Mechanisms of tubular atrophy in renal disease

描述（由申请方提供）：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。细胞凋亡的原始术语是“皱缩坏死”，基于对细胞质体积减少的描述。凋亡细胞也发展胞质酸化，其促进半胱天冬酶活化。初步数据表明，质膜Na+/H+转运蛋白NHE 1通过Na+/H+交换保护细胞体积和pHi来促进RTC存活。此外，凋亡或渗透压应激激活信号级联，其将NHE 1胞质结构域连接至ezrin、radixin、膜突蛋白（ERM）蛋白，其将NHE 1拴系至皮质肌动蛋白，随后下游激活细胞存活激酶、PIS激酶和Akt。已发表的报告和初步数据也涉及Rho依赖性激酶（ROCK）和磷脂酰肌醇4，5-二磷酸（PIP 2）的途径。此外，一个强大的死亡刺激与caspase-3降解NHE 1，细胞皱缩和胞质酸化。在体内，NHE 1缺陷小鼠表现出增加RTC凋亡后，阿霉素或链脲佐菌素诱导肾病。假设是NHE 1激活刺激ROCK活性和ERM募集到NHE 1胞质结构域，以在富含PIP 2的质膜微环境内形成细胞存活信号复合物。肾小管萎缩和肾脏疾病进展需要NHE 1失活，这是由于NHE 1胞质尾区的半胱天冬酶裂解。该假设将以下列具体目标为依据：（1）使用生物化学和免疫定位技术表征NHE 1调节的细胞存活信号复合物的组装，其被渗透/凋亡应激激活，（2）通过鉴定切割NHE 1的半胱天冬酶，通过绘制NHE 1半胱天冬酶切割位点，以及通过测试抗切割NHE 1突变体表达对细胞存活的影响，以及（3）为了测试NHE 1在体内RTC存活中的作用，将在诱导进行性肾病后在NHE 1缺陷小鼠与对照小鼠中确定肾表型。所提出的实验将证明NHE 1是关键的细胞存活因子和半胱天冬酶靶标。将NHE 1确立为细胞存活或死亡的仲裁者将保证设计治疗策略以稳定NHE 1调节的存活信号复合物和/或抑制NHE 1的半胱天冬酶切割。

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.