Expression of sFlt1 and its function in the glomerular endothelium

描述(由申请人提供)：在大鼠和小鼠体内注射抗血管内皮生长因子抗体或可溶性血管内皮生长因子受体拮抗剂sFlt1，会导致严重的蛋白尿和高血压，并伴有肾小球内皮增生症。这种综合征概括了在接受抗血管内皮生长因子单抗或酪氨酸激酶抑制剂化疗的患者中常见的蛋白尿和高血压，并为研究血管内皮生长因子及其受体在肾小球内皮细胞信号转导和损伤中的作用提供了一个模型。肾小球内皮细胞增生症也伴随着毒血症中的大量蛋白尿，被认为是其决定性的病理损害。中毒症妇女的血清刺激肾小球内皮细胞(GEC)释放内皮素-1(ET-1)等可溶性因子，继而诱导足细胞中nevinin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sFlt1在中毒血症中表达增加的机制，并确定sFlt1亚型对肾小球内皮细胞功能的影响。我们已经发现了新的sFtl1亚型，它们在毒血症中增加，并受缺氧的调节。我们计划研究Flt1初级转录本的交替处理，重点是低氧和血管紧张素II 1型(AT1)受体的激活，这两条途径可以增加sFlt1的表达。我们还将研究从中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转录后调节sFlt1来刺激sFlt1的表达。我们还假设Flt1基因座中的顺式元件调节Flt1和sFlt1转录本的丰度。这些顺式元件被预测位于Flt1基因5‘侧翼区，协同调控Flt1和sFlt1的转录，并位于Flt1基因内含子13和相邻外显子之间，相互调控flt1和sFlt1。我们推测，sFlt1抑制肾小球内皮型一氧化氮合酶(ENOS)和NO的释放，进而增加ET-1的表达和GEC的释放。我们还假设，中毒血清诱导GEC释放ET-1的作用是由于血清中sFlt1的增加所致。为了验证我们的假设，我们提出了以下具体目标：(I)建立缺氧刺激sFlt1mRNA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。我们将比较主要的sFlt1亚型在原代培养的GEC中的作用，并评估中毒血清增加肾小球ET-1释放的作用是否通过增加sFlt1介导。了解sFlt1在GEC中的调控过程和信号通路的阐明，对于蛋白尿肾脏疾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.