Exosomal transfer from human renal proximal tubule cells to distal tubule and collecting duct cells

Abstract

Objectives Exosomes are 50-90 nm extracellular membrane particles that may mediate trans-cellular communication between cells and tissues. We have reported that human urinary exosomes contain miRNA that are biomarkers for salt sensitivity and inverse salt sensitivity of blood pressure. This study examines exosomal transfer between cultured human renal proximal tubule cells (RPTCs) and from RPTCs to human distal tubule and collecting duct cells. Design and methods For RPTC-to-RPTC exosomal transfer, we utilized 5 RPTC lines producing exosomes that were fluorescently labeled with exosomal-specific markers CD63-EGFP or CD9-RFP. Transfer between RPTCs was demonstrated by co-culturing CD63-EGFP and CD9-RFP stable clones and performing live confocal microscopy. For RPTC-to-distal segment exosomal transfer, we utilized 5 distal tubule and 3 collecting duct immortalized cell lines. Results Time-lapse videos revealed unique proximal tubule cellular uptake patterns for exosomes and eventual accumulation into the multi-vesicular body. Using culture supernatant containing exosomes from 3 CD9-RFP and 2 CD63-EGFP RPTC cell lines, all 5 distal tubule cell lines and all 3 collecting duct cell lines showed exosomal uptake as measured by microplate fluorometry. Furthermore, we found that RPTCs stimulated with fenoldopam (dopamine receptor agonist) had increased production of exosomes, which upon transfer to distal tubule and collecting duct cells, reduced the basal reactive oxygen species (ROS) production rates in those recipient cells. Conclusion Due to the complex diversity of exosomal contents, this proximal-to-distal vesicular inter-nephron transfer may represent a previously unrecognized trans-renal communication system.

Isolation and characterization of urinary extracellular vesicles: implications for biomarker discovery

“…Importantly, these effects were lost upon RNase treatment of microvesicles or knockdown of Dicer in progenitor cells. Exosomes labelled with fluorescent exosome-specific fusion proteins from multiple proximal tubule cell lines were taken up by distal tubule cells and collecting duct cells 143 . Using dopamine receptor-specific agonists (fenoldopam), these exosomes reduced the generation of reactive oxygen species in distal tubular and collecting duct cells, although transfer of RNA was not examined 143 .…”

Exosomes and microvesicles in normal physiology, pathophysiology, and renal diseases

“…Glomerular endothelial cell-derived microvesicles exposing the kinin B1 receptor and interleukin 8 (IL-8) on their surface attracted neutrophils [127, 128]. Proximal tubular cells cultured in the presence of fenoldopam (a dopamine receptor agonist) released exosomes that reduced the production of reactive oxygen species in distal tubule and collecting duct cells [129], indicating the transfer of an anti-inflammatory response.…”

Exosomal GAPDH from Proximal Tubule Cells Regulate ENaC Activity

“…Another mechanism for communication from the proximal tubule to the distal tubule may involve exosomes [2]. Exosomes are specialized nanometer-scale membranous vesicles derived from cells and are present in biological fluids including blood and urine.…”