Urinary Extracellular Vesicles for Renal Tubular Transporters Expression in Patients With Gitelman Syndrome.

Chih-Chien Sung,Yi-Jia Lin,Min-Hsiu Chen,Shih-Hua Lin,Sung-Sen Yang,Yu-Chun Lin,Yi-Chang Lin

doi:10.3389/fmed.2021.679171

Chih-Chien Sung, Yi-Jia Lin + Show 5 more

Open Access

https://doi.org/10.3389/fmed.2021.679171

Copy DOI

Abstract

Background: The utility of urinary extracellular vesicles (uEVs) to faithfully represent the changes of renal tubular protein expression remains unclear. We aimed to evaluate renal tubular sodium (Na+) or potassium (K+) associated transporters expression from uEVs and kidney tissues in patients with Gitelman syndrome (GS) caused by inactivating mutations in SLC12A3.Methods: uEVs were isolated by ultracentrifugation from 10 genetically-confirmed GS patients. Membrane transporters including Na+-hydrogen exchanger 3 (NHE3), Na+/K+/2Cl− cotransporter (NKCC2), NaCl cotransporter (NCC), phosphorylated NCC (p-NCC), epithelial Na+ channel β (ENaCβ), pendrin, renal outer medullary K1 channel (ROMK), and large-conductance, voltage-activated and Ca2+-sensitive K+ channel (Maxi-K) were examined by immunoblotting of uEVs and immunofluorescence of biopsied kidney tissues. Healthy and disease (bulimic patients) controls were also enrolled.Results: Characterization of uEVs was confirmed by nanoparticle tracking analysis, transmission electron microscopy, and immunoblotting. Compared with healthy controls, uEVs from GS patients showed NCC and p-NCC abundance were markedly attenuated but NHE3, ENaCβ, and pendrin abundance significantly increased. ROMK and Maxi-K abundance were also significantly accentuated. Immunofluorescence of the representative kidney tissues from GS patients also demonstrated the similar findings to uEVs. uEVs from bulimic patients showed an increased abundance of NCC and p-NCC as well as NHE3, NKCC2, ENaCβ, pendrin, ROMK and Maxi-K, akin to that in immunofluorescence of their kidney tissues.Conclusion: uEVs could be a non-invasive tool to diagnose and evaluate renal tubular transporter adaptation in patients with GS and may be applied to other renal tubular diseases.

Highlights

Gitelman syndrome (GS) is one of the most common inherited tubulopathy with a prevalence ranging from 0.25 to 4/10,000 per population
The defective NCC function caused by different classes of SLC12A3 mutations leads to the reduced sodium chloride (NaCl) reabsorption in distal convoluted tubules (DCT) with increased luminal NaCl delivery to downstream collecting ducts (CD) responsible for NaCl reabsorption via epithelial Na+ channel (ENaC) and K+ secretion via renal outer medullary K1 channel (ROMK) and largeconductance, voltage-activated and Ca2+-sensitive K+ channel (Maxi-K)
The expression of ENaCβ, ROMK and MaxiK in mouse GS model has been reported to be significantly increased in both immunoblotting and immunofluorescence of mouse kidney [6], the adaptive response of upstream and downstream Na+ and K+ associated transporters in response to renal Na+ and K+ wasting in GS patients remains unknown

Summary

Introduction

Gitelman syndrome (GS) is one of the most common inherited tubulopathy with a prevalence ranging from 0.25 to 4/10,000 per population. It is caused by biallelic inactivating mutations in the SLC12A3 gene encoding thiazide-sensitive sodium-chloride cotransporter (NCC) expressed in the apical membrane of distal convoluted tubules (DCT) [1, 2]. We aimed to evaluate renal tubular sodium (Na+) or potassium (K+) associated transporters expression from uEVs and kidney tissues in patients with Gitelman syndrome (GS) caused by inactivating mutations in SLC12A3

Objectives

Methods

Results

Conclusion