Comparative proteomic analysis of kidney distal convoluted tubule and cortical collecting duct cells following long-term hormonal stimulation

The distal convoluted tubule (DCT) and the cortical collecting ducts (CCD) are portions of renal tubule that are partly responsible for maintaining the systemic concentrations of potassium, sodium, calcium and magnesium. Despite being structurally similar, DCT and CCD cells have different transport capabilities due to a variety of different membrane-associated transport proteins. However, DCT and CCD cells appear to be modulated via the same hormones. The objective of this study was assess the differential response of DCT and CCD cells to long-term exposure to the hormones vasopressin or angiotensin II, both of which modulate DCT and CCD cells differently. Mass spectrometry based quantitative proteomics was used to profile the differential proteome between DCT and CCD. Mouse kidney distal convoluted tubule cells (mpkDCT) were cultured in heavy SILAC medium (Lys+6, Arg+10) while cortical collecting duct cells (mpkCCD) were cultured in light SILAC medium (Lys+0, Arg+0). Four passages of labelled cells were used to generate four biological replicates for statistical analysis. In addition to control conditions, cells were also stimulated for 4 days with the vasopressin type II receptor agonist 1-desamino-8-D-arginine vasopressin (dDAVP, 1nM) or angiotensin II (ANGII, 1nM). Cells were harvested, equally pooled and subjected to offline high-pH fractionation based two dimensional LC-MS/MS analysis (Q-Exactive). Identification and quantification of proteins was performed by MaxQuant. Proteins that had at least three ratios out of four biological replicates were defined as quantifiable proteins. Proteins that had at least three iBAQ values out of four biological replicates in light or heavy channel and zero iBAQ values in the other channel were defined as unique proteins in a particular cell type. Out of the some four thousand quantifiable proteins under basal, dDAVP and AngII conditions, 1101, 1566 and 1294 proteins passed the quantitative Benjamini-Hochberg (BH) FDR 1% threshold, respectively. The mpkDCT or mpkCCD specific proteins were defined based on being an up-regulated protein that passed the 1% BH FDR threshold in one cell type plus the unique proteins in this cell type. These 1025 mpkDCT specific proteins and 1211 mpkCCD specific proteins under the three conditions were subjected to further bioinformatics analyses including Panther and DAVID gene ontology analyses, E3 ligase and deubiquitinating enzyme identification, and kinase and transcription factor predictions, with the aim to identify cell specific proteins that define tubule-specific biological processes. Preliminary data suggests that one specific gene CHIP in mpkCCD might be involved in the regulation of AQP2.