Wright, Joshua

Cux1 is a homeobox gene involved in cell cycle regulation and kidney development. Cux1 is upregulated in the kidneys of both mice and humans with autosomal dominant polycystic kidney disease (ADPKD). However, Cux1 transgenic mice do not develop cystic kidneys, indicating that upregulation of Cux1 alone is insufficient to develop PKD. Mice carrying a deletion of the C‐terminus of Cux1 (Cux1 tm2Ejn‐/‐ ) express a truncated protein that does not localize to the nucleus, resulting in functional loss of Cux1 transcriptional activity. To test the hypothesis that Cux1 is required to develop PKD, we generated collecting duct specific Pkd1 mutant mice (Pkd1 CD ) that were also homozygous for the Cux1 mutation. When kidneys isolated from newborn Pkd1 CD /Cux1 tm2Ejn ‐/‐ mice were examined, we found a marked reduction in cystic dilations compared to the kidneys isolated from newborn Pkd1 CD mice. We confirmed the Cre activity by crossing the Pkd1 CD and Pkd1 CD / Cux1 tm2Ejn ‐/‐ mice to the membrane‐targeted Tomato/Green Fluorescent Protein (mT/mG) double fluorescent reporter mouse strain (Gt(ROSA)26Sor tm4(ACTB‐tdTomato,‐EGFP)Luo /J), which carries a loxP‐flanked Tomato cassette. Immunofluorescence of kidney sections indicated that collecting duct derived cysts were positive for the excision event in the Pkd1 CD mice. In contrast, although collecting ducts were positive for the excision event in Pkd1 CD / Cux1 tm2Ejn ‐/‐ mice, none of the positive tubules were cystic. Taken together, our results suggest that Cux1 gene dosage influences the progression of cystic disease in an ADPKD mouse model.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common monogenic hereditary disorders in humans characterized by fluid-filled cysts, primarily in the kidneys. Cux1, a cell cycle regulatory gene highly expressed during kidney development, is elevated in the cyst-lining cells of Pkd1 mutant mice, and in human ADPKD cells. However, forced expression of Cux1 is insufficient to induce cystic disease in transgenic mice or to induce rapid cyst formation after cilia disruption in the kidneys of adult mice. Here we report a double mutant mouse model that has a conditional deletion of the Pkd1 gene in the renal collecting ducts together with a targeted mutation in the Cux1 gene (Pkd1CD;Cux1tm2Ejn). While kidneys isolated from newborn Pkd1CDmice exhibit cortical and medullary cysts, kidneys isolated from newborn Pkd1CD;Cux1tm2Ejn−/−mice did not show any cysts. Because Cux1tm2Ejn−/−are perinatal lethal, we evaluated Pkd1CDmice that were heterozygote for the Cux1 mutation. Similar to the newborn Pkd1CD;Cux1tm2Ejn−/−mice, newborn Pkd1CD;Cux1tm2Ejn+/−mice did not show any cysts. Comparison of Pkd1CDand Pkd1CD;Cux1tm2Ejn+/−mice at later stages of development showed a reduction in the severity of PKD in the Pkd1CD;Cux1tm2Ejn+/−mice. Moreover, we observed an increase in expression of the cyclin kinase inhibitor p27, a target of Cux1 repression, in the rescued collecting ducts. Taken together, our results suggest that Cux1 expression in PKD is not directly involved in cystogenesis but promotes cell proliferation required for expansion of existing cysts, primarily by repression of p27.