iTRAQ-based proteomics and in vitro experiments reveals essential roles of ACE and AP-N in the renin-angiotensin system-mediated congenital ureteropelvic junction obstruction

Abstract

Objective: Ureteropelvic junction obstruction (UPJO) is a common renal obstructive disorder, but its pathogenic mechanisms remain largely unclear. We aimed to investigate the potential involvement of the renin-angiotensin system in congenital UPJO pathogenesis.

Methods: Differentially expressed proteins in exosomes isolated from amniotic fluid of patients with congenital UPJO were characterized using iTRAQ (isobaric tags for relative and absolute quantification)-based proteomics. The expressions of angiotensin-converting enzyme (ACE) and aminopeptidase N (AP-N) in HK2 cells were inhibited by quinapril and siRNA, respectively. Cell proliferation and reactive oxygen species were measured by EdU staining and flow cytometry, respectively. Gene expression was detected by Western blot or qRT-PCR. The inflammatory factors were measured through ELISA. Mice that underwent unilateral ureteral obstruction were used as the animal model.

Results: The identity of exosomes from amniotic fluids was confirmed by the expression of CD9 and CD26. In total, 633 differentially expressed proteins were identified in the amniotic fluid-derived exosomes from patients with UPJO, including 376 up- and 257 down-regulated proteins associated with multiple biological processes. Of them, ACE and AP-N were significantly decreased in the amniotic fluid exosomes. Inhibition of ACE and AP-N resulted in suppressed cell proliferation; repressed IARP, AT1R, and MAS1 expression; elevated ROS production; and increased IL-1β, TNF-α, and IL-6 levels in HK2 cells. Decreased ACE expression and elevated IL-1β levels were also observed in the mouse model.

Conclusion: Suppression of ACE and AP-N expression mediates congenital UPJO pathogenesis by repressing renal tubular epithelial proliferation, promoting ROS production, and enhancing inflammatory factor expression.

Keywords: ACE; AP-N; Congenital ureteropelvic junction obstruction; Renin–angiotensin system; iTRAQ.