MOLECULAR ASPECTS OF ALPORT RENAL DISEASE PROGRESSION

说明： Alport综合征是一种相对常见的遗传性疾病(每5000人中有1人)，会导致进行性肾脏疾病，并伴有听力损失和视网膜斑点。在过去的十年里，导致该综合征的基因已经被确定，并且在理解进行性肾小球和肾小管间质疾病的分子机制方面取得了重大进展。这项建议是一个有重点的方法，旨在定义可能有助于肾小球发病机制的特定方面。在初步结果部分，我们提出了证据表明，基质金属蛋白酶-2、基质金属蛋白酶-9、基质金属弹性酶-12和基质金属蛋白酶-14(MT1-基质金属蛋白酶)可能以不同的方式参与肾小球的发病。目前对MMPs在肾小球发病机制中的作用尚无深入探讨。我们认为，升高的基质金属蛋白酶-2和金属硫蛋白-1基质金属蛋白酶起到保护作用，而金属弹性蛋白酶起到破坏性作用。从分离的肾小球制备的RNA的实时定量PCR分析将被用于原位杂交和免疫荧光研究，以确定这些MMPs在体内肾小球细胞中的时空调节。细胞培养研究将被用来探索小鼠模型系统中基质金属蛋白酶表达升高的机制。将使用双基因敲除和三基因敲除小鼠模型，试图从功能上确定这些MMPs在Alport肾小球发病机制中的作用。在第三个目的中，我们探讨了Alport和整合素α1缺失的Alport小鼠中基质金属蛋白酶失调的机制。我们提供的证据表明，在整合素α1缺失的小鼠中，MAP激酶信号通路被激活。这一信号通路与其他系统中的基质金属蛋白酶失调有关。我们将使用pp38和perk的特定抑制剂以及整合素特异的中和抗体来剖析整合素和MAP激酶激活在整合素A1缺失的Alport小鼠肾小球基质金属蛋白酶异常调节中的作用。在初步结果中，我们发现抑制基质金属蛋白酶-12可以阻止Alport小鼠肾脏疾病的进展。我们已经发现在Alport肾小球中GM-CSF和MCP-1的表达显著增加，这两种细胞因子已知在其他系统中诱导基质金属蛋白酶-12的表达。我们发现，这些细胞因子的受体(分别为α-GMR和CCR2)在Alport肾小球和培养的肾小球足细胞上都有表达。我们将使用体内和细胞培养系统来确定这一途径是否支持Alport肾小球中MMP-12的激活。这些研究可能会成为其他肾脏疾病模型相关研究的基础，在其他肾脏疾病模型中，这些相同的系统已被牵连。

DESCRIPTION: Alport syndrome is a relatively common (1 in 5000) genetic disorder that results in progressive renal disease with hearing loss and retinal flecks. In the past decade, the genes responsible for the syndrome have been identified, and significant progress has been made towards understanding the molecular mechanisms underlying the progressive glomerular and tubulointerstitial disease. This proposal is a focused approach aimed at defining specific aspects that may contribute towards mechanisms underlying glomerular pathogenesis. In the preliminary results section we present evidence that MMP-2, MMP-9, MMP-12 (metalloelastase) and MMP-14 (MT1-MMP) may contribute to glomerular pathogenesis in distinct ways. The role of MMPs in glomerular pathogenesis has been only marginally explored. We suggest dual contributory roles where elevated MMP-2 and MT-1 MMP are protective, while metalloelastase plays a destructive role. Real time PCR analysis of RNA from isolated glomerular preparations will be used for in situ hybridization and immunofluorescence studies to identify the temporal and spatial regulation of these MMPs in glomerular cells in vivo. Cell culture studies will be employed to probe the mechanism underlying elevated MMP expression in mouse model systems. Double and triple knockout mouse models will be used in an attempt to functionally define the roles of these MMPs in Alport glomerular pathogenesis. In the third aim we explore the mechanism of MMP dysregulation in Alport and integrin alpha1-null Alport mice. We provide evidence that the MAP kinase-signaling pathway is activated in integrin alpha1-null mice. This signaling pathway has been linked to MMP dysregulation in other systems. We will employ specific inhibitors of pp38 and pERK as well as integrin-specific neutralizing antibodies to dissect the role of integrins and MAP kinase activation in MMP dysregulation in glomeruli from integrin a1-null Alport mice. In the preliminary results, we show MMP-12 inhibition arrests the progression of renal disease in Alport mice. We have identified markedly elevated expression of GM-CSF and MCP- 1 in Alport glomeruli, cytokines known to induce expression of MMP-12 in other systems. We show that the receptors for these cytokines (alpha-GMR and CCR2, respectively) are expressed both in Alport glomeruli and on cultured glomerular podocytes. We will employ in vivo and cell culture systems to determine whether this pathway underlies MMP-12 activation in Alport glomeruli. These studies will likely form the basis for related studies in other renal disease models where these same systems have been implicated.