Polycystic kidney diseases (PKDs) are primarily characterized by the growth of fluid-filled cysts in renal tubules leading to end-stage renal disease1,2,3. Mutations in the PKD1 or PKD2 genes lead to autosomal dominant PKD (ADPKD), a slowly developing adult form4,5. Autosomal recessive polycystic kidney disease results from mutations in the PKHD1 gene, affects newborn infants and progresses very rapidly6,7. No effective treatment is currently available for PKD. A previously unrecognized site of subcellular localization was recently discovered for all proteins known to be disrupted in PKD: primary cilia8,9. Because ciliary functions seem to be involved in cell cycle regulation, disruption of proteins associated with cilia or centrioles may directly affect the cell cycle and proliferation, resulting in cystic disease10,11,12. We therefore reasoned that the dysregulated cell cycle may be the most proximal cause of cystogenesis, and that intervention targeted at this point could provide significant therapeutic benefit for PKD. Here we show that treatment with the cyclin-dependent kinase (CDK) inhibitor (R)-roscovitine does indeed yield effective arrest of cystic disease in jck and cpk mouse models of PKD. Continuous daily administration of the drug is not required to achieve efficacy; pulse treatment provides a robust, long-lasting effect, indicating potential clinical benefits for a lifelong therapy. Molecular studies of the mechanism of action reveal effective cell-cycle arrest, transcriptional inhibition and attenuation of apoptosis. We found that roscovitine is active against cysts originating from different parts of the nephron, a desirable feature for the treatment of ADPKD, in which cysts form in multiple nephron segments. Our results indicate that inhibition of CDK is a new and effective approach to the treatment of PKD.
To determine the potential of CDK inhibition as a new therapeutic approach for the treatment of PKD we employed (R)-roscovitine (CYC202), a potent inhibitor of Cdk2–cyclin E with a 50% inhibitory concentration (IC50) of 0.1 μM as well as Cdk7–cyclin H (IC50 0.4 μM), Cdk9–cyclin T1 (IC500.8 μM) and Cdk5–p35–p25 (IC50 0.16 μM)13,14. It is currently in clinical trials as an anticancer agent15,16. We tested the effect of roscovitine on slowly progressive renal cystic disease in jck mice. The development of PKD in the jck mice resembles human disease in many ways, as described previously17. Thus, similarly to ADPKD, jck mice develop cysts in multiple nephron segments and display gender dimorphism with more aggressive disease in males. jck/jck males were injected daily with 50 and 150 mg kg-1 roscovitine from day 26 to day 64 for a total of 5 weeks (Fig. 1a, b). The drug was well tolerated with no weight loss, mortality or toxicity (Supplementary Table 1). This is not surprising because roscovitine seems to be well tolerated in preclinical mouse models up to 2,000 mg kg-1 orally and in phase I/II clinical trials in solid tumours16. The roscovitine-treated group showed a significant decrease in the ratio of kidney weight to body weight and in cystic volumes, in a dose-dependent manner (Fig. 1a, b, and Supplementary Table 1). In addition, blood urea nitrogen (BUN) was significantly decreased in the 50 mg kg-1 roscovitine-treated group and normalized with 150 mg kg-1 roscovitine (Fig. 1a). Roscovitine also effectively inhibited PKD in jck/jck females, in which the disease is less severe than in males (Supplementary Table 1).
Given the prominent inhibition of cystogenesis in jck mice with daily dosing of roscovitine for 5 weeks, we examined whether continuous administration of the drug is required for efficacy, or whether pulse therapy with roscovitine will produce a long-lasting effect. We administered 150 mg kg-1 roscovitine daily to jck/jck males for 3 weeks, followed by 2 weeks without treatment (Fig. 1c; 3/2 schedule). A long-lasting anti-cystic effect was detected with normalized BUN levels. We also employed a different schedule in which the drug was administered daily for 1 week on treatment followed by 1 week off treatment for a total of 5 weeks (Fig. 1c; 1/1 schedule). Again, a significant decrease in cystogenesis was detected, whereas the decrease in BUN was not statistically significant. Roscovitine therefore shows a long-lasting treatment effect after drug withdrawal and is also efficacious with intermittent dosing. This potential for pulse therapy may prove extremely important for the treatment of human PKD and other chronic kidney diseases for which lifelong safe therapy is required.
Because different animal models each manifest different facets of human PKD, the efficacy of a therapy needs to be assessed in more than one model. We have analysed the effect of roscovitine on aggressive PKD in cpk mice. Daily treatment of cpk mice with 100 mg kg-1 roscovitine from day 7 to day 21 resulted in a significant decrease in PKD (Fig. 2 and Supplementary Table 2). Thus, roscovitine effectively inhibited renal cystic disease in two models of PKD, namely slowly progressive jck and aggressive cpk.
It is possible that cysts originating from functionally different nephron segments may respond differently to therapy. We have previously shown that similarly to ADPKD, jck mice develop cysts in multiple parts of the nephron17. We show that roscovitine is highly effective against cysts of different origins: the cortical collecting duct (CD), the distal tubule (DT) and the loop of Henle (LH) (Supplementary Fig. S2).
To understand the mode of action of roscovitine in PKD, we analysed its effect on the proliferation of cystic epithelia. First, with proliferating cell nuclear antigen (PCNA) immunostaining we confirmed high proliferation rates for human ADPKD cyst-lining epithelia (Fig. 3a). Similarly, a large number of PCNA-positive cells were seen in jck cysts (Fig. 3b). In contrast, a quantitative decrease in PCNA-positive cells was detected in roscovitine-treated jck kidney (Fig. 3b, c). Next we examined the effects of roscovitine treatment on the cell cycle. Two kinases, Cdk4/6–cyclin D and Cdk2–cyclin E, and the transcription complex that includes retinoblastoma protein (Rb) are pivotal in controlling the G1/S checkpoint (Fig. 3e). Because a primary target of roscovitine is the Cdk2–cyclin E complex that phosphorylates and inactivates Rb tumour suppressor, we examined Rb phosphorylation (Rb-p) in wild-type (WT), jck and roscovitine-treated kidneys (Fig. 3d, e). The Rb-p form was significantly increased in cystic kidneys. Treatment with roscovitine resulted in the dephosphorylation of Rb, suggesting an effective G1/S cell-cycle block. In addition, treatment with roscovitine normalized the levels of the Rb-associated proteins Rb2 and retinoblastoma-binding protein (Fig. 3d). Although the level of cyclin D1 was not substantially increased in cystic kidneys, its phosphorylation (at Thr 286) was significantly decreased (Fig. 3d). Inhibition of cyclin D1 phosphorylation in the jck kidney probably prevents its degradation through the ubiquitin–proteasome pathway18. Roscovitine normalized the phosphorylation level of cyclin D1 (Fig. 3d). The levels of cyclin D2 and D3 expression were increased in cystic kidneys and downregulated by roscovitine. Roscovitine also affected signalling from Ras–Raf to MEK–ERKs, known to regulate cyclin D1 by decreasing the level of ERK 1/2 activation (Fig. 3d). This effect is likely to be mediated by direct targeting of ERK 2 by roscovitine19. The importance of MEK–ERK signalling in PKD has been confirmed by showing that MEK inhibition slows cystogenesis in vivo20.
Roscovitine also inhibits Cdk7 and Cdk9, which regulate transcription by phosphorylating the carboxy-terminal domain of RNA polymerase II (RNA pol II). Inhibition of RNA pol II-dependent transcription may generate additional benefits in slowing cystogenesis, as described previously for cancer15. Moderately increased levels of Cdk7 and Cdk9 detected in cystic kidneys resulted in greatly increased phosphorylation of RNA pol II (Fig. 3d). In contrast, roscovitine-treated samples were almost indistinguishable from wild-type controls, showing markedly decreased levels of RNA pol II phosphorylation. Thus, roscovitine attenuates cystogenesis through cell-cycle arrest and transcriptional inhibition. Roscovitine also targets Cdk5, a unique kinase activated by complexing with activator molecules p35 and mainly its cleaved product p25 (ref. 13). Abnormal Cdk5 activity has been shown to have a function in neurodegenerative diseases, and cleavage of p35 to p25 by calpain has been linked to activation of Cdk5 and cell death21. We detected increased Cdk5 expression in jck kidneys in comparison with wild-type controls, accompanied by the generation of p25 cleaved product, whereas p35 was predominantly seen in wild-type kidney (Fig. 3d). Treatment with roscovitine resulted in the downregulation of Cdk5–p25 and normalized levels of p35 (Fig. 3d). It is possible that targeting Cdk5 with roscovitine may affect apoptotic pathways in cystic disease.
Apoptosis is causally linked to cystogenesis: deletion of anti-apoptotic genes encoding Bcl-2 and AP-2β and overexpression of pro-apoptotic c-Myc in mice results in renal cystic disease22,23. Treatment with roscovitine decreased the number of TdT-mediated dUTP nick end labelling (TUNEL)-positive cells, suggesting a potent anti-apoptotic effect (Fig. 4a). Caspase-2, a key initiator of the mitochondrial pathway of apoptosis, was found to be greatly induced in jck kidney (Fig. 4b). The increased level of ApaF1 activated by mitochondrial protein release in response to apoptotic signals was detected in jck kidneys (Fig. 4b). The expression of caspase-2 and ApaF1 in roscovitine-treated jck kidneys was indistinguishable from that of wild-type controls. Furthermore, because ApaF1 activates the caspase cascade downstream of caspase-9, we found inactive caspase-3 to be downregulated in jck kidneys but restored by treatment with roscovitine (Fig. 4b). Bcl-2 and Bcl-xL proteins were upregulated with treatment, suggesting effective inhibition of apoptosis. It is possible that Cdk5 inhibition by roscovitine might have a function in its blockade of apoptosis in cystic kidneys. Such a mechanism might be responsible for anti-apoptotic effects of roscovitine in neurodegenerative diseases24,25. This activity is different from roscovitine action in tumour cells, in which it causes induction of apoptosis15,26. Roscovitine treatment therefore resulted in effective reduction of apoptosis in cystic kidneys. Recently, (R)-roscovitine was shown to interact with pyridoxal kinase19. We show that pyridoxal kinase is not responsible for anti-cystic activity of roscovitine (Supplementary Fig. S5). Although the molecular basis for the long-lasting therapeutic effect of roscovitine remains to be explained, it is possible that restoration of the cell-cycle–apoptosis balance promotes a more normal epithelial phenotype. The observed effects of roscovitine on cystogenesis are probably mediated by a combination of its targets, including the possible regulation of calcium signalling, making the analysis of each molecular target an aim of future studies. Although no effective PKD therapy is currently available, new therapeutic approaches are beginning to emerge27,28,29,30. Multiple treatment options are needed to target PKD, and it is likely that combination therapies may prove most effective.
Our study shows for the first time the therapeutic potential for cell-cycle inhibition for the treatment of polycystic kidney diseases. Roscovitine is a selective inhibitor for CDKs with minimal off-target kinase activities19, making it a clinical candidate for ADPKD. We show that roscovitine effectively inhibited cystogenesis and improved renal function in jck and cpk models of PKD, with a long-lasting effect. Analysis of the molecular targets of roscovitine in cystogenesis showed that it acts through blockade of the cell cycle, transcriptional regulation and inhibition of apoptosis.
Jck and cpk mouse models of PKD and experimental design
C57BL/6J jck/+ and C57BL/6J cpk/+ mice were obtained from the Jackson Laboratory and were used to establish a breeding colony maintained at Biomedical Research Models and Genzyme. (R)-Roscovitine (6-benzylamino-2-((R)-1-ethyl-2-hydroxyethylamino)-9-isopropylpurine) was obtained from A.G. Scientific. Cystic jck/jck mice were identified by a custom genotyping assay as described previously17; they were divided into control and treated groups and injected intraperitoneally with either vehicle or drug at specified concentrations. Details are given in Supplementary Methods.
Morphometric analysis of cystogenesis
Longitudinal and transverse kidney sections (4 μm) were stained with haematoxylin and eosin, slides were digitized with an ACIS system (Clarient) and processed with the MetaMorph Imaging Series software (Molecular Devices Corp.) and Scion Image software (Scion Corp.). Cystic percentage was measured as a ratio of the cystic area to the total section area and was used in calculations of cystic volumes (percentage of body weight) as described previously30.
Immunohistochemical, immunofluorescence and western blot analyses
Detailed protocols are given in Supplementary Methods.
Data are expressed as means ± s.d. Comparisons were made by two-tailed t- test and significance was accepted at the 0.05 level of probability (P < 0.05).
We thank R. Russo, T. Barry and A. Taylor for expert technical assistance, the staff of the Genzyme Department of Comparative Medicine and Histology unit for help with in vivo studies and sample preparations. We are grateful to L. Meijer and H. Galons (CNRS, Roscoff) for providing N6-methyl-(R)-roscovitine. We thank A. Smith, R. Gregory, T. Natoli, H. Husson and J. Leonard for helpful discussions and comments on this manuscript.
About this article
Anatomical Science International (2017)