Urinary Extracellular Vesicles and Salt-Losing Tubulopathies: A Proteomic Approach

Abstract

Renal tubular cells release urinary extracellular vesicles (uEV) that are considered a promising source of molecular markers for renal dysfunction and injury. We investigated uEV proteomes of patients with hereditary salt-losing tubulopathies (SLTs), focusing on those caused by Gitelman and Bartter (BS) syndromes, to provide potential markers for differential diagnosis. Second morning urine was collected from patients with genetically proven SLTs and uEV were isolated by the ultracentrifugation-based protocol. The uEV proteome was run through a diagonal bidimensional electrophoresis (16BAC/SDS-PAGE), to improve hydrophobic protein resolution. Sixteen differential spots from the proteome of two variants (BS2 and BS3) were analysed by nLC-ESI-MS/MS after in-gel tryptic digestion. A total of 167 protein species were identified from 7 BS2 spots and 9 BS3 spot. Most of these proteins were membrane-associated proteins, in particular transmembrane proteins, and were related to typical renal functions. The differential content of some uEV was then validated by immunoblotting. Our work suggests that uEV proteomics represents a promising strategy for the identification of differential SLT proteins. This could play a role in understanding the pathophysiological disease mechanisms and may support the recognition of different syndromes.

Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles

“…These results were the foundation for a prostate cancer diagnostic test that has been extensively validated in two prospective multi‐center US studies (McKiernan et al., 2016; McKiernan et al., 2018). Altogether, these promising results inspired the search for uEV‐based biomarkers for other urogenital tract pathologies such as polycystic kidney disease, cystinuria, diabetic nephropathy, acute kidney injury/ renal ischemia‐reperfusion injury, glomerulonephritis, renal interstitial fibrosis/ chronic kidney disease, lupus nephritis, nephronophthisis‐related ciliopathies, tubulopathies and primary and secondary hypertension (Abe et al., 2018; Bourderioux et al., 2015; Chun‐Yan et al., 2018; Corbetta et al., 2015; Gonzalez‐Calero et al., 2017; Kwon et al., 2017; La Salvia et al., 2020; Morikawa et al., 2018; Qi et al., 2016; Raimondo et al., 2016; Raimondo et al., 2020; Salih et al., 2016; Salih et al., 2018; Sonoda et al., 2009; Stokman et al., 2019; Tangtanatakul et al., 2018; van der Lubbe et al., 2012; Williams et al., 2020; Wolley et al., 2017; Zubiri et al., 2014). Many of the newly identified candidate biomarkers have not yet been validated in large independent cohorts or in additional laboratories, but nevertheless these examples highlight the enormous potential for uEV analyses as readouts for pathophysiological alterations within the urogenital and other systems.…”

Comprehensive strategy for identifying extracellular vesicle surface proteins as biomarkers for chronic kidney disease

“…NKCC2 levels are altered in OCKD ( Riazi et al, 2006 ; Wu et al, 2021 ). Raimondo et al, proposed NKCC2 levels in uEVs as potential biomarker for kidney injury Raimondo et al (2020) .…”

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

“…The absence of sodium-potassium-chloride cotransporter 2 (NKCC2) in urine exosomes from patients with Bartter syndrome was also reported in this study [ 115 ]. 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 ].…”