Gautam Bhave - US grants

DESCRIPTION (provided by applicant): Diabetic nephropathy (DN) is the leading cause of end-stage renal disease in the U.S. A central derangement in diabetic nephropathy is excessive generation of reactive oxygen species (ROS). Superoxide and its product, hydrogen peroxide (H2O2), are ROS that have garnered greatest attention in DN. We recently discovered that peroxidasin, a protein embedded in extracellular matrix, converts H2O2 to highly reactive hypobromous (HOBr) and hypochlorous (HOCl) acid, collectively denoted as hypohalous acids (HOX). Peroxidasin uses HOX to form novel sulfilimine bonds (S=N) in basement membrane collagen IV, the first identified function for peroxidasin. But, in a diabetic milieu saturated wit H2O2 substrate, peroxidasin may produce excessive HOX. HOX are highly reactive and oxidatively halogenate proteins to adversely affect cell function. In preliminary work using a mouse model of diabetic nephropathy, we have found that glomerular and tubular peroxidasin expression and HOX mediated protein damage are increased. Based on these findings, we hypothesize that peroxidasin plays a pathogenic role in diabetic nephropathy by generating toxic HOX that halogenate proteins to alter cell function and promote extracellular matrix expansion. To test this hypothesis, Aim 1 will determine how peroxidasin functions within basement membranes examining the interaction between collagen IV and peroxidasin. In Aim 2, we will examine the role of peroxidasin and HOX in hyperglycemia induced renal cell dysfunction and matrix accumulation as an in vitro model of diabetic nephropathy. Using innovative mass spectrometry, we will identify specific sites of HOX mediated protein halogenation associated with peroxidasin upregulation. In Aim 3, we will directly test the role of peroxidasin and HOX in a mouse model of diabetic nephropathy. Building upon the mass spectrometry studies in Aim 2, we will identify protein halogenation sites in vivo. Furthermore, we will pharmacologically block peroxidasin and determine whether the intervention ameliorates diabetic nephropathy. The proposed work holds promise to provide insight into the pathogenesis of diabetic nephropathy and establish peroxidasin as a potential therapeutic target. Environment The proposed studies will be conducted at the Vanderbilt University Medical Center within the Division of Nephrology. The division has 14 NIH funded laboratories with over 13 million dollars in annual NIH funding and is one of only nine NIH George O'Brien Kidney Research Centers in the U.S. thus providing Dr. Bhave ample financial, administrative, and scientific support. Dr. Bhave is primarily mentored by Dr. Billy Hudson, a well- established scientist with nearly 40 years of NIH funding. His expertise resides in basement membrane and type IV collagen biochemistry and his seminal work includes the identification of the Goodpasture antigen and the development of pyridoxamine as a glycation inhibitor for the treatment of diabetic nephropathy. Dr. Bhave is also supported by Drs. Raymond Harris and Roy Zent as co-mentors to help develop an independent line of investigation and career path. Dr. Harris is Chief of the Division of Nephrology and his experience in models of diabetic nephropathy is crucial to the proposed work. Dr. Zent heads a well-funded group studying renal development and integrins and will provide expertise in the investigation of peroxidasin and cell-matrix interactions. Taken together, Dr. Bhave's mentors collectively represent over 75 years of experience in career development, mentoring, and scientific investigation. Candidate Dr. Gautam (Jay) Bhave is currently an Instructor in the Division of Nephrology at Vanderbilt with >80% of his time dedicated to bench research. He completed his M.D. and Ph.D. degrees at Baylor College of Medicine with High Honors and clinical training at Johns Hopkins Hospital and Vanderbilt. His graduate work examined sensitization of sensory neurons with tissue injury and inflammation and garnered high-profile, first author publications in Nature Neuroscience, Neuron, and PNAS. After clinical training, Dr. Bhave joined the lab of Dr. Billy Hudson as a research fellow and began investigating how a newly discovered sulfilimine (S=N) cross-link in the collagen IV protein network is formed. The work has reached fruition with the discovery of a catalyzing enzyme, peroxidasin, and a first author manuscript describing this work was recently published in Nature Chemical Biology accompanied by a commentary and cover illustration. Ultimately, Dr. Bhave hopes to translate his seminal biochemical discovery into an independent line of investigation examining the role of peroxidasin in matrix remodeling and expansion in renal disease.

The excessive generation of reactive oxygen species (ROS) significantly contributes to maladaptive renal inflammation and fibrosis. While most attention has been directed towards superoxide, hydrogen peroxide (H2O2), and other ROS, our recent work has focused on hypobromous acid (HOBr) as an important ROS. Animal heme peroxidases produce HOBr using H2O2 and Br- as substrates. HOBr, a chemical cousin of bleach (HOCl), is a powerful oxidant capable of damaging proteins, lipids, and nucleic acids and typically promotes tissue injury. We recently discovered that an extracellular matrix (ECM) protein known as peroxidasin (Pxdn) also generates HOBr. Pxdn uses HOBr as an anabolic, reactive intermediate to form novel sulfilimine (S=N) cross- links in collagen IV, a prominent constituent of basement membranes (BM), such as glomerular BM (GBM). In Drosophila, we found that loss of Pxdn and sulfilimine cross-links compromised basement membrane and tissue integrity leading to larval lethality. However, Pxdn knock-out (KO) mice are viable but demonstrate reduced sulfilimine cross-links and BM strength. Based on this phenotype, we expected that unilateral ureteral obstruction (UUO) and increased intratubular pressures would lead to greater tubular BM stretch and mechanical stress on tubular cells in Pxdn KO mice. Consequently, Pxdn KO mice would exhibit increased renal inflammation and fibrosis. Paradoxically, Pxdn KO mice demonstrated less renal inflammation and fibrosis with diminished HOBr mediated oxidative damage. Based on these data, we hypothesize that Pxdn normally uses HOBr to cross-link collagen IV and support tissue integrity, but when dysregulated, Pxdn generates excessive HOBr leading to oxidative damage and tissue injury. To test this hypothesis, in Aim 1, we will determine whether Pxdn loss of function protects against renal injury that varies in extent of inflammation and localization contrasting glomerular and tubulointerstitial disease. Aim 2 will examine how HOBr alters renal cell behavior to promote tubulointerstitial fibrosis and inflammation, including the use of mass spectrometry to identify HOBr mediated oxidative modifications of proteins. In Aim 3, we will mechanistically address how Pxdn uses HOBr to cross-link collagen IV yet avoid collateral damage to other biomolecules. Taken together, this proposal aims to define a novel role for HOBr in renal inflammation and fibrosis. These studies hope to rationalize a strategy to target HOBr to treat chronic kidney disease.