The <scp>miRNA</scp> Expression of Urinary Extracellular Vesicles in Patients With Gitelman Syndrome: The Role of hsa‐let‐7d‐3p

Gitelman syndrome (GS) is caused by an inactivating mutation in the SLC12A3, encoding the thiazide-sensitive sodium chloride cotransporter (NCC), leading to salt wasting and electrolyte imbalance. Our aim is to identify microRNA (miRNA) expression and explore their role from urinary extracellular vesicles (uEVs) in GS patients. In this study, both uEVs from 23 genetically confirmed GS patients and renal biopsied tissues from another 3 GS patients were extracted for small RNA sequencing. Small RNA sequencing identified 358 miRNAs from uEVs and 652 miRNAs from renal biopsied tissues. Among differentially expressed miRNAs, 20 were upregulated and 23 downregulated in uEVs, while renal biopsied tissues showed 30 upregulated and 23 downregulated miRNAs. Four miRNAs (hsa-let-7d-3p, hsa-miR-362-5p, hsa-miR-30c-5p, and hsa-miR-30b-5p) overlapped from uEVs and renal tissues. In particular, the terms of the distinct upregulated hsa-let-7d-3p target genes were related to ion transport and membrane depolarization, especially in neural precursor cell expressed, developmentally downregulated 4-like (NEDD4L). Real-time PCR of uEVs from another 11 GS patients confirmed significantly elevated hsa-let-7d-3p compared to healthy controls. The decrease in the Nedd4l expression in the collecting duct was also confirmed in Ncc^S707X/S707X knock-in mice. Dual luciferase assays further demonstrated that hsa-let-7d-3p negatively regulated the expression of NEDD4L. These findings concluded that differentially expressed miRNAs could be identified from uEVs in GS patients, and hsa-let-7d-3p, the only upregulated miRNA, negatively regulates NEDD4L expression in the collecting duct.