Specificity of Calcineurin Signaling in the Kidney

说明(申请人提供)：钙调神经磷酸酶是一种钙依赖的磷酸酶，已成为肾脏中重要的信号分子。钙调神经磷酸酶是环孢菌素A(CsA)等药物的靶标，其治疗作用受到相关肾毒性的限制。目前已知钙调神经磷酸酶在Ag11、IGF-I和TGFbeta等关键分子的信号转导中发挥作用。然而，钙调神经磷酸酶的作用是细胞特有的，在肾脏中至少有两种不同的钙调神经磷酸酶作用模式。首先，我们已经证明，在培养的系膜细胞(MC)中，钙调神经磷酸酶是转化生长因子β介导的细胞外基质积累所必需的，抑制钙调神经磷酸酶可在体内保护肾小球免受与I型糖尿病相关的细胞外基质聚集。相反，CsA在体外诱导ECM在体外的肾小管上皮细胞(TECs)和体内的肾小管间质中积聚。此外，用钙调神经磷酸酶抑制皮质小管间质中的钙调神经磷酸酶对糖尿病引起的细胞外基质积聚没有明显的保护作用。了解钙调神经磷酸酶在肾脏中的信号特异性机制是靶向抑制钙调神经磷酸酶以防止细胞外基质在肾小球积聚和避免CsA介导的肾小管间质毒性的关键。钙调神经磷酸酶在肾脏细胞中的信号转导机制尚不清楚，在肾脏中钙调神经磷酸酶的作用靶点也很少。我们的工作已经确定了钙调神经磷酸酶下游的候选通路，这可能是细胞特异性调控细胞外基质蛋白的关键。此外，我们发现钙调神经磷酸酶A亚型在糖尿病肾脏中有不同的调节，这表明这可能是肾脏细胞特异性信号的进一步水平。因此，我们的假设如下：钙调神经磷酸酶在肾脏中的细胞特异性作用是钙调神经磷酸酶下游信号特异性、细胞特异性靶标去磷酸化和/或不同钙调神经磷酸酶亚型作用的结果。首先，我们将描述可能参与调控MCs和TECs中ECM积聚的下游信号通路。接下来，我们将确定钙调神经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Aα亚型和钙调神经磷酸酶Aβ亚型在MCs和TECs中对ECM积聚的调节作用。这些实验的目的是区分钙调神经磷酸酶对肾小球细胞外基质的调节机制和钙调神经磷酸酶导致CsA肾毒性的作用机制。

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.