WISP-1在肾脏纤维化中的功能定位及其机制研究

Renal fibrosis is the final common pathway of a wide variety of chronic kidney diseases leading to the end-stage of renal disease. However, no effective therapies are available so far. Study has shown that WNT-inducible signaling protein 1 (WISP-1) involves in heart and lung fibrosis but the role of WISP-1 in renal fibrosis has not been explored. TGF-β/Smads signaling plays a critical role in renal fibrosis. By microarray and real-time PCR analysis, we firstly identified WISP-1 expression is upregulated in TGF-β1-induced renal fibrosis in tubular epithelial cells (TECs) (P<0.01) and in diseased kidneys from mouse models of type I diabetic nephropathy (1.5 fold), Anti-GBM glomerulonephritis (2.1 fold), Adriamycin nephropathy (1.6 fold) and obstructive nephropathy (1.6 fold), suggesting that WISP-1 may be a key downstream protein of TGF-β/Smads signaling induced renal fibrosis. To test this hypothesis, the functional role of WISP-1 in TGF-β1-induced renal fibrosis will be determined in vitro by overexpression or knockdown of WISP-1 in kidney cells. It will be further examined that the signaling mechanisms by which TGF-β signaling through Smad2/3 to regulate the WISP-1 expression in renal fibrosis. Moreover, the therapeutic potential of WISP-1 in renal fibrosis will be examined in Unilateral Ureteral Obstruction (UUO) mouse model by a novel gene therapy transfer system, ultrasound-microbubble-mediated WISP-1 shRNA transfer. In conclusion, targeting WISP-1 may be an effective therapeutic approach to combat renal fibrosis.

肾脏纤维化是各种慢性肾脏病进展至终末期肾病的共同通路。目前无有效的治疗方法。TGF-β/Smad信号通路在肾脏纤维化中有重要作用。本课题组首次发现在肾脏纤维化的细胞及动物模型中WISP-1表达升高，且与TGF-β/Smad信号通路活化有关。由此我们推测：WISP-1可能作为致纤维化因子，受TGF-β/Smad信号通路调控；而抑制WISP-1在肾脏的表达可降低肾脏纤维化。为验证此推测，首先利用质粒转染及ShRNA方法上调或抑制WISP-1在肾脏细胞中的表达，明确WISP-1在肾脏纤维化中的功能，并深入探讨TGF-β信号通路调控WISP-1的机制。进一步利用UUO小鼠模型，采用超声微泡介导质粒转染技术，将WISP-1 shRNA质粒特异性转染入小鼠肾脏，探索抑制WISP-1对肾脏纤维化的预防和治疗作用。本课题将首次阐明WISP-1在肾脏纤维化中的功能和机制，为肾脏纤维化防治提供新的思路和靶点。

肾脏纤维化是所有慢性肾脏病发展至终末的共同通路，是导致终末期肾病的主要原因之一，但缺乏有效的干预措施和治疗方法。最新研究表明，Wnt1诱导分泌蛋白-1（Wnt1-induced secreted protein 1，WISP-1/CCN4）参与心脏、肺组织纤维化，但它在肾脏纤维化中的表达及其作用无相关报道研究。本项目通过一系列的临床实验和体内、外实验，检测WISP-1在肾脏纤维化中的表达水平，揭示其表达与肾纤维化的关系，明确WISP-1对肾脏纤维化的功能作用，探讨其分子机制。本研究首先通过临床实验和体内、外实验，发现在TGF-β1诱导大鼠肾小管上皮细胞、UUO小鼠模型梗阻侧肾组织、肾穿刺证实有肾纤维化CKD患者的肾活检组织（糖尿病肾病、局灶节段性硬化性肾小球肾炎、局灶增生型IgA肾病）及血清中，WISP-1均一致性的升高；进一步通过免疫组化发现WISP-1在UUO小鼠及纤维化人肾组织的表达均主要集中在肾间质部位；通过对上述CKD患者肾活检肾组织纤维化进行病理分析，结果发现CKD患者血清WISP-1的表达水平与纤维化程度呈正相关；其次，通过体外实验揭示过表达WISP-1可加重TGF-β1诱导大鼠肾小管上皮细胞纤维化，相反，抑制WISP-1表达可以减轻纤维化；并通过体内实验证实通过注射抗WISP-1抗体干预UUO模型小鼠，发现抑制WISP-1表达可以减轻肾脏纤维化。最后，我们通过体内外实验证实WISP-1的表达与TGF-β信号通路有关。上述一系列研究结果提示 WISP-1可能是促进肾纤维化的重要细胞因子，与肾纤维化的程度有关；干预WISP-1的表达可以减轻肾脏纤维化。该研究成果为肾脏纤维化的干预和防治提供了新的思路和作用靶点。