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.     

Functional polycystin-1 dosage governs autosomal dominant polycystic kidney disease severity

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations to PKD1 or PKD2, triggering progressive cystogenesis and typically leading to end-stage renal disease in midlife. The phenotypic spectrum, however, ranges from in utero onset to adequate renal function at old age. Recent patient data suggest that the disease is dosage dependent, where incompletely penetrant alleles influence disease severity. Here, we have developed a knockin mouse model matching a likely disease variant, PKD1 p.R3277C (RC), and have proved that its functionally hypomorphic nature modifies the ADPKD phenotype. While Pkd1^+/null mice are normal, Pkd1^RC/null mice have rapidly progressive disease, and Pkd1^RC/RC animals develop gradual cystogenesis. These models effectively mimic the pathophysiological features of in utero–onset and typical ADPKD, respectively, correlating the level of functional Pkd1 product with disease severity, highlighting the dosage dependence of cystogenesis. Additionally, molecular analyses identified p.R3277C as a temperature-sensitive folding/trafficking mutant, and length defects in collecting duct primary cilia, the organelle central to PKD pathogenesis, were clearly detected for the first time to our knowledge in PKD1. Altogether, this study highlights the role that in trans variants at the disease locus can play in phenotypic modification of dominant diseases and provides a truly orthologous PKD1 model, optimal for therapeutic testing.     

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.     

Identification and localization of polycystin,the PKD1 gene product.

Polycystin, the product of autosomal dominant polycystic kidney disease (ADPKD) 1 gene (PKD1) is the cardinal member of a novel class of proteins. As a first step towards elucidating the function of polycystin and the pathogenesis of ADPKD, three types of information were collected in the current study: the subcellular localization of polycystin, the spatial and temporal distribution of the protein within normal tissues and the effects of ADPKD mutations on the pattern of expression in affected tissues. Antisera directed against a synthetic peptide and two recombinant proteins of different domains of polycystin revealed the presence of an approximately 400-kD protein (polycystin) in the membrane fractions of normal fetal, adult, and ADPKD kidneys. Immunohistological studies localized polycystin to renal tubular epithelia, hepatic bile ductules, and pancreatic ducts, all sites of cystic changes in ADPKD, as well as in tissues such as skin that are not known to be affected in ADPKD. By electron microscopy, polycystin was predominantly associated with plasma membranes. Polycystin was significantly less abundant in adult than in fetal epithelia. In contrast, polycystin was overexpressed in most, but not all, cysts in ADPKD kidneys.     

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.     

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.     

Macrophage migration inhibitory factor promotes cyst growth in polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by renal cyst formation, inflammation, and fibrosis. Macrophages infiltrate cystic kidneys, but the role of these and other inflammatory factors in disease progression are poorly understood. Here, we identified macrophage migration inhibitory factor (MIF) as an important regulator of cyst growth in ADPKD. MIF was upregulated in cyst-lining epithelial cells in polycystin-1–deficient murine kidneys and accumulated in cyst fluid of human ADPKD kidneys. MIF promoted cystic epithelial cell proliferation by activating ERK, mTOR, and Rb/E2F pathways and by increasing glucose uptake and ATP production, which inhibited AMP-activated protein kinase signaling. MIF also regulated cystic renal epithelial cell apoptosis through p53-dependent signaling. In polycystin-1–deficient mice, MIF was required for recruitment and retention of renal macrophages, which promoted cyst expansion, and Mif deletion or pharmacologic inhibition delayed cyst growth in multiple murine ADPKD models. MIF-dependent macrophage recruitment was associated with upregulation of monocyte chemotactic protein 1 (MCP-1) and inflammatory cytokine TNF-α. TNF-α induced MIF expression, and MIF subsequently exacerbated TNF-α expression in renal epithelial cells, suggesting a positive feedback loop between TNF-α and MIF during cyst development. Our study indicates MIF is a central and upstream regulator of ADPKD pathogenesis and provides a rationale for further exploration of MIF as a therapeutic target for 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.     

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.     

Deficiency of FLCN in Mouse Kidney Led to Development of Polycystic Kidneys and Renal Neoplasia

The Birt–Hogg–Dubé (BHD) disease is a genetic cancer syndrome. The responsible gene, BHD, has been identified by positional cloning and thought to be a novel tumor suppressor gene. BHD mutations cause many types of diseases including renal cell carcinomas, fibrofolliculomas, spontaneous pneumothorax, lung cysts, and colonic polyps/cancers. By combining Gateway Technology with the Ksp-Cre gene knockout system, we have developed a kidney-specific BHD knockout mouse model. BHD^flox/flox/Ksp-Cre mice developed enlarged kidneys characterized by polycystic kidneys, hyperplasia, and cystic renal cell carcinoma. The affected BHD^flox/flox/Ksp-Cre mice died of renal failure at approximate three weeks of age, having blood urea nitrogen levels over tenfold higher than those of BHD ^flox/+/Ksp-Cre and wild-type littermate controls. We further demonstrated that these phenotypes were caused by inactivation of BHD and subsequent activation of the mTOR pathway. Application of rapamycin, which inhibits mTOR activity, to the affected mice led to extended survival and inhibited further progression of cystogenesis. These results provide a correlation of kidney-targeted gene inactivation with renal carcinoma, and they suggest that the BHD product FLCN, functioning as a cyst and tumor suppressor, like other hamartoma syndrome–related proteins such as PTEN, LKB1, and TSC1/2, is a component of the mTOR pathway, constituting a novel FLCN-mTOR signaling branch that regulates cell growth/proliferation.     

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.     

Characterization of microsatellite markers to diagnose ADPKD

Autosomal dominant polycystic kidney disease (ADPKD) maps to chromosome 16p13.3 (PKD1) and to chromosome 4q21-23 (PKD2), with the likelihood of a third unmapped locus. The size and genomic complexity of the PKD1 gene make it impractical to detect mutations for prenatal diagnosis. Therefore, pedigree-based linkage analysis remains useful for diagnosis of ADPKD. Since, the complete genome sequences of chromosome 16p13.3 and 4q21-23 including PKD1 and PKD2, respectively, were reported very recently, in order to do more precise diagnosis of ADPKD, we tried to find microsatellite markers. We performed database searches of 2000 kb of genome sequence across the 16p13.3 and the 4q21-23. To determine the distribution of alleles and the degree of polymorphism of the microsatellites, genotyping experiments were performed on 48 Korean individuals. We found novel 14 microsatellite markers around ADPKD that are more polymorphic and closer to PKD1 or PKD2 than the known markers. The novel microsatellite markers were applied to diagnose ADPKD families. These novel microsatellite markers are not only useful for presymptomatic and prenatal diagnosis of ADPKD, but also applicable in the study of positional cloning, human evolution and tumor biology.     

The antifibrotic effects of plasminogen activation occur via prostaglandin E2 synthesis in humans and mice

Plasminogen activation to plasmin protects from lung fibrosis, but the mechanism underlying this antifibrotic effect remains unclear. We found that mice lacking plasminogen activation inhibitor–1 (PAI-1), which are protected from bleomycin-induced pulmonary fibrosis, exhibit lung overproduction of the antifibrotic lipid mediator prostaglandin E₂ (PGE₂). Plasminogen activation upregulated PGE₂ synthesis in alveolar epithelial cells, lung fibroblasts, and lung fibrocytes from saline- and bleomycin-treated mice, as well as in normal fetal and adult primary human lung fibroblasts. This response was exaggerated in cells from Pai1^–/– mice. Although enhanced PGE₂ formation required the generation of plasmin, it was independent of proteinase-activated receptor 1 (PAR-1) and instead reflected proteolytic activation and release of HGF with subsequent induction of COX-2. That the HGF/COX-2/PGE₂ axis mediates in vivo protection from fibrosis in Pai1^–/– mice was demonstrated by experiments showing that a selective inhibitor of the HGF receptor c-Met increased lung collagen to WT levels while reducing COX-2 protein and PGE₂ levels. Of clinical interest, fibroblasts from patients with idiopathic pulmonary fibrosis were found to be defective in their ability to induce COX-2 and, therefore, unable to upregulate PGE₂ synthesis in response to plasmin or HGF. These studies demonstrate crosstalk between plasminogen activation and PGE₂ generation in the lung and provide a mechanism for the well-known antifibrotic actions of the fibrinolytic pathway.     

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.     

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.