Early Molecular Events Mediating Loss of Aquaporin-2 during Ureteral Obstruction in Rat

Abstract

Background: Ureteral obstruction is marked by disappearance of the vasopressin-dependent water channel aquaporin-2 (AQP2) in the renal collecting duct and polyuria upon reversal. Most studies of unilateral ureteral obstruction (UUO) models have examined late time points, obscuring the early signals that trigger loss of AQP2.

Methods: We performed RNA-Seq on microdissected rat cortical collecting ducts (CCDs) to identify early signaling pathways after establishment of UUO.

Results: Vasopressin V2 receptor (AVPR2) mRNA was decreased three hours after UUO, identifying one cause of AQP2 loss. Collecting duct principal cell differentiation markers were lost, including many not regulated by vasopressin. Immediate-early genes in CCDs were widely induced three hours post-UUO, including Myc, Atf3, and Fos (confirmed at the protein level). Simultaneously, expression of NF-κB signaling response genes known to repress Aqp2 increased. RNA-seq for CCDs at an even earlier time point (30 mins) showed widespread mRNA loss, indicating a 'stunned' profile. Immunocytochemical labeling of markers of mRNA-degrading P-bodies DDX6 and 4E-T indicated an increase in P-body formation within 30 minutes.

Conclusions: Immediately after establishment of UUO, collecting ducts manifest a 'stunned' state with broad disappearance of mRNAs. Within 3 hours follows upregulation of immediate-early and inflammatory genes and disappearance of the V2 vasopressin receptor, resulting in loss of AQP2 (confirmed by lipopolysaccharide administration). The inflammatory response seen rapidly after UUO establishment may be relevant to UUO-induced polyuria as well as to the long-term development of fibrosis in UUO kidneys.