肾小球系膜细胞钙库操纵性钙内流在糖尿病肾病中的肾脏保护作用

The early features of diabetic nephropathy (DN) include glomerular/mesangial cell (MCs) hypertrophy with accumulation of extracellular matrix (ECM) proteins. Reactive oxygen species (ROS) produced by NADPH oxidases have been recognized as a principal component in the progression of the early diabetic changes in glomeruli. However, recent in vivo studies suggest that ROS may also play a protective role against kidney damage. Stromal interaction molecule 1 (STIM1) and Orai1-mediated store-operated Ca2+ entry (SOCE) plays a pivotal role in many physiological and pathological processes. This Ca2+ signaling can promote or suppress cell growth, depending on cell types and downstream pathways. However, whether and how this Ca2+ signaling in MCs influences the development of DN is completely unknown. In recent pilot studies, we found that ROS increased Orai1 protein expression in MCs, and the STIM1/Orai1-mediated SOCE in MCs suppressed ECM protein expression and MC hypertrophy. We, thereby propose that in addition to the established deleterious process of ROS, diabetes simultaneously activates a beneficial ROS/SOCE pathway in MCs as a self-protective or compensatory mechanism to protect kidneys. Our hypothesis is that 1) diabetic hyperglycemia enhances STIM1/Orai1-mediated SOCE in MCs through an NADPH oxidase/ROS mechanism, and 2) the SOCE in diabetic MCs suppresses MC hypertrophy and accumulation of ECM proteins by inhibiting the SMAD pathway. To test this hypothesis, SOCE will be pharmacologically and biologically upregulated or downregulated in cultured MCs. Importantly, we will use the recently-established nanoparticle- and recombinant associate adenovirus-delivery systems to selectively introduce siRNA/shRNA or expression plasmids of STIM1/Orai1 into MCs of normal and diabetic animals. The information obtained from this novel study will provide a new therapeutic strategy for progression of DN.

糖尿病肾病（DN）早期病理特征是肾小球肥大和系膜区细胞外基质（ECM）聚积。肾小球系膜细胞（MC）是产生ECM的主要细胞。以STIM1和Orai1介导的钙库操纵性钙内流（SOCE）在不同类型细胞中起促进或抑制蛋白合成的作用。然而，MC的SOCE在DN发生发展中的作用还从未探讨过。另外，虽然NADPH氧化酶及其产物ROS在DN中对肾脏的损害已被广泛认知，但最近研究表明它们也可能有肾脏保护作用，其具体机制不明。我们前期研究发现SOCE可抑制ECM表达和MC肥大，且ROS可以增加Orai1表达。故提出DN时产生的ROS同时启动了肾脏内在的保护机制，即增加MC中的SOCE，从而通过SMAD旁路抑制MC肥大和ECM增生。我们将以药理学和分子生物学方法调节体外培养的MC的SOCE，并用新颖的纳米技术和重组腺相关病毒基因转移技术，在体内实验中将shRNA/siRNA或表达质粒特异性引入MC，来验证假设。

糖尿病肾病（DN）的早期病理特征是肾小球肥大和系膜区细胞外基质（ECM）聚积。肾小球系膜细胞（MC）是产生ECM的主要细胞。以STIM1和Orai1介导的钙库操纵性钙内流（SOCE）在不同类型细胞中其促进或抑制蛋白合成的作用。然而，MC的SOCE在DN发生发展中的作用还从未探讨过。我们前期研究发现，SOCE可抑制ECM表达和MC肥大，故提出DN时SOCE具有肾脏保护作用。我们以药理学和分子生物学方法调节体外培养的MC的SOCE，并用新颖的纳米技术和重组病毒基因转移技术，在体内试验中将shRNA/siRNA特异性引入MC，探讨高糖或糖尿病时，SOCE与MCs肥大和ECM聚集的关系，以及DN时MCs的SOCE和SMAD信号通路的相关性。结果发现，SOC可抑制高糖和AngII诱导的HMCs ECM蛋白的表达。敲除小鼠MCs 的Orai1能增加肾皮质和肾小球ECM蛋白的表达，诱导基质增生。应用rAAV转移系统将STIM1shRNA引入大鼠模型，可增加ECM蛋白的表达，大鼠尿白蛋白排泄率增加，发生肾损害。在培养的人MCs中，SOCE可减少Smad1蛋白表达并可抑制Smad1的磷酸化。体内试验，应用NP转移系统选择性将Orai1siRNA 引入正常小鼠模型。敲除小鼠MCs 的Orai1，能增加肾小球Smad1蛋白的表达，并显著增加肾皮质Col IV 的表达。我们的研究提示，高糖或DN中，SOCE可通过SMAD旁路抑制MC肥大和ECM增生，具有肾脏保护作用。基于我们的研究，可以考虑在应用RAS抑制剂治疗时，选择性保护甚至促进SOCE的作用。这种新的治疗策略，可能对DN产生更好的治疗效果。