Mechanism of Renal Cell Injury

点击翻译按钮获取中文摘要

 DESCRIPTION (provided by applicant): Hyperglycemia and increased transforming growth factor-β (TGFβ) expression activate mTOR (mechanistic target of rapamycin), which acts as a key node to promote glomerular and tubular hypertrophy and matrix protein fibronectin expression. We have reported that high glucose and TGFβ activate both mTOR complexes (C1 and C2) in mesangial and proximal tubular epithelial (PTE) cells and in kidneys of type 2 and type 1 diabetic mice. Blocking mTOR activity with rapamycin, we showed reduction in albuminuria, renal hypertrophy and matrix expansion in these models of diabetes. Complete inhibition of mTOR by rapamycin can be detrimental as the activity of this kinase is required for maintaining renal cell homeostasis. Also, rapamycin changes gene expression profile, which causes adverse effects. Therefore, alternative therapeutic approach is necessary to block mTOR kinase. Our data demonstrate that the expression of deptor, a component of both mTOR complexes, and inhibitor of their kinase activities, is reduced in kidneys of diabetic rodents and in mesangial and PTE cells treated with high glucose or TGFβ. Exploiting how deptor is suppressed to increase mTOR activity can identify novel molecular targets to block diabetic renal complications. In our preliminary data, we find reduction in deptor levels concomitant with increased expression of EZH2 (enhancer of zeste homolg 2), the catalytic subunit of the polycomb repressor complex 2, which trimethylates histone H3 at lysine- 27 to block transcription of a specific gene. Moreover, we show enhanced expression of the E3 ubiquitin ligase βTrCP (transducin repeat containing protein) by high glucose and TGFβ in renal cells. βTrCP targets deptor for degradation. Furthermore, we demonstrate high glucose and TGFβ increase the expression of a family of microRNA, miR-181 (a,b,c,d), and miR-221. These microRNAs are also increased in kidneys of type 1 and type 2 diabetic mice and target deptor for downregulation. In this proposal, using cultured mesangial and PTE cells and renal tissues from diabetic OVE26 and db/db mice, we will test the hypothesis that inappropriate deptor downregulation contributes to renal hypertrophy and matrix expansion in diabetic kidney disease. Probing the specificities of underlying mechanisms, we will define the regulatory modules of deptor suppression. In the first specific aim, we will investigate EZH2 as a candidate for transcriptional suppression of deptor, hypertrophy and, fibronectin and PAI-1 (plasminogen activator inhibitor-1) expression. In the second aim, the role of βTrCP in deptor protein degradation/mTORC1/C2 activation and its cross talk with TGFβ signaling in forcing mesangial and PTE cell hypertrophy and, fibronectin and PAI-1 expression will be examined. In specific aim 3, we will investigate the contribution of miR-181 family and miR-221 to hypertrophy and, fibronectin and PAI-1 expression in response to high glucose and TGFβ in mesangial and PTE cells and in diabetic mice kidneys. To address these aims, techniques including immunoblotting, immunoprecipitation, qRT-PCR, morphometry, immunohistochemistry, reporter transfection assays, chromatin immunoprecipitation assays and siRNA-mediated downregulation of proteins will be used.