Cyclin-Dependent Kinase 1 Activity Is a Driver of Cyst Growth in Polycystic Kidney Disease

Abstract

Background: Mutations in PKD1 and PKD2, which encode the transmembrane proteins polycystin-1 and polycystin-2, respectively, cause autosomal dominant polycystic kidney disease (ADPKD). Polycystins are expressed in the primary cilium, and disrupting cilia structure significantly slows ADPKD progression following inactivation of polycystins. The cellular mechanisms of polycystin- and cilia-dependent cyst progression in ADPKD remain incompletely understood.

Methods: Unbiased transcriptional profiling in an adult-onset Pkd2 mouse model before cysts formed revealed significant differentially expressed genes (DEGs) in Pkd2 single-knockout kidneys, which were used to identify candidate pathways dysregulated in kidneys destined to form cysts. In vivo studies validated the role of the candidate pathway in the progression of ADPKD. Wild-type and Pkd2/Ift88 double-knockout mice that are protected from cyst growth served as controls.

Results: The RNASeq data identified cell proliferation as the most dysregulated pathway, with 15 of 241 DEGs related to cell cycle functions. Cdk1 appeared as a central component in this analysis. Cdk1 expression was similarly dysregulated in Pkd1 models of ADPKD, and conditional inactivation of Cdk1 with Pkd1 markedly improved the cystic phenotype and kidney function compared with inactivation of Pkd1 alone. The Pkd1/Cdk1 double knockout blocked cyst cell proliferation that otherwise accompanied Pkd1 inactivation alone.

Conclusions: Dysregulation of Cdk1 is an early driver of cyst cell proliferation in ADPKD due to Pkd1 inactivation. Selective targeting of cyst cell proliferation is an effective means of slowing ADPKD progression caused by inactivation of Pkd1.

Keywords: ADPKD; proliferation; transcriptional profiling.

Figure 1.

Increased cell cycle signatures in ADPKD models shown by RNA sequencing analysis. (A) Venn diagram of DEGs. The center circle shows the 241 DEGs with the same direction change in Pkd2 single knockouts relative to both the wild type and Ift88/Pkd2 double-knockout controls. Ten-week-old mice were used (Supplemental Figure 1); n=3 for each genotype. (B) KEGG pathway analysis using 241 DEGs. Only pathways that pass FDR<0.05 are shown. (C) Volcano plots of statistical significance against fold change for the indicated genotype pairs. Above the horizontal dashed lines, FDR is <0.05; the vertical dashed lines mark the two-fold change threshold. The final group of 241 DEGs identified that are significantly dysregulated in the same direction of change between Pkd2 single knockouts and both the wild type and Ift88/Pkd2 knockouts is labeled with blue dots. Representative DEGs in the KEGG cell cycle pathway analysis are marked with the gene names and red dots. (D) Gene interaction analysis using 15 of 241 DEGs included in the KEGG cell cycle pathway. Cdk1 is one of the central nodes (red box); the labeled representative DEGs in (C) are shown in blue boxes. (E, left panel) Quantitative real-time PCR validation of Cdk1 expression in Pkd1 mouse models: wild type (WT), Pkd1^fl/fl;Pax8^rtTA;TetO-cre (Pkd1), and Kif3a^fl/fl;Pkd1^fl/fl;Pax8^rtTA;TetO-cre (Kif3a/Pkd1); n=3 for each group. Gene expression was normalized to Gapdh. One-way ANOVA was followed by Tukey multiple comparison adjustment; mean ± SEM. *P=0.05; **P=0.01. (E, right panel) Immunoblot showing increased Cdk1 protein in 10-week-old Pkd1^fl/fl;Pax8^rtTA;TetO-cre mice compared with the wild-type (WT) control. The upper band of the Cdk1 doublet is the phosphorylated form. Hsp90 was used as the loading control.

Figure 2.

Cdk1 inactivation slows cyst progression in an early-onset model of ADPKD. (A) Representative images of kidneys from mice at P24 with the indicated genotypes. (B) Aggregate quantitative data for the kidney-body wt ratio. (C) Aggregate quantitative data for cystic index (percentage of cystic area). (D) Aggregate quantitative data for serum urea nitrogen (SUN). The SUN data of some samples are missing due to a technical problem while obtaining blood from mice. The biologic samples that missed SUN data are highlighted with filled boxes in Supplemental Figure 3. Colored shapes in (A) correspond to data in (B–D). Biologic replicate numbers are shown below each data plot. Male mice are represented as open symbols, whereas female mice are shown as filled symbols. One-way ANOVA was followed by Tukey multiple comparison adjustment; mean ± SEM. *P=0.05; ***P<0.001; ****P<0.001. Scale bar, 250 µm.

Figure 3.

Cdk1 inactivation slows cyst growth in an adult model of ADPKD. (A) Representative images of kidneys from mice with the specified genotypes at 18 weeks of age, 12 weeks after the end of doxycycline induction. Aggregate quantitative data for (B) kidney-body wt ratio, (C) cystic index (percentage of cystic area), and (D) serum urea nitrogen (SUN). The SUN data of some samples are missing due to technical difficulties with collecting blood; the kidneys lacking SUN data are marked in Supplemental Figure 4, which shows all kidney images used in this figure. The color key in (A) corresponds to data in (B–D). The numbers of animals in each group are indicated. Male mice are show as open symbols, whereas female mice are shown as filled symbols. One-way ANOVA was followed by Tukey multiple comparison adjustment; mean ± SEM. *P=0.05; ***P<0.001; ****P<0.001. Scale bar, 250 µm.

Figure 4.

Loss of Cdk1 reduces cell proliferation of cyst-lining cells by inhibiting G1-S phase. (A) The aggregate quantitative data at P24 along with the representative images of the percentage of EdU-positive nuclei (red) in DBA-positive collecting duct cells (green) in the early-onset model with the indicated color-coded genotypes. (B and C) The aggregate quantitative data along with the representative images at 18 weeks for the percentage of EdU-positive nuclei (red) in (B) DBA-positive collecting duct cells (green) and (C) LTA-positive proximal tubule cells (green) with the indicated color-coded genotypes. Quantification in (A–C) was done by counting the number of EdU-positive nuclei among at least 1000 DBA-positive or LTA-positive cells from each mouse. The biologic replicate numbers are noted in each panel. (D) Immunoblotting for Cdk1, PCNA, and phosphohistone 3 (p-H3) in the adult Pax8^rtTA;TetO-cre models at 18 weeks. The upper band of the doublet in the Cdk1 blot is the phosphorylated form of Cdk1. The lower band in the doublet on the p-H3 blot is the nonphosphorylated form of histone 3. Hsp90 was used as loading control. (E) G1-S–phase markers Cyclin D1 and Cyclin E1 in the adult Pax8^rtTA;TetO-cre models at 18 weeks. Hsp90 was used as loading control. (F) Quantitative real-time RT-PCR of multiple S-phase genes in 18-week-old adult model kidneys. All of the gene expressions were normalized to Gapdh. One-way ANOVA followed by Tukey multiple comparison adjustment was used in (A–C and F); mean ± SEM. Cdk1 + Pkd1, Cdk1^fl/fl;Pkd1^fl/fl;Pax8^rtTA;TetO-cre; Pkd1, Pkd1^fl/fl;Pax8^rtTA;TetO-cre; WT, wild type. *P=0.05; **P=0.01; ***P<0.001; ****P<0.001. Scale bar, 40 µm.