MOLECULAR ASPECTS OF ALPORT RENAL DISEASE PROGRESSION

ALPORT 肾病进展的分子方面

• 批准号: 6968892
• 负责人: Dominic E. Cosgrove
• 金额: $ 27万
• 依托单位: FATHER FLANAGAN'S BOYS' HOME
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-15 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6968892
• 关键词: Alport syndrome; biological signal transduction; colony stimulating factor; elastases; enzyme mechanism; gene targeting; genetically modified animals; immunofluorescence technique; in situ hybridization; integrins; kidney disorder; laboratory mouse; metalloendopeptidases; mitogen activated protein kinase; polymerase chain reaction; renal glomerulus; tissue /cell culture

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.

产品说明： Alport综合征是一种相对常见的（1/5000）遗传性疾病，可导致进行性肾脏疾病伴听力丧失和视网膜震颤。在过去的十年中，已经确定了导致该综合征的基因，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。这一建议是一个集中的方法，旨在定义可能有助于肾小球发病机制的具体方面。在初步结果部分，我们提出的证据表明，MMP-2，MMP-9，MMP-12（金属弹性蛋白酶）和MMP-14（MT 1-MMP）可能有助于肾小球发病机制的不同方式。基质金属蛋白酶在肾小球发病机制中的作用仅得到了很少的探讨。我们认为，MMP-2和MT-1 MMP升高的双重作用是保护性的，而金属弹性蛋白酶起着破坏性的作用。来自分离的肾小球制备物的RNA的真实的时间PCR分析将用于原位杂交和免疫荧光研究以鉴定这些MMP在体内肾小球细胞中的时间和空间调节。将采用细胞培养研究来探测小鼠模型系统中MMP表达升高的潜在机制。将使用双和三基因敲除小鼠模型，试图从功能上定义这些MMP在Alport肾小球发病机制中的作用。在第三个目标中，我们探索MMP失调的Alport和整合素α 1缺失的Alport小鼠的机制。我们提供的证据表明，MAP激酶信号通路激活整合素α 1-null小鼠。该信号通路与其他系统中的MMP失调有关。我们将采用pp 38和pERK的特异性抑制剂以及整合素特异性中和抗体来剖析整合素和MAP激酶激活在整合素α 1缺失的Alport小鼠肾小球MMP失调中的作用。在初步结果中，我们表明MMP-12抑制剂阻止了Alport小鼠肾脏疾病的进展。我们已经确定了Alport肾小球中GM-CSF和MCP- 1的表达显著升高，这些细胞因子已知在其他系统中诱导MMP-12的表达。我们发现，这些细胞因子（α-GMR和CCR 2，分别）的受体表达在Alport肾小球和培养的肾小球足细胞。我们将采用在体内和细胞培养系统，以确定是否该途径的基础MMP-12激活Alport肾小球。这些研究可能会形成其他肾脏疾病模型相关研究的基础，这些模型涉及相同的系统。