基于足细胞RAGE/Heparanase途径探讨“利湿化瘀通络”消肾方对糖尿病肾病早期肾小球基底膜保护作用机制研究

Diabetic nephropathy (DN) is the major cause for the end-stage renal diseases（ESRD）and there is no ideal therapeutic method for treatment of DN till now. Glumerular basement memberance (GBM) damages is closely associated with the onset of albuminuria in the early stage of DN. Based on the pathogenesis “Collaterals blockage by dampness and stagnancy” hold in Traditional Chinese Medicine about DN, we observed for the first time that XiaoShen Formulation (XSF) with the effect of “Removing dampness, dissolving stagnancy and relaxing collaterals” had notable clinical therapeutic effect on proteinuria in patients at early stage of DN. In the present studies,the multiple molecular methods such as immunoelectron transmission microscopy, dual immune-staining, knockdown, over-expression, promoter-driven luciferase reporter gene expression and chromatin immunoprecitation (ChIP) and serum pharmacology were adopted to investigate the protective role of XSF on GBM damages mediated by podocyte RAGE/heparanase pathway and the underlying molecular machanisms on the STZ-induced DBA/2 Type 1 DN mice model, db/db Type 2 DN mice model and cultured podocytes. Our studies could offer some novel insights to explaining the pathophysiological mechanisms for DN. Furthermore, it could supply experimental basis and scientific evidence for elucidating the “Removing dampness, dissolving stagnancy and relaxing collaterals” effect on DN in Traditional Chinese Medicine and expending the comprehensive clinical usage of XSF.

糖尿病肾病(DN)是临床终末期肾病的主要原因，目前仍无理想治疗方法。肾小球基底膜（GBM）损伤是DN蛋白尿形成的关键环节，足细胞糖基化终末产物受体（RAGE）调控乙酰肝素酶（Heparanase）表达是介导DN早期GBM损伤的始动机制。基于中医理论对DN “湿瘀阻络”基本病机的认识和我们观察到“利湿化瘀通络”消肾方对DN患者早期蛋白尿显著临床疗效的基础上，本课题拟利用STZ诱导DBA/2小鼠1型DN模型、db/db小鼠2型DN模型和离体培养足细胞系，应用免疫电镜、免疫双标、siRNA、over-expression、荧光报告基因和ChIP等分子生物学技术和血清药理学方法，深入探讨消肾方对足细胞RAGE/Heparanase途径介导DN早期GBM损伤的保护作用机制。本研究可为中医药防治DN策略提供新的思路，为消肾方的临床应用提供实验依据，有助于阐明“利湿化瘀通络”法治疗DN蛋白尿的科学内涵。

糖尿病肾病(Diabetic nephropathy, DN)是糖尿病最主要的微血管并发症，是临床终末期肾病（ESRD）的首要病因。蛋白尿不仅是DN早期的主要临床症状和诊断指标，而且是引起肾功能持续损害的重要因素。足细胞RAGE/Heparanase途径激活介导肾小球基底膜（GBM）损伤是DN早期肾脏病理损伤的关键机制，也是DN早期蛋白尿生成的始动环节，但目前为止仍没有干预该靶点的成熟有效药物运用于DN的临床治疗。根据中医理论对DN“湿瘀阻络”基本病机的认识，我们在前期临床研究中证实具有“利湿化瘀通络”功效的消肾方（XSF）对DN早期患者蛋白尿和肾脏损伤有显著疗效，但其作用机制尚不明确。本课题通过在体实验观察到XSF对STZ诱导DBA/2小鼠1型DN早期模型和db/db小鼠2型DN模型的尿蛋白水平（尿mAlb/Cr和24h-UTP）和DN相关肾脏病理学损伤均有显著的减轻作用，具有一定量效依赖性关系，而对血压和糖脂代谢指标无明显影响。同时XSF可抑制DN小鼠足细胞RAGE和Heparanase表达上调，减少系膜外基质沉积和系膜增生程度，减轻GBM增厚和Heparan sulfate破坏程度。XSF抑制足细胞RAGE/Heparanase途径激活是其减轻DN早期GBM损伤和蛋白尿的重要的分子药理机制。在此基础上，进一步体外实验观察到消肾方含药血清（XSF-CS）预处理对RAGE配体羧甲基赖氨酸白蛋白（CML-BSA）诱导离体培养足细胞Heparanase表达升高有显著抑制作用，该作用与下调Heparanase启动子的转录活性水平有关。XSF-CS对CML-BSA诱导足细胞Heparanase表达升高的干预作用是通过抑制RAGE下游的NF-κB信号途径实现的，而与p38MAPK或PI3K/Akt信号途径无关。XSF对足细胞RAGE/Heparanase途径的抑制作用是通过NF-κB信号途径介导的，初步明确了XSF干预作用的细胞分子机制。本研究应用1型、2型DN小鼠模型和离体培养足细胞，应用多种分子生物学、免疫学和病理形态学技术，从整体、细胞和分子等不同水平明确了XSF对DN早期足细胞RAGE/Heparanase途径介导GBM损伤的治疗作用机理，揭示了中医“利湿化瘀通络”法对DN早期蛋白尿和肾脏损伤治疗作用的科学内涵，并为XSF广泛应用于临床防治DN提供扎实的科学证据。