title: Quantification of angiotensin II-regulated proteins in urine of patients with polycystic and other chronic kidney diseases by selected reaction monitoring.
abstract: 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.
  
  
  RESULTS
  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 <15 % of the final concentrations. Urine excretion rates of proteins BST1, LAMB2, LYPA1, RHOB and TSP1 were significantly different (p < 0.05, one-way ANOVA) between patients with CKD, those with ADPKD and healthy controls. Urine protein excretion rates were highest in CKD patients and lowest in ADPKD patients. Univariate analysis demonstrated significant association between urine protein excretion rates of most proteins and disease group (p < 0.05, ANOVA) as well as sex (p < 0.05, unpaired t test). Multivariate analysis across protein concentration, age and sex demonstrated good separation between ADPKD and CKD patients.
  
  
  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.
authors: Division of Nephrology, Department of Medicine, Toronto General Hospital, University Health Network, University of Toronto, 11-PMB-189, 585 University Avenue, Toronto, ON M5G 2N2 Canada ; Toronto General Research Institute, University Health Network, Toronto, Canada.
authors: Ana
authors: A
authors: Konvalinka
authors: Department of Laboratory Medicine and Pathobiology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Canada.
authors: Ihor
authors: I
authors: Batruch
authors: Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, Canada.
authors: Tomas
authors: T
authors: Tokar
authors: Department of Laboratory Medicine and Pathobiology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Canada.
authors: Apostolos
authors: A
authors: Dimitromanolakis
authors: Toronto General Research Institute, University Health Network, Toronto, Canada.
authors: Shelby
authors: S
authors: Reid
authors: Division of Genomic Medicine, University Health Network, University of Toronto, Toronto, Canada.
authors: Xuewen
authors: X
authors: Song
authors: Division of Nephrology, Department of Medicine, Toronto General Hospital, University Health Network, University of Toronto, 11-PMB-189, 585 University Avenue, Toronto, ON M5G 2N2 Canada ; Toronto General Research Institute, University Health Network, Toronto, Canada.
authors: York
authors: Y
authors: Pei
authors: Department of Laboratory Medicine and Pathobiology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Canada.
authors: Andrei P
authors: AP
authors: Drabovich
authors: Department of Laboratory Medicine and Pathobiology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Canada ; Department of Clinical Biochemistry, University Health Network, University of Toronto, Toronto, Canada.
authors: Eleftherios P
authors: EP
authors: Diamandis
authors: Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, Canada ; Departments of Medical Biophysics and Computer Science, University Health Network, University of Toronto, Toronto, Canada.
authors: Igor
authors: I
authors: Jurisica
authors: Division of Nephrology, Department of Medicine, Toronto General Hospital, University Health Network, University of Toronto, 11-PMB-189, 585 University Avenue, Toronto, ON M5G 2N2 Canada ; Toronto General Research Institute, University Health Network, Toronto, Canada.
authors: James W
authors: JW
authors: Scholey
keywords: Angiotensin II; Autosomal dominant polycystic kidney disease; Chronic kidney disease; Polycystic kidney disease; Proteomics; Renin angiotensin system; Selected reaction monitoring; Urine