidney tissues); 2) Resource for Pre-Clinical Studies of AKI (animal models, small animal imaging and physiology); 3) Bioanalytical Core (bioenergetics, biomarkers, small molecule analysis). A Biostatistical Resource will provide support for the cores and pilot projects. The Center also includes an active and productive PAF program designed to attract new investigators into the field and yield a high return on investment in terms of extramural funding and publications. In the prior 4 years of funding, the Center has galvanized our research community and created a robust collaborative environment particularly for AKI research integrating two institutions and resulting in 220 publications. The Internal Research Base includes 100 investigators (from UAB and UCSD) with NIDDK funding of $9.6 million in annual direct costs and 88 investigators from other institutions that form an Extended Research Base. Twelve pilot awardees were funded, and we propose to fund 3-4 pilots/year in this renewal cycle. The Center will continue to promote a seamless integration of expertise and resources at both institutions for innovative and productive research needed for the translation of new insights into novel therapies for patients with AKI.

Acute kidney injury (AKI) is a common and serious complication of medical and surgical diseases that has significant attributable morbidity and mortality in critically ill patients. Analysis of outcomes data reported that patients who develop AKI during a hospitalization are at substantial risk for the development of chronic kidney disease within 1 year. Mononuclear phagocytes (MP), which consist of macrophages and dendritic cells, have long been known to exist within the kidney. They are actively involved in maintenance of renal homeostasis and, more importantly, the restoration of homeostasis after injury. We propose to study the role of MP in AKI with the guiding hypothesis that the course and outcome of AKI is a function of the involvement of specific subpopulations of MP, delineated on the basis of their embryonic origin and expression of genes associated with macrophage function with respect to time post-injury. Intrarenal resident MP are a unique F4/80HiCD11bInt subpopulation that constitute 50% of renal MP in normal kidneys and are distinct from monocyte derived MP that arrive from the peripheral circulation. They initially arise from the fetal yolk sac, colonizing the kidney during embryonic days 8.5-11 in mice and receive little to no further input from the circulation in normal kidneys. Other subpopulations of renal MP arise from hematopoietic stem cells in the fetal liver and bone marrow. Very little is known about the role of these F4/80HiCD11bInt resident renal MP in kidney homeostasis and disease. Our preliminary bulk and single cell RNAseq data, flow cytometry analyses and morphological studies indicate that resident renal MP follow a developmental program which encompasses a developmental switch in the resident MP, exemplified by turning on expression of major histocompatibility complex (MHC) class II between 7-21 days after birth. Notably, there is a reversion of resident renal MP to the MHC negative phenotype shortly after AKI and preliminary RNAseq data indicate that resident renal MP secrete Wnt glycoproteins, which are known to be intimately involved with kidney embryonic development. These findings suggest there is at least partial recapitulation of a developmental program after injury, which has been previously proposed, but never proven. Our central hypothesis is that renal resident MP undergo a developmental program that is recapitulated, at least in part, following AKI. This developmental program is a component of the mechanism of recovery from injury and could be involved in a failure to re-establish homeostasis (i.e. unsuccessful or deranged repair) leading to CKD. To test this hypothesis, we will execute the following specific aims: 1) To test the hypothesis that renal resident MP are an independent, self-renewing subpopulation that receives no input from the peripheral circulation after AKI; 2) To test the hypothesis that renal MP recapitulate a developmental program after AKI; 3) To test the hypothesis that the transition of AKI to CKD involves a failure of resident MP transcriptional programming to return to the homeostatic state, resulting in inappropriate expression of nephrogenic and fibrogenic gene products.

The overarching goal of this competitive revision application is to extend the mission of the University of Alabama at Birmingham (UAB)-University of California, San Diego (UCSD) O?Brien Center for Acute Kidney Injury (AKI) Research by defining the impact of AKI on the excess burden of chronic kidney disease (CKD) in the Southeastern US. The clustering of major CKD risk factors such as black race, obesity and diabetes in our region is a key driver of the high prevalence of CKD in the Southeastern US. As such, identifying potential interventions to reduce the incidence and progression of CKD in these vulnerable populations is critical for reducing the substantial social and financial costs associated with CKD and related health disparities (stroke, heart disease, death) in our region. AKI affects as many as 20% of hospitalized patients and is a recognized risk factor for the development of CKD, cardiovascular disease and death. These outcomes may be modifiable by care provided in the post-AKI period, yet optimum care for care of AKI survivors is not well defined and is often fragmented after discharge. Disadvantaged patients are at especially high risk for inappropriate follow-up and low quality of care, suggesting that disparities in AKI outcomes contribute to the excess risk of incident and progressive CKD in high-risk populations in our region. However, progress in this area is hampered by a lack of data on the degree to which differences in AKI outcomes contribute to CKD disparities in the Southeastern US, and potential interventions that may target these disparities. Accordingly, the primary focus of this application is to define the impact of AKI during acute hospitalization on incident and progressive CKD; how these associations differ by race, obesity and diabetes status; and pilot a dedicated AKI follow-up clinic as a potential intervention to address disparities in these outcomes. Specifically, Aim 1 will leverage an established, ongoing regional CTSA collaboration?the Southeastern Shared Health Research Information Network (SE-SHRINE)?to examine all hospitalized AKI patients within four centers (UAB, MUSC, UAMS, U Kentucky) and link them to the US Renal Data Systems to define disparities in AKI outcomes ([i] severity and duration of AKI; [ii] recovery of dialysis-dependent AKI or development of ESRD after discharge; and [iii] associated health disparities such as death and cardiovascular disease) by race, obesity and diabetes. Aim 2 will assess the feasibility and collect key efficacy measures of a dedicated AKI follow-up clinic for patients with AKI discharged to the community at three Southeastern CTSA hubs (UAB, Vanderbilt, U Kentucky). We will leverage the resources available in O?Brien Cores to integrate existing intellectual and technological resources of UAB and facilitate the success of this project. This project will help build a collaborative research network for AKI research that could serve as a platform for multi-center, transdisciplinary science and clinical trials focused on reducing the burden of CKD and related health disparities in the Southeastern US.