Exosomal GAPDH from Proximal Tubule Cells Regulate ENaC Activity

Abstract

Exosomes are nanometer-scale, cell-derived vesicles that contain various molecules including nucleic acids, proteins, and lipids. These vesicles can release their cargo into adjacent or distant cells and mediate intercellular communication and cellular function. Here we examined the regulation of epithelial sodium channels in mpkCCD cells and distal tubule Xenopus 2F3 cells by exosomes isolated from proximal tubule LLC-PK1 cells. Cultured mpkCCD cells were stained with CTX coupled to a green fluorophore in order to label the cell membranes and freshly isolated exosomes from LLC-PK1 cells were labeled with the red lipophilic dye PKH26 in order to visualize uptake of exosomes into the cells. Single-channel patch clamp recordings showed the open probability of ENaC in Xenopus 2F3 cells and in freshly isolated split-open tubules decreased in response to exogenous application of exosomes derived from LLC-PK1 proximal tubule cells. Active GAPDH was identified within exosomes derived from proximal tubule LLC-PK1 cells. The effect on ENaC activity in Xenopus 2F3 cells was blunted after application of exosomes transfected with the GAPDH inhibitor heptelidic acid. Also, we show GAPDH and ENaC subunits associate in mpkCCD cells. These studies examine a potential role for exosomes in the regulation of ENaC activity and examine a possible mechanism for communication from proximal tubule cells to distal tubule and collecting duct cells.

Lipidomic analysis of urinary exosomes from hereditary α‐tryptasemia patients and healthy volunteers

“…Exosome concentration and size was determined by nanoparticle tracking analysis using a NanoSight NS300 (Malvern Instruments) as previously described. 13,25,28 For Western blot analysis, an aliquot of the exosomes was first mixed in an equal volume of radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher) and then sonicated for two 10 second intervals on ice. Protein concentration was determined using the bicinchoninic acid protein assay (Thermo Fisher).…”

“…Exosomes are nano‐sized vesicles derived from the endosomal network that have been shown to play an important role in various biological processes including intercellular communication, cell differentiation, proliferation, and intracellular signaling . The physiological and pathophysiological roles of exosomes are mediated by the packaged material that they carry which include mRNAs, miRNAs, lipids, and various types of proteins .…”

Hydrogen Peroxide Stimulates Exosomal Cathepsin B Regulation of the Receptor for Advanced Glycation End‐Products (RAGE)

“…Exosomes were isolated from conditioned media as described by [Jella et al, 2016] with the following modifications. The conditioned media from R3/1 cells cultured in tissue culture flasks and treated with vehicle or hydrogen peroxide was collected every other day for up to 2 weeks.…”

“…Exosomes, membrane‐bound vesicles of 30–150 nM in diameter that are released into the extracellular space after fusion with the plasma membrane, are present in most biological fluids [Zeringer et al, ]. Exosomes serve as vehicles for cell‐to‐cell communication, notably spanning a wide range of distances, in which cargo (e.g., nucleic acids, proteins, prions, etc …) modulate recipient cells [Jella et al, ]. The composition of the exosome varies depending upon cell type and context, and multiple studies converge to illustrate that exosomes affect cell function [Lang et al, ; Menay et al, ; Wang et al, ; Zhang et al, ].…”

Extracellular vesicles in motion

“…Furthermore, EVs are found to play key roles when studied in relation to physiology, oncology as well as immunology [8] [9] [10] [11] [12]. When EV morphology is studied with cryo-electron microscopy (cryo-EM), most vesicles are round, but they can also have multiple other shapes [13] [14] [15] [16] [17].…”