Kidney Histomorphology and Molecular Biology Laboratory, Nephrology Unit, Department of Medicine-DIMED, University of Padua, Via Giustiniani n° 2, 35128 Padua, Italy.
Department of Molecular Medicine, University of Padua, 35121 Padua, Italy.
Int J Mol Sci. 2023 Jan 9;24(2):1313. doi: 10.3390/ijms24021313.
Dent disease (DD1) is a rare tubulopathy caused by mutations in the CLCN5 gene. Glomerulosclerosis was recently reported in DD1 patients and ClC-5 protein was shown to be expressed in human podocytes. Nephrin and actin cytoskeleton play a key role for podocyte functions and podocyte endocytosis seems to be crucial for slit diaphragm regulation. The aim of this study was to analyze whether ClC-5 loss in podocytes might be a direct consequence of the glomerular damage in DD1 patients. Three DD1 kidney biopsies presenting focal global glomerulosclerosis and four control biopsies were analyzed by immunofluorescence (IF) for nephrin and podocalyxin, and by immunohistochemistry (IHC) for ClC-5. ClC-5 resulted as down-regulated in DD1 vs. control (CTRL) biopsies in both tubular and glomerular compartments (p < 0.01). A significant down-regulation of nephrin (p < 0.01) in DD1 vs. CTRL was demonstrated. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Caspase9) gene editing of CLCN5 in conditionally immortalized human podocytes was used to obtain clones with the stop codon mutation p.(R34Efs*14). We showed that ClC-5 and nephrin expression, analyzed by quantitative Reverse Transcription/Polymerase Chain Reaction (qRT/PCR) and In-Cell Western (ICW), was significantly downregulated in mutant clones compared to the wild type ones. In addition, F-actin staining with fluorescent phalloidin revealed actin derangements. Our results indicate that ClC-5 loss might alter podocyte function either through cytoskeleton disorganization or through impairment of nephrin recycling.
Dent 病（DD1）是一种由 CLCN5 基因突变引起的罕见的管状病变。最近有研究报道 DD1 患者存在肾小球硬化，并且 ClC-5 蛋白在人足细胞中表达。裂孔隔膜的调节对于足细胞功能至关重要，而 Nephrin 和肌动蛋白细胞骨架在其中发挥着关键作用。本研究旨在分析足细胞中 ClC-5 的缺失是否是 DD1 患者肾小球损伤的直接后果。通过免疫荧光（IF）和免疫组化（IHC），对 3 例表现为局灶性全球肾小球硬化的 DD1 肾活检和 4 例对照活检进行了 Nephrin 和 Podocalyxin 的分析，同时还对 ClC-5 进行了分析。结果显示，DD1 肾活检中 ClC-5 的表达在管状和肾小球区域均低于对照组（p<0.01）。与对照组相比，DD1 组 Nephrin 的表达显著下调（p<0.01）。利用条件永生化人足细胞的 CRISPR/Cas9（Clustered Regularly Interspaced Short Palindromic Repeats/Caspase9）基因编辑技术，获得了具有 p.(R34Efs*14) 终止密码突变的克隆。我们通过定量逆转录/聚合酶链反应（qRT/PCR）和细胞内 Western（ICW）分析显示，ClC-5 和 Nephrin 的表达在突变克隆中明显下调，与野生型克隆相比。此外，用荧光鬼笔环肽进行 F-肌动蛋白染色显示肌动蛋白排列紊乱。我们的结果表明，ClC-5 的缺失可能通过细胞骨架紊乱或 Nephrin 回收受损而改变足细胞的功能。
