Exosomal GAPDH from Proximal Tubule Cells Regulate ENaC Activity.

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

Identification of Glyceraldehyde-3-phosphate dehydrogenase by LC-MS/MS.

<p>The peptide was detected as doubly charged with a mass-to-charge ratio of 778.9126, which represents an error of 5 ppm. The tandem mass spectrum matched the following sequence, VPTPNVSVVDLT<u>C</u>R. The detection was made with Mascot with ion score 87.8.</p

Effect of exogenous application of apical exosomes from LLC-PK1 cells on amiloride-sensitive transepithelial current measurement in mpkCCD cells.

<p>Cells were cultured for 10 days to allow the formation of tight junctions and the measurement of resistances and voltages across confluent monolayers. Exosomes isolated from conditioned media present on the apical side of LLC-PK1 cells cultured on permeable transwell inserts were applied to the apical side of mpkCCD cell monolayers. Application of these exosomes resulted in a time and dose dependent decrease in transepithelial current. Each point represents the mean ± s.e. from 3 separate inserts containing cells (N = 3).</p

Uptake of fluorescently labeled exosomes from LLC-PK1 cells in mouse cortical collecting duct principal cells.

<p>mpkCCD cells were incubated with red PKH26-labeled exosomes at 37°C for one hour. The cell surface was labeled with the green CTX fluorescent dye. Arrows indicate exosomes taken up by the cells after a 1 hour incubation at 37°C. Twelve z stack images were taken in total and an orthogonal view (XZ and YZ axis) is shown.</p

Size characterization of intact exosomes isolated from the apical side of LLC-PK1 cells.

<p>(A) Electron micrograph of exosomes purified from LLC-PK1 cells. Negative staining transmission electron microscopy shows a field of exosomes ranging from 30–100 nm in size. Bar, 100nm. (B) Confirmation of exosomes size distribution and concentration by NanoSight analysis. Samples were prepared to a 1:1000 dilution in 1X phosphate buffered saline before being loaded onto a NanoSight NS300 with a high sensitivity camera and green 532nm laser. The size distribution profiles for the purified exosomes showed a peak at 50 nm. The concentration of the exosomes with a diameter of 50 nm in size was 2–2.5x10^7 exosomes/ml.</p

Exosomal GAPDH from Proximal Tubule Cells Regulate ENaC Activity - Fig 2

<p>Coomassie blue–stained sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis (A) and mass spectrometry analysis (B) of lysed exosomes isolated from conditioned media in the apical compartment of LLC-PK1 cells. Coomassie blue–stained sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis (C) and mass spectrometry analysis (D) of lysed exosomes isolated from conditioned media in the basolateral compartment of LLC-PK1 cells. Molecular weight markers (MWM) are shown in the first lane. Peptides listed in (B) and (D) are signature peptides corresponding to GAPDH.</p

Coimmunoprecipation and Western blot analysis showing an association between GAPDH and ENaC subunits.

<p>Polyclonal ENaC alpha, beta, and gamma antibodies were used to immunoprecipatate each subunit from mpkCCD cellular lysates and pull-down protein binding partners. The eluent from the immunoprecipitated complexes were separated by SDS-PAGE and the blots were probed with GAPDH polyclonal antibody. (A) Western blot analysis showing an immunoreactive band corresponding to GAPDH protein at 35 kDa from both Xenopus 2F3 cells and mpkCCD cells. (B) IP-Western showing specific antibodies for ENaC alpha, beta, and gamma subunits pull-down GAPDH from mpkCCD cell lysates. Heavy and light chains of IgG are indicated by arrows. IP refers to immunoprecipitation. IgG refers to immunoglobulin.</p

Effect of exosomes isolated from conditioned media on the basolateral side of LLC-PK1 cells on ENaC activity in Xenopus 2F3 cells.

<p>(A): representative single-channel recording using the cell-attached configuration shows no appreciable change in ENaC activity after application of basolateral plasma membrane LLC-PK1 exosomes to the apical side of Xenopus 2F3 cells. The dashed lines denote open and closed levels (o, open and c, closed). The number of current levels represents the number of channels in the patch. The arrow indicates the time point at which the exosomes were applied to the cells. (B): summary line graph showing the open probability (<i>P</i>_<b>o</b>) of ENaC did not change after applying exosomes to the apical surface of Xenopus 2F3 cells. Each point represents the mean ± s.e. and the data shown are from 10 separate patches (N = 10); P = 0.33.</p

GAPDH activity in exosomes isolated from LLC-PK1 cells.

<p>Three batches of exosomes (n = 3) isolated from conditioned media of LLC-PK1 cells present in the apical compartment or basolateral compartment of permeable transwell inserts were lysed in RIPA buffer. The ΔOD was calculated for the GAPDH activity in each sample by taking the difference in absorbance readings between two time points that fell within an NADH standard curve generated using six different concentrations of NADH. All sample readings were corrected for by subtracting the background reading from measuring the OD of the background control mix alone. The NADH amount generated by GAPDH activity during the reaction time was calculated for each sample. GAPDH activity is expressed as U/ml where one unit of GAPDH is the amount of enzyme that will generate 1.0 μmol of NADH per minute at pH 7.2 at 37°C. OD represents optical density. Data is presented as mean ± s.e. from 3 separate batches of exosomes (N = 3).</p