Human kidney primary proximal tubule cell LC-MSMS

Angiotensin II (AngII), the effector of the renin angiotensin system causes kidney disease progression by signalling through AT-1 receptor, but there are no measures of AngII activity in the kidney. Accordingly, we sought to define an AngII-regulated proteome in primary human proximal tubular epithelial cells (PTEC) in order to identify potential markers of AngII activity in the kidney. PTECs were labeled with SILAC heavy arginine(+6) and lysine(+8) OR light arginine and lysine for 6 doubling times. Heavy-labeled PTECs were stimulated with AngII and light-labeled PTECs were stimulated with the control medium. AngII-treated and control-treated PTEC lysates were mixed in 1:1 total protein ratio and their proteomes were compared. We generated 5 biological replicates (1 supernatant, 1 replicate with reverse labeling). LTQ-Orbitrap mass spectrometer was used for LC-MSMS analysis. Of 4618 quantified proteins, 83 were differentially regulated by AngII in 4 biological replicates of PTEC lysates. We subsequently confirmed and verified 18 differentially regulated proteins by using SRM, RT-PCR and ELISA. We also went on to validate the main functional, enriched networks by using systems biology approach and an in vivo mouse model.