Department of Nephrology, The First Affiliated Hospital, Sun Yat-Sen University, 58th, Zhongshan Road II, 510080, Guangzhou, People's Republic of China.
Diabetologia. 2014 Aug;57(8):1726-36. doi: 10.1007/s00125-014-3282-0. Epub 2014 Jun 9.
AIMS/HYPOTHESIS: The contribution of aberrantly expressed microRNAs (miRNAs) to diabetic nephropathy in vivo is poorly understood.
Integrated comparative miRNA array profiling was used to examine the expression of serum miRNAs in patients with diabetic nephropathy. The abundance of miRNA-135a (miR-135a) was measured by real-time quantitative PCR in the serum and kidney tissues of patients with diabetic nephropathy. The luciferase assay combined with mutation and immunoblotting was used to screen and verify the bioinformatically predicted miRNAs. Ca(2+) entry or intracellular Ca(2+) ([Ca(2+)]i) was performed by imaging Fura-2/AM-loaded cells using a fluorescence microscopy system. The role of miR-135a in vivo was explored with locked nucleic acid antisense oligonucleotides.
MiR-135a was markedly upregulated in serum and renal tissue from patients with diabetic nephropathy, as well from db/db mice, and this was associated with the development of microalbuminuria and renal fibrosis. Furthermore, we identified transient receptor potential cation channel, subfamily C, member 1 (TRPC1) as a target of miR-135a during renal injury. We demonstrated that overexpression of TRPC1 was able to reverse the pathological effects of miR-135a on promoting proliferation of mesangial cells and increasing synthesis of extracellular matrix proteins. Moreover, miR-135a attenuated store depletion-induced Ca(2+) entry into cells by regulating TRPC1. Importantly, knockdown of miR-135a in diabetic kidneys restored levels of TRPC1 and reduced synthesis of fibronectin and collagen I in vivo. Suppressing TRPC1 levels to prevent Ca(2+) entry into cells may be a mechanism whereby miR-135a promotes renal fibrosis in diabetic kidney injury.
CONCLUSIONS/INTERPRETATION: These findings suggest an important role for miR-135a in renal fibrosis and inhibition of miR-135a might be an effective therapy for diabetic nephropathy.
目的/假设：异常表达的 microRNAs（miRNAs）在糖尿病肾病中的作用还不太清楚。
采用整合比较 miRNA 芯片分析方法检测糖尿病肾病患者血清中 miRNAs 的表达。采用实时定量 PCR 检测糖尿病肾病患者血清和肾脏组织中 miRNA-135a（miR-135a）的含量。采用荧光显微镜系统检测 Fura-2/AM 负载细胞的 Ca2+内流或细胞内 Ca2+浓度([Ca2+]i)。采用荧光素酶报告基因检测和突变及免疫印迹实验筛选和验证生物信息学预测的 miRNAs。采用锁定核酸反义寡核苷酸探索 miR-135a 在体内的作用。
miR-135a 在糖尿病肾病患者血清和肾脏组织中以及 db/db 小鼠中显著上调，与微量白蛋白尿和肾纤维化的发生相关。此外，我们发现瞬时受体电位阳离子通道亚家族 C，成员 1（TRPC1）是 miR-135a 在肾脏损伤过程中的靶基因。我们证实，TRPC1 的过表达能够逆转 miR-135a 促进系膜细胞增殖和增加细胞外基质蛋白合成的病理作用。此外，miR-135a 通过调节 TRPC1 抑制储存耗竭诱导的 Ca2+内流。重要的是，在糖尿病肾脏中敲低 miR-135a 可恢复 TRPC1 水平并减少体内纤维连接蛋白和 I 型胶原的合成。抑制 TRPC1 水平以阻止 Ca2+进入细胞可能是 miR-135a 促进糖尿病肾病肾纤维化的机制之一。
这些发现表明 miR-135a 在肾脏纤维化中起重要作用，抑制 miR-135a 可能是治疗糖尿病肾病的有效方法。
