Quantification of Angiotensin II-regulated Proteins in Urine of Patients With Polycystic and Other Chronic Kidney Diseases by Selected Reaction Monitoring

摘要

BACKGROUND: Angiotensin-II (Ang II) mediates progression of autosomal-dominant polycystic kidney disease (ADPKD) and other chronic kidney diseases (CKD). However, markers of kidney Ang II activity are lacking. We previously defined 83 Ang II-regulated proteins in vitro, which reflected kidney Ang II activity in vivo. METHODS: In this study, we developed selected reaction monitoring (SRM) assays for quantification of Ang II-regulated proteins in urine of ADPKD and CKD patients. We demonstrated that 47 of 83 Ang II-regulated transcripts were differentially expressed in cystic compared to normal kidney tissue. We then developed SRM assays for 18 Ang II-regulated proteins overexpressed in cysts and/or secreted in urine. Methods that yielded CV ≤ 6 % for control proteins, and recovery ~100 % were selected. Heavy-labeled peptides corresponding to 13 identified Ang II-regulated peptides were spiked into urine samples of 17 ADPKD patients, 9 patients with CKD predicted to have high kidney Ang II activity and 11 healthy subjects. Samples were then digested and analyzed on triple-quadrupole mass spectrometer in duplicates. RESLUTS: Calibration curves demonstrated linearity (R(2) > 0.99) and within-run CVs < 9 % in the concentration range of 7/13 peptides. Peptide concentrations were normalized by urine creatinine. Deamidated peptide forms were monitored, and accounted for CONCLUSIONS: We have optimized methods for quantification of Ang II-regulated proteins, and we demonstrated that they reflected differences in underlying kidney disease in this pilot study. High urine excretion of Ang II-regulated proteins in CKD patients likely reflects high kidney Ang II activity. Low excretion in ADPKD appears related to lack of communication between cysts and tubules. Future studies will determine whether urine excretion rate of Ang II-regulated proteins correlates with kidney Ang II activity in larger cohorts of chronic kidney disease patients.