Kidney-specific inactivation of Ofd1 leads to renal cystic disease associated with upregulation of the mTOR pathway

Abstract

The oral-facial-digital type I syndrome (OFDI; MIM 311200) is a rare syndromic form of inherited renal cystic disease. It is transmitted as an X-linked dominant, male lethal disorder and is caused by mutations in the OFD1 gene. Previous studies demonstrated that OFDI belongs to the growing number of disorders ascribed to dysfunction of primary cilia. We generated a conditional inactivation of the mouse Ofd1 gene using the Ksp-Cre transgenic line, which resulted in a viable model characterized by renal cystic disease and progressive impairment of renal function. The study of this model allowed us to demonstrate that primary cilia initially form and then disappear after the development of cysts, suggesting that the absence of primary cilia is a consequence rather than the primary cause of renal cystic disease. Immunofluorescence and western blotting analysis revealed upregulation of the mTOR pathway in both dilated and non-dilated renal structures. Treatment with rapamycin, a specific inhibitor of the mTOR pathway, resulted in a significant reduction in the number and size of renal cysts and a decrease in the cystic index compared with untreated mutant animals, suggesting that dysregulation of this pathway in our model is mTOR-dependent. The animal model we have generated could thus represent a valuable tool to understand the molecular link between mTOR and cyst development, and eventually to the identification of novel drug targets for renal cystic disease.

Figure 1.

Ofd1 inactivation in mutant animals. (A) RT–PCR analysis showed the presence of the mutant Ofd1^Δ4-5 allele (369 bp) only in the kidney of Ofd1^fl/y;cre^Ksp and Ofd1^fl/+;cre^Ksp mutant animals. The mutated allele is absent in the lung of mutants and in the kidney and lung of wt (Ofd1^+/+) mice. All samples showed the presence of the wt allele (469 bp). No amplification was observed in the negative control. M = marker. (B) Quantitative RT–PCR with primers that specifically amplified the wt Ofd1 allele on genomic DNA (primers A and A′) and on RNA (primers B and B′). The position of the primers is displayed in the upper scheme. The analysis was performed on genomic DNA and RNA from kidneys of conditional mutants (Ofd1^fl/y;cre^Ksp, Ofd1^fl/+;cre^Ksp) and controls (Ofd1^+/+). Significant differences between mutants and controls (P < 0.05) are indicated (asterisk). Error bars represent standard error of the mean.

Figure 2.

Characterization of Ofd1 mutant animals. (A) Gross appearance of kidneys from Ofd1^+/+ (left), Ofd1^fl/+;cre^Ksp (center) and Ofd1^fl/y;cre^Ksp (right) mice at P70. (B) Hematoxylin/eosin staining of kidneys sections from Ofd1^fl/y;cre^Ksp mutants at different stages. At P7, no cysts are visible. Dilated tubules appeared at P14 and cysts (black arrows), rapidly increased in size and number. Bar = 500 µm. (C) GFR at different stages showed a reduced renal function in the kidney from mutant males (Ofd1^fl/y;cre^Ksp) and females (Ofd1^fl/+;cre^Ksp) compared with control littermates (Ofd1^+/+). Bars = standard error of the mean. Data were analyzed by Student's t-test. *P < 0.05.

Figure 3.

Cysts origin in Ofd1^fl/y;cre^Ksp mutant animals. Kidneys from Ofd1^fl/y;cre^Ksp mutants at P21 were stained for Tamm–Horsfall protein (THP), Arachis hypogaea lectin (LAH) and Na-PiII co-transporter (green, NaPiII). The cysts were positive for THP and LAH, thus indicating a distal tubular origin of cysts. Na-PiII transporter, a marker of proximal tubules, did not label any cysts (Cy) at this stage. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue). PAS staining on kidneys from Ofd1^fl/y;cre^Ksp excluded the presence of fibrosis. Bar = 50 µm for THP, LAH and NapiII panels. Bar = 500 µm for PAS staining.

Figure 4.

Analysis of primary cilia in renal tubules of Ofd1^fl;cre^Ksp mutant animals. Immunostaining with anti-acetylated tubulin (red) and SEM analysis revealed the presence of cilia in Ofd1^fl;cre^Ksp and control littermates (Ofd1^+/+) at P7 in the kidney distal tubules [arrowheads in (A–C), arrows in (D–F)]. Immunostaining with anti-acetylated tubulin (red) showed the absence of cilia in the cells lining the cysts (Cy) and the presence of cilia in non-dilated tubules (arrowheads) from Ofd1^fl/+;cre^Ksp (H) and Ofd1^fl/y;cre^Ksp (I) mutants at P14. Cilia are normally seen in Ofd1^+/+ control animals (G). SEM analysis confirm the absence of cilia from cells lining cystic dilations in both Ofd1^fl/+;cre^Ksp (M) and Ofd1^fl/y;cre^Ksp (N) mutants at P14, whereas cilia are present in control littermates at the same stage (arrows in L). Bars = 10 µm (A–C, G–I); 5 µm (D–F, L–N). Tubules are outlined with dotted lines.

Figure 5.

Upregulation of the mTOR pathway in Ofd1^fl;cre^Ksp mutant animals at P21. Immunostaining and western blot analysis with anti-phospho S6 (P-S6). The immunofluorescence revealed an higher level of P-S6 (green) on kidney sections from Ofd1^fl/+;cre^Ksp (C and D) and Ofd1^fl/y;cre^Ksp (E–G) mutant animals when compared with Ofd1^+/+ controls (A and B). In (B), (D) and (F), nuclei were counterstained with DAPI (blue). Fews cysts do not display staining with anti P-S6 (asterisks in D and F). Upregulation of P-S6 (arrowheads) is observed also in non-dilated tubules in both Ofd1^fl/+;cre^Ksp (D) and Ofd1^fl/y;cre^Ksp (magnification in G) mutant animals. Bars (A–G) = 50 µm. (H) Western blot analysis of total S6 (lower panel) and P-S6 (upper panel) on total protein extracts of kidneys from Ofd1^+/+, Ofd1^fl/+;cre^Ksp and Ofd1^fl/y;cre^Ksp animals confirmed the upregulation of the mTOR pathway.

Figure 6.

Rapamycin treatment significantly improves the renal cystic phenotype observed in Ofd1^fl/y;cre^Ksp animals (B) when compared with mutant animals treated with vehicle alone (A). Bottom panels in (A) and (B) represent higher magnification of the boxed images. In (C), kidneys from wt littermates treated with rapamycin or vehicle alone are shown as a control. (D) Cystic indices were calculated based on representative renal sections from non-treated and treated Ofd1^fl/y;cre^Ksp mice (n = 3 kidneys). Bars in (A), (B) and (C) = 1.2 mm. Data were analyzed by Student's t-test. ***P < 0.005.