MSU通过RIPK1/RIPK3/MLKL通路诱导肾小管损伤的分子机制研究

Although hyperuricemia (HUA) and gout have been identified as common etiological factor renal tubular injury, the molecular mechanisms underlying renal tubular impairments caused by HUA remain evasive. Our previous study showed monosodium urate (MSU) crystal deposition, ROS production increased and renal tubular necrosis in spontaneous HUA mouse model. Further study showed that RIPK1/RIPK3/MLKL expression levels and phosphorylation levels were up-regulated, and the enhanced phosphorylation level of RIPK1 could be inhibited by ROS scavenger. Therefore, we speculated that MSU may up-regulate the phosphorylation level of RIPK1 via inducing the production of ROS , and ultimately lead to renal tubular injury. In the present study, we plan to (1) examine the effects of MSU on mitochondrial function and Nox4 expression level, two main pathway of ROS production, and the effects of ROS on RIPK1 signaling pathway and cell damage in HK-2 cells and primary renal tubular epithelial cells, after intervention with ROS modulators NAC or BSO, key molecule-specific inhibitors of the RIPK1 signaling pathway and RNAi. (2) verify the role of RIPK1 signaling pathway actived by ROS in MSU-induced tubular injury at the animal level in RIPK1 conditional gene knockout spontaneous HUA mice. By unraveling the molecular mechanism of MSU-regulated renal tubular injury through oxidative stress, this study will provide scientific basis for early treatment and prevention of MSU-induced urate nephropathy.

高尿酸血症（HUA）已成为肾小管损伤的常见病因，但分子机制不清。课题组前期发现，自发HUA小鼠肾脏出现尿酸钠（MSU）晶体沉积，肾小管坏死，ROS生成增多，RIPK1磷酸化水平上调；ROS清除剂能够显著抑制MSU引起的RIPK1磷酸化及肾小管上皮细胞损伤。推测MSU通过诱导ROS生成，调控RIPK1信号通路，导致肾小管损伤。本课题拟：⑴在HK-2细胞和原代肾小管上皮细胞检测MSU对ROS主要生成途径线粒体功能与Nox4表达水平的影响，并应用ROS诱导剂或清除剂、RIPK1信号通路及ROS生成关键分子抑制剂和RNAi干预，明确MSU诱导生成的ROS对RIPK1信号通路及肾小管上皮细胞损伤的影响；⑵应用RIPK1条件性基因敲除自发HUA小鼠，在动物水平验证ROS对RIPK1信号通路的激活在MSU诱导肾小管损伤中的作用。本研究将明确MSU引起肾小管损伤的分子机制，为尿酸性肾病的早期防治提供依据。

高尿酸血症（Hyperuricemia，HUA）是由嘌呤代谢紊乱引起的代谢异常综合征。长期高尿酸血症将导致单钠尿酸盐（Monosodium Urate，MSU）晶体的形成，并沉积在关节滑膜、滑囊、软骨及肾脏等组织中，引起炎症及靶器官损伤。目前，我国HUA与痛风患病率呈现明显上升和年轻化趋势，其中HUA总体患病率为13.3%，患病人数超1.2亿，痛风患病率为1%~3%，患病人数超过1700万，高尿酸血症与痛风均已成为严重危害国民健康的常见代谢病。有研究显示，HUA患者肾小管MSU晶体沉积远早于痛风性关节炎的首次发作。HUA与痛风引起MSU晶体在肾间质沉积及血管损伤，出现间歇性蛋白尿、血尿、肾浓缩功能受损等临床表现，导致急性肾病、慢性间质性肾炎等尿酸性肾病。本项目前期发现，肾小管上皮细胞功能受损在MSU晶体引起尿酸性肾病发病过程中发挥关键作用，但MSU晶体引起肾小管上皮细胞损伤的分子机制不清。本项目发现，尿酸氧化酶基因敲除（UOX-KO）小鼠血尿酸、肌酐、尿素氮及尿尿酸/肌酐显著升高，尿液pH值显著降低；小鼠肾小管损伤标记物KIM-1及NGAL表达水平显著升高；肾脏多普勒超声及HE染色均显示UOX-KO小鼠肾小管损伤。体外实验发现，HK-2细胞经MSU（500 μg/mL）刺激后，MTT检测结果及FDA/PI染色均显示MSU能够诱导生小管上皮细胞损伤。深入研究发现，UOX-KO小鼠肾脏TNF-α、TNF-R1、RIP3、MLKL及p-MLKL的表达显著升高；HK2细胞经MSU刺激以后，RIP3、MLKL、TNF-R1及TRIF的表达水平显著升高，表明MSU通过RIP3/MLKL诱导肾小管上皮细胞发生坏死性凋亡，导致肾小管损伤。由于MSU在肾脏的沉积与肾小管管腔的pH密切相关，本项目进一步研究了不同pH条件下MSU对肾小管的损伤。结果显示，随着pH的下降（pH=5.5），细胞的损伤明显加重；而MSU合并低pH环境进一步加重了肾小管的损伤。本研究初步揭示了MSU及MSU合并低pH环境引起肾小管损伤的分子机制，为尿酸性肾病的早期防治提供了依据。