Expression of sFlt1 and its function in the glomerular endothelium

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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.