Steroid regulation of the ENaC recycling pathway: a proteomic analysis

The epithelial Na channel (ENaC) recycles to the apical membrane in kidney cortical collecting duct (CCD) cells through sequential endosomal compartments. We have demonstrated ENaC flow through early endosomal antigen (EEA1) and Rab11 positive compartments. The size and function of the ENaC recycling compartment diminishes in the absence of steroids (SD) and is reconstituted by steroid repletion (SR). We propose that steroids regulate specific components of the recycling pathway and have used an unbiased proteomic approach to examine the relative proteome of EEA1 and Rab11 endosomes under conditions of steroid depletion and repletion. Two approaches were used. Immunoisolated EEA1 or Rab11 vesicles from SD or SR cells were in‐solution digested prior to LC‐MS/MS and proteins were quantitatively compared using iTRAQ labeling technique. Alternatively, endosomes were in‐gel digested following 1D SDS‐PAGE and analyzed. Over 100 proteins were identified in each endosome population and there was roughly 20% overlap between EEA1 and Rab11 proteomes. Several proteins were identified as up‐regulated in SR cells by iTRAQ in both EEA1and Rab11 endosomes and have been confirmed by Western Blot. A more robust proteome was revealed in the 1D gel approach than in iTRAQ analysis suggesting that greater precision might be achieved with quantitative analysis of whole protein iTRAQ labeling, 1D gel separation and MS analysis.