Urinary extracellular vesicles and tubular transport

Abstract

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 noninvasive approach to probing tubular transport in health and disease. Here, we 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 must still be addressed. The future will tell whether uEV analysis will mainly be a powerful tool to study tubular physiology in humans or whether it will move forward to become a diagnostic bedside test.

Keywords: aldosterone; aquaporin; biomarker; chronic kidney disease; exosomes; hypertension.

FIGURE 1:

uEVs: from excretion to analysis. a, Cells lining the kidney tubules excrete uEVs, either by outward budding of the cell membrane (microvesicles or apoptotic bodies) or through multivesicular bodies (MVB) that excrete exosomes after fusion with the cell membrane. b, uEVs are isolated before characterization, usually by a differential ultracentrifugation (UC) or ultrafiltration (UF) protocol. The isolated uEVs can be further purified by density gradient or size exclusion chromatography. c, Crude but faster alternatives for uEV isolation include precipitation, immunocapture, or acoustic trapping. d, After isolation, uEVs can be characterized by electron microscopy (with or without immunogold labeling), immunoblotting, liquid chromatography coupled with mass spectrometry (LC-MS), polymerase chain reaction (PCR), or RNA sequencing. e, Other techniques allow direct uEV/particle characterization, including nanoparticle tracking analysis (which may also be used to detect labeled uEVs); TRFIA (a sandwich immunoassay that uses a capture antibody and a secondary visualization antibody); or EVQuant, in which uEVs are immobilized in a clear gel, and then visualized by confocal microscopy, allowing for characterization of multiple proteins per single uEV. Not shown here are high-resolution flow cytometry techniques. f, After characterization, uEVs can be normalized (especially when using spot urine) with different approaches, including the use of an uEV marker (e.g. CD9, TSG101, ALIX), Z/quantile normalization (in the case of LC-MS or RNAseq), timed excretion, or creatinine normalization.

FIGURE 2:

Overview of the major apical tubular transporters identified in uEVs, including disease associations. uEVs are secreted by the tubular epithelial cells of all nephron segments and will therefore carry segment-specific transporters (depicted by the corresponding colors). The analysis of these tubular transport has been pursued in various disorders, including tubulopathies, hypertension, and kidney injury. The figure was created with BioRender.com.