Abstract 38: Exosomes Secreted From Human Renal Proximal Tubule Cells Can Transfer Their Contents to Proximal Tubule, Distal Tubule and Collecting Duct Cells

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Abstract

Exosomes are 50-90 nm extracellular membrane particles that may mediate trans-cellular communication between cells and tissues. We have previously found that urinary exosomes contain miRNA that are biomarkers for salt sensitivity and inverse salt sensitivity of blood pressure. We also found that urinary exosomes contain dopamine 1 receptor and G protein-coupled receptor kinase type 4. We have also shown that exosomes can transfer between cultured human renal proximal tubule cells (RPTC). Here, we further explored RPTC-to-RPTC exosomal transfer using 5 RPTC lines containing exosomes that were fluorescently labelled with either exosomal-specific marker CD63-EGFP or CD9-mCherry. Transfer between RPTCs was confirmed by co-culturing CD63-EGFP and CD9-mCherry stable RPTC clones and performing live confocal microscopy. Dual color time-lapse videos reveal unique cellular secretion and uptake patterns for exosomes and eventual accumulation into the multi-vesicular body.

We hypothesized that proximal tubule-derived exosomes could also be taken up by the distal tubule (DT). Five DT and 3 collecting duct (CD) cell lines were isolated and immortalized by infecting with hTert-containing lentiviral particles. DT cells were isolated from human kidney by collagenase digestion and biotinylated lectin or immuno-magnetic affinity separation (using dolichos biflorus agglutinin (DBA) or anti-Tamm-Horsfall protein antibody). CD cells were similarly isolated with anti-L1CAM antibody. Using sterile-filtered cell culture supernatant from 3 different CD9-mCherry RPTC lines, all 5 DT cell lines showed exosome transfer at 24 hours as measured by microplate fluorometry (10,406 ± 1132 SEM RFU above background, p<0.0001, n=30). Similarly, all 3 CD cell lines showed significant exosome transfer (8773 ± 1558 SEM RFU, p<0.0001, n=18). CD63-EGFP exosomes from 2RPTC lines also transferred to all of the DT cell lines (1713 ± 1131 RFU above background, p<0.0001, n=30) and CD cell lines (1007 ± 315 RFU, p<0.01, n=18).

In conclusion, intra-nephron cellular exosomal transfer is demonstrated in RPTCs and between RPTCs and distal tubule and collecting duct cells. This proximal to distal nephron transfer may represent a new trans-cellular communication within the human kidney.

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