Inhibition of Hedgehog signaling does not mitigate polycystic kidney disease severity in a Pkd1 mutant mouse model

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic disorder caused by mutations in PKD1 or PKD2, encoding polycystin-1 and polycystin-2, respectively. These polycystins form a cilia-localized complex that, when mutated, fails to inhibit an uncharacterized cilia-dependent cyst activation (CDCA) signal. This leads to progressive bilateral cyst growth and ultimately compromised renal function. Previous in vitro and in vivo studies from our group have demonstrated that Hedgehog (Hh) signaling inhibition reduces renal cystic severity in PKD models. To further investigate, we inactivated several Hh pathway components (Gli1, Gli2, Gli3 and Smo) in a Pkd1 hypomorphic mouse model through conditional deletion by tamoxifen-induced Cre-Lox recombination. We assessed cystic severity using kidney weight assessment and a microcomputed tomography (micro-CT)-based 3D imaging assay. Contrary to expectations, inactivation of Gli1 and Smo significantly increased cystogenesis. These findings suggest that Hh signaling does not mediate the CDCA signal.

Keywords: Hedgehog signaling; Mouse disease models; Polycystic kidney disease.

Figure 1

Pkd1^RC/RC kidneys progressively develop cysts 90 days postnatally. (A) Micro-CT cross sections from representative Pkd1^RC/RC kidneys from each time point (P21, P44, P67, P90) in the time series analysis with a P90 Pkd1^+/+ control. (B) Average cystic volume percentage (CV %) and (C) kidney weight-to-body weight (KW/BW) ratio of cystic kidneys at each time point with standard deviation. (D) CV % and KW/BW % plotted for each mouse to assess correlation between the two metrics. P21 mice excluded due to weak correlation, likely reflecting early disease stage and minimal cystic burden. n=11 P21; n=20 P44; n=12 P67; n=13 P90.

Figure 2

Attenuated Hedgehog signaling through Gli1 inactivation increases cystic severity in Pkd1^RC/RC mice. (A) KW/BW ratio and (B) CV % of 90-day Pkd1^RC/RC mouse kidneys with and without Gli1 expression. Comparison of the datasets were done using the Mann-Whitney test and presented as mean ± standard error of mean (SEM). ***P=0.0001; ****P<0.0001.

Figure 3

Attenuated Hedgehog signaling through Gli2 and Gli3 inactivation has no effect on cystic severity in Pkd1^RC/RC mice. (A) KW/BW ratio and (B) CV % of mice with and without Cre recombinase expression. Gli2^flox/flox; Gli3^flox/flox; Pkd1^RC/RC mice with or without R26CreER were administered tamoxifen at P13.5 to induce the deletion of Gli2 and Gli3. Kidneys were analyzed at 90 days. Comparison of the datasets were done using the Mann-Whitney test, presented as mean ± SEM, and demonstrated no significant differences.

Figure 4

Attenuated Hedgehog signaling through Smo inactivation increased cystic severity in Pkd1^RC/RC mice. (A) KW/BW ratio and (B) CV % of mice with and without Cre recombinase expression. Smof^lox/flox; Pkd1^RC/RC; mice with or without R26CreER were administered tamoxifen at P13.5 to induce the deletion of Smo. Kidneys were analyzed at 90 days. Comparison of the datasets were done using the Mann-Whitney test and presented as mean ± SEM. **P=0.0093; ***P=0.0001.