Role of Peroxidasin in Glomerular Matrix Homeostasis

描述（由申请人提供）：糖尿病肾病（DN）是美国终末期肾脏疾病的主要原因，糖尿病肾病的中心紊乱是活性氧（ROS）的过量产生。超氧化物及其产物过氧化氢（H2O2）是DN中最受关注的活性氧。我们最近发现，过氧化物酶是一种嵌入细胞外基质的蛋白质，可将H2O2转化为高活性的次溴酸（HOBr）和次氯酸（HOCl），统称为次卤酸（HOX）。Peroxidasin利用HOX在基底膜胶原IV中形成新的亚砜亚胺键（S=N），这是Peroxidasin第一个被发现的功能。但是，在饱和H2O2底物的糖尿病环境中，过氧化物酶可能产生过量的HOX。HOX是高活性和氧化卤化蛋白，对细胞功能产生不利影响。在使用糖尿病肾病小鼠模型的初步工作中，我们发现肾小球和小管过氧化物酶表达和HOX介导的蛋白损伤增加。基于这些发现，我们假设过氧化物酶通过产生有毒的HOX在糖尿病肾病中起致病作用，HOX使蛋白质卤化改变细胞功能并促进细胞外基质扩张。为了验证这一假设，目的1将确定过氧化物酶如何在基底膜内发挥作用，检查胶原IV和过氧化物酶之间的相互作用。在Aim 2中，我们将研究过氧化物酶和HOX在高血糖诱导的肾细胞功能障碍和基质积累中的作用，作为糖尿病肾病的体外模型。使用创新的质谱法，我们将确定与过氧化物酶上调相关的HOX介导的蛋白质卤化的特定位点。在Aim 3中，我们将直接测试过氧化物酶和HOX在a中的作用

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.