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

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

doi:10.3389/fmed.2021.679171

Cited by 9 publications

(10 citation statements)

References 58 publications

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“…To isolate uEVs, the collected urine was centrifuged at 17 000× g and 37°C for 10 min, and the supernatant was ultracentrifuged at 200 000× g for 2 h at 4°C. The pellet was resuspended in Laemmli buffer with dithiothreitol for immunoblotting (IB) 37,38 …”

Section: Methodsmentioning

confidence: 99%

Generation and analysis of pseudohypoaldosteronism type II knock‐in mice caused by a nonsense KLHL3 mutation in the Kelch domain

et al. 2022

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Mutations in the Kelch‐like 3 (KLHL3) gene are the most common cause of inherited pseudohypoaldosteronism type II (PHAII) featuring thiazide‐sensitive hypertension and hyperkalemic metabolic acidosis. Although Klhl3R528H/+ knock‐in (KI) mice carrying a missense mutation in the Kelch repeat domain have been reported, nonsense KLHL3 mutations in the same domain that cause PHAII have not been fully investigated in vivo. We generated and analyzed Klhl3 KI mice harboring a nonsense W523X mutation (corresponding to the human KLHL3 W470X mutation). Both heterozygous and homozygous Klhl3W523X/+ KI mice exhibited typical PHAII with low‐renin hypertension, hyperkalemia with reduced renal potassium excretion, and hyperchloremic metabolic acidosis. Their kidney tissues showed the presence of Klhl3 mRNA and increased Klhl3 protein levels along with enhanced downstream Wnk1/4‐Spak/Osr1‐N(k)cc phosphorylation. Increased protein expression of total Spak, phosphor(p‐)Spak, total Ncc, and p‐Ncc from urinary extracellular vesicles (uEVs) also confirmed the activation of the Wnk‐mediated Ncc pathway. In vitro studies showed that the human KLHL3 W470X mutation resulted in increased KLHL3 protein stability and disrupted its binding affinity for WNK1/4, leading to the attenuated degradation and increased abundance of total WNKs. In conclusion, nonsense Klhl3W523X/+ mice recapitulating PHAII phenotypes exhibit Klhl3 protein stability, abrogating its binding to Wnks, with enhanced Ncc expression in the kidney tissue and even in uEVs. Activation of the WNK‐mediated Na+‐Cl− co‐transporter reiterated the in vivo pathogenic role of nonsense KLHL3 mutations in PHAII.

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Section: Methodsmentioning

confidence: 99%

Generation and analysis of pseudohypoaldosteronism type II knock‐in mice caused by a nonsense KLHL3 mutation in the Kelch domain

et al. 2022

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“…In patients with Gitelman syndrome, uEV analysis also reflects the changes that occur in unaffected nephron segments to compensate for salt wasting. Although NCC was absent or reduced, NHE3, β-ENaC, and pendrin were increased both in uEVs and in kidney biopsies of patients with Gitelman syndrome [37]. Conversely, uEV abundances of NCC and pNCC were increased in patients with familial hyperkalemic hypertension, a mirror image of Gitelman syndrome caused by genetic variants in NCC regulating kinases or ligases, including WNK1, WNK4, KLHL3, or cullin-3 [3,38].…”

Section: Inherited Tubulopathiesmentioning

confidence: 96%

Urinary extracellular vesicles and tubular transport

Rudolphi

Blijdorp

Willigenburg

et al. 2022

Nephrology Dialysis Transplantation

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Tubular transport is a key function of the kidney to maintain electrolyte and acid-base homeostasis. Urinary extracellular vesicles (uEVs) harbor water, electrolyte, and acid-base transporters expressed at the apical plasma membrane of tubular epithelial cells. Within the uEV proteome, the correlations between kidney and uEV protein abundances are strongest for tubular transporters. Therefore, uEVs offer a non-invasive approach to probe tubular transport in health and disease. Here, we will review how kidney tubular physiology is reflected in uEVs and, conversely, how uEVs may modify tubular transport. Clinically, uEV tubular transporter profiling has been applied to rare diseases such as inherited tubulopathies, but also to more common conditions such as hypertension and kidney disease. Although uEVs hold the promise to advance the diagnosis of kidney disease to the molecular level, several biological and technical complexities still need to be addressed. The future will tell if uEV analysis will mainly be a powerful tool to study tubular physiology in humans or if it will move forward to become a diagnostic bedside test.

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“…In addition, Raimondo et al investigated the proteome of uEVs and set up a panel of 5 proteins, including carbonic anhydrase (CA2), vacuolar-type ATPase B subunit 1 (VATB1), Annexin A2 (ANXA2), sodium chloride cotransporter (NCC), and NKCC2, which can differentiate Gitelman and Bartter syndromes (salt-losing tubulopathies) [ 116 ]. Sung et al also found that NCC and phosphorylated-NCC were decreased in uEVs from patients with Gitelman syndrome, while Na + -hydrogen exchanger 3 (NHE3), epithelial Na + channel β (ENaCβ), and pendrin were increased [ 117 ]. By investigating urinary microvesicles, Rood et al found the increase of lysosome membrane protein 2 (LIMP-2) from patients with idiopathic membranous nephropathy, and the upregulation of LIMP-2 was identified in renal biopsies that the protein was co-localized with IgG along the glomerular basement membrane [ 118 ].…”

Section: Proteomics-based Studies Of Exosomes/sevs For Kidney Diseasesmentioning

confidence: 99%

Exosomal proteomics in kidney disease: From technical approaches to clinical applications

Brahmadhi¹,

Chuang²,

Wang³

et al. 2022

Journal of Food and Drug Analysis

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Exosomes are small extracellular vesicles (sEVs) secreted from cells and have a general diameter ranging from 30–150 nm. It was reported that exosomes have essential roles in intercellular communication and can be targeted as biomarkers of disease or as therapeutic agents. Among the different techniques used for exosome investigation, the mass spectrometry-based proteomics approach has accelerated the unraveling of the molecular composition of exosomes and has contributed to improved knowledge of molecular processes in various diseases. In this review, we focused on proteomics-based studies of exosomes and clinical applications in kidney diseases. A general introduction of exosomes, isolation and characterization techniques, and proteomics-based study workflows are included in this article. We also categorized applications in acute kidney injury, chronic kidney disease, renal transplantation, congenital kidney disease, and malignant kidney disorder to show the important findings from proteomics-based exosomal investigations.

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

Cited by 9 publications

References 58 publications

Generation and analysis of pseudohypoaldosteronism type II knock‐in mice caused by a nonsense KLHL3 mutation in the Kelch domain

Generation and analysis of pseudohypoaldosteronism type II knock‐in mice caused by a nonsense KLHL3 mutation in the Kelch domain

Urinary extracellular vesicles and tubular transport

Exosomal proteomics in kidney disease: From technical approaches to clinical applications

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