Specificity of Calcineurin Signaling in the Kidney

肾脏中钙调神经磷酸酶信号传导的特异性

• 批准号: 6919997
• 负责人: JENNIFER L GOOCH
• 金额: $ 25.17万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6919997
• 关键词: SDS polyacrylamide gel electrophoresis; biological signal transduction; calcineurin; cyclosporines; extracellular matrix proteins; fibronectins; gel mobility shift assay; gene expression; genetic promoter element; genetic transcription; genetically modified animals; immunofluorescence technique; insulin dependent diabetes mellitus; kidney cell; kidney metabolism; laboratory mouse; phosphorylation; protein binding; protein isoforms; protein metabolism; protein structure function; renal glomerulus; renal toxin; renal tubule; transforming growth factors

DESCRIPTION (provided by applicant): Calcineurin is a calcium-dependent phosphatase that has emerged as an important signaling molecule in the kidney. Calcineurin is the target of drugs such as cyclosporin A (CsA), whose therapeutic use is limited by associated nephrotoxicity. Currently, calcineurin is known to function in signal transduction of key molecules including Agll, IGF-I and TGFbeta. However, action of calcineurin is cell-specific and there are at least two distinct models of calcineurin action in the kidney. First, we have shown that calcineurin is required for TGFbeta-mediated ECM accumulation in cultured mesangial cells (MCs) and inhibition of calcineurin protects glomeruli from ECM accumulation associated with type I diabetes in vivo. Conversely, CsA induces ECM accumulation in tubule epithelial cells (TECs) in vitro and in the tubulointerstitium in vivo. Furthermore, there is no significant protection from diabetes-induced ECM accumulation with calcineurin inhibition in the cortical tubulointerstitium. Understanding mechanisms of calcineurin signaling specificity in the kidney is key to targeting inhibition of calcineurin to prevent ECM accumulation in glomeruli and avoid CsA-mediated nephrotoxicity in the tubulointerstitium. Signaling mechanisms of calcineurin in renal cells are poorly understood and few targets of calcineurin phosphatase action have been described in the kidney. Our work has identified candidate pathways downstream of calcineurin that may be critical to cell-specific regulation of ECM proteins. Moreover, we have discovered that calcineurin A isoforms are differentially regulated in the diabetic kidney, suggesting that this may be a further level of cell-specific signaling in the kidney. Therefore, our hypothesis is the following: Cell-specific action of calcineurin in the kidney is the result of signaling specificity downstream of calcineurin, dephosphorylation of cell-specific targets, and/or action of different calcineurin isoforms. First, we will delineate downstream signaling pathways that may be involved in regulation of ECM accumulation in MCs and TECs. Next, we will identify cell-specific targets of calcineurin dephosphorylation. Finally, we will evaluate the specific contribution of calcineurin A alpha isoform and calcineurin A beta isoform to regulation of ECM accumulation in MCs and TECs. The goal of these experiments is to distinguish mechanisms of calcineurin-mediated regulation of ECM in glomeruli from calcineurin action that contributes to CsA-nephrotoxicity.

描述（由申请人提供）：钙调磷酸酶是一种钙依赖性磷酸酶，已成为肾脏中重要的信号分子。钙调磷酸酶是环孢素A （CsA）等药物的靶点，其治疗用途受到相关肾毒性的限制。目前已知钙调磷酸酶参与Agll、IGF-I和tgf - β等关键分子的信号转导。然而，钙调磷酸酶的作用是细胞特异性的，至少有两种不同的钙调磷酸酶在肾脏中的作用模式。首先，我们已经证明，在培养的系膜细胞（MCs）中，tgf - β介导的ECM积累需要钙调神经磷酸酶，抑制钙调神经磷酸酶可以保护肾小球免受体内与1型糖尿病相关的ECM积累。相反，CsA在体外诱导ECM在小管上皮细胞（TECs）和体内小管间质中积累。此外，皮质小管间质钙调磷酸酶抑制对糖尿病诱导的ECM积累没有显著的保护作用。了解肾中钙调神经磷酸酶信号特异性的机制是靶向抑制钙调神经磷酸酶以防止肾小球内ECM积聚和避免csa介导的肾小管间质肾毒性的关键。钙调磷酸酶在肾细胞中的信号传导机制尚不清楚，在肾脏中很少有钙调磷酸酶作用的靶点被描述。我们的工作已经确定了钙调磷酸酶下游的候选途径，这可能对ECM蛋白的细胞特异性调节至关重要。此外，我们发现钙调磷酸酶A亚型在糖尿病肾脏中受到差异调节，这表明这可能是肾脏中细胞特异性信号传导的进一步水平。因此，我们的假设如下：肾中钙调神经磷酸酶的细胞特异性作用是钙调神经磷酸酶下游信号特异性、细胞特异性靶点去磷酸化和/或不同钙调神经磷酸酶同种异构体作用的结果。首先，我们将描述可能参与调控MCs和tec中ECM积累的下游信号通路。接下来，我们将确定钙调磷酸酶去磷酸化的细胞特异性靶点。最后，我们将评估钙调神经磷酸酶A α和钙调神经磷酸酶A β异构体对MCs和tec中ECM积累的调节作用。这些实验的目的是区分钙调磷酸酶介导的肾小球ECM调节机制和钙调磷酸酶作用导致csa肾毒性的机制。