Enzymatic Regulation of the Podocyte

描述(申请人提供)：肾小球肾脏疾病呈上升趋势，目前还没有针对蛋白尿肾病的明确治疗方法。在过去的几年里，我们已经证明，一种新的胞浆组织蛋白L变异体(CCatL)可以特异性地诱导足细胞足突(FP)消失、蛋白尿和肾脏疾病的进展。CCatL 作为肾小球损伤过程中诱导的调节性蛋白水解酶，裂解TGTP酶动力素、突触素和CD2AP。这些卵裂事件导致了一个新的概念，即FP消退是足细胞酶促疾病。在这次竞争性更新中，我们寻求阐明中和cCatL的创新方法，以努力更有效地治疗肾小球疾病。我们将验证抑制足细胞中cCatL活性将改善肾小球疾病的假设。为了验证这一假设，我们将进行硅胶和细胞培养研究的组合，并利用我们的新小鼠模型，该模型可诱导特定足细胞表达cCatL。在诱导足细胞cCatL后，该小鼠出现足细胞FP消失和蛋白尿。我们将利用这只小鼠，研究一组名为蛇的酶对cCatL的抑制作用。我们最初的重点将是SERPINB13(Hurpin)，它经典地位于皮肤细胞的胞浆中，已知是组织蛋白酶L的特异性抑制剂。我们发现在足细胞中可以诱导表达，并创建了SERPINB13的足细胞特异性敲除动物，并获得了SERPINB13转基因小鼠，以研究这些酶在面对高cCatL时的表现。我们还将分析足细胞碱化对cCatL活性的影响，因为正常足细胞内的pH接近7.3，但疾病时更接近cCatL活性的最适pH值在7.0。我们有初步数据显示，在脂多糖小鼠模型中，使用例如L-谷氨酰胺补充剂的碱化足细胞可以防止蛋白尿。总而言之，生化、细胞培养和遗传动物模型将被用于一种新的多方面的方法来解决蛋白尿肾脏疾病。

DESCRIPTION (provided by applicant): Glomerular kidney diseases are on the rise with no definitive treatment for proteinuric renal disorders available yet. Over the past years, we have shown that podocyte foot process (FP) effacement, proteinuria and kidney disease progression can be specifically induced by the action of a novel cytosolic cathepsin L variant (cCatL). cCatL hereby acts as regulatory protease that is induced during glomerular injury cleaving th GTPase dynamin, synaptopodin as well as CD2AP. These cleavage events have led to a novel concept that characterizes FP effacement as podocyte enzymatic disease. In this competitive renewal, we seek to clarify innovative ways to neutralize cCatL in an effort to treat glomerular disease more effectively. We will test the hypothesis that inhibition of cCatL activity in podocytes will improve glomerular disease. To test this hypothesis, we will perform a combination of in silico and cell culture studies and make use of our novel mouse model that is inducible for specific podocyte-expression of cCatL. Upon induction of podocyte cCatL, this mouse develops podocyte FP effacement and proteinuria. We will utilize this mouse and study inhibition of cCatL by a group of enzymes called serpins. Our initial focus will be on SERPINB13 (hurpin) which is classically located in the cytosol of skin cels and known to be a specific inhibitor for cathepsin L. We found inducible expression in podocytes and have created podocyte-specific knockout animals for SERPINB13 and also possess SERPINB13 transgenic mice to study how these enzymes will perform in the face of high cCatL. We will also analyze the effects of podocyte alkalinization on cCatL activity as normal podocyte intracellular pH is close to 7.3 but in disease is closer to the pH optimum for cCatL activity at p 7.0. We have preliminary data showing that alkalinized podocytes using for example L-glutamine supplementation are protected from proteinuria in the LPS mouse model. In sum, biochemical, cell culture and genetic animal models will be utilized in a novel multifaceted approach to tackle proteinuric renal diseases.