Recent Discoveries in Epigenetic Modifications of Polycystic Kidney Disease.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a heritable renal disease that results in end-stage kidney disease, due to the uncontrolled bilateral growth of cysts throughout the kidneys. While it is known that a mutation within a PKD-causing gene is required for the development of ADPKD, the underlying mechanism(s) causing cystogenesis and progression of the disease are not well understood. Limited therapeutic options are currently available to slow the rate of cystic growth. Epigenetic modifications, including DNA methylation, are known to be altered in neoplasia, and several FDA-approved therapeutics target these disease-specific changes. As there are many similarities between ADPKD and neoplasia, we (and others) have postulated that ADPKD kidneys contain alterations to their epigenetic landscape that could be exploited for future therapeutic discovery. Here we summarise the current understanding of epigenetic changes that are associated with ADPKD, with a particular focus on the burgeoning field of ADPKD-specific alterations in DNA methylation.

Chemical Synthesis of the PAX Protein Inhibitor EG1 and Its Ability to Slow the Growth of Human Colorectal Carcinoma Cells.

Colorectal cancer is primarily a disease of the developed world. The incidence rate has continued to increase over time, reflecting both demographic and lifestyle changes, which have resulted in genomic and epigenomic modifications. Many of the epigenetic modifications occur in genes known to be closely associated with embryonic development and cellular growth. In particular, the paired box (PAX) transcription factors are crucial for correct tissue development during embryogenesis due to their role in regulating genes involved in proliferation and cellular maintenance. In a number of cancers, including colorectal cancer, the PAX transcription factors are aberrantly expressed, driving proliferation and thus increased tumour growth. Here we have synthesized and used a small molecule PAX inhibitor, EG1, to inhibit PAX transcription factors in HCT116 colorectal cell cultures which resulted in reduced proliferation after three days of treatment. These results highlight PAX transcription factors as playing an important role in the proliferation of HCT116 colorectal cancer cells, suggesting there may be a potential therapeutic role for inhibition of PAX in limiting cancer cell growth.

Extensive Inter-Cyst DNA Methylation Variation in Autosomal Dominant Polycystic Kidney Disease Revealed by Genome Scale Sequencing.

Autosomal dominant polycystic kidney disease (ADPKD) is a heritable disease characterized by bilateral renal enlargement due to the growth of cysts throughout the kidneys. Inheritance of a disease-causing mutation is required to develop ADPKD, which results in end-stage kidney disease and is associated with a high morbidity. The pathology underlying cyst formation is not well understood. To address this, we have previously shown the global methylome is altered in ADPKD tissue, suggesting a role of DNA methylation in disease-state renal tissue. As cysts are believed to arise independently, we hypothesize that DNA methylation changes vary accordingly. Here we further investigate the role of DNA methylation within independent cysts to characterize key intra-individual changes. We demonstrate that fragments within CpG islands and gene bodies harbor the greatest amount of variation across the ADPKD kidney, while intergenic fragments are comparatively stable. A proportion of variably methylated genes were also differentially methylated in ADPKD tissue. Our data provide evidence that individual molecular mechanisms are operating in the development of each cyst.

Targeted Therapies for Autosomal Dominant Polycystic Kidney Disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common life-threatening genetic disease in humans, affecting approximately 1 in 500 people. ADPKD is characterized by cyst growth in the kidney leading to progressive parenchymal damage and is the underlying pathology in approximately 10% of patients requiring hemodialysis or transplantation for end-stage kidney disease. The two proteins that are mutated in ADPKD, polycystin-1 and polycystin-2, form a complex located on the primary cilium and the plasma membrane to facilitate calcium ion release in the cell. There is currently no Food and Drug Administration (FDA)-approved therapy to cure or slow the progression of the disease. Rodent ADPKD models do not completely mimic the human disease, and therefore preclinical results have not always successfully translated to the clinic. Moreover, the toxicity of many of these potential therapies has led to patient withdrawals from clinical trials. RESULTS: Here, we review compounds in clinical trial for treating ADPKD, and we examine the feasibility of using a kidney-targeted approach, with potential for broadening the therapeutic window, decreasing treatment-associated toxicity and increasing the efficacy of agents that have demonstrated activity in animal models. We make recommendations for integrating kidney- targeted therapies with current treatment regimes, to achieve a combined approach to treating ADPKD. CONCLUSION: Many compounds are currently in clinical trial for ADPKD yet, to date, none are FDA-approved for treating this disease. Patients could benefit from efficacious pharmacotherapy, especially if it can be kidney-targeted, and intensive efforts continue to be focused on this goal.

Genome-Scale Single Nucleotide Resolution Analysis of DNA Methylation in Human Autosomal Dominant Polycystic Kidney Disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of fluid-filled cysts in the kidney and end stage renal disease by the fourth or fifth decade of life. Mutations in the PKD1 gene account for 85% of all cases of ADPKD. No curative therapy exists for patients affected by this disease and an underexplored avenue for the treatment of ADPKD is the targeting of epigenetic changes that occur during cystogenesis. Limited data exists on alterations in DNA methylation that are associated with ADPKD. Given the similarities between cyst growth and neoplasia, and the fact that 2 DNA methylation inhibitors are already Food and Drug Administration-approved for the treatment of myelodysplastic syndrome, we hypothesized that global DNA methylation patterns in ADPKD would parallel that observed in neoplasia, and which may provide an opportunity to treat ADPKD with epigenetic inhibitors. To address this hypothesis, we undertook a global DNA methylation analysis of human ADPKD kidney. METHODS: We generated single nucleotide resolution methylomes of cortical kidney tissue from individuals with ADPKD, and from non-ADPKD kidney tissue, using reduced representation bisulfite sequencing. Using quantitative reverse transcription polymerase chain reaction, we investigated expression of the PKD1 gene in both ADPKD and non-ADPKD kidney. RESULTS: We have shown that ADPKD-derived genomic DNA exhibits global hypomethylation when compared with non-ADPKD kidney, a pattern commonly observed in DNA methylation studies of tumor-derived tissue. We have also identified 13 discrete regions that are significantly differentially methylated in ADPKD compared to non-ADPKD, and 8 of these are gene specific. The PKD1 gene shows an increase in methylation at the 3' end of the gene body, but in contrast to previously published data, this is not associated with a decrease in PKD1 mRNA expression. CONCLUSION: This genome-scale single nucleotide resolution analysis of DNA methylation in human polycystic kidneys suggests that DNA methylation differences at specific loci are associated with ADPKD. Further exploration into the significance of these preliminary results may shed light on the disease process, and potentially reveal new therapeutic possibilities.

SMAD proteins directly suppress PAX2 transcription downstream of transforming growth factor-beta 1 (TGF-ß1) signalling in renal cell carcinoma.

Canonical TGF-ß1 signalling promotes tumor progression by facilitating invasion and metastasis, whereby release of TGF-ß1, by (for example) infiltrating immune cells, induces epithelial to mesenchymal transition (EMT). PAX2, a member of the Paired box family of transcriptional regulators, is normally expressed during embryonic development, including in the kidney, where it promotes mesenchymal to epithelial transition (MET). PAX2 expression is silenced in many normal adult tissues. However, in contrast, PAX2 is expressed in several cancer types, including kidney, prostate, breast, and ovarian cancer. While multiple studies have implicated TGF-ß superfamily members in modulating expression of Pax genes during embryonic development, few have investigated direct regulation of Pax gene expression by TGF-ß1. Here we have investigated direct regulation of PAX2 expression by TGF-ß1 in clear cell renal cell carcinoma (CC-RCC) cell lines. Treatment of PAX2-expressing 786-O and A498 CC-RCC cell lines with TGF-ß1 resulted in inhibition of endogenous PAX2 mRNA and protein expression, as well as expression from transiently transfected PAX2 promoter constructs; this inhibition was abolished in the presence of expression of the inhibitory SMAD, SMAD7. Using ChIP-PCR we showed TGF-ß1 treatment induced SMAD3 protein phosphorylation in 786-O cells, and direct SMAD3 binding to the human PAX2 promoter, which was inhibited by SMAD7 over-expression. Overall, these data suggest that canonical TGF-ß signalling suppresses PAX2 transcription in CC-RCC cells due to the direct binding of SMAD proteins to the PAX2 promoter. These studies improve our understanding of tumor progression and epithelial to mesenchyme transition (EMT) in CC-RCC and in other PAX2-expressing cancer types.

Polycystic kidney disease - where gene dosage counts.

Gene dosage effects have emerged as playing a central role in the pathogenesis of polycystic kidney disease. Yet, how gene dosage can ultimately have an impact on the formation of kidney cysts remains unknown. In this commentary we review the evidence for the role of gene dosage effects versus the "2-hit" mutation model in polycystic kidney disease (PKD), and also discuss how gene networks may potentially make intertwined contributions to PKD.

Rapamycin-mediated suppression of renal cyst expansion in del34 Pkd1-/- mutant mouse embryos: an investigation of the feasibility of renal cyst prevention in the foetus.

AIM: Polycystic kidney disease (PKD) in humans involves kidney cyst expansion beginning in utero. Recessive PKD can result in end-stage renal disease (ESRD) within the first decade, whereas autosomal dominant PKD (ADPKD), caused by mutations in the PKD1 or PKD2 gene, typically leads to ESRD by the fifth decade of life. Inhibition of mTOR signalling was recently found to halt cyst formation in adult ADPKD mice. In contrast, no studies have investigated potential treatments to prevent cyst formation in utero in recessive PKD. Given that homozygous Pkd1 mutant mice exhibit cyst formation in utero, we decided to investigate whether mTOR inhibition in utero ameliorates kidney cyst formation in foetal Pkd1 homozygous mutant mice. METHODS: Pregnant Pkd1(+/-) female mice (mated with Pkd1(+/-) male mice) were treated with rapamycin from E14.5 to E17.5. Foetal kidneys were dissected, genotyped and evaluated by cyst size as well as expression of the developmental marker, Pax2. RESULTS: Numerous cysts were present in Pkd1(-/-) kidneys, which were twice the weight of wild-type kidneys. Cyst size was reduced by a third in rapamycin-treated Pkd1(-/-) kidney sections and kidney mass was reduced to near wild-type levels. However, total cyst number was not reduced compared with control embryos. Pax2 expression and kidney development were unaltered in rapamycin-treated mice but some lethality was observed in Pkd1(-/-) null embryos. CONCLUSION: Rapamycin treatment reduces cyst formation in Pkd1(-/-) mutant mice; therefore, the prevention of kidney cyst expansion in utero by mTOR inhibition is feasible. However, selective rapamycin-associated lethality limits its usefulness as a treatment in utero.

Pax2 gene dosage influences cystogenesis in autosomal dominant polycystic kidney disease.

Mutations in PKD1 cause dominant polycystic kidney disease (PKD), characterized by large fluid-filled kidney cysts in adult life, but the molecular mechanism of cystogenesis remains obscure. Ostrom et al. [Dev. Biol., 219, 250-258 (2000)] showed that reduced dosage of Pax2 caused increased apoptosis, and ameliorated cystogenesis in Cpk mutant mice with recessive PKD. Pax2 is expressed in condensing metanephrogenic mesenchyme and arborizing ureteric bud, and plays an important role in kidney development. Transient Pax2 expression during fetal kidney mesenchyme-to-epithelial transition, as well as in nascent tubules, is followed by marked down-regulation of Pax2 expression. Here, we show that in humans with PKD, as well as in Pkd1(del34/del34) mutant mice, Pax2 was expressed in cyst epithelial cells, and facilitated cyst growth in Pkd1(del34/del34) mutant mice. In Pkd1(del34/del34) mutant kidneys, the expression of Pax2 persisted in nascent collecting ducts. In contrast, homozygous Pkd1(del34/del34) fetal mice carrying mutant Pax2 exhibited ameliorated cyst growth, although reduced cystogenesis was not associated with increased apoptosis. Pax2 expression was attenuated in nascent collecting ducts and absent from remnant cysts of Pkd1(del34/del34)/Pax2(1Neu/+) mutant mice. To investigate whether the Pkd1 gene product, Polycystin-1, regulates Pax2, MDCK cells were engineered constitutively expressing wild-type Pkd1; Pax2 protein levels and promoter activity were both repressed in MDCK cells over-expressing Pkd1, but not in cells without transgenic Pkd1. These data suggest that polycystin-1-deficient tubular epithelia persistently express Pax2 in ADPKD, and that Pax2 or its pathway may be an appropriate target for the development of novel therapies for ADPKD.

Ureteric bud apoptosis and renal hypoplasia in transgenic PAX2-Bax fetal mice mimics the renal-coloboma syndrome.

In humans, PAX2 haploinsufficiency causes renal-coloboma syndrome (RCS) involving eye abnormalities, renal hypoplasia, and renal failure in early life. The authors previously showed that heterozygous mutant Pax2 mice have smaller kidneys with fewer nephrons, associated with elevated apoptosis in the ureteric bud (UB). However, PAX2 may have a variety of developmental functions such as effects on cell fate and differentiation. To determine whether apoptosis alone is sufficient to cause a UB branching deficit, the authors targeted a pro-apoptotic gene (Baxalpha) to the embryonic kidney under the control of human PAX2 regulatory elements. The exogenous PAX2 promoter directed Baxalpha gene expression specifically to the developing kidney UB, eye, and mid/hindbrain. At E15.5 PAX2Promoter-Baxalpha fetal mice exhibited renal hypoplasia, elevated UB apoptosis, and retinal defects, mimicking the phenotype observed in RCS. The kidneys of E15.5 PAX2Promoter-Baxalpha fetal mice were 55% smaller than those of wild-type fetal mice, and they contained 70% of the normal level of UB branching. The data indicate that loss of Pax2 anti-apoptotic activity is sufficient to account for the reduced UB branching observed in RCS and suggest that elevated UB apoptosis may be a key process responsible for renal hypoplasia. The authors propose a morphogenic unit model in which cell survival influences the rate of UB branching and determines final nephron endowment.

Magnetic resonance imaging assessment of a murine model of recessive polycystic kidney disease.

PURPOSE: The pathogenesis of polycystic kidney disease (PKD) has not been firmly established; however, our current knowledge of cystogenesis and human cystic disease has been greatly advanced by a variety of animal models of PKD. To study the onset and degree of cyst formation in PKD mouse models without requiring sacrifice of these animals, we have initiated magnetic resonance imaging (MRI) studies of the juvenile cystic kidney (jck) mouse model. METHODS: The MRI experiments were performed by use of a Bruker 8.5 T system, on seven-week-old mice that were homozygous for the recessive jck mutation and that manifested PKD. Kidney volume was measured, using three-dimensional segmentation postprocessing techniques. RESULTS: The MR images of the enlarged kidneys from affected mice had regions of high signal intensity, with a radial distribution, which reflected the dilated collecting ducts observed in the corresponding histologic slices. The volume of PKD-affected kidney was about 4 times greater than that of the normal kidney. CONCLUSIONS: Magnetic resonance imaging has the ability to non-invasively assess cystic disease in mouse models of PKD. Of considerable importance is the opportunity to characterize this disease without sacrificing the animal. The three-dimensional MRI segmentation method provides accurate calculation of renal volume.

Tissue and cellular localization of a novel polycystic kidney disease-like gene product, polycystin-L.

Polycystin-L (PCL), the third member of the polycystin family of proteins, functions as a Ca2+-modulated nonselective cation channel when expressed in Xenopus oocytes. Polycystin-1 and -2 are mutated in autosomal-dominant polycystic kidney disease (ADPKD), but the role of PCL in disease has not been determined. In this study, an anti-peptide polyclonal antiserum was generated against the carboxyl terminal domain of human PCL and used to determine the patterns of expression and distribution of PCL by indirect immunofluorescence in both developing and adult mice. The results show that PCL is predominantly expressed in adult mouse tissues and has a more restricted pattern of expression than either polycystin-1 or -2. In the kidney, PCL expression was first detected at E16, and levels increased into adulthood. Localization of PCL was predominantly found in the apical region of the principal cells of inner medullary collecting ducts. PCL was also found in discrete cell types of the retina, testis, liver, pancreas, heart, and spleen, but it was not detected in the lung. These data in combination with evidence of PCL channel activity are crucial for elucidating the physiologic role of this novel cation channel and may shed light on the function of inner medullary collecting ducts and polycystins. The expression pattern of PCL suggests that it is unlikely to be a candidate gene for ADPKD, but it remains a potential candidate for other as yet unmapped human cystic disorders.