Mechanisms of tubular atrophy in renal disease

肾脏疾病中肾小管萎缩的机制

• 批准号: 7096016
• 负责人: JEFFREY R SCHELLING
• 金额: $ 31.56万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7096016
• 关键词: apoptosis; cell line; chronic renal failure; clinical research; cysteine endopeptidases; enzyme activity; enzyme substrate; epithelium; human genetic material tag; laboratory mouse; laboratory rat; molecular pathology; protein structure function; renal tubule; serine threonine protein kinase; sodium hydrogen exchanger

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.

描述（由申请人提供）：肾小管上皮细胞（RTC）凋亡引起管状萎缩，这是慢性肾脏疾病的标志。基于细胞质体积减少的描述，凋亡的原始术语是“收缩坏死”。凋亡细胞还会发展出胞质酸化，从而促进胱天蛋白酶激活。初步数据表明，质膜Na+/H+转运蛋白NHE1通过捍卫细胞体积和PHI通过Na+/H+交换来促进RTC存活。此外，凋亡或渗透应激激活了将NHE1胞质结构域与Ezrin，radixin，Moesin（ERM）蛋白联系起来的信号传导级联，该蛋白将NHE1绑扎到皮质肌动蛋白，然后在下游细胞生存激酶，Pis Kinase，Pis Kinase和akt的下游激活。已发表的报告和初步数据也暗示了途径中的Rho依赖性激酶（岩石）和磷脂酰肌醇4,5-双磷酸（PIP2）。此外，强大的死亡刺激与NHE1的caspase-3降解有关，细胞收缩和胞质酸化。在体内，NHE1缺陷小鼠表现出用阿霉素或链霉菌素诱导肾脏疾病后的RTC凋亡增加。假设是NHE1激活刺激岩石活性和ERM募集到NHE1细胞质结构域，以在富含PIP2的质膜微环境中形成细胞存活信号。肾小管萎缩和肾脏疾病进展需要NHE1由于NHE1细胞质尾巴的裂解而失活。该假设将以以下具体目的提出：（1）使用生化和免疫定位技术来表征NHE1调节的细胞存活信号的组装，该基于渗透/凋亡应激被激活，（2）通过caspase suptrate（Caspase suppase）（caspase）的作用（2）来确定CASPase suppase（CASPASE）的作用。 NHE1 caspase裂解位点，以及通过测试抗裂解的NHE1突变体对细胞存活的影响，（3）测试NHE1在体内RTC生存中的作用，将在NHE1-Deecient VS.对照小鼠中确定肾脏表型在诱导型肾脏肾脏疾病后的肾脏表型。提出的实验将证明NHE1是关键细胞存活因子和caspase靶标。建立NHE1作为细胞存活或死亡的仲裁者将需要设计治疗策略，以稳定NHE1调节的生存信号和/或抑制NHE1的caspase裂解。