The miRNA Expression of Urinary Extracellular Vesicles in Patients With Gitelman Syndrome: The Role of hsa-let-7d-3p

Abstract

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.

Keywords: Gitelman syndrome; NEDD4L; extracellular vesicle; hsa‐let‐7d‐3p; microRNA; thiazide‐sensitive sodium chloride cotransporter.

FIGURE 1

Characteristics of urinary extracellular vesicles (uEVs). uEVs were confirmed by immunoblotting (TSG101, CD9, and NSE) (A), nanoparticle tracking analysis with an average size distribution of 115.3 nm (B) and real‐time monitoring image (C), and electron micrography (scale bar 100 nm) (D).

FIGURE 2

Differential expression miRNAs. The volcano plot showed the differential expression miRNAs (red points) from urinary extracellular vesicles (uEVs) (n = 358) (A) and renal biopsy tissues (n = 652) (B) when comparing GS patients to the healthy control (HC) (Log₂[GS UMIs/HC UMIs]). p values as indicated, Student's paired t‐test.

FIGURE 3

Validation of increased hsa‐let‐7d‐3p and decreased Nedd4l expression in mice. (A) Increased hsa‐let‐7d‐3p from Ncc^S707x/S707x uEVs using real‐time PCR. (B) Decreased Nedd4l expression was confirmed in Ncc^S707X/S707X mice with Aqp2 used as a tubular marker of collecting duct (CD). (C) Three pairs of tissues from wild‐type mice and Ncc^S707x/S707x were shown.

FIGURE 4

Real‐time PCR of hsa‐let‐7d‐3p from human uEVs. (A) Amplification plots of hsa‐let‐7d‐3p from real‐time PCR of uEVs samples from Gitelman syndrome patients (GS, red) and healthy controls (HC, blue) (GS, n = 11; HC, n = 11). ΔRn, Δ Rn value. (B) Relative quantification data of hsa‐let‐7d‐3p abundance in panel A. p values as indicated, unpaired t test. Error bars indicate SD.

FIGURE 5

The miRNA hsa‐let‐7d‐3p targeting the NEDD4L mRNA through a targeting sequence located at 3′‐UTR. (A) The miRNA recognition element (MRE), located at position 3744–3750 of the NEDD4L 3′UTR (untranslated region), binds to the miRNA sequence. The binding coding (CD) sequence (CGUAUAAA) is paired with hsa‐let‐7d‐3p (GCAUAUC). This site was used in the luciferase assay with the pMIR‐REPORT system (B). Dual‐luciferase reporter assays were performed to test the interaction of hsa‐let‐7d‐3p and its targeting sequence in the NEDD4L 3′‐UTR using constructs containing the predicted targeting sequence (pMIR‐REPORT‐NEDD4L‐WT) and mutated targeting sequence (pMIR‐REPORT‐NEDD4L‐Mut) cloned into the 3′‐UTR of the reporter gene (B). Data represents three independent experiments with triplicate measurements. *Indicates p < 0.05.

FIGURE 6

Proposed role of uEVs hsa‐let‐7d‐3p in regulating NEDD4L expression in Gitelman syndrome (GS). (A) Thiazide‐sensitive sodium chloride co‐transporter (NCC) and NEDD4L that promote ENaC degradation in distal tubules involve the balancing sodium reabsorption from ENaC in healthy control (HC). (B) In GS patients, a mutation in SLC12A3 reduces NCC activity in the distal convoluted tubule resulting in salt wasting. Increased uEVs hsa‐let‐7d‐3p will negatively regulate NEDD4L, that will reduce ENaC degradation, leading to reabsorption of sodium from ENaC.