A1AR保护糖尿病肾小管周微环境的非管球反馈机制

Diabetic nephropathy(DN) is the leading cause of End Stage Renal Disease(ESRD). But there is still no effective interventions to slow the progression of diabetes-induced CKD. Therefore, studies to identify novel therapeutic interventions to prevent DN or slow its progression are urgently needed. It has been reasonable to infer that peritubular capillary (PTC) failure is common pathway and an independent risk factor of kidney survival in both non-diabetic and DN. In the present study, we try to investigate the functional roles of A1AR during renal peritubular microenvironment protection of DN, by combining genetic and pharmacologic approaches.First in DN patients and A1AR-/-Akita Diabetes mice and A1AR-/-db/db mice , the relationship between renal peritubular microenvironment ( RPM, including tubular, interstitial and PTC,) injury and the proteuria or GFR will be evaluated.Then the Adora2b receptor antagonist will be used to observe the co-operation of A1AR. To identify the core of extracellular adenosine generation and signaling through tubular-expressed A1AR in RPM protection, the megalin, cubilin mediated endocytosis, Treg cells infiltration and tight junction changes will be studied in vivo and vitro. Rho/Rho kinase pathway and activation of NF-kB will be studied by antagonist and agonist in vitro cultured tubular cells with high glucose. claudin-1 target siRNA will be used to identify the cooperation of transmembrane protein and scaffolding protein in tubular tight junction.Take together, the present study would like to indentify the protect effect and mechanism of A1AR independent Tubulo-glomerular feedback pathway, implicate a new idea of DN theraphy.

糖尿病肾病（DN）是ESRD的重要原因，目前还没有有效的方法遏制其进展，因此探讨DN发病机制,寻找新的干预位点有重要的现实意义。而肾小管周毛细血管（PTC）损伤被认为是DN和非DN进展的共同途径和预测肾脏结局的独立危险因素。本研究在DN患者、已经建立的腺苷1型受体（A1AR）基因敲除的1型和2型DM（Akita,db/db）小鼠模型和体外实验中，从分子、细胞、组织和器官水平,①观察A1AR缺失对DN肾小管周微环境（肾小管、间质和肾小管周毛细血管）非依赖于管球反馈的损伤，与GFR和蛋白尿的关系; ②观察A1AR与Adora2b受体的协同作用；③确定DN，A1AR活化在上调Treg细胞抑制活性、保护刷状缘转运蛋白Megalin与细胞间紧密连接的稳定性3方面的核心地位，观察A1AR下游Rho/Rho 激酶及NF-kB活化的调节机制，进而为DN防治提供新的思路和潜在的干预位点。

糖尿病肾病（DN）是终末期肾病的重要病因，肾小管周毛细血管（PTC）损伤是慢性肾脏病进展的共同途径并能预测肾脏不良结局，探讨DN中PTC损伤及肾小管周微环境稳态破坏的具体机制，对于寻找糖尿病进展的新干预位点具有重要意义。本研究通过建立DN及多种近端肾小管功能障碍疾病患者队列，以及不同基因背景的DN小鼠模型，从基因、mRNA表达、蛋白功能不同维度，深入到炎症通路、水盐代谢、能量代谢具体机制，观察到：. 1、DN患者及不同类型DN小鼠模型，均存在近端肾小管转运蛋白megalin/cubilin表达下降，与白蛋白尿水平负相关，伴PTC损伤与肾脏纤维化，DN小鼠肾脏腺苷1型受体（A1AR）表达升高，A1AR基因敲除后上述现象加重，提示A1AR通过保护近端肾小管转运蛋白及PTC，维持肾小管周微环境的稳定性，在DN蛋白尿及肾脏纤维化中发挥保护作用。. 2、深入探讨肾小管周微环境稳态破坏的具体机制，观察到野生DN小鼠中：①p-ERK/NLRP3/IL-1β炎症小体通路表达下调，与megalin损伤及蛋白尿水平一致；②肾小管周细胞（PDGFR）激活，淋巴管（Podoplanin）增生，周细胞与内皮细胞分离后分泌TGFβ等促纤维化因子，参与肾脏纤维化发生；③血压先降低后逐渐升高，肾脏水盐调节相关的离子转运蛋白NCC、NKCC2、ENaC表达显著增加。A1AR基因敲除后上述3条通路均进一步加重，首次证实A1AR通过调节肾小管周微环境的稳态在DN中发挥保护作用，具体机制包括3方面：①A1AR抑制炎症小体通路（p-ERK/NLRP3/IL-1β）改善DN近端肾小管megalin损伤相关的蛋白尿；②A1AR通过周细胞和淋巴管回流途径（PDGFR/ Podoplanin）减轻DN管周微血管损伤导致的间质纤维化；③A1AR调节肾小管钠离子通道表达参与DN水盐代谢紊乱相关高血压。. 3、拓展至近端肾小管功能障碍疾病，募集家族性肾性糖尿（SGLT2相关基因变异所致）家系，建立遗传性肾小管疾病二代测序平台及肾糖阈检测方法进行新基因筛选及功能评估；在范可尼综合征患者及小鼠模型中观察到近端肾小管megalin/cubilin表达降低，证实了异位生发中心形成、Treg/Th17调节紊乱及能量代谢异常参与近端肾小管损伤。