内环境调控SCF/c-kit信号通路影响老年肾间质纤维化的机制研究

Renal interstitial fibrosis is regarded as an important pathological basis for kidney aging and the progression of senile nephropathy. In previous studies, we have found that the young internal environment can significantly reduce the renal interstitial fibrosis of elderly mice by establishing a complex animal model for parabiosis and unilateral ureteral obstruction (UUO). We also demonstrated the important role of SCF/c-kit in the progression of senile renal interstitial fibrosis with the mutant mice which carried a missense mutation of c-kit gene. This study will further focus on the expression and location between SCF/c-kit and the makers of inflammation, autophagy, apoptosis and fibrosis in the aged kidney tissue of UUO mice using a Microenvironment Multi-criteria Whole Landscape analysis system; and also tests on the regulating role of NF-κB or STAT1 for the expression of c-kit based on the ChIP-PCR, luciferase expression vector and other experimental techniques. In addition, renal tubular epithelial cells overexpressing and knocking down of c-kit gene would be constructed to investigate the effects of SCF/c-kit on inflammation, autophagy, apoptosis and transdifferentiation in renal tubular epithelial cells; and a mice model carried tissue-specific knockout of c-kit gene for renal tubular epithelial cells would be applied to demonstrate that SCF/c-kit acts directly on renal tubular epithelial cells and plays an important role in promoting renal interstitial fibrosis in the elderly mice. This study will enrich the theoretical connotation of renal aging and renal interstitial fibrosis, so is of great scientific significance.

肾间质纤维化是肾脏衰老和老年肾病进展的重要病理基础。前期研究利用连体动物模型及其叠加单侧输尿管梗阻（UUO）模型发现青年内环境改善老年肾间质纤维化，利用SCF配体c-kit基因错义突变小鼠证实SCF/c-kit在老年肾间质纤维化进展中的重要作用。本项研究将应用组织微环境多标全景观分析系统分析老年小鼠UUO模型肾组织的SCF/c-kit与炎症、自噬、凋亡和肾间质纤维化标志的定位与表达关系，利用ChIP-PCR、荧光素酶表达载体等实验技术证实NFκB或STAT1调控c-kit表达作用，构建过表达和敲低c-kit表达的肾小管上皮细胞模型，研究SCF/c-kit对肾小管上皮细胞炎症、自噬、凋亡和转分化中作用，构建肾小管上皮细胞特异性敲除c-kit小鼠，证实SCF/c-kit直接作用肾小管上皮细胞，促进老年肾间质纤维化的重要作用。从而丰富肾脏衰老和肾间质纤维化理论内涵，具有重要科学价值。

老年人是慢性肾脏病（CKD）的发病主要人群，防治老年CKD 发生是减少CKD 危害的关键环节，是重要的公共卫生课题；并且，肾脏衰老及其肾间质纤维化是老年CKD 与终末期肾脏疾病高发的最主要原因。本项目在前期利用异龄连体动物模型证实年轻化内环境改善老年肾间质纤维化，并筛选出关键内环境因子——干细胞因子（SCF）的基础上，通过体内外实验，明确了青年与老年小鼠单侧输尿管梗阻损（UUO）模型的肾功能演变和肾组织损伤的差异；发现血液和肾组织SCF表达与增龄和肾间质纤维化密切相关；发现c-Kit基因突变抑制NF-κB，减轻UUO肾组织损伤和肾间质纤维化，促进UUO后肾功能恢复；成功构建肾小管特异性敲除c-Kit 小鼠叠加肾间质纤维化模型进行验证。通过体外实验证实，TGF-β1可下调miR-133b表达，miR-133b下调CTGF表达；双荧光素酶报告分析证实CTGF是miR-133b直接靶基因；miR-133b抑制TGF-β1-诱导HK2细胞上皮间充质细胞转分化（EMT），而过表达CTGF可逆转这种效应；并且通过体内实验证实了miR-133b靶向抑制CTGF表达，改善TGF-β1诱导的HK2细胞EMT及老龄UUO小鼠的肾间质纤维化。此外，完成细胞外囊泡在啮齿动物急性肾损伤模型中疗效的荟萃分析；证实外源性生物肾脏支持改善小鼠横纹肌溶解引起的急性肾损伤，并初步解析其机制；构建了横纹肌溶解患者肾脏替代治疗需求的人工智能早期预测模型，以及个性化的死亡风险人工智能预测模型。项目研究结果将为老年肾间质纤维化的诊断和治疗提供新的生物学标志物和干预靶标，具有较好的科学价值和潜在临床应用价值。项目合计发表SCI论文10篇，中文核心期刊论文7篇。