Expression of sFlt1 and its function in the glomerular endothelium

描述（由申请人提供）：在大鼠和小鼠中输注抗VEGF抗体或可溶性VEGF受体拮抗剂sFlt1，可导致严重蛋白尿和高血压伴肾小球内皮增生。该综合征概括了抗VEGF单克隆抗体或酪氨酸激酶抑制剂化疗患者常出现的蛋白尿和高血压，并为研究VEGF及其受体在肾小球内皮细胞信号传导和损伤中的作用提供了模型。肾小球内皮增生也伴随大量蛋白尿出现在毒血症中，被认为是肾小球内皮增生的决定性病理病变。毒血症妇女的血清刺激肾小球内皮细胞（GEC）释放可溶性因子，如内皮素-1 (ET-1)，从而诱导足细胞中肾素脱落和肌动蛋白细胞骨架重排，强调肾小球内皮细胞在诱导蛋白尿中的作用。该项目的主要目标是确定毒血症中sFlt1表达增加的机制，并确定sFlt1亚型对肾小球内皮细胞功能的影响。我们已经确定了新的sft1亚型，在毒血症中增加，并受缺氧调节。我们计划研究FLT1主要转录本的替代加工，重点关注缺氧和血管紧张素II型1 （AT1）受体激活，这两种途径可以增加sFlt1的表达。我们还将研究从毒血症患者获得的血浆的影响，以及新型sFlt1亚型对GEC前内皮素-1表达和ET-1释放的影响。我们假设缺氧主要通过sFlt1的转录后调控来刺激sFlt1的表达。我们还假设FLT1位点内的顺式元件调节FLT1和sFlt1转录本的丰度。预计这些顺式元件位于FLT1的5'侧翼区域，协调调节FLT1和sFlt1的转录，并位于FLT1的内含子13和邻近的外显子内，相互调节FLT1和sFlt1。我们假设sFlt1抑制肾小球内皮一氧化氮合酶（eNOS）和NO的释放，从而增加ET-1的表达和GEC的释放。我们还假设，毒血症血清诱导GEC释放ET-1的作用可归因于血清中sFlt1的增加。为了验证我们的假设，我们提出了以下具体目标：(i)建立导致缺氧刺激sFlt1 mRNA表达的机制；（ii）研究Flt1的转录调控；（iii）确定sFlt1亚型对GEC一氧化氮合成和内皮素-1释放的影响。我们将比较主要sFlt1亚型在培养的原代GEC中的作用，并评估毒血症血清增加肾小球ET-1释放的作用是否通过增加sFlt1介导。了解sFlt1的调控过程和阐明GEC中的信号通路对蛋白尿肾病的研究具有广泛的意义，并可能为高血压和蛋白尿的治疗提供新的治疗靶点。

DESCRIPTION (provided by applicant): Infusing an anti-VEGF antibody or a soluble VEGF receptor antagonist, sFlt1, in rats and mice lead to the development of severe proteinuria and hypertension accompanied by glomerular endotheliosis. This syndrome recapitulates the proteinuria and hypertension frequently seen in patients undergoing chemotherapy with anti-VEGF monoclonal antibodies or with tyrosine kinase inhibitors and provides a model to study the role of VEGF and its receptors in glomerular endothelial cell signaling and injury. Glomerular endotheliosis also accompanies the heavy proteinuria seen in toxemia and is considered its defining pathological lesion. Serum from toxemic women stimulates the glomerular endothelial cell (GEC) to release soluble factors such as endothelin-1 (ET-1), which in turn induce nephrin shedding and actin cytoskeletal rearrangement in the podocyte, emphasizing the role of the glomerular endothelial cell in inducing proteinuria. The primary goals of this project are to identify the mechanisms that underlie the increased expression of sFlt1 in toxemia and to determine the effect of sFlt1 isoforms on glomerular endothelial cell function. We have identified novel sFtl1 isoforms that are increased in toxemia and are regulated by hypoxia. We plan to study alternate processing of the primary transcripts of FLT1, focusing on hypoxia and angiotensin II type 1 (AT1) receptor activation, two pathways that increase sFlt1 expression. We will also study the effect of plasma obtained from patients with toxemia and the effect of novel sFlt1 isoforms on GEC preproendothelin-1 expression and ET-1 release. We hypothesize that hypoxia differentially stimulates sFlt1 expression, primarily by post-transcriptional regulation of sFlt1. We also hypothesize that cis-elements within FLT1 locus regulates the abundance of Flt1 and sFlt1 transcripts. These cis-elements are predicted to be in the 5' flanking region of FLT1 coordinately regulating the transcription of Flt1 and sFlt1 and within intron 13 of FLT1 and neighboring exons reciprocally regulating Flt1 and sFlt1. We hypothesize that sFlt1 inhibits glomerular endothelial nitric oxide synthase (eNOS) and NO release which in turn increases ET-1 expression and release from GEC. We also hypothesize that the effect of toxemic serum to induce ET-1 release from GEC is attributable to the increase sFlt1 in serum. To test our hypotheses we propose the following specific aims: (i)establish the mechanisms that lead to the stimulation of sFlt1 mRNA expression by hypoxia; (ii)study the transcriptional regulation of Flt1; and (iii)determine the effect of sFlt1 isoforms on GEC nitric oxide synthesis and endothelin-1 release. We will compare the effects of the principal sFlt1 isoforms in cultured primary GEC and we will assess whether the effect of toxemic serum to increase glomerular ET-1 release is mediated through increased sFlt1. An understanding of the processes that regulate sFlt1 and the elucidation of signaling pathways in GEC has broad implication for the study of proteinuric kidney diseases and may provide new therapeutic targets for treatment of hypertension and proteinuria. PUBLIC HEALTH RELEVANCE: Proteinuria and hypertension are signs of glomerular disease and increasing proteinuria and poorly controlled hypertension are independent correlates of progressive kidney disease. Although proteinuria is recognized as a modifiable risk factor for some kinds of renal disease, therapies to control proteinuria are non-specific and not always effective. This research proposal is focused on the study of a protein called sFlt1 that appears to injure the glomerular endothelial cell which in turn aberrantly signals to a second glomerular cell, the podocyte, leading to proteinuria. An understanding of the processes that regulate sFlt1 and the elucidation of the effects of sFlt1 on signaling pathways in glomerular endothelial cells has broad implication for the study of proteinuric kidney diseases and may provide new therapies for treatment of hypertension and proteinuria.