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: 7092931
• 负责人: JENNIFER L GOOCH
• 金额: $ 18.58万
• 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/7092931
• 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）：钙调神经磷酸酶是一种钙依赖性磷酸酶，已成为肾脏中的重要信号分子。钙调神经磷酸酶是药物如环孢素A（CsA）的靶点，其治疗用途受到相关肾毒性的限制。目前，已知钙调磷酸酶在包括AgII、IGF-I和TGF β的关键分子的信号转导中起作用。 然而，钙调神经磷酸酶的作用是细胞特异性的，并且在肾脏中存在至少两种不同的钙调神经磷酸酶作用模型。 首先，我们已经表明，钙调磷酸酶是所需的TGF β介导的ECM积累在培养的系膜细胞（MC）和钙调磷酸酶的抑制保护肾小球ECM积累与I型糖尿病在体内。 相反，CsA在体外诱导小管上皮细胞（TEC）中和在体内诱导小管上皮细胞中的ECM积累。此外，在皮质微管中，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。 了解肾脏中钙调神经磷酸酶信号传导特异性的机制是靶向抑制钙调神经磷酸酶以防止肾小球中ECM积聚和避免小管中CsA介导的肾毒性的关键。 钙调神经磷酸酶在肾细胞中的信号传导机制知之甚少，在肾脏中钙调神经磷酸酶作用的靶点很少。 我们的工作已经确定了钙调磷酸酶下游的候选途径，这可能是细胞特异性调节ECM蛋白的关键。 此外，我们发现钙调神经磷酸酶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：钙调磷酸酶在肾脏中的细胞特异性作用是钙调磷酸酶下游信号传导特异性、细胞特异性靶点的去磷酸化和/或不同钙调磷酸酶亚型的作用的结果。 首先，我们将描绘下游信号通路，可能参与调节ECM积累的MC和TEC。 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亚型和钙调神经磷酸酶A β亚型对MCs和TEC中ECM积累的调节的具体贡献。 这些实验的目的是区分钙调神经磷酸酶介导的调节ECM在肾小球钙调神经磷酸酶的作用，有助于CsA肾毒性的机制。