Genetics and mechanisms of hepatic cystogenesis

Polycystic liver disease (PLD) is a heterogeneous genetic condition. PKD1 and PKD2 germline mutations are found in patients with autosomal dominant polycystic kidney disease (ADPKD). Autosomal dominant polycystic liver disease (ADPLD) is associated with germline mutations in PRKCSH, SEC63, LRP5, and recently ALG8 and SEC61. GANAB mutations are found in both patient groups. Loss of heterozygosity of PLD-genes in cyst epithelium contributes to the development of hepatic cysts. A genetic interaction network is implied in hepatic cystogenesis that connects the endoplasmic glycoprotein control mechanisms and polycystin expression and localization. Wnt signalling could be the major downstream signalling pathway that results in hepatic cyst growth. PLD in ADPLD and ADPKD probably results from changes in one common final pathway that initiates cyst growth. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.     

Post-translational modifications of the polycystin proteins

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited cause of kidney failure and affects up to 12 million people worldwide. Germline mutations in two genes, PKD1 or PKD2, account for almost all patients with ADPKD. The ADPKD proteins, polycystin-1 (PC1) and polycystin-2 (PC2), are regulated by post-translational modifications (PTM), with phosphorylation, glycosylation and proteolytic cleavage being the best described changes. A few PTMs have been shown to regulate polycystin trafficking, signalling, localisation or stability and thus their physiological function. A key challenge for the future will be to elucidate the functional significance of all the individual PTMs reported to date. Finally, it is possible that site-specific mutations that disrupt PTM could contribute to cystogenesis although in the majority of cases, confirmatory evidence is awaited.     

Epigenetics and cell cycle regulation in cystogenesis

The role of genetic mutations in the development of polycystic kidney disease (PKD), such as alterations in PKD1 and PKD2 genes in autosomal dominant PKD (ADPKD), is well understood. However, the significance of epigenetic mechanisms in the progression of PKD remains unclear and is increasingly being investigated. The term of epigenetics describes a range of mechanisms in genome function that do not solely result from the DNA sequence itself. Epigenetic information can be inherited during mammalian cell division to sustain phenotype specifically and physiologically responsive gene expression in the progeny cells. A multitude of functional studies of epigenetic modifiers and systematic genome-wide mapping of epigenetic marks reveal the importance of epigenomic mechanisms, including DNA methylation, histone/chromatin modifications and non-coding RNAs, in PKD pathologies. Deregulated proliferation is a characteristic feature of cystic renal epithelial cells. Moreover, defects in many of the molecules that regulate the cell cycle have been implicated in cyst formation and progression. Recent evidence suggests that alterations of DNA methylation and histone modifications on specific genes and the whole genome involved in cell cycle regulation and contribute to the pathogenesis of PKD. This review summarizes the recent advances of epigenetic mechanisms in PKD, which helps us to define the term of “PKD epigenetics” and group PKD epigenetic changes in three categories. In particularly, this review focuses on the interplay of epigenetic mechanisms with cell cycle regulation during normal cell cycle progression and cystic cell proliferation, and discusses the potential to detect and quantify DNA methylation from body fluids as diagnostic/prognostic biomarkers. Collectively, this review provides concepts and examples of epigenetics in cell cycle regulation to reveal a broad view of different aspects of epigenetics in biology and PKD, which may facilitate to identify possible novel therapeutic intervention points and to explore epigenetic biomarkers in PKD.     

A tumor necrosis factor-alpha-mediated pathway promoting autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is caused by heterozygous mutations in either PKD1 or PKD2, genes that encode polycystin-1 and polycystin-2, respectively. We show here that tumor necrosis factor-alpha (TNF-alpha), an inflammatory cytokine present in the cystic fluid of humans with ADPKD, disrupts the localization of polycystin-2 to the plasma membrane and primary cilia through a scaffold protein, FIP2, which is induced by TNF-alpha. Treatment of mouse embryonic kidney organ cultures with TNF-alpha resulted in formation of cysts, and this effect was exacerbated in the Pkd2(+/-) kidneys. TNF-alpha also stimulated cyst formation in vivo in Pkd2(+/-) mice. In contrast, treatment of Pkd2(+/-) mice with the TNF-alpha inhibitor etanercept prevented cyst formation. These data reveal a pathway connecting TNF-alpha signaling, polycystins and cystogenesis, the activation of which may reduce functional polycystin-2 below a critical threshold, precipitating the ADPKD cellular phenotype.     

Polycystic kidney disease: novel insights into polycystin function

Autosomal dominant polycystic kidney disease (ADPKD) is a life-threatening monogenic disease caused by mutations in PKD1 and PKD2 that encode polycystin 1 (PC1) and polycystin 2 (PC2). PC1/2 localize to cilia of renal epithelial cells, and their function is believed to embody an inhibitory activity that suppresses the cilia-dependent cyst activation (CDCA) signal. Consequently, PC deficiency results in activation of CDCA and stimulates cyst growth. Recently, re-expression of PCs in established cysts has been shown to reverse PKD. Thus, the mode of action of PCs resembles a ‘counterbalance in cruise control’ to maintain lumen diameter within a designated range. Herein we review recent studies that point to novel arenas for future PC research with therapeutic potential for ADPKD.     

CDK inhibitors R-roscovitine and S-CR8 effectively block renal and hepatic cystogenesis in an orthologous model of ADPKD

Autosomal dominant polycystic kidney disease (ADPKD) and other forms of PKD are associated with dysregulated cell cycle and proliferation. Although no effective therapy for the treatment of PKD is currently available, possible mechanism-based approaches are beginning to emerge. A therapeutic intervention targeting aberrant cilia-cell cycle connection using CDK-inhibitor R-roscovitine showed effective arrest of PKD in jck and cpk models that are not orthologous to human ADPKD. To evaluate whether CDK inhibition approach will translate into efficacy in an orthologous model of ADPKD, we tested R-roscovitine and its derivative S-CR8 in a model with a conditionally inactivated Pkd1 gene (Pkd1 cKO). Similar to ADPKD, Pkd1 cKO mice developed renal and hepatic cysts. Treatment of Pkd1 cKO mice with R-roscovitine and its more potent and selective analog S-CR8 significantly reduced renal and hepatic cystogenesis and attenuated kidney function decline. Mechanism of action studies demonstrated effective blockade of cell cycle and proliferation and reduction of apoptosis. Together, these data validate CDK inhibition as a novel and effective approach for the treatment of ADPKD.     

A loss-of-function model for cystogenesis in human autosomal dominant polycystic kidney disease type 2.

Autosomal dominant polycystic kidney disease (ADPKD) is genetically heterogeneous, with at least three chromosomal loci (PKD1, PKD2, and PKD3) that account for the disease. Mutations in the PKD2 gene, on the long arm of chromosome 4, are expected to be responsible for approximately 15% of cases of ADPKD. Although ADPKD is a systemic disease, it shows a focal expression, because <1% of nephrons become cystic. A feasible explanation for the focal nature of events in PKD1, proposed on the basis of the two-hit theory, suggests that cystogenesis results from the inactivation of the normal copy of the PKD1 gene by a second somatic mutation. The aim of this study is to demonstrate that somatic mutations are present in renal cysts from a PKD2 kidney. We have studied 30 renal cysts from a patient with PKD2 in which the germline mutation was shown to be a deletion that encompassed most of the disease gene. Loss-of-heterozygosity (LOH) studies showed loss of the wild-type allele in 10% of cysts. Screening of six exons of the gene by SSCP detected eight different somatic mutations, all of them expected to produce truncated proteins. Overall, >/=37% of the cysts studied presented somatic mutations. No LOH for the PKD1 gene or locus D3S1478 were observed in those cysts, which demonstrates that somatic alterations are specific. We have identified second-hit mutations in human PKD2 cysts, which suggests that this mechanism could be a crucial event in the development of cystogenesis in human ADPKD-type 2.     

Autosomal dominant polycystic kidney disease: Disrupted pathways and potential therapeutic interventions

Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic inherited renal cystic disease that occurs in different races worldwide. It is characterized by the development of a multitude of renal cysts, which leads to massive enlargement of the kidney and often to renal failure in adulthood. ADPKD is caused by a mutation in PKD1 or PKD2 genes encoding the proteins polycystin-1 and polycystin-2, respectively. Recent studies showed that cyst formation and growth result from deregulation of multiple cellular pathways like proliferation, apoptosis, metabolic processes, cell polarity, and immune defense. In ADPKD, intracellular cyclic adenosine monophosphate (cAMP) promotes cyst enlargement by stimulating cell proliferation and transepithelial fluid secretion. Several interventions affecting many of these defective signaling pathways have been effective in animal models and some are currently being tested in clinical trials. Moreover, the stem cell therapy can improve nephropathies and according to studies were done in this field, can be considered as a hopeful therapeutic approach in future for PKD. This study provides an in-depth review of the relevant molecular pathways associated with the pathogenesis of ADPKD and their implications in development of potential therapeutic strategies.     

Phosphorylation, protein kinases and ADPKD

Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disease characterized by renal cyst formation and caused by mutations in the PKD1 and PKD2 genes, which encode polycystin-1(PC-1) and -2 (PC-2) proteins, respectively. PC-1 is a large plasma membrane receptor involved in the regulation of several biological functions and signaling pathways including the Wnt cascade, AP-1, PI3kinase/Akt, GSK3β, STAT6, Calcineurin/NFAT and the ERK and mTOR cascades. PC-2 is a calcium channel of the TRP family. The two proteins form a functional complex and prevent cyst formation, but the precise mechanism(s) involved remains unknown. This article is part of a Special Issue entitled: Polycystic Kidney Disease.     

Construction of a transgenic pig model overexpressing polycystic kidney disease 2 (PKD2) gene

Autosomal dominant polycystic kidney disease (ADPKD) is a common human genetic disease, affecting millions of people worldwide. The progressive growth of cysts in kidneys eventually leads to renal failure in 50 % of patients, and there is currently no effective treatment. Various murine models have been studied to elucidate the disease mechanisms, and much information has been acquired. However, the course of the disease cannot be fully recapitulated using these models. The pig is a suitable model for biomedical research, and pig PKD2 has high similarity to the human ortholog at the molecular level. Here, a mini-pig PKD2 transgenic model was generated, driven by a ubiquitous cytomegalovirus enhancer/promoter. Using somatic cell nuclear transfer, four transgenic pigs with approximately 10 insertion events each were generated. Quantitative real-time PCR and western blotting showed that PKD2 was more highly expressed in transgenic pigs than in wild-type counterparts. Because of the chronic nature of ADPKD, blood urea nitrogen and serum creatinine levels were continuously measured to assess the pig kidney function. The transgenic pigs continue to show no significant alteration in kidney function; it is estimated that 1–2 more years may be required for manifestation of renal cystogenesis in these pigs.     

Overexpression of PKD1 causes polycystic kidney disease

The pathogenetic mechanisms underlying autosomal dominant polycystic kidney disease (ADPKD) remain to be elucidated. While there is evidence that Pkd1 gene haploinsufficiency and loss of heterozygosity can cause cyst formation in mice, paradoxically high levels of Pkd1 expression have been detected in the kidneys of ADPKD patients. To determine whether Pkd1 gain of function can be a pathogenetic process, a Pkd1 bacterial artificial chromosome (Pkd1-BAC) was modified by homologous recombination to solely target a sustained Pkd1 expression preferentially to the adult kidney. Several transgenic lines were generated that specifically overexpressed the Pkd1 transgene in the kidneys 2- to 15-fold over Pkd1 endogenous levels. All transgenic mice reproducibly developed tubular and glomerular cysts and renal insufficiency and died of renal failure. This model demonstrates that overexpression of wild-type Pkd1 alone is sufficient to trigger cystogenesis resembling human ADPKD. Our results also uncovered a striking increased renal c-myc expression in mice from all transgenic lines, indicating that c-myc is a critical in vivo downstream effector of Pkd1 molecular pathways. This study not only produced an invaluable and first PKD model to evaluate molecular pathogenesis and therapies but also provides evidence that gain of function could be a pathogenetic mechanism in ADPKD.     

An Efficient and Comprehensive Strategy for Genetic Diagnostics of Polycystic Kidney Disease

Renal cysts are clinically and genetically heterogeneous conditions. Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent life-threatening genetic disease and mainly caused by mutations in PKD1. The presence of six PKD1 pseudogenes and tremendous allelic heterogeneity make molecular genetic testing challenging requiring laborious locus-specific amplification. Increasing evidence suggests a major role for PKD1 in early and severe cases of ADPKD and some patients with a recessive form. Furthermore it is becoming obvious that clinical manifestations can be mimicked by mutations in a number of other genes with the necessity for broader genetic testing. We established and validated a sequence capture based NGS testing approach for all genes known for cystic and polycystic kidney disease including PKD1. Thereby, we demonstrate that the applied standard mapping algorithm specifically aligns reads to the PKD1 locus and overcomes the complication of unspecific capture of pseudogenes. Employing careful and experienced assessment of NGS data, the method is shown to be very specific and equally sensitive as established methods. An additional advantage over conventional Sanger sequencing is the detection of copy number variations (CNVs). Sophisticated bioinformatic read simulation increased the high analytical depth of the validation study and further demonstrated the strength of the approach. We further raise some awareness of limitations and pitfalls of common NGS workflows when applied in complex regions like PKD1 demonstrating that quality of NGS needs more than high coverage of the target region. By this, we propose a time- and cost-efficient diagnostic strategy for comprehensive molecular genetic testing of polycystic kidney disease which is highly automatable and will be of particular value when therapeutic options for PKD emerge and genetic testing is needed for larger numbers of patients.     

CDK inhibitors R-roscovitine and S-CR8 effectively block renal and hepatic cystogenesis in an orthologous model of ADPKD

Autosomal dominant polycystic kidney disease (ADPKD) and other forms of PKD are associated with dysregulated cell cycle and proliferation. Although no effective therapy for the treatment of PKD is currently available, possible mechanism-based approaches are beginning to emerge. A therapeutic intervention targeting aberrant cilia-cell cycle connection using CDK-inhibitor R-roscovitine showed effective arrest of PKD in jck and cpk models that are not orthologous to human ADPKD. To evaluate whether CDK inhibition approach will translate into efficacy in an orthologous model of ADPKD, we tested R-roscovitine and its derivative S-CR8 in a model with a conditionally inactivated Pkd1 gene (Pkd1 cKO). Similar to ADPKD, Pkd1 cKO mice developed renal and hepatic cysts. Treatment of Pkd1 cKO mice with R-roscovitine and its more potent and selective analog S-CR8 significantly reduced renal and hepatic cystogenesis and attenuated kidney function decline. Mechanism of action studies demonstrated effective blockade of cell cycle and proliferation and reduction of apoptosis. Together, these data validate CDK inhibition as a novel and effective approach for the treatment of ADPKD.     

Polycystins as components of large multiprotein complexes of polycystin interactors

Naturally occurring mutations in two separate genes, PKD1 and PKD2, are responsible for the vast majority of all cases of autosomal dominant polycystic kidney disease (ADPKD), one of the most common genetic diseases affecting 1 in 1000 Americans. The hallmark of ADPKD is the development of epithelial cysts in the kidney, liver, and pancreas. PKD1 encodes a large plasma membrane protein (PKD1, PC1, or Polycystin-1) with a long extracellular domain and has been speculated to function as an atypical G protein coupled receptor. PKD2 encodes an ion channel of the Transient Receptor Potential superfamily (TRPP2, PKD2, PC2, or Polycystin-2). Despite the identification of these genes more than 20 years ago, the molecular function of their encoded proteins and the mechanism(s) by which mutations in PKD1 and PKD2 cause ADPKD remain elusive. Genetic, biochemical, and functional evidence suggests they form a multiprotein complex present in multiple locations in the cell, including the plasma membrane, endoplasmic reticulum, and the primary cilium. Over the years, numerous interacting proteins have been identified using directed and unbiased approaches, and shown to modulate function, cellular localization, and protein stability and turnover of Polycystins. Delineation of the molecular composition of the Polycystin complex can have a significant impact on understanding their cellular function in health and disease states and on the identification of more specific and effective therapeutic targets.