Novel Endogenous Pkd1 Reexpression Allele and Effects of Mosaic Reexpression of Pkd1 in ADPKD

Background: Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease caused by mutations in PKD1 and PKD2. Somatic ‘second hit’ mutations involving the normal allele in renal tubular cells are sufficient to start cyst formation. We previously reported that reactivation of Pkd2 and Pkd1 expressed from BAC transgenes can reverse ADPKD in mouse models. In this study we developed a single copy endogenous Pkd1 reactivation system to more closely mimic the human ‘two-hit’ model. Methods: To produce an endogenous Pkd1 inducible reactivation allele, we inserted a transcriptional stop sequence flanked by FRT sites into the first intron of mouse Pkd1 in the same location as we had previously used in the Pkd1 BAC re-expression allele. This Pkd1-FSF-V5 allele was additionally modified with a V5 epitope tag sequence in frame before the termination codon. Pkd1-FSF-V5, which is functionally null in the absence of Flp recombinase activation, was combined to obtain Pkd1flox/FSF-V5;Pax8rtTA;TetOCre;R26FlpoER mice (a.k.a., Pkd1FSF-V5). Results: Pkd1FSF-V5 mice were treated with doxycycline at postnatal days 28 to 42 to inactivate Pkd1flox, followed by tamoxifen for 7 days beginning at 13 weeks of age to induce re-expression of Pkd1-FSF-V5. Kidneys examined at 14, 16 and 24 weeks age showed a full recovery of the polycystic phenotype (kidney/body weight ratio [KW:BW], cystic index [CI]) and renal function (BUN). Late re-expression at 16 weeks (7 days of tamoxifen) also showed rapid and progressive recovery of cystogenesis and renal function at 17, 19 and 24 weeks of age. We next examined the effects of mosaic reactivation using a single dose of tamoxifen instead of 7 doses. A single dose of tamoxifen at 13 weeks (KW:BW 7.5%, CI 44%, BUN 66 mg/dL) showed no significant differences in KW:BW 5.4%, CI 38%, BUN 44 mg/dL when examined at 16 weeks which in turn differed significantly (P<0.01) from 16 week kidneys without re-expression (KW:BW 12.8%, CI 55%, BUN 81 mg/dL). A single dose of tamoxifen resulted in mosaic re-expression in 20-40% of tubule cells compared to 7 days of tamoxifen when assessed by an mT/mG Flp-reporter allele. Conclusion: ADPKD is reversible by re-expressing single endogenous Pkd1 allele and mosaic (partial) re-expression has significant preclinical therapeutic benefit in ADPKD mice models based on Pkd1. Funding: NIDDK Support