高胱氨酸尿对大鼠肾脏自噬水平的抑制作用

摘要 目的：探讨胱氨酸尿症中高胱氨酸浓度对大鼠肾脏自噬水平的影响。方法：通过液相色谱串联质谱（LC-MS/MS）测定Slc7a9基因敲除大鼠24小时尿液胱氨酸浓度确定高尿胱氨酸；通过IHC（免疫组织化学）染色筛选无结石产生的胱氨酸尿症大鼠、观察肾脏组织结构有无明显变化；通过Western blot测定肾脏组织中的LC3-I、LC3-II、p62和mTOR的蛋白相对表达量，以检测自噬水平的变化，并探索变化原因；通过组织切片Masson染色法检测肾脏髓质纤维化程度。结果：10只无结石胱氨酸尿症大鼠尿液胱氨酸显著高于对照组；未发现有胱氨酸结石的生成与肾脏结构性变化；Masson染色提示胱氨酸尿症大鼠发现轻度肾脏纤维化过程；肾脏组织自噬标记蛋白LC3-I、LC3-II蛋白相对表达量、LC3-II/LC3-I比值以及自噬当量p62相对表达较对照组均显著降低，mTOR相对表达量显著升高。以上差异均有统计学意义（P＜0.05）。结论：在胱氨酸尿症大鼠模型上，发现无结石形成情况下的尿高胱氨酸水平可通过mTOR途径抑制大鼠肾脏组织的自噬水平，自我保护作用减弱，由此参与胱氨酸尿症的肾脏损伤过程。

Inhibited Renal Autophagy Activity Caused by High Urinary Cystine in Cystinuria Rats

ABSTRACT Objective: To investigate the effect on renal autophagy activity of cystinuria rat models. Methods: 24-hour urine was determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) to confirm high content of cystine in urine. IHC was conducted to make sure no stone formatted and no obvious organizational changes. Masson staining of kidney tissue sections was done to detect renal fibrosis level and to evaluate degree of kidney damage. Expression of autophagy marker, LC3-I, LC3-II, p62 and mTOR protein was, measured by Western blotting to detect changes in the pathway of autophagy and the reason of variations. Results: The urinary cystine of 10 cystinuria rats obtained was significantly higher than those of the wild type group rats, and the generation of cystine stones was not detected. Masson staining suggested that mild renal fibrosis was found in cystinuria rats. The relative expression of LC3-I, LC3-II and p62 proteins and the LC3-II/LC3-I ratio in the kidney tissues of stone-free cystinuria rats were significantly lower than those of the wild type group rats. Expression of mTOR raised significantly. All those differences were statistically significant(P<0.05). Conclusion: Cystinuria rats inhibited renal autophagy activity significantly by upregulating mTOR level, and hence lead decreasing of self-protection to the kidney, even without stone formation. And inhibited autophagy activity may involve in the process of kidney damage in cystinuria.