Mechanisms of tubular atrophy in renal disease

描述（由申请人提供）：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一个标志。细胞凋亡最初的术语是“收缩性坏死”，基于对胞质体积减少的描述。凋亡细胞也会发生胞质酸化，从而促进半胱天冬酶的激活。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，凋亡或渗透应激激活信号级联，将NHE1细胞质结构域连接到ezrin， radixin, moesin （ERM）蛋白，这些蛋白将NHE1连接到皮质肌动蛋白，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暗示rho依赖性激酶（ROCK）和磷脂酰肌醇4,5-二磷酸（PIP2）参与该途径。此外，强大的死亡刺激与NHE1的caspase-3降解、细胞收缩和细胞质酸化有关。在体内，阿霉素或链脲佐菌素诱导肾脏疾病后，NHE1缺陷小鼠的RTC凋亡增加。假设NHE1激活刺激ROCK活性和ERM募集到NHE1细胞质结构域，从而在富含pip2的质膜微环境中形成细胞存活信号通路。由于半胱天冬酶切割NHE1细胞质尾部，小管萎缩和肾脏疾病的进展需要NHE1失活。这一假设将有以下具体目标：(1)描述装配NHE1-regulated细胞生存signalplex,由渗透/激活凋亡压力,使用生化和immunolocalization技术,(2)确定的角色NHE1半胱天冬酶底物,通过识别半胱天冬酶(s)切割NHE1,通过映射NHE1半胱天冬酶乳沟网站,并通过测试的影响cleavage-resistant NHE1突变表达对细胞生存,和(3)测试的角色NHE1 RTC体内生存,在诱导进行性肾脏疾病后，将确定nhe1缺陷小鼠与对照小鼠的肾脏表型。我们提出的实验将证明NHE1是一个关键的细胞存活因子和caspase靶点。确定NHE1作为细胞存活或死亡的仲裁者，将保证设计治疗策略来稳定NHE1调节的存活信号plex和/或抑制NHE1的caspase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.