Pkd1 regulates immortalized proliferation of renal tubular epithelial cells through p53 induction and JNK activation

Autosomal dominant polycystic kidney disease (ADPKD) is the most common human monogenic genetic disorder and is characterized by progressive bilateral renal cysts and the development of renal insufficiency. The cystogenesis of ADPKD is believed to be a monoclonal proliferation of PKD-deficient (PKD(-/-)) renal tubular epithelial cells. To define the function of Pkd1, we generated chimeric mice by aggregation of Pkd1(-/-) ES cells and Pkd1(+/+) morulae from ROSA26 mice. As occurs in humans with ADPKD, these mice developed cysts in the kidney, liver, and pancreas. Surprisingly, the cyst epithelia of the kidney were composed of both Pkd1(-/-) and Pkd1(+/+) renal tubular epithelial cells in the early stages of cystogenesis. Pkd1(-/-) cyst epithelial cells changed in shape from cuboidal to flat and replaced Pkd1(+/+) cyst epithelial cells lost by JNK-mediated apoptosis in intermediate stages. In late-stage cysts, Pkd1(-/-) cells continued immortalized proliferation with downregulation of p53. These results provide a novel understanding of the cystogenesis of ADPKD patients. Furthermore, immortalized proliferation without induction of p53 was frequently observed in 3T3-type culture of mouse embryonic fibroblasts from Pkd1(-/-) mice. Thus, Pkd1 plays a role in preventing immortalized proliferation of renal tubular epithelial cells through the induction of p53 and activation of JNK.     

Failure to ubiquitinate c-Met leads to hyperactivation of mTOR signaling in a mouse model of autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder that is caused by mutations at two loci, polycystin 1 (PKD1) and polycystin 2 (PKD2). It is characterized by the formation of multiple cysts in the kidneys that can lead to chronic renal failure. Previous studies have suggested a role for hyperactivation of mammalian target of rapamycin (mTOR) in cystogenesis, but the etiology of mTOR hyperactivation has not been fully elucidated. In this report we have shown that mTOR is hyperactivated in Pkd1-null mouse cells due to failure of the HGF receptor c-Met to be properly ubiquitinated and subsequently degraded after stimulation by HGF. In Pkd1-null cells, Casitas B-lineage lymphoma (c-Cbl), an E3-ubiquitin ligase for c-Met, was sequestered in the Golgi apparatus with α₃β₁ integrin, resulting in the inability to ubiquitinate c-Met. Treatment of mouse Pkd1-null cystic kidneys in organ culture with a c-Met pharmacological inhibitor resulted in inhibition of mTOR activity and blocked cystogenesis in this mouse model of ADPKD. We therefore suggest that blockade of c-Met is a potential novel therapeutic approach to the treatment of ADPKD.     

Sirtuin 1 inhibition delays cyst formation in autosomal-dominant polycystic kidney disease

Autosomal-dominant polycystic kidney disease (ADPKD) is caused by mutations in either PKD1 or PKD2 and is characterized by the development of multiple bilateral renal cysts that replace normal kidney tissue. Here, we used Pkd1 mutant mouse models to demonstrate that the nicotinamide adenine dinucleotide–dependent (NAD-dependent) protein deacetylase sirtuin 1 (SIRT1) is involved in the pathophysiology of ADPKD. SIRT1 was upregulated through c-MYC in embryonic and postnatal Pkd1-mutant mouse renal epithelial cells and tissues and could be induced by TNF-α, which is present in cyst fluid during cyst development. Double conditional knockouts of Pkd1 and Sirt1 demonstrated delayed renal cyst formation in postnatal mouse kidneys compared with mice with single conditional knockout of Pkd1. Furthermore, treatment with a pan-sirtuin inhibitor (nicotinamide) or a SIRT1-specific inhibitor (EX-527) delayed cyst growth in Pkd1 knockout mouse embryonic kidneys, Pkd1 conditional knockout postnatal kidneys, and Pkd1 hypomorphic kidneys. Increased SIRT1 expression in Pkd1 mutant renal epithelial cells regulated cystic epithelial cell proliferation through deacetylation and phosphorylation of Rb and regulated cystic epithelial cell death through deacetylation of p53. This newly identified role of SIRT1 signaling in cystic renal epithelial cells provides the opportunity to develop unique therapeutic strategies for ADPKD.     

Autosomic dominant polycystic kidney disease and metformin: Old knowledge and new insights on retarding progression of chronic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most common congenital kidney disorder, generally caused by mutations in the PKD1 and PKD2 genes, coding for polycystins 1 and 2. Its pathogenesis is accompanied by alterations of the cAMP, mTOR, MAPK/ERK, and JAK/STAT pathways. ADPKD is clinically characterized by the formation of many growing cysts with kidney enlargement and a progressive damage to the parenchyma, up to its complete loss of function, and the onset of end-stage renal disease (ESRD). The current aim of ADPKD therapy is the inhibition of cyst development and retardation of chronic kidney disease progression. Several drugs have been recently included as potential therapies for ADPKD including metformin, the drug of choice for the treatment of type 2 diabetes mellitus, according to its potential inhibitory effects on cystogenesis. In this review, we summarize preclinical and clinical evidence endorsing or rejecting metformin administration in ADPKD evolution and pathological mechanisms. We explored the biology of APDKD and the role of metformin in slowing down cystogenesis searching PubMed and Clinical Trials to identify relevant data from the database inception to December 2020. From our research analysis, evidence for metformin as emerging cure for ADPKD mainly arise from preclinical studies. In fact, clinical studies are still scanty and stronger evidence is awaited. Its effects are likely mediated by inhibition of the ERK pathway and increase of AMPK levels, which are both linked to ADPKD pathogenesis.     

Molecular genetics of autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is a common Mendelian disorder, occurring in approximately 1 in 1000 births and accounting for 8% to 10% of cases of end-stage renal disease (ESRD). Mutations of 2 genes, PKD1 and PKD2, account for the disease in approximately 80% to 85% and 10% to 15% of families respectively. The gene products (polycystin 1 and 2) of PKD1 and PKD2 are plasma membrane proteins and components of a novel signalling pathway that regulates epithelial cell growth and differentiation. Significant inter- and intrafamilial renal disease variability in ADPKD has been well documented and is influenced by both germline and somatic genetic events. Specifically, genetic locus heterogeneity and 2 rare Mendelian syndromes have been shown to strongly influence the variability of interfamilial renal disease, and as-yet-unknown genetic and environmental factors likely modify both inter- and intrafamilial renal disease severity. Furthermore, individual cyst formation in ADPKD represents an aberration of monoclonal growth triggered by somatic PKD1 or PKD2 mutations within individual epithelial cells. Current studies are in progress to identify major genetic and environmental modifiers of renal disease variability. A thorough knowledge of these determinants will allow better patient risk assessment and development of mechanism-based therapy in ADPKD.     

Recent advances in the molecular diagnosis of polycystic kidney disease

Introduction: Polycystic kidney disease (PKD) is clinically and genetically heterogeneous and constitutes the most common heritable kidney disease. Most patients are affected by the autosomal dominant form (ADPKD) which generally is an adult-onset multisystem disorder. By contrast, the rarer recessive form ARPKD usually already manifests perinatally or in childhood. In some patients, however, ADPKD and ARPKD can phenotypically overlap with early manifestation in ADPKD and only late onset in ARPKD. Progressive fibrocystic renal changes are often accompanied by severe hepatobiliary changes or other extrarenal abnormalities.

Areas covered: A reduced dosage of disease proteins disturbs cell homeostasis and explains a more severe clinical course in some PKD patients. Cystic kidney disease is also a common feature of other ciliopathies and genetic syndromes. Genetic diagnosis may guide clinical management and helps to avoid invasive measures and to detect renal and extrarenal comorbidities early in the clinical course.

Expert Commentary: The broad phenotypic and genetic heterogeneity of cystic and polycystic kidney diseases make NGS a particularly powerful approach. Interpretation of data becomes the challenge and bench and bedside benefit from digitized multidisciplinary interrelationships.     

Practical genetics for autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most common mendelian disorder of the kidney and accounts for ~5% of end-stage renal disease in North America. It is characterized by focal development of renal cysts which increase in number and size with age. Mutations of PKD1 and PKD2 account for most cases. Although the clinical manifestations of both gene types overlap completely, PKD1 is associated with more severe disease than PKD2, with larger kidneys and earlier onset of end-stage renal disease. Furthermore, marked within-family renal disease variability is well documented in ADPKD and suggests a strong modifier effect from as yet unknown genetic and environmental factors. In turn, the significant inter- and intra-familial renal disease variability poses a challenge for diagnosis and genetic counseling. In general, renal ultrasonography is commonly used for the diagnosis, and age-dependent criteria have been defined for subjects at risk of PKD1. However, the utility of the PKD1 ultrasound criteria in the clinical setting is unclear since their performance characteristics have not been defined for the milder PKD2 and the gene type for most test subjects is unknown. Recently, highly predictive ultrasound diagnostic criteria have been derived for at-risk subjects of unknown gene type. Additionally, both DNA linkage and gene-based direct sequencing are available for the diagnosis of ADPKD, especially in subjects with equivocal imaging results, a negative or indeterminate family history, or in younger at-risk individuals being evaluated as potential living related kidney donor. This review will highlight the utility and limitations of clinical predictors of gene types, imaging- and molecular-based diagnostic tests, and present an integrated approach for evaluating individuals suspected to have ADPKD.     

常染色体显性多囊肾病的预后评估及治疗

常染色体显性多囊肾病(autosomal dominant polycystic kidney disease, ADPKD)患病率为1‰~2‰, 属于罕见病, 临床主要表现为双侧肾囊肿且逐渐发展, 肾脏体积进行性增大, 肾功能逐步降低。PKD1基因突变约占81%, PKD2基因突变约占10.5%~22%。血管加压素(arginine vasopressin, AVP)和环磷酸腺苷(cyclic adenosine monophosphate, cAMP)信号通路在ADPKD囊肿发展过程中发挥重要作用。近年来发表的梅奥风险评估模型和PROPKD(predicting renal outcome in polycystic kidney disease)评分是ADPKD较好的预后评估模型, 已成为临床医生决策的重要依据。通过拮抗AVP受体, 抑制cAMP通路的托伐普坦已成为ADPKD首个特异治疗药物, 可有效抑制总肾脏体积的增长和保护肾功能。药物的长期安全性仍需进一步研究。     

The polycystin-1 C-terminal fragment triggers branching morphogenesis and migration of tubular kidney epithelial cells

Mutations of either PKD1 or PKD2 cause autosomal dominant polycystic kidney disease, a syndrome characterized by extensive formation of renal cysts and progressive renal failure. Homozygous deletion of Pkd1 or Pkd2, the genes encoding polycystin-1 and polycystin-2, disrupt normal renal tubular differentiation in mice but do not affect the early steps of renal development. Here, we show that expression of the C-terminal 112 amino acids of human polycystin-1 triggers branching morphogenesis and migration of inner medullary collecting duct (IMCD) cells, and support in vitro tubule formation. The integrity of the polycystin-2-binding region is necessary but not sufficient to induce branching of IMCD cells. The C-terminal domain of polycystin-1 stimulated protein kinase C-alpha (PKC-alpha), but not the extracellular signal-regulated kinases ERK1 or ERK2. Accordingly, inhibition of PKC, but not ERK, prevented polycystin-1-mediated IMCD cell morphogenesis. In contrast, HGF-mediated morphogenesis required ERK activation but was not dependent on PKC. Our findings demonstrate that the C-terminal domain of polycystin-1, acting in a ligand-independent fashion, triggers unique signaling pathways for morphogenesis, and likely plays a central role in polycystin-1 function.     

The pathogenesis of autosomal dominant polycystic kidney disease

In individuals with autosomal dominant polycystic kidney disease (ADPKD), renal function deteriorates as the kidneys become replaced by multitudes of fluid-filled cysts. Although the PKD genes were identified a decade ago, the pathway(s) leading from mutation to disease remain the subject of intense investigation. As a result of this work, it has become apparent that the polycystins are multifunctional proteins that, in the broadest sense, appear to be involved in the transduction of a number of environmental cues into appropriate cellular responses. It is likely that the central pathogenetic pathway for cystogenesis stems from de-differentiation of tubular epithelial cells. Available evidence indicates that loss of polycystin activity leads to subtle derangements of cell calcium regulation through several possible pathways. Abnormal cell calcium homeostasis might then lead to altered differentiation in affected cells. The study of the polycystins has revealed some entirely novel insights into fundamental cell biology but these have not yet been satisfactorily integrated into a verified pathogenetic pathway for the development of ADPKD.     

Therapies to slow polycystic kidney disease

Advances in the understanding of cystogenesis and availability of animal models orthologous to human autosomal dominant polycystic kidney disease (ADPKD) and recessive polycystic kidney disease (ARPKD) will likely facilitate the development of treatments for these diseases. Proteins mutated in ADPKD and ARPKD, as well as in several animal models, are localized to renal primary cilia. These are thought to have a sensory function and contribute to the regulation of the intracellular calcium ([Ca2+]i). It seems likely that the maintenance of a differentiated renal epithelial phenotype, characterized by controlled fluid secretion and cell proliferation, requires precise functional coordination of cAMP and Ras/Raf/MEK/ERK signaling by [Ca2+]i. [Ca2+]i alterations, linked to genetic defects causing polycystic kidney disease, may hinder negative feedback mechanisms that control cAMP and Ras/Raf/MEK/ERK signaling, and result in increased fluid secretion and cell proliferation. cAMP levels, Raf kinase activities and ERK phosphorylation are increased in polycystic kidneys. There is also evidence of abnormal cross-talk between cAMP and MAPK pathways, that can be reproduced in wild-type cells by altering [Ca2+]i. While cAMP inhibits Ras-Raf-1-stimulated phosphorylation of ERK in normal kidney cells, it markedly increases B-Raf kinase activity and ERK phosphorylation in polycystic kidney cells. Treatment strategies should probably be aimed at increasing [Ca2+]i, inhibiting Ras/Raf/MEK/ERK signaling or lowering cAMP in the distal nephron and collecting duct. Vasopressin is the major adenylyl cyclase agonist in the collecting duct principal cells via a V2 receptor. OPC31260, a V2 receptor antagonist, lowers renal cAMP and markedly inhibits cystogenesis in four animal models of polycystic kidney disease, three of which are orthologous to human diseases (PCK rat, ARPKD; pcy mouse, adolescent nephronophthisis; Pkd2WS25/- mouse, ADPKD). The renal selectivity and safety profile of this class of drugs make it an excellent candidate for clinical trials.     

Improving recognition of inherited renal disease

Polycystic kidney disease and Alport's syndrome are the most common causes of inherited renal disease in the UK. An average GP practice is likely to have at least six patients with autosomal dominant polycystic kidney disease (ADPKD). The disorder is characterised by the formation of fluid-filled cysts in the kidneys resulting in progressive renal impairment. Mutations in two genes have been identified. The PKD1 gene abnormality is responsible for 85% of cases of ADPKD, patients with PKD2 mutations typically present later and progress more slowly. Patients with ADPKD can present with a positive family history, hypertension, flank pain, haematuria, renal insufficiency or proteinuria. The diagnosis has traditionally been based on ultrasound imaging. Screening will reduce the incidence of a late diagnosis when renal disease is advanced but a normal ultrasound scan in those under 30 years old is not conclusive. It is not recommended that children are screened. The key to minimising the rate of progressive disease is tight BP control. ACE inhibitors are recommended as the initial antihypertensive agent unless contraindicated. Alport's syndrome is a disorder characterised by abnormal type IV collagen which is found in the kidney, eyes, skin and ears. Around one in ten practices are likely to have a patient with Alport's syndrome. Eighty per cent of patients have the X-linked form of the disease. All first-degree relatives of a patient with confirmed Alport's syndrome should be offered screening. The combination of reduced hearing and urinary abnormalities in a young boy should alert GPs to consider this as a possible diagnosis and initiate referral. Diagnosis can be confirmed by renal or skin biopsy.     

Towards understanding the polycystins

Autosomal dominant polycystic kidney disease (ADPKD) is a very common inherited disease caused by mutations in PKD1 or PKD2 genes characterized by progressive enlargement of fluid-filled cysts and loss of renal function [1]. Previous studies proposed a role for human polycystin-1 in renal morphogenesis acting as a matrix receptor in focal adhesions and for polycystin-2 as a putative calcium channel [2, 3]. The genome of Caenorhabditis elegans contains 2 new members of the polycystin family: lov-1, the homolog for PKD1; and pkd-2, the homolog for PKD2 [4; this paper]. Mutation analysis in C. elegans showed similarly compromised male mating behaviors in all single and double lov-1 and pkd-2 mutants, indicating their participation in a single genetic pathway. Expression analysis localized LOV-1 and PKD-2 to the ends of sensory neurons in male tails and to the tips of CEM neurons in the head, consistent with functions as chemo- or mechanosensors. Human and C. elegans PKD1 and PKD2 homologs, transfected into mammalian renal epithelial cells, co-localized with paxillin in focal adhesions suggesting function in a single biological pathway. Based on the role of polycystins in C. elegans sensory neuron function and the conservation of PKD pathways we suggest that polycystins act as sensors of the extracellular environment, initiating, via focal adhesion assembly, intracellular transduction events in neuronal or morphogenetic processes.     

Altered trafficking and stability of polycystins underlie polycystic kidney disease

The most severe form of autosomal dominant polycystic kidney disease occurs in patients with mutations in the gene (PKD1) encoding polycystin-1 (PC1). PC1 is a complex polytopic membrane protein expressed in cilia that undergoes autoproteolytic cleavage at a G protein–coupled receptor proteolytic site (GPS). A quarter of PKD1 mutations are missense variants, though it is not clear how these mutations promote disease. Here, we established a cell-based system to evaluate these mutations and determined that GPS cleavage is required for PC1 trafficking to cilia. A common feature among a subset of pathogenic missense mutations is a resulting failure of PC1 to traffic to cilia regardless of GPS cleavage. The application of our system also identified a missense mutation in the gene encoding polycystin-2 (PC2) that prevented this protein from properly trafficking to cilia. Using a Pkd1-BAC recombineering approach, we developed murine models to study the effects of these mutations and confirmed that only the cleaved form of PC1 exits the ER and can rescue the embryonically lethal Pkd1-null mutation. Additionally, steady-state expression levels of the intramembranous COOH-terminal fragment of cleaved PC1 required an intact interaction with PC2. The results of this study demonstrate that PC1 trafficking and expression require GPS cleavage and PC2 interaction, respectively, and provide a framework for functional assays to categorize the effects of missense mutations in polycystins.     

A role for microRNA in cystic liver and kidney diseases

The polycystic liver and kidney diseases are a family of disorders with heterogeneous etiologies. Proposed mechanisms of disease include ciliary dysfunction, excess cell proliferation, and altered cell-cell or cell-matrix interactions. In this issue of the JCI, Lee and colleagues provide data to support a novel mechanism for cystogenesis involving microRNA (miRNA) (see the related article beginning on page 3714). They demonstrate that levels of the miRNA miR15a are decreased in livers of patients with autosomal recessive and autosomal dominant polycystic kidney disease (ARPKD and ADPKD, respectively) and congenital hepatic fibrosis as well as in the PKC rat model of ARPKD. This results in increased expression of the cell-cycle regulator Cdc25A, which is a direct target of miR15a, and increased cellular proliferation and cystogenesis in vitro. These findings suggest that other miRNAs may also participate in the molecular pathogenesis of cystic liver and kidney diseases.     

Molecular diagnosis of autosomal dominant polycystic kidney disease

Introduction: Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease that accounts for 5–10% of end-stage renal disease in developed countries. Mutations in PKD1 and PKD2 account for a majority of cases. Mutation screening of PKD1 is technically challenging largely due to the complexity resulting from duplication of its first 33 exons in six highly homologous pseudogenes (i.e. PKD1P1-P6). Protocol using locus-specific long-range and nested PCR has enabled comprehensive PKD1 mutation screening but is labor-intensive and costly. Here, the authors review how recent advances in Next Generation Sequencing are poised to transform and extend molecular diagnosis of ADPKD.

Areas covered: Key original research articles and reviews of the topic published in English identified through PubMed from 1957–2017.

Expert commentary: The authors review current and evolving approaches using targeted resequencing or whole genome sequencing for screening typical as well as challenging cases (e.g. cases with no detectable PKD1 and PKD2 mutations which may be due to somatic mosaicism or other cystic disease; and complex genetics such as bilineal disease).     

Autosomal dominant polycystic kidney disease: recent advances in pathogenesis and treatment

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder affecting 1 in 1,000 people in the general population and accounts for up to 10% of all patients on renal replacement therapy. Numerous fluid-filled epithelial cysts arise from different nephron segments as spherical dilatations or small out-pouchings, enlarge progressively and eventually become disconnected from the rest of the renal tubule. The development of cysts is accompanied by destruction of the renal parenchyma, interstitial fibrosis, cellular infiltration and loss of functional nephrons. ADPKD is not only a kidney disease but also a systemic disorder associated with intracranial arterial aneurysms, cardiac valvular defects, colonic diverticulosis and cyst formation in other organs such as the liver, spleen and pancreas. The identification of PKD1 and PKD2 together with the drive to elucidate the functions of their encoded proteins, polycystin-1 (PC1) and polycystin-2 (PC2), has led to an explosion of clinical and scientific interest in this common disorder. The aim of this review is to highlight recent advances in our understanding of ADPKD pathogenesis which are leading to exciting new treatment strategies.     

The molecular genetic analysis of polycystic kidney disease]

Autosomal dominant polycystic kidney disease (ADPKD) is one of common single gene disorders. The development of molecular genetic techniques has shown that mutant PKD1 gene assigned to ADPKD was closely linked to alpha-globin on the short arm of chromosome 16. This location was established when genetic linkage was found between ADPKD and a highly polymorphic region at the 3' end of the alpha-globin cluster (3' HVR). The discover of genetic linkage markers such as 3' HVR probe has provided a diagnostic test in presymptomatic stage. We performed this diagnostic test using DNA probes in 3 patients with ADPKD of one Japanese family. They also showed PKD1 gene linkage as previously described by Reeders et al. Linkage analysis of the PKD1 gene might be available to diagnostic test of ADPKD. DNA diagnosis of ADPKD however has to be performed carefully because of an ethical standpoint.