Introduction: Cystogenesis in Autosomal Dominant Polycystic Kidney Disease (ADPKD) is caused by a mutation in the genes encoding polycystin-1 or 2 (Pkd1/Pkd2), and involves dysregulated fluid transport in the kidney tubules. ADPKD patients present with a urinary concentrating defect, even prior to decreased glomerular filtration rate despite higher plasma vasopressin levels. Hence, we investigated whether vasopressin-mediated aquaporin-2 (AQP2) trafficking and water balance are dysregulated in ADPKD and lead to cyst expansion.

Methods: Doxycycline-inducible, kidney-tubule-specific Pkd1 knock-out (KO) mice (Pax8; tetO-Cre; Pkd1^flox/flox) and control littermates (Pax8; tetO-Cre; Pkd1^wt/wt) were treated with doxycycline on postnatal (PN) day 10, 11 and 12. On PN day 21, dDAVP (0.1 µg/kg), a synthetic analog of vasopressin, was administered as a single intraperitoneal (IP) injection and mice were euthanized after 6 hours (acute studies). For long term studies, dDAVP (0.1µg/kg) was administered IP for 5 days (PN day 17 to 21). Urine samples were collected for osmolality measurements, and kidneys were harvested for western blotting and immunohistochemistry (IHC). A novel polycystin-1 deficient mouse cortical collecting duct cell line (mpkCCD-Pkd1^-/-) was developed using the CRISPR/Cas9 technique, and validated through PCR, western blotting, 3D cell morphogenesis, and cAMP assays.

Results: At 3 weeks of age, KO mice (n=3) had lower urine osmolality at baseline compared with control mice (n=5) (481.7 versus 1874 mOsm/Kg; p≤ 0.01). Urine osmolality increased with dDAVP administration in the control mice (p≤ 0.001), whereas no response was noted in the KO mice. In long-term studies, AQP2 expression increased and was apically targeted with dDAVP treatment in the renal cortex and inner medulla of both control (n=4) and KO mice (n=4). However, AQP2 expression was lower in the inner medulla of both saline (p<0.01) and dDAVP-treated (p<0.01) KO mice compared to controls. IHC indicated that NKCC2 and NHE3 protein expression were reduced in the whole kidneys of KO mice. Large cysts were observed in the cortex of KO mice. When grown in a basement membrane matrix (cultrex), mpkCCD-Pkd1^-/- cells grew as cysts while the control-transduced mpkCCD cells grew as tubules, providing a phenotypical confirmation of the cell lines. mpkCCD-Pkd1^-/- cells exhibited significantly lower dDAVP-induced AQP2 protein expression (n=3, p≤ 0.05) when compared to control cells, although both mpkCCD-Pkd1^-/- and control cells displayed dDAVP-induced cAMP production.

Conclusion: ADPKD mice have a urine concentrating defect, which may be explained by the downregulation of AQP2 in the inner renal medulla, and sodium transporters important for countercurrent multiplication such as NKCC2 and NHE3. In mpkCCD-Pkd1^-/- cells, AQP2 is downregulated despite dDAVP-induced cAMP elevation, suggesting AQP2 dysregulation.  Further investigations are required to understand AQP2 trafficking in these models.

Ethical approval:  All protocols of the animal studies were approved by the Animal Experiments Inspectorate of the Ministry of Food, Agriculture and Fisheries in Denmark.