Mechanisms of tubular atrophy in renal disease

描述(申请人提供)：肾小管上皮细胞(RTC)凋亡导致肾小管萎缩，这是慢性肾脏疾病的标志。细胞凋亡最初的术语是“萎缩坏死”，这是基于对细胞质体积缩小的描述。凋亡的细胞也会发生胞浆酸化，这会促进caspase的激活。初步数据表明，质膜Na+/H+转运体NHE1通过Na+/H+交换保护细胞体积和phi，从而促进RTC存活。此外，细胞凋亡或渗透应激激活了将NHE1胞浆结构域连接到Ezrin、Radioxin、Moesin(ERM)蛋白的信号级联，从而将NHE1与皮质肌动蛋白捆绑在一起，随后下游激活了细胞存活蛋白、PIS激酶和Akt。已发表的报告和初步数据还涉及Rho依赖的激酶(ROCK)和磷脂酰肌醇4，5-二磷酸(PIP2)在该途径中的作用。此外，强劲的死亡刺激与NHE1的caspase-3降解、细胞收缩和胞浆酸化有关。在体内，NHE1缺陷小鼠在阿霉素或链脲佐菌素诱导的肾脏疾病后表现出RTC凋亡增加。假设NHE1激活刺激ROCK活性和ERM募集到NHE1细胞质区域，在富含PIP2的质膜微环境中形成细胞生存信号复合体。肾小管萎缩和肾脏疾病进展需要NHE1失活，因为NHE1胞浆尾部的caspase裂解。该假说将出于下列特定目的：(1)利用生化和免疫定位技术鉴定NHE1调控的细胞生存信号复合体的组装；(2)确定NHE1作为caspase底物的作用；通过鉴定裂解NHE1的caspase(S)，通过绘制NHE1 caspase裂解位点以及通过测试NHE1切割抗性突变表达对细胞生存的影响；以及(3)为了测试NHE1在体内RTC存活中的作用，将在进行性肾脏疾病诱导后，确定NHE1缺陷小鼠的肾脏表型。建议的实验将证明NHE1是一个关键的细胞生存因子和caspase靶标。将NHE1确立为细胞生存或死亡的仲裁者将需要设计治疗策略来稳定NHE1调节的生存信号复合体和/或抑制NHE1的caspase裂解。

DESCRIPTION (provided by applicant): Renal tubular epithelial cell (RTC) apoptosis causes tubular atrophy, a hallmark of chronic renal disease. The original term for apoptosis was "shrinkage necrosis", based upon descriptions of reduced cytoplasmic volume. Apoptotic cells also develop cytosolic acidification, which promotes caspase activation. Preliminary data demonstrate that the plasma membrane Na+/H+ transporter, NHE1, promotes RTC survival by defending cell volume and pHi through Na+/H+ exchange. In addition, apoptotic or osmotic stress activates a signaling cascade that links the NHE1 cytosolic domain to ezrin, radixin, moesin (ERM) proteins, which tether NHE1 to cortical actin, followed by downstream activation of the cell survival kinases, PIS kinase and Akt. Published reports and preliminary data also implicate Rho-dependent kinase (ROCK) and phosphatidylinositol 4,5-bisphosphate (PIP2) in the pathway. Furthermore, a robust death stimulus is associated with caspase-3 degradation of NHE1, cell shrinkage and cytosol acidification. In vivo, NHE1- deficient mice demonstrate increased RTC apoptosis following renal disease induction with adriamycin or streptozotocin. The hypothesis is that NHE1 activation stimulates ROCK activity and ERM recruitment to the NHE1 cytoplasmic domain, to form a cell survival signalplex within a PIP2-rich plasma membrane microenvironment. Tubular atrophy and renal disease progression require NHE1 inactivation due to caspase cleavage of the NHE1 cytoplasmic tail. The hypothesis will be pursued with the following specific aims: (1) To characterize assembly of the NHE1-regulated cell survival signalplex, which is activated by osmotic/apoptotic stress, using biochemical and immunolocalization techniques, (2) To determine the role of NHE1 as a caspase substrate, by identification of the caspase(s) that cleave NHE1, by mapping the NHE1 caspase cleavage sites, and by testing the effect of cleavage-resistant NHE1 mutant expression on cell survival, and (3) To test the role of NHE1 in RTC survival in vivo, kidney phenotypes will be determined in NHE1-deficient vs. control mice following induction of progressive renal diseases. The proposed experiments will prove that NHE1 is a critical cell survival factor and caspase target. Establishing NHE1 as an arbiter of cell survival or death would warrant the design of therapeutic strategies to stabilize the NHE1- regulated survival signalplex and/or inhibit caspase cleavage of NHE1.