Yu Min, Hu Jian, Ju Tao, Wang Ren, Wang Meiqiu, Gao Chunlin, Xia Zhengkun
Department of Neonatology, Taizhou People's Hospital, Nanjing Medical University, Taizhou, Jiangsu 225300, China.
Department of Pediatrics, The Affiliated Huai'an No.1 People's Hospital of Nanjing Medical University, Huai'an, Jiangsu 223300, China.
Cell Mol Biol (Noisy-le-grand). 2024 May 27;70(5):284-288. doi: 10.14715/cmb/2024.70.5.42.
Podocyte injury plays a vital role in focal segmental glomerulosclerosis (FSGS), and apoptosis is one of its mechanisms. The transient receptor potential channel 6 (TRPC6) is highly expressed in podocytes and mutations mediate podocyte injury. We found TRPC6 gene mutation (N110S) was a new mutation and pathogenic in the preliminary clinical work. The purpose of this study was to investigate the potential mechanism of mutation in TRPC6 (TRPC6-N110S) in the knock-in gene mouse model and in immortalized mouse podocytes (MPC5). Transmission electron microscopy was used to evaluate renal injury morphology. We measured 24-hour urinary albumin-to-creatinine ratios and major biochemical parameters such as serum albumin, urea nitrogen, and total cholesterol. The results of CCK-8 assay and apoptosis experiments showed that the TRPC6-N110S overexpression group had slower proliferative activity and increased apoptosis than the control group. FluO-3 assay revealed increased calcium influx in the TRPC6-N110S overexpression group. Podocin level was decreased in TRPC6-N110S group, while TRPC6 and desmin levels were increased in TRPC6-N110S group. The 24 h uACR at 6 weeks was significantly higher in the pure-zygotes group than in the WT and heterozygotes groups, and this difference was found at 8 and 10 weeks.TRPC6 levels showed no significant difference between homozygote and WT mice. Compared to homozygote group, expression of podocin and nephrin were increased in WT, but levels of desmin was decreased in WT. Our results suggest that this new mutation causes podocyte injury probably by enhancing calcium influx and podocyte apoptosis, accompanied by increased proteinuria and decreased expression of nephrin and podocin.
足细胞损伤在局灶节段性肾小球硬化（FSGS）中起着至关重要的作用，而细胞凋亡是其机制之一。瞬时受体电位通道6（TRPC6）在足细胞中高度表达，其突变介导足细胞损伤。在初步临床工作中，我们发现TRPC6基因突变（N110S）是一种新的突变且具有致病性。本研究的目的是在基因敲入小鼠模型和永生化小鼠足细胞（MPC5）中研究TRPC6（TRPC6-N110S）突变的潜在机制。采用透射电子显微镜评估肾脏损伤形态。我们测量了24小时尿白蛋白与肌酐比值以及主要生化参数，如血清白蛋白、尿素氮和总胆固醇。CCK-8检测和凋亡实验结果表明，与对照组相比，TRPC6-N110S过表达组的增殖活性较慢且凋亡增加。FluO-3检测显示TRPC6-N110S过表达组的钙内流增加。TRPC6-N110S组的足突蛋白水平降低，而TRPC6和结蛋白水平在TRPC6-N110S组中升高。纯合子组在6周时的24小时尿白蛋白与肌酐比值显著高于野生型和杂合子组，在8周和10周时也发现了这种差异。纯合子小鼠与野生型小鼠的TRPC6水平无显著差异。与纯合子组相比，野生型小鼠的足突蛋白和nephrin表达增加，但野生型小鼠的结蛋白水平降低。我们的结果表明，这种新突变可能通过增强钙内流和足细胞凋亡导致足细胞损伤，同时伴有蛋白尿增加以及nephrin和足突蛋白表达降低。
