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Polycystins, focal adhesions and extracellular matrix interactions
Abstract
Polycystic kidney disease is the most common heritable disease in humans. In addition to epithelial cysts in the kidney, liver and pancreas, patients with autosomal dominant polycystic kidney disease (ADPKD) also suffer from abdominal hernia, intracranial aneurysm, gastrointestinal cysts, and cardiac valvular defects, conditions often associated with altered extracellular matrix production or integrity. Despite more than a decade of work on the principal ADPKD genes, PKD1 and PKD2, questions remain about the basis of cystic disease and the role of extracellular matrix in ADPKD pathology. This review explores the links between polycystins, focal adhesions, and extracellular matrix gene expression. These relationships suggest roles for polycystins in cell-matrix mechanosensory signaling that control matrix production and morphogenesis. This article is part of a Special Issue entitled: Polycystic Kidney Disease.
... Indeed, besides flow sensing, PC1 is implicated in a plethora of cellular activities, including adhesion, extracellular matrix (ECM) regulation, cell signaling, and metabolism. [24][25][26] In accordance with these biological activities, PC1 is seen in a variety of non-epithelial cell types and disrupting Pkd1 in these tissues leads to defective organogenesis or malfunction in a number of organ systems. [27][28][29][30] In the kidney, less well recognized is the function of PC1 in stromal cells and their derivatives, which show dynamic PC1 expression in both developing and adult kidneys. ...
... 32 Multiple lines of evidence suggest that Pkd1-encoded PC1 is involved in ECM regulation in a variety of organ systems. 25,32,58,59 The extracellular domain of PC1 physically interacts with most components of ECM, including collagen, laminin and fibronectin. 25,59 Thus, PC1 has been proposed as a receptor for ECM components. ...
... 25,32,58,59 The extracellular domain of PC1 physically interacts with most components of ECM, including collagen, laminin and fibronectin. 25,59 Thus, PC1 has been proposed as a receptor for ECM components. Our data support this view, as disruption of Pkd1 in stromal cells leads to significant ECM reduction in the stromal compartment. ...
Autosomal dominant polycystic kidney disease (ADPKD), caused by PKD1 and PKD2 gene mutations, is one of the most common genetic diseases, affecting up to 1 in 500 people. Mutations of PKD1 account for over 85% of ADPKD cases. However, mechanisms of disease progression and explanations for the wide range in disease phenotype remain to be elucidated. Moreover, functional roles of PKD1 in the renal stromal compartment are poorly understood. In this work, we tested if Pkd1 is essential for development and maintenance of the renal stromal compartment and if this role contributes to pathogenesis of polycystic kidney disease using a novel tissue-specific knockout mouse model. We demonstrate that deletion of Pkd1 from renal stromal cells using Foxd1-driven Cre causes a spectrum of defects in the stromal compartment, including excessive apoptosis/proliferation and extracellular matrix deficiency. Renal vasculature was also defective. Further, mutant mice showed epithelial changes and progressive cystogenesis in adulthood modeling human ADPKD. Altogether, we provide robust evidence to support indispensable roles for Pkd1 in development and maintenance of stromal cell derivatives by using a novel ADPKD model. Moreover, stromal compartment defects caused by Pkd1 deletion might serve as an important mechanism for pathogenesis of ADPKD.
... 1 Distinctive features of ADPKD are the alteration of epithelial extracellular matrix (ECM) 2,3 and the progressive renal fibrosis, which parallels the course of the disease. 4 The involvement of the ECM in the pathogenesis of ADPKD has long been suspected 5,6 and is supported by the observations that the altered expression of the ECM components laminin-a5 and proteoglycans 7,8 or the focal adhesion protein tensin lead to renal cystogenesis. 9 ECM is essential in not only maintaining the normal tubule architecture, but also wound repair and developmental processes as well as the regulation of cell growth, polarity, migration, and differentiation. ...
... 21,22 PC1 colocalizes within integrins at the focal adhesions, suggesting that it may affect the interaction of integrins with their ECM ligands. 2,6,23,24 Growing evidence indicates that integrins expressed on cilia mediate the mechanical stimulation by ECM components through cues that may involve PC1. 20,[25][26][27][28] The upregulation of integrin in ADPKD alters the adhesion and motility properties of cystic cells, possibly triggering a stimulatory loop that can be reinforced by the aberrant deposition of ECM. ...
... When plated on collagen type I in the presence of Mn 2+ , F1/Pkd1 2/2 cells showed an enhanced Akt phosphorylation (Ser473) compared with F1/wild-type (WT) cells, thus confirming the higher integrinmediated signaling of the PC1-depleted cells. Overall, these results confirmed the association between the loss of PC1 and the changes in ECM deposition 4,6 and suggested that a positive feedback loop between Intb1 and ECM may potentiate the cystic mechanisms in the absence of PC1. 34,37 Reduced Cystogenesis and Restored Renal Function in Pkd1 Knockout Kidneys Lacking Intb1 To assess the role of b1 integrin in ADPKD cystogenesis, we generated a conditional double knockout (DKO) mouse model in which the floxed Pkd1 35 and floxed Itgb1 38 genes are inactivated specifically in the collecting ducts by aquaporin-2 (Aqp2) promoter-driven Cre recombinase. ...
Dysregulation of polycystin-1 (PC1) leads to autosomal dominant polycystic kidney disease (ADPKD), a disorder characterized by the formation of multiple bilateral renal cysts, the progressive accumulation of extracellular matrix (ECM), and the development of tubulointerstitial fibrosis. Correspondingly, cystic epithelia express higher levels of integrins (ECM receptors that control various cellular responses, such as cell proliferation, migration, and survival) that are characteristically altered in cystic cells. To determine whether the altered expression of ECM and integrins could establish a pathologic autostimulatory loop, we tested the role of integrin-β1 in vitro and on the cystic development of ADPKD in vivo. Compared with wild-type cells, PC1-depleted immortalized renal collecting duct cells had higher levels of integrin-β1 and fibronectin and displayed increased integrin-mediated signaling in the presence of Mn(2+). In mice, conditional inactivation of integrin-β1 in collecting ducts resulted in a dramatic inhibition of Pkd1-dependent cystogenesis with a concomitant suppression of fibrosis and preservation of normal renal function. Our data provide genetic evidence that a functional integrin-β1 is required for the early events leading to renal cystogenesis in ADPKD and suggest that the integrin signaling pathway may be an effective therapeutic target for slowing disease progression.
... Previously, the polycystins have been implicated in epithelial differentiation and establishment of tubules (Boletta, 2003, Grimm et al., 2006. Further, polycystins have a noted relationship with the microenvironment (Cruz et al., 2017, Drummond, 2011, but there is not a current model system to determine the immediacy of these changes in response to gene function loss in relevant epithelial structures. While decades of work have led to exciting advances in our understanding of disease pathology and the first FDA approved treatments, the initiating mechanisms underlying progressive cystogenesis remains undefined in renal epithelial cells (Dixon and Woodward, 2018, Torres and Harris, 2019, Brill and Ehrlich, 2020. ...
... Loss of function mutations in either PKD1 or PKD2 are associated with downstream changes, including increased proliferation, changes in apicobasolateral organization, alterations in cell-matrix interactions, and fluid secretion and accumulation (Terryn et al., 2011, Drummond, 2011, Cornec-Le Gall et al., 2019. ...
Cystogenesis is a morphological consequence of numerous genetic diseases of the epithelium. In the kidney, the pathogenic mechanisms underlying the program of altered cell and tubule morphology are obscured by secondary effects of cyst expansion. Here, we developed a new 3D tubuloid system to isolate the rapid changes in protein localization and gene expression that correlate with altered cell and tubule morphology during cyst initiation. Mouse renal tubule fragments were pulsed with a cell differentiation cocktail including glial-derived neurotrophic factor (GDNF) to yield collecting duct-like tubuloid structures with appropriate polarity, primary cilia, and gene expression. Using the 3D tubuloid model with an inducible Pkd2 knockout system allowed the tracking of morphological, protein, and genetic changes during cyst formation. Within hours of inactivation of Pkd2 and loss of polycystin-2, we observed significant progression in tubuloid to cyst morphology that correlated with 35 differentially expressed genes, many related to cell junctions, matrix interactions, and cell morphology previously implicated in cystogenesis.
This article has an associated First Person interview with the first author of the paper .
... [10][11][12][13][14][15][16] Further, a genetic syndrome, Autosomal-Dominant Polycystic Kidney Disease (ADPKD), predisposes patients to IA formation (although the vast majority of familial and sporadic IA patients do not have ADPKD or other defined syndromes). 17 Nearly all cases of ADPKD are caused by mutations in either PDK1 or PDK2 that have a range of functions from forming a mechanosensor that regulates intracellular calcium levels to being involved in focal adhesions and the extracellular matrix. ...
... 32 Further, THSD1 shares some similarities to PDK1 and PDK2, two genes whose mutation causes Autosomal-Dominant Polycystic Kidney Disease (ADPKD) and increases IA risk. 17 In addition to a role as a mechanosensor, PDK1 and PDK2 form a complex that is involved in focal adhesion and cell-extracellular matrix interactions. 33 The significance of THSD1 mutations in other aneurysm types or more broadly in cardiovascular pathogenesis remains unclear. ...
Background and Purpose— A ruptured intracranial aneurysm (IA) is the leading cause of a subarachnoid hemorrhage. This study seeks to define a specific gene whose mutation leads to disease.
Methods— More than 500 IA probands and 100 affected families were enrolled and clinically characterized. Whole exome sequencing was performed on a large family, revealing a segregating THSD1 (thrombospondin type 1 domain containing protein 1) mutation. THSD1 was sequenced in other probands and controls. Thsd1 loss-of-function studies in zebrafish and mice were used for in vivo analyses and functional studies performed using an in vitro endothelial cell model.
Results— A nonsense mutation in THSD1 was identified that segregated with the 9 affected (3 suffered subarachnoid hemorrhage and 6 had unruptured IA) and was absent in 13 unaffected family members (LOD score 4.69). Targeted THSD1 sequencing identified mutations in 8 of 507 unrelated IA probands, including 3 who had suffered subarachnoid hemorrhage (1.6% [95% confidence interval, 0.8%–3.1%]). These THSD1 mutations/rare variants were highly enriched in our IA patient cohort relative to 89 040 chromosomes in Exome Aggregation Consortium (ExAC) database ( P <0.0001). In zebrafish and mice, Thsd1 loss-of-function caused cerebral bleeding (which localized to the subarachnoid space in mice) and increased mortality. Mechanistically, THSD1 loss impaired endothelial cell focal adhesion to the basement membrane. These adhesion defects could be rescued by expression of wild-type THSD1 but not THSD1 mutants identified in IA patients.
Conclusions— This report identifies THSD1 mutations in familial and sporadic IA patients and shows that THSD1 loss results in cerebral bleeding in 2 animal models. This finding provides new insight into IA and subarachnoid hemorrhage pathogenesis and provides new understanding of THSD1 function, which includes endothelial cell to extracellular matrix adhesion.
... Activation of integrin receptors leads to increased PI3K, Ca 2+ level and Ras/Raf/Erk signaling pathways, all of which may be associated with ADPKD. Furthermore, PC1 colocalizes with integrin proteins in focal adhesions, cell-cell contact, and cilium in renal epithelial cells [18,34,35]. It is also reported that polycystic cells exhibit increased adhesion to laminin proteins through integrins [24], further supporting that PC1 exerts integrin-ECM interaction. ...
Mutations of PKD1 coding for polycystin-1 (PC1) account for most cases of autosomal-dominant polycystic kidney disease (ADPKD). The extracellular region of PC1 contains many evolutionarily conserved domains for ligand interactions. Among these are the leucine-rich repeats (LRRs) in the far N-terminus of PC1. Using zebrafish (Danio rerio) as an in vivo model system, we explored the role of LRRs in the function of PC1. Zebrafish expresses two human PKD1 paralogs, pkd1a and pkd1b. Knockdown of both genes in zebrafish by morpholino antisense oligonucleotides produced phenotypes of dorsal-axis curvature and pronephric cyst formation. We found that overexpression of LRRs suppressed both phenotypes in pkd1-morphant zebrafish. Purified recombinant LRR domain inhibited proliferation of HEK cells in culture and interacted with the heterotrimeric basement membrane protein laminin-511 (α5β1γ1) in vitro. Mutations of amino acid residues in LRRs structurally predicted to bind laminin-511 disrupted LRR–laminin interaction in vitro and neutralized the ability of LRRs to inhibit cell proliferation and cystogenesis. Our data support the hypothesis that the extracellular region of PC1 plays a role in modulating PC1 interaction with the extracellular matrix and contributes to cystogenesis of PC1 deficiency.
... Furthermore, PC1 colocalizes with integrin proteins in focal adhesions, cellcell contact and cilium in renal epithelial cells. (26,43,44) It is also reported that polycystic cells exhibit increased adhesion to laminin proteins through integrins, (34) further supporting that PC1 exerts integrin-ECM interaction. Along the same line, laminin-332 and its binding partner integrin α6ß4 are altered in PKD. ...
Mutations of PKD1 coding for polycystin-1 (PC1) account for most of the autosomal dominant polycystic kidney disease (ADPKD). The extracellular region of PC1 contains many evolutionarily conserved domains for ligand interactions. Among these is the leucine-rich repeats (LRR) in the far N-terminus of PC1. Using zebrafish (Danio rerio) as an in vivo model system, we explored the role of LRR in the function of PC1. Zebrafish expresses two human PKD1 paralogs, pkd1a and pkd1b. Knockdown of both genes in zebrafish by morpholino antisense oligonucleotides produced phenotypes of dorsal axis curvature and pronephric cyst formation. We found that overexpression of LRR suppressed both phenotypes in pkd1-morphant zebrafish. Purified recombinant LRR domain inhibited proliferation of HEK cells in culture and interacted with the heterotrimeric basement membrane protein laminin-511 (α5β1γ1) in vitro. Mutations of amino acid residues in LRR structurally predicted to bind laminin-511 disrupted LRR-laminin interaction in vitro and neutralized the ability of LRR to inhibit cell proliferation and cystogenesis. Our data support the hypothesis that the extracellular region of PC1 plays a role in modulating PC1 interaction with the extracellular matrix and contributes to cystogenesis of PC1-deficiency.
... Qian et al. found no association between PE and renal dysfunction and suggested that defects in polycystin-1, a large integral glycoprotein encoded by PKD1-gen, and in polycystin-2 protein, a non-selective calcium channel encoded by PKD2-gen, are directly involved into the molecular pathogenesis of PE [2,8]. Polycystins are expressed not only in renal tubules but also in other epithelial and endothelial cells throughout the body, and have important roles in cell adhesion and extracellular matrix regulation explaining the risk for connective tissue defects in ADPKD patients [9]. ...
Background
Autosomal dominant polycystic kidney disease (ADPKD) has numerous extrarenal manifestations. Pericardial effusion (PE) may be an under-recognized complication with a reported prevalence of up to 35%. Our study is the first to systematically evaluate the prevalence of PE and associated risk factors in an ADPKD cohort outside the U.S.
Methods
Clinically stable ADPKD patients from a specialized outpatient clinic were evaluated retrospectively. Magnetic resonance tomography and computed tomography scans were analysed regarding the presence of PE (≥4 mm). Imaging results were linked to clinical characteristics.
Results
Out of 286 ADPKD patients, 208 had computed tomography or magnetic resonance imaging suitable for evaluation of PE. In this group, we detected PE in 17 patients (8.2%). The overall prevalence of PE was 6.3% with more females being affected (prevalence of PE 7.8% in female and 3.8% in male patients). PE mean size was 6.8 ± 3.3 mm. The prevalence of autoimmune diseases was higher in the patients with PE (11.8% versus 2.1%, P = 0.022), while presence and size of PE was not associated with signs of rapid progressive disease, ADPKD genotype, patient age, BMI, and other clinical parameters. Exploratory investigation of individual characteristics of PE patients by regression tree analysis suggested renal functional impairment, sex, and proteinuria as candidate variables.
Conclusions
PE prevalence in our cohort was lower than previously reported and showed a clear female preponderance. Our data suggest that patients with PE size > 10 mm deserve further attention, as they may have additional non-ADPKD related pathologies.
... PC1 also exhibits the ability to interact with the extracellular matrix (ECM) through intermediate filaments. The co-localization of PC1 with α 2 β 1 integrins supports its role in the ECM-cell interplay [30]. Moreover, integrins are known to act as receptor proteins binding to ECM. ...
Developmental cysts are pathological epithelial-lined cavities arising in various organs as a result of systemic or hereditary diseases. Molecular mechanisms involved in the formation of developmental odontogenic cysts (OCs) are not fully understood yet; the cystogenesis of renal cysts originating from the autosomal dominant polycystic kidney disease (ADPKD) has been, however, explored in much greater detail. This narrative review aimed i) to summarize molecular and cellular processes involved in the formation and growth of developmental OCs, especially dentigerous cysts (DCs) and odontogenic keratocysts (OKCs), ii) to find if there are any similarities in their cystogenesis to ADPKD cysts, and, based on that, iii) to suggest potential factors, candidate molecules, and mechanisms that could be involved in the DC formation, thus proposing further research directions. Here we suggest a possible association of developmental OCs with primary cilia disruption and with hypoxia, which have been previously linked with cyst formation in ADPKD patients. This is illustrated on the imagery of tissues from an ADPKD patient (renal cyst) and from developmental OCs, supporting the similarities in cell proliferation, apoptosis, and primary cilia distribution in DC/OKC/ADPKD tissues. Based on all that, we propose a novel hypothesis of OCs formation suggesting a crucial role of mutations associated with the signaling pathways of primary cilia (in particular, Sonic Hedgehog). These can lead to excessive proliferation and formation of cell agglomerates, which is followed by hypoxia-driven apoptosis in the centers of such agglomerates (controlled by molecules such as Hypoxia-inducible factor-1 alpha), leading to cavity formation and, finally, the OCs development. Based on this, we propose future perspectives in the investigation of OC pathogenesis.
... Abnormal ECM and fibrosis are observed in ciliopathies and neurodegenerative diseases including Alzheimer's disease. Polycystin 1 and its interaction with ECM is implicated in cyst formation [56,85]. EVs are components of the ECM and EVs themselves may carry ECM proteins and ECM-modifying enzymes [86,87]. ...
The cilium acts as an antenna receiving and sending signals, the latter via extracellular vesicles (EVs). In C. elegans and mammals, the Autosomal Dominant Polycystic Kidney Disease (ADPKD) gene products polycystin-1 (PC1) and polycystin-2 (PC2) localize to both cilia and EVs, act in the same genetic pathway, and function in a sensory capacity, suggesting ancient conservation. However, the functions of the polycystins on cilia and EVs remain enigmatic. We used our C. elegans model and endogenously fluorescent-tagged LOV-1/polycystin-1 to study LOV-1 processing, trafficking, transport, EV biogenesis, and function in living animals. Super resolution, real time imaging reveals that LOV-1 is processed into N-terminal (NTM) and C-terminal (CTM) forms via a conserved GPCR proteolytic site (GPS). The LOV-1 NTM is secreted into the extracellular matrix and not localized to ciliary tip EVs. In contrast, LOV-1 CTM and PKD-2 are co-trafficked, co-transported, and co-localized in cilia and on environmentally released ciliary EVs. LOV-1 CTM requires PKD-2 for ciliary EV localization, while PKD-2 localizes to ciliary EVs independent of LOV-1. We find that LOV-1 but not PKD-2 is required for chemosensation of an ascaroside mating pheromone. These findings indicate that the polycystins LOV-1 and PKD-2 function together and independently and provide insight to how cargo is selected and packaged in ciliary EVs.
... The tubular shape of bioprinted constructs allowed us to examine Pkd1 knockout-induced deviations from this geometry, of which the formation of side branches was the most striking manifestation. The pathogenetic chain of events after second hit-induced PC1 loss can conceptually be broken down into loss of polarity, disruption of cellmatrix interactions and fluid accumulation 71,72 . The observed alterations in tubule geometry may thus reflect earliest disease manifestations and could be caused by disturbed cell-cell or cell-matrix interactions and a partially disorientated growth behavior. ...
Renal tubular cells frequently lose differentiation markers and physiological properties when propagated in conventional cell culture conditions. Embedding cells in 3D microenvironments or controlling their 3D assembly by bioprinting can enhance their physiological properties, which is beneficial for modeling diseases in vitro.
A potential cellular source for modeling renal tubular physiology and kidney diseases in vitro are directly reprogrammed induced renal tubular epithelial cells (iRECs). iRECs were cultured in various biomaterials and as bioprinted tubular structures. They showed high compatibility with the embedding substrates and dispensing methods. The morphology of multicellular aggregates was substantially influenced by the 3D microenvironment. Transcriptomic analyses revealed signatures of differentially expressed genes specific to each of the selected biomaterials. Using a new cellular model for autosomal-dominant polycystic kidney disease, Pkd1−/− iRECs showed disrupted morphology in bioprinted tubules and a marked upregulation of the Aldehyde dehydrogenase 1a1 (Aldh1a1). In conclusion, 3D microenvironments strongly influence the morphology and expression profiles of iRECs, help to unmask disease phenotypes, and can be adapted to experimental demands. Combining a direct reprogramming approach with appropriate biomaterials will facilitate construction of biomimetic kidney tubules and disease models at the microscale.
... These alterations include apical-basal polarity, planar cell polarity, increased extracellular matrix production, and cellular metabolism, in conjunction with essential cellular functions such as fluid transport, proliferation, apoptosis, cell adhesion, and differentiation. [54][55][56] The cAMP-CREB-miR-21-PDCD4 Axis cAMP signaling has been shown to play a key role in kidney cyst growth, with evidence showing that cAMP levels were increased in ADPKD and inhibition of this pathway slowed cyst growth in both mice and humans. 57 A study derived from kidney samples of patients with ADPKD and orthologous mice models of PKD illustrated that cAMP signaling transactivated miR-21 promoter and promoted miR-21 expression. ...
Important advances have been made regarding the diagnosis and management of polycystic kidney diseases. Care of patients with polycystic kidney diseases has moved beyond supportive care for complications and chronic kidney disease to new potentially disease-modifying therapies. Recently, the role of noncoding RNAs, in particular microRNAs, has been described in polycystic kidney diseases. microRNAs are involved in the regulation of gene expression, in which PKD1, PKD2, and other genes that contribute to the pathogenesis of polycystic kidney diseases are considerable participants. Seminal studies have highlighted the potential importance of microRNAs as new therapeutic targets and innovative diagnostic and/or prognostic biomarkers. Furthermore, an anti–miR-17 drug has advanced through preclinical autosomal dominant polycystic disease studies, and an anti–miR-21 drug has already cleared a phase 1 clinical trial. Most probably, new drugs in the microRNA research field will be yielded as a result of ongoing and planned therapeutic trials. To provide a foundation for understanding microRNA functions as a disease-modifying therapeutic drug in novel targeted therapies, in this narrative review we present an overview of the current knowledge of microRNAs in the pathogenesis of polycystic kidney diseases.
... Overexpression of extracellular matrix (ECM) proteins has been observed in human ADPKD cells and ADPKD animal models. In addition, the cysts lining epithelial cells show elevated adhesiveness to type I and type IV collagen in response to growth factors (28). ...
ABSTRACT Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic renal disease, which is caused by pathogenic mutations of either PKD1 (85%) or PKD2 (15%) genes, encoding for polycystin-1 (PC1) or polycystin-2 (PC2), respectively. These two proteins hetero-dimerize in renal primary cilia to act as a calcium channel. Primary cilia that protrude from cell membranes have a microtubule-based finger-like structure and are found on most mammalian cells. Primary cilia in the kidney have no motility but act as mechanosensors to sense fluid flow through renal tubules. In addition, various signaling proteins related to Hedgehog (Hh) and platelet-derived growth factor receptor alpha (PDGFRα) are localized to the cilia to detect changes in the extracellular environment. Recent studies have demonstrated that many ADPKD animal models have defective cilia in the epithelial cells that line the cysts. Also, animal models targeting ciliary genes show abnormal phenotypes such as polycystic kidneys and developmental defects. These findings reveal that ciliary malfunction is sufficient to cause ADPKD. In this chapter, we will review the putative roles of cilia in cyst formation and development in ADPKD.
... Ciliary mastigonemes, including the structurally more complex tripartite mastigonemes, are present in various groups of protists, but homologues of MST1 are limited in distribution to green algae (Bouck, 1971;Bouck et al., 1978). However, several lines of evidence suggest a relationship between cilia, PKD2, and the ECM (Drummond, 2011;Seeger-Nukpezah and Golemis, 2012). For example, the ciliated endings of mechanosensory neurons in Caenorhabditis elegans and the modified cilia in mechanosensory insect sensilla, the sense organs for hearing and tactile sensation, are embedded in the ECM (Garcia-Anoveros and Corey, 1997;Keil, 2012). ...
Mutations in the channel protein PKD2 cause autosomal dominant polycystic kidney disease, but the function of PKD2 in cilia remains unclear. Here, we show that PKD2 targets and anchors mastigonemes, filamentous polymers of the glycoprotein MST1, to the extracellular surface of Chlamydomonas cilia. PKD2-mastigoneme complexes physically connect to the axonemal doublets 4 and 8, positioning them perpendicular to the plane of ciliary beating. pkd2 mutant cilia lack mastigonemes, and mutant cells swim with reduced velocity, indicating a motility-related function of the PKD2-mastigoneme complex. Association with both the axoneme and extracellular structures supports a mechanosensory role of Chlamydomonas PKD2. We propose that PKD2-mastigoneme arrays, on opposing sides of the cilium, could perceive forces during ciliary beating and transfer these signals to locally regulate the response of the axoneme.
... Mangos et al. showed that knockout of Pkd1 or Pkd2 orthologs in zebrafish resulted in ectopic and persistent expression of multiple collagen mRNAs and proteins, including Col2a1, in the notochord and caused dorsal axis curvature, a PKD phenotype in zebrafish [17]. Gene knockdown of Col2a1 or inhibition of collagen crosslinking restored a normal phenotype, suggesting that abnormalities in ECM composition or amount are developmental defects that are directly linked to polycystin function and not a consequence of tissue damage or inflammation [40]. This evidence supports the hypothesis that cystic transformation and aberrant ECM production may be caused by arrested development of the cystic epithelial cells [41]. ...
In autosomal dominant polycystic kidney disease (ADPKD), the inexorable growth of numerous fluid-filled cysts leads to massively enlarged kidneys, renal interstitial damage, inflammation, and fibrosis, and progressive decline in kidney function. It has long been recognized that interstitial fibrosis is the most important manifestation associated with end-stage renal disease; however, the role of abnormal extracellular matrix (ECM) production on ADPKD pathogenesis is not fully understood. Early evidence showed that cysts in end-stage human ADPKD kidneys had thickened and extensively laminated cellular basement membranes, and abnormal regulation of gene expression of several basement membrane components, including collagens, laminins, and proteoglycans by cyst epithelial cells. These basement membrane changes were also observed in dilated tubules and small cysts of early ADPKD kidneys, indicating that ECM alterations were early features of cyst development. Renal cystic cells were also found to overexpress several integrins and their ligands, including ECM structural components and soluble matricellular proteins. ECM ligands binding to integrins stimulate focal adhesion formation and can promote cell attachment and migration. Abnormal expression of laminin-332 (laminin-5) and its receptor α6β4 stimulated cyst epithelial cell proliferation; and mice that lacked laminin α5, a component of laminin-511 normally expressed by renal tubules, had an overexpression of laminin-332 that was associated with renal cyst formation. Periostin, a matricellular protein that binds αVβ3- and αVβ5-integrins, was found to be highly overexpressed in the kidneys of ADPKD and autosomal recessive PKD patients, and several rodent models of PKD. αVβ3-integrin is also overexpressed by cystic epithelial cells, and the binding of periostin to αVβ3-integrin activates the integrin-linked kinase and downstream signal transduction pathways involved in tissue repair promoting cyst growth, ECM synthesis, and tissue fibrosis. This chapter reviews the roles of the ECM, integrins, and focal adhesion signaling in cyst growth and fibrosis in PKD.
... 28 It has been hypothesized that AWH result from the combination of altered matrix integrity and increased abdominal pressure from cyst burden. 29,30 While the latter seems unlikely in DNAJB11 patients, who generally present with non-enlarged polycystic kidneys and mild liver involvement, reduced mature PC1 might cause altered extracellular matrix organization. Interestingly, AWH and arterial phenotypes are also described in hereditary connective tissue disorders such as vascular Ehlers-Danlos syndrome (vEDS), an autosomal dominant disorder due to mutations of COL3A1 gene. ...
Monoallelic mutations of DNAJB11 were recently described in seven pedigrees with atypical clinical presentations of autosomal dominant polycystic kidney disease. DNAJB11 encodes one of the main cofactors of the endoplasmic reticulum chaperon BiP, a heat-shock protein required for efficient protein folding and trafficking. Here we conducted an international collaborative study to better characterize the DNAJB11-associated phenotype. Thirteen different loss-of-function variants were identified in 20 new pedigrees (54 affected individuals) by targeted next-generation sequencing, whole-exome sequencing or whole-genome sequencing. Amongst the 77 patients (27 pedigrees) now in total reported, 32 reached end stage kidney disease (range, 55-89 years, median age 75); without a significant difference between males and females. While a majority of patients presented with non-enlarged polycystic kidneys, renal cysts were inconsistently identified in patients under age 45. Vascular phenotypes, including intracranial aneurysms, dilatation of the thoracic aorta and dissection of a carotid artery were present in four pedigrees. We accessed Genomics England 100,000 genomes project data, and identified pathogenic variants of DNAJB11 in nine of 3934 probands with various kidney and urinary tract disorders. The clinical diagnosis was cystic kidney disease for eight probands and nephrocalcinosis for one proband. No additional pathogenic variants likely explaining the kidney disease were identified. Using the publicly available GnomAD database, DNAJB11 genetic prevalence was calculated at 0.85/10.000 individuals. Thus, establishing a precise diagnosis in atypical cystic or interstitial kidney disease is crucial, with important implications in terms of follow-up, genetic counseling, prognostic evaluation, therapeutic management, and for selection of living kidney donors.
... The most likely explanation proposed is an increase in extracellular matrix deposition due to an increase in the production and/or deposition of collagen IIα2 57 . Early changes in ECM deposition have been observed in the ADPKD kidney and are likely to be a component of the multiple and complex mechanisms underlying cystogenesis 58,59 . Three chemical classes could be defined from the compounds identified from the Spectrum library: steroids, coumarins and flavonoids (Table S1). ...
Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic cause of end-stage renal failure in humans and results from germline mutations in PKD1 or PKD2. Despite the recent approval of tolvaptan, safer and more effective alternative drugs are clearly needed to slow disease progression. As a first step in drug discovery, we conducted an unbiased chemical screen on zebrafish pkd2 mutant embryos using two publicly available compound libraries (Spectrum, PKIS) totalling 2,367 compounds to identify novel treatments for ADPKD. Using dorsal tail curvature as the assay readout, three major chemical classes (steroids, coumarins, flavonoids) were identified from the Spectrum library as the most promising candidates to be tested on human PKD1 cystic cells. Amongst these were an androgen, 5α−androstane 3,17-dione, detected as the strongest enhancer of the pkd2 phenotype but whose effect was found to be independent of the canonical androgen receptor pathway. From the PKIS library, we identified several ALK5 kinase inhibitors as strong suppressors of the pkd2 tail phenotype and in vitro cyst expansion. In summary, our results identify ALK5 and non-canonical androgen receptors as potential therapeutic targets for further evaluation in drug development for ADPKD.
... These findings are consistent with observations in cystic kidneys. For example, Drummond's [39] review of the characteristics of fibrosis observed in cystic kidney diseases provided evidence of altered interactions between epithelial cells and stromal fibroblasts, followed by accumulation of ECM (collagen, specific laminins, and other proteins) and enhanced expression of matrix metalloproteases and transforming growth factor β. Okada et al. [40] investigated the phenotype of collagen-producing cells in the cystic kidneys of DBA/2pcy mice and characterized the spectrum of interstitial cells associated with renal fibro-genesis. Furthermore, Lee et al. [41] provided genetic evidence that a functional integrin-β1 is required for the early events leading to renal cystogenesis in ADPKD and suggested that the integrin signaling pathway may represent an effective therapeutic target for slowing disease progression. ...
Background/aims:
Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic form of kidney disease. High-throughput microarray analysis has been applied for elucidating key genes and pathways associated with ADPKD. Most genetic profiling data from ADPKD patients have been uploaded to public databases but not thoroughly analyzed. This study integrated 2 human microarray profile datasets to elucidate the potential pathways and protein-protein interactions (PPIs) involved in ADPKD via bioinformatics analysis in order to identify possible therapeutic targets.
Methods:
The kidney tissue microarray data of ADPKD patients and normal individuals were searched and obtained from NCBI Gene Expression Omnibus. Differentially expressed genes (DEGs) were identified, and enriched pathways and central node genes were elucidated using related websites and software according to bioinformatics analysis protocols. Seven DEGs were validated between polycystic kidney disease and control kidney samples by quantitative real-time polymerase chain reaction.
Results:
Two original human microarray datasets, GSE7869 and GSE35831, were integrated and thoroughly analyzed. In total, 6,422 and 1,152 DEGs were extracted from GSE7869 and GSE35831, respectively, and of these, 561 DEGs were consistent between the databases (291 upregulated genes and 270 downregulated genes). From 421 nodes, 34 central node genes were obtained from a PPI network complex of DEGs. Two significant modules were selected from the PPI network complex by using Cytotype MCODE. Most of the identified genes are involved in protein binding, extracellular region or space, platelet degranulation, mitochondrion, and metabolic pathways.
Conclusions:
The DEGs and related enriched pathways in ADPKD identified through this integrated bioinformatics analysis provide insights into the molecular mechanisms of ADPKD and potential therapeutic strategies. Specifically, abnormal decorin expression in different stages of ADPKD may represent a new therapeutic target in ADPKD, and regulation of metabolism and mitochondrial function in ADPKD may become a focus of future research.
... 48 Öte yandan özellikle ODPBH genetiğine bağlı olarak ekstrasellüler matriks üretimindeki defekt nedeniyle zaten abdominal herni prevelansının yüksek olduğu bilinmektedir. 49 Bu nedenle önceden belirtilen diğer risklerin aksine,abdominal herni riskinin ODPBH hastalarında anlamlı derecede yüksek olduğuna, ancak bunun periton diyalizine devam edilmesini önemli ölçüde engellemediğine dair ikna edici kanıtlar vardır. 47 Böbrek ve karaciğer volümü ile periton diyalizinin sürdürülebilirliği arasındaki ilişkinin araştırıldığı bir çalışmada, toplam böbrek ve karaciğer volümü (TV) en yüksek olan bir hastada (8912 ml) sızıntı nedeniyle periton diyalizinin sonlandırıldığı, ancak TV biraz daha düşük (ortalama 7597 ± 1431 ml) hastalarda ise ortalama diyalizat hacmi 1500cc düzeyinde iken sızıntı gelişmediği bildirilmiştir. ...
Periton diyalizi, renal replasman tedavisi seçenekleri içerisinde etkinliği ve güvenliği kan-ıtlanmış, rezidüel renal fonksiyonların korunması ve tedavi modalitesindeki esnekliği nedeniyle hemodiyalize göre bazı hasta grupları için bir takım avantajlar sağlayan bir tedavi yöntemidir. Bu-nunla birlikte, tedavinin primer olarak hasta tarafından uygulanması ve teknik açıdan kendine özgü nitelikleri barındırması bakımından periton diyalizinin bazı hasta gruplarında uygulanabilirliği ve sürdürülebilirliği ile ilgili çekinceler gündeme gelebilmektedir. Bu derlemede peritoneal yapışıklık, fiziksel veya zihinsel engellilik, batın içi inflamatuar hastalıklar ve kanser, stomalı hastalar, abdominal herniler, obezite, ventriküloperitoneal şant, amiloidoz ve polikistik böbrek hastalığı olgu-larında periton diyalizinin uygulanabilirliği ve dikkat edilmesi gereken hususlar ile ilgili bilimsel veriler irdelenmiştir.
... Polycystins exert several cellular functions through which they mediate mechanotransduction. PC1, either alone or with PC2, is located in cell-to-cell and cell-to-ECM protein complexes, and therefore it regulates the interactions among adjacent cells, and between cells and their surrounding ECM (Figure 1) [9][10][11][12]. However, PC1 does not only transduce extracellular physical cues into the cell interior as a receptor, but also applies direct transcriptional regulation through its C-terminal end. ...
Alterations in the process of mechanotransduction have been implicated in the pathogenesis of several diseases such as genetic diseases, osteoporosis, cardiovascular anomalies, and cancer. Several studies over the past twenty years have demonstrated that polycystins (polycystin-1, PC1; and polycystin-2, PC2) respond to changes of extracellular mechanical cues, and mediate pathogenic mechanotransduction and cyst formation in kidney cells. However, recent reports reveal the emergence of polycystins as key proteins that facilitate the transduction of mechano-induced signals in various clinical entities besides polycystic kidney disease, such as cancer, cardiovascular defects, bone loss, and deformations, as well as inflammatory processes like psoriasis. Herewith, we discuss data from recent studies that establish this role with potential clinical utility.
... Polycystins are known to mediate cell-to-cell and cell-to-ECM interactions and associate with focal adhesion and ECM proteins that become deregulated in oncogenesis [24]. They also regulate apoptosis, differentiation, cell orientation/migration, cell cycle, and tissue morphogenesis [25]. ...
Cell and extracellular matrix (ECM) biomechanics emerge as a distinct feature during the development and progression of colorectal cancer (CRC). Polycystins are core mechanosensitive protein molecules that mediate mechanotransduction in a variety of epithelial cells. Polycystin-1 (PC1) and polycystin-2 (PC2) are engaged in signal transduction mechanisms and during alterations in calcium influx, which regulate cellular functions such as proliferation, differentiation, orientation, and migration in cancer cells. Recent findings implicate polycystins in the deregulation of such functions and the formation of CRC invasive phenotypes. Polycystins participate in all aspects of the cell’s biomechanical network, from the perception of extracellular mechanical cues to focal adhesion protein and nuclear transcriptional complexes. Therefore, polycystins could be employed as novel biomarkers and putative targets of selective treatment in CRC.
... The polycystin proteins form multimeric protein complexes that modulate several signalling pathways, such as Ca 2+ , cAMP, mechanistic target of rapamycin (mTOR), WNT, vascular endothelial growth factor (VEGF) and Hippo signalling. Consequently, numerous cellular changes have been observed in cyst-lining cells, including alterations in apical-basal polarity, planar cell polarity (PCP), increased extracellular matrix production and cellular metabolism, involving essential cellular functions such as fluid transport, proliferation, apoptosis, cell adhesion and differentiation [81][82][83][84][85] (FIG. 4). ...
Cystic kidneys are common causes of end-stage renal disease, both in children and in adults. Autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD) are cilia-related disorders and the two main forms of monogenic cystic kidney diseases. ADPKD is a common disease that mostly presents in adults, whereas ARPKD is a rarer and often more severe form of polycystic kidney disease (PKD) that usually presents perinatally or in early childhood. Cell biological and clinical research approaches have expanded our knowledge of the pathogenesis of ADPKD and ARPKD and revealed some mechanistic overlap between them. A reduced ‘dosage’ of PKD proteins is thought to disturb cell homeostasis and converging signalling pathways, such as Ca²⁺, cAMP, mechanistic target of rapamycin, WNT, vascular endothelial growth factor and Hippo signalling, and could explain the more severe clinical course in some patients with PKD. Genetic diagnosis might benefit families and improve the clinical management of patients, which might be enhanced even further with emerging therapeutic options. However, many important questions about the pathogenesis of PKD remain. In this Primer, we provide an overview of the current knowledge of PKD and its treatment.
... Polycystins constitute a family of proteins that have recently emerged as chief mechanosensors and mediators of mechanotransduction [13]. The main representatives of this family are polycystin-1 (PC1) and polycystin-2 (PC2), which have been detected in focal adhesion and ECM complexes [14]. PC1 is a transmembrane protein with a large, pliable, mechanosensitive N-extracellular end, and a C-terminus that produces transcriptionally active cleavages. ...
Psoriatic plaques tend to localize to the knees and elbows, areas that are particularly subject to mechanical stress resulting from bending and friction. Moreover, plaques often develop at sites of mechanical trauma or injury (Koebner phenomenon). Nevertheless, mechanotransduction has never been linked to psoriasis. Polycystins (polycystin-1, PC1; polycystin-2, PC2) are mechanosensitive molecules that function as key regulators of cellular mechanosensitivity and mechanotransduction. The aim of this in vitro study was to investigate the role of polycystins in the development of psoriasis. We showed that PC1 knockdown in HaCaT cells led to an elevated mRNA expression of psoriasis-related biomarkers Ki-67, IL-6, TNF-α, VEGF and Bcl-2, while PC1 functional inhibition was accompanied by increased cell proliferation and migration of HaCaT cells. In addition, PC1 knockdown via siRNA in HaCaT cells was followed by activation of critical molecules of the mTOR and MAPK pathways and this mTOR pathway activation was ERK-dependent. Furthermore, loss of PC1 protein expression and elevated levels of activated mTOR substrates were also observed in human samples of psoriatic plaques. Overall, our study suggests that the PC1/ERK/mTOR signaling axis represents a novel potential mechanism in psoriasis pathogenesis.
... Furthermore, it was recently observed that the defects that arise from the combined knockdown of the PKD1 paralogs Pkd1a and Pkd1b in zebrafish, such as dorsal axis curvature, were a result of dysregulation of collagen synthesis in these mutants. Based on the results of this study, it was suggested that Pkd1a/b and Pkd2 interact to regulate extracellular matrix (ECM) secretion or assembly and that altered matrix integrity may be a primary defect underlying ADPKD tissue pathologies (5,26). ...
... An unanswered question from our present study concerns the specific mechanism by which PKD1 regulates LARG function. Along this line, we note that previous studies have linked PKD1 to trimeric G proteins, integrins, and cilia function (Chapin and Caplan 2010;Drummond 2011). Whether any of these entities is required for PKD1-mediated regulation of LARG activity requires future studies. ...
Autosomal dominant polycystic kidney disease (ADPKD) is an inherited disorder caused by mutations in PKD1 or PKD2 and affects one in 500-1000 humans. Limited treatment is currently available for ADPKD. Here we identify the Hippo signaling effector YAP and its transcriptional target, c-Myc, as promoters of cystic kidney pathogenesis. While transgenic overexpression of YAP promotes proliferation and tubule dilation in mouse kidneys, loss of YAP/TAZ or c-Myc suppresses cystogenesis in a mouse ADPKD model resulting from Pkd1 deficiency. Through a comprehensive kinase inhibitor screen based on a novel three-dimensional (3D) culture of Pkd1 mutant mouse kidney cells, we identified a signaling pathway involving the RhoGEF (guanine nucleotide exchange factor) LARG, the small GTPase RhoA, and the RhoA effector Rho-associated kinase (ROCK) as a critical signaling module between PKD1 and YAP. Further corroborating its physiological importance, inhibition of RhoA signaling suppresses cystogenesis in 3D culture of Pkd1 mutant kidney cells as well as Pkd1 mutant mouse kidneys in vivo. Taken together, our findings implicate the RhoA-YAP-c-Myc signaling axis as a critical mediator and potential drug target in ADPKD.
... After then, kidney function declines rapidly. Because polycystin-1 and polycystin-2 are part of multiple protein complexes with wide cellular distribution, dysregulation of either in the renal tubule affects many pathways, including apicobasal and planar cell polarity, cell proliferation, cell metabolism, fluid secretion, and the extracellular matrix [112][113][114][115]. In PKD cysts form in the renal tubular epithelium where some cells reactivate normally quiescent proliferation pathways and begin to divide. ...
Detoxification is a fundamental function for all living organisms that need to excrete catabolites and toxins to maintain homeostasis. Kidneys are major organs of detoxification that maintain water and electrolyte balance to preserve physiological functions of vertebrates. In insects, the renal function is carried out by Malpighian tubules and nephrocytes. Due to differences in their circulation, the renal systems of mammalians and insects differ in their functional modalities, yet carry out similar biochemical and physiological functions and share extensive genetic and molecular similarities. Evolutionary conservation can be leveraged to model specific aspects of the complex mammalian kidney function in the genetic powerhouse Drosophila melanogaster to study how genes interact in diseased states. Here, we compare the human and Drosophila renal systems and present selected fly disease models.
... Cystogenesis in autosomal dominant polycystic kidney disease (ADPKD) is an extremely complicated process. The mechanism of cystogenesis associated with ADPKD can be conceptually parsed into several key components, including loss of polycystin protein, changes in apical and basolateral organization, disruptions in cell-matrix interactions, increases in proliferation, and fluid secretion/accumulation (5,10,41,46). In practice, experimentally teasing apart the stages of cystogenesis is extremely difficult. ...
Novel technologies, new understanding of the basement membrane composition, and better comprehension of the embryonic development of the mammalian kidney have led to explosive growth in the use of three dimensional (3D) in vitro models to study a range of human disease pathologies(6, 35). The development of these effective model systems represents a new tool to study the progressive cystogenesis of autosomal dominant polycystic kidney disease (ADPKD). ADPKD is a prevalent and complex monogenetic disease, characterized by the pathological formation of fluid fill cysts in renal tissue(17, 40). ADPKD cystogenesis is attributed to loss of function mutations in either PKD1 or PKD2, which encode for two transmembrane proteins, polycystin-1 and polycystin-2, and progresses with loss of both copies of either gene through a proposed two-hit mechanism with secondary somatic mutations(8, 33, 43). The exaggerated consequences of large fluid filled cysts result in fibrosis and nephron injury, leading initially to functional compensation, but ultimately to dysfunction(16, 32, 36). The complicated disease progression has scattered focus and resources across the spectrum of ADPKD research.
... Once the newly formed cysts separate from the renal tubule, they continue growing by not only cell proliferation, but also by a change in the transporting properties of the epithelium, which favors fluid secretion into the cyst over fluid reabsorption [218]. Other processes that accompany cyst growth include increased apoptosis, changes in lateral cell polarity, enhancement of cell migration, defects in the function of the cell primary cilium, remodeling and abnormal deposition of the extracellular matrix proteins, inflammatory changes, and interstitial fibrosis [219][220][221]. ...
Ouabain and other cardenolides are steroidal compounds originally discovered in plants. Cardenolides were first used as poisons, but after finding their beneficial cardiotonic effects, they were rapidly included in the medical pharmacopeia. The use of cardenolides to treat congestive heart failure remained empirical for centuries and only relatively recently, their mechanisms of action became better understood. A breakthrough came with the discovery that ouabain and other cardenolides exist as endogenous compounds that circulate in the bloodstream of mammals. This elevated these compounds to the category of hormones and opened new lines of investigation directed to further study their biological role. Another important discovery was the finding that the effect of ouabain was mediated not only by inhibition of the activity of the Na,K-ATPase (NKA), but by the unexpected role of NKA as a receptor and a signal transducer, which activates a complex cascade of intracellular second messengers in the cell. This broadened the interest for ouabain and showed that it exerts actions that go beyond its cardiotonic effect. It is now clear that ouabain regulates multiple cell functions, including cell proliferation and hypertrophy, apoptosis, cell adhesion, cell migration, and cell metabolism in a cell and tissue type specific manner. This review article focuses on the cardenolide ouabain and discusses its various in vitro and in vivo effects, its role as an endogenous compound, its mechanisms of action, and its potential use as a therapeutic agent; placing especial emphasis on our findings of ouabain as a pro-cystogenic agent in autosomal dominant polycystic kidney disease (ADPKD).
... PC-1 interacts with PC-2, a non-selective calcium channel, to regulate a number of signalling pathways involving cyclic adenosine monophosphate (cAMP) and Ca 2þ homeostasis [5][6][7][8]. Defects in polycystin expression or function result in changes to a number of cellular pathways, such as proliferation, apoptosis, fluid secretion, differentiation and cell adhesion [9][10][11][12][13][14]. Other cellular abnormalities reported include ciliary dysfunction, planar cell polarity, centrosome number and inflammation [15][16][17][18][19][20]. ...
Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited cause of end-stage renal failure. Understanding the molecular and cellular pathogenesis of ADPKD could help to identify new targets for treatment. The classic cellular cystic phenotype includes changes in proliferation, apoptosis, fluid secretion, extracellular matrix and cilia function. Hoever, recent research, suggests that the cellular cystic phenotype could be broader and that changes, such as altered metabolism, autophagy, inflammation, oxidative stress and epigenetic modification, could play important roles in the processes of cyst initiation, cyst growth or disease progression. Here we review these newer cellular pathways, describe evidence for their possible links to cystic pathogenesis or different stages of disease and discuss the options for developing novel treatments.
... Des expériences de ciliogenèse et de morphogenèse épithéliale en culture 3D de cellules rénales suggèrent des défauts de polarité, principalement d'organisation apicale ; ceux-ci sont associés à la formation de structures épithéliales à lumières multiples [8,9,42,43]. Ces données suggèrent, entre autres, un rôle de ces protéines dans la sécrétion et l'assemblage de la matrice extracellulaire, et le dysfonctionnement de ce processus pourrait être une cause de formation de kystes dans le rein [44]. De plus, in vitro, PC1 régule l'organisation du cytosquelette d'actine lors de la migration directionnelle de cellules embryonnaires de reins [45], et ce cytosquelette est également désorganisé in vivo chez les patients atteints de polykystose (ADPKD). ...
Advances in genomics, bioinformatics and the creation of model organisms have identified many genes associated with polycystic kidney diseases. Historically, these genes were not necessarily associated with ciliopathies, but it appeared that many connections can be made between the cystic kidney disease and function of the primary cilium. Indeed, the proteins encoded by these genes are localized to the cilium itself, to the basal body or are known to regulate the expression and localization of ciliary proteins. The goal of this article is to describe the multiple cellular processes that may lead to the development of renal cysts if they are deregulated. These include changes in proliferation rate, cell polarity or signaling pathways involved in embryonic kidney development. To highlight the role of the primary cilium in cystogenesis, I will discuss several studies investigating the function of ciliary genes and cilia in the kidneys of different model organisms.
© 2014 médecine/sciences – Inserm.
WT 9-12 is one of the cell lines commonly used for autosomal dominant polycystic kidney disease (ADPKD) studies. Previous studies had described the PKD gene mutations and polycystin expression in WT 9-12. Nonetheless, the mutations occurring in other ADPKD-associated genes have not been investigated. This study aims to revisit these mutations and protein profile of WT 9-12. Whole genome sequencing verified the presence of truncation mutation at amino acid 2556 (Q2556X) in PKD1 gene of WT 9-12. Besides, those variations with high impacts included single nucleotide polymorphisms (rs8054182, rs117006360, and rs12925771) and insertions and deletions (InDels) (rs145602984 and rs55980345) in PKD1L2; InDel (rs1296698195) in PKD1L3; and copy number variations in GANAB. Protein profiles generated from the total proteins of WT 9-12 and HK-2 cells were compared using isobaric tags for relative and absolute quantitation (iTRAQ) analysis. Polycystin-1 was absent in WT 9-12. The gene ontology enrichment and reactome pathway analyses revealed that the upregulated and downregulated proteins of WT 9-12 relative to HK-2 cell line leaded to signaling pathways related to immune response and amino acid metabolism, respectively. The ADPKD-related mutations and signaling pathways associated with differentially expressed proteins in WT 9-12 may help researchers in cell line selection for their studies.
Ciliopathies are a group of rare genetic disorders caused by defects to the structure or function of the primary cilium. They often affect multiple organs, leading to brain malformations, congenital heart defects, and anomalies of the retina or skeletal system. Kidney abnormalities are among the most frequent ciliopathic phenotypes manifesting as smaller, dysplastic, and cystic kidneys that are often accompanied by renal fibrosis. Many renal ciliopathies cause chronic kidney disease and often progress to end-stage renal disease, necessitating replacing therapies. There are more than 35 known ciliopathies; each is a rare hereditary condition, yet collectively they account for a significant proportion of chronic kidney disease worldwide. The primary cilium is a tiny microtubule-based organelle at the apex of almost all vertebrate cells. It serves as a “cellular antenna” surveying environment outside the cell and transducing this information inside the cell to trigger multiple signaling responses crucial for tissue morphogenesis and homeostasis. Hundreds of proteins and unique cellular mechanisms are involved in cilia formation. Recent evidence suggests that actin remodeling and regulation at the base of the primary cilium strongly impacts ciliogenesis. In this review, we provide an overview of the structure and function of the primary cilium, focusing on the role of actin cytoskeleton and its regulators in ciliogenesis. We then describe the key clinical, genetic, and molecular aspects of renal ciliopathies. We highlight what is known about actin regulation in the pathogenesis of these diseases with the aim to consider these recent molecular findings as potential therapeutic targets for renal ciliopathies.
Objective:
To perform an integrated analysis in identifying novel hub genes that could facilitate the diagnosis and targeted therapy of ameloblastoma.
Design:
The expression profiling dataset, GSE38494, was obtained from the Gene Expression Omnibus database. Differentially expressed genes were identified through GEO2R online tool and characterised via Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The protein-protein interaction network and hub genes were screened using the STRING database and Cytoscape software. Subsequently, an upregulated gene was selected for further validation using the GSE132472 dataset. Further, immunohistochemistry was performed to assess the expression of the selected gene in ameloblastomas, odontogenic keratocysts, dentigerous cysts, and gingival tissues. The diagnostic and therapeutic utility of the selected hub genes were further verified by receiver operating characteristic analysis and the DGIdb database.
Results:
We identified six hub genes in ameloblastoma, among which the upregulated gene PKD2 and its related gene PKD1 were further validated. GO functional annotation revealed that PKD2 is involved in cell-cell junction, extracellular exosome, cytoplasm, endoplasmic reticulum, and calcium ion transport. The immunohistochemical analysis showed that the expression of polycystin-1 and polycystin-2, encoded by the PKD1 and PKD2 genes, respectively, was upregulated in ameloblastoma. PKD1 and PKD2 had a high diagnostic utility for ameloblastoma, and allopurinol interacted with the PKD2 gene.
Conclusion:
Our research indicates that polycystins are highly expressed in ameloblastoma and might be involved in the oncogenesis of ameloblastoma, thus offering a new perspective on the molecular mechanisms and targeted therapies on ameloblastoma.
Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder that leads to chronic kidney disease and end-stage kidney disease (ESKD). Polycystic liver disease (PCLD) is the most common extrarenal manifestation of ADPKD. Though isolated PCLD and PCLD due to ADPKD are genetically distinct, they follow a similar clinical course of hepatomegaly from multiple cysts with preserved liver function. Tolvaptan use in ADPKD can slow down the deterioration of renal function and growth of cysts. Somatostatin analogs can slow the growth of polycystic livers but the effect is short-lived. The only curative therapy for PCLD is liver transplantation. Renal transplantation can significantly improve survival in patients with ESKD due to ADPKD.
The mechanobiological aspects of glioblastoma (GBM) pathogenesis are largely unknown. Polycystin-1 (PC1) is a key mechanosensitive protein which perceives extracellular mechanical cues and transforms them into intracellular biochemical signals that elicit a change in cell behaviour. The aim of the present study was to investigate if and how PC1 participates in GBM pathogenesis under a mechanically induced microenvironment. Therefore, we subjected T98G GBM cells to continuous hydrostatic pressure (HP) and/or PC1 blockade and evaluated their effect on cell behaviour, the activity of signalling pathways and the expression of mechano-induced transcriptional regulators and markers associated with properties of cancer cells. According to our data, PC1 and HP affect GBM cell proliferation, clonogenicity and migration; the diameter of GBM spheroids; the phosphorylation of mechanistic target of rapamycin (mTOR), extracellular signal-regulated kinase (ERK) and focal adhesion kinase (FAK); the protein expression of transcription cofactors YES-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ); and the mRNA expression of markers related to anti-apoptosis, apoptosis, angiogenesis, epithelial to mesenchymal transition (EMT) and proliferation. Together, our in vitro results suggest that PC1 plays an important role in GBM mechanobiology.
Background
Causative mutations in the GANAB gene have been described in only 14 families, 9 diagnosed with late-onset Autosomal Dominant Polycystic Kidney Disease (ADPKD) and 5 with Autosomal Dominant Polycystic Liver Disease (ADPLD).CaseDiagnosis of ADPKD was made in a 45-year old man during screening for hernia repair. CT scan showed enlarged cystic kidneys, nephrolithiasis and normal-sized liver with multiple cysts. Hematuria, hypertension and aortic root dilatation were also documented. Renal function was normal. Molecular analysis of PKD genes disclosed a heterozygous p.R839W GANAB variant inherited from the mother. Both his elderly parents presented normal-sized bilateral cystic kidneys but normal renal function. The GANAB-ADPKD mother had no liver cysts. The father was screened for PKD-related genes and no variant was found.Genetic analysisWe describe a new family with late-onset ADPKD due to the p.R839W GANAB variant, previously reported in a severe ADPLD patient, requiring liver transplantation.DiscussionSince ADPKD-GANAB is an ultrarare, recently described disease, reporting further patients may help unraveling gene-related phenotype. In our patients the p.R839W GANAB variant was not related to severe ADPLD, as previously reported, but with mild ADPKD and a plethora of renal and extrarenal manifestations, usually described in PKD1/PKD2 patients. The evidence that the GANAB variant may cause both ADPKD and ADPLD of variable severity supports that renal and hepatic cystogenesis are the result of a common defective polycystin-1 pathway.Graphic abstract
ADPKD is the most common genetic disease of the kidney leading to end-stage renal disease necessitating renal replacement therapy at any time between the 1st and 8th decades of life due to widely variable rates of disease progression. This presents significant patient anxiety and a significant prognostic and therapeutic challenge. Tolvaptan is the only approved drug licensed to slow ADPKD progression by reducing renal cystic expansion but side-effects can limit its efficacy.To address the need to identify new biomarkers to monitor progression of ADPKD and to evaluate the therapeutic effects of Tolvaptan, proteomic analysis was conducted on defined (40-100nm) urinary exosomes isolated from ADPKD patients phenotyped and clinically monitored over a 10-year period. Comparative Gene Ontology analysis of Tandem Mass Tag labelled mass spectrometry-derived protein profiles from urinary exosomes from ADPKD patients with rapid (>10ml/min/5 years decline in estimated glomerular filtration rate) versus slow progression showed distinctive patterns of pathway up-regulation. Clear discrimination between rapid and slowly-progressive profiles were seen in all stages functional decline in ADPKD patients whether with mild (>70ml/min), moderate (50-69ml/min) or severe (
Polycystin-1 (PC-1) is a transmembrane protein, encoded by the PKD1 gene, mutated in Autosomal Dominant Polycystic Kidney Disease (ADPKD). This common genetic disorder, characterized by cyst formation in both kidneys, ultimately leading to renal failure, is still waiting for a definitive treatment. The overall function of PC-1 and the molecular mechanism responsible for cyst formation are slowly coming to light, but they are both still intensively studied. In particular, PC-1 has been proposed to act as a mechanosensor, although the precise signal that activates the mechanical properties of this protein has been long debated and questioned. In this Review we report studies and evidences of PC-1 function as a mechanosensor, starting from the peculiarity of its structure, through the long journey that progressively shed new light on the potential initiating events of cystogenesis, concluding with the description of PC-1 recently shown ability to sense the mechanical stimuli provided by the stiffness of the extracellular environment. These new findings have potentially important implications for the understanding of ADPKD pathophysiology and potentially for designing new therapies.
The major hallmark of Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the formation of many fluid-filled cysts in the kidneys, which ultimately impairs the normal renal structure and function, leading to end-stage renal disease (ESRD). A large body of evidence suggests that injury-repair mechanisms are part of ADPKD progression. Once cysts have been formed, proliferation and fluid secretion contribute to the cyst size increase, which eventually causes stress on the surrounding tissue resulting in local injury and fibrosis. In addition, renal injury can cause or accelerate cyst formation.
In this review, we will describe the various mechanisms activated during renal injury and tissue repair and show how they largely overlap with the molecular mechanisms activated during PKD progression. In particular, we will discuss molecular mechanisms such as proliferation, inflammation, cell differentiation, cytokines and growth factors secretion, which are activated following the renal injury to allow the remodelling of the tissue and a proper organ repair. We will also underline how, in a context of PKD-related gene mutations, aberrant or chronic activation of these developmental pathways and repair/remodelling mechanisms results in exacerbation of the disease.
Urinary tract infection (UTI) is a broad term referring to an infection of the kidneys, ureters, bladder and/or urethra. Due to its prevalence, frequent recurrence and rising resistance to antibiotics, UTI has become a challenge in clinical practice. Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic disorder of the kidney and is characterized by the growth of fluid-filled cysts in both kidneys. Progressive cystic enlargement, inflammation and interstitial fibrosis result in nephron loss with subsequent decline in kidney function. ADPKD patients frequently develop UTI; however, the cellular and molecular mechanisms responsible for the high UTI incidence in ADPKD patients remain virtually unaddressed. Emerging evidence suggests that α-intercalated cells (α-ICs) of the collecting ducts function in the innate immune defense against UTI. α-ICs inhibit bacterial growth by acidifying urine and secreting neutrophil gelatinase-associated lipocalin (NGAL) that chelates siderophore-containing iron. It is necessary to determine, therefore, if ADPKD patients with recurrent UTI have a reduced number and/or impaired function of α-ICs. Identification of the underlying cellular and molecular mechanisms may lead to the development of novel strategies to reduce UTI in ADPKD.
The hormone ouabain has been shown to enhance the cystic phenotype of autosomal dominant polycystic kidney disease (ADPKD). Among other characteristics, the ADPKD phenotype includes cell de-differentiation and epithelial to mesenchymal transition (EMT). Here, we determined whether physiological concentrations of ouabain induces EMT in human renal epithelial cells from patients with ADPKD. We found that ADPKD cells respond to ouabain with a decrease in expression of the epithelial marker E-cadherin and increase in the expression of the mesenchymal markers N-cadherin, α smooth muscle actin (αSMA) and collagen-I; and the tight junction protein occludin and claudin-1. Other adhesion molecules, such as ZO-1, β-catenin and vinculin were not significantly modified by ouabain. At the cellular level, ouabain stimulated ADPKD cell migration, reduced cell-cell interaction, and the ability of ADPKD cells to form aggregates. Moreover, ouabain increased the transepithelial electrical resistance of ADPKD cell monolayers, suggesting that the paracellular transport pathway was preserved in the cells. These effects of ouabain were not observed in normal human kidney (NHK) cells. Altogether these results show a novel role for ouabain in ADPKD, inducing changes that lead to a partial EMT phenotype in the cells. These effects further support the key role that ouabain has as a factor that promotes the cystic characteristics of ADPKD cells.
Tissues of the body possess intrinsic control mechanisms for regulating hemodynamics at the local level. These mechanisms largely occur independently of innervation and circulating factors and allow blood flow to be matched to local metabolic requirements. In the case of the myogenic response, or pressure-induced vasoconstriction, this mechanism is dependent on the cells of the vessel wall detecting and responding to a mechanical stimulus. This mechanical event then must be conveyed across the smooth muscle cell membrane to the contractile proteins to affect an appropriate contractile response. Diabetes and related metabolic disorders that lead to hyperglycemia may interfere with this process by either alterations to the vessel wall (for example, remodeling events, increased stiffness, decreased distensibility) or impairment of signal transduction mechanisms (including ion channel function, Ca2+ handling and contractile protein interactions). This chapter reviews the cellular mechanisms underlying myogenic contraction and how these may be altered in hyperglycemic states.
The endothelium consists of a single layer of vascular endothelial cells (ECs) and serves as a selective barrier between the blood and arteries. ECs are constantly exposed to blood flow- and pulsatile blood pressure-induced hemodynamic forces. The cells are able to convert these mechanical stimuli into biochemical signals and then transmit the signals into the cell interior to affect cellular functions. These mechanical stimuli are detected by multiple mechanosensors in ECs that activate signaling pathways through their associated adaptor proteins, eventually leading to the maintenance of vascular homeostasis or the development of the pathogenesis of vascular disorders. These mechanosensors are distributed in different parts of the ECs, including the cell membrane, cell-to-cell junctions, the cytoplasm, and the nucleus. This review attempts to bring together recent findings on these mechanosensors and presents a conceptual framework for understanding the regulation of endothelial mechanosensors in response to hemodynamic forces. With verification by in vitro and in vivo evidence, endothelial mechanosensors have been demonstrated to contribute to health and disease by regulating physiological and pathophysiological processes in response to mechanical stimuli.
Epidemiological studies have revealed that intracranial arterial aneurysms (IAAs) are responsible for 10–15 % of hemorrhagic stroke in children and young adults. If one differentiates the pattern and location of intracranial hemorrhage, the importance of IAAs as a cause of hemorrhagic stroke in children is even more apparent. For example, IAAs account for as much as 57 % of pure spontaneous SAH in the pediatric population. In the non-adult population, the incidence of hemorrhagic stroke due to IAAs is highest in patients 15–19 years of age. This is more than five times that observed in younger age groups. It has been proposed that conventional vascular risk factors, such as tobacco use and cocaine abuse, are introduced in late adolescence and likely account for the sharp increase in aneurysmal disease in the latter half of the second decade (Jordan et al. 2009).
Berenice Reed-Gitomer Division of Renal Diseases and Hypertension, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Abstract: Autosomal dominant polycystic kidney disease (ADPKD) is the most common potentially lethal genetic disorder, accounting for 2%–8% of end-stage renal disease worldwide. While development of renal cysts is the major characteristic of ADPKD, several disease-related complications contribute significantly to morbidity and mortality. Since introduction of renal replacement therapies, cardiovascular disease is the leading cause of death among ADPKD patients. Loss of renal function requiring renal replacement therapy occurs in 50% of patients by the age of 60 years. The results of recent large epidemiological studies in ADPKD have shown little progress in delaying onset of renal failure despite an improvement in patient mortality. Significant progress has been made over the past decade in elucidating the genetics of the disorder. Genetic testing is now available in most countries, and development of more reliable methods for prenatal diagnosis of ADPKD has increased the sensitivity of testing. While there are no approved drugs for treatment of ADPKD within the USA, the first agent targeting renal disease progression in this disorder was recently approved for clinical use in Japan. Furthermore, several additional drugs for treatment of ADPKD are currently under clinical investigation. Overall, this presents an optimistic future for new therapeutic interventions in this disease. This paper reviews the current knowledge related to the epidemiology, genetics, and treatment of ADPKD. Keywords: autosomal dominant polycystic kidney disease, hereditary renal disease, renal cyst, genetics
Peroxisome proliferator-activated receptor γ agonists have been shown to inhibit angiotensin II (AngII)-induced experimental abdominal aortic aneurysms. Macrophage infiltration to the vascular wall is an early event in this pathology, and therefore we explored the effects of the peroxisome proliferator-activated receptor γ agonist pioglitazone on AngII-treated macrophages. Using microarray-based expression profiling of phorbol ester-stimulated THP-1 cells, we found that a number of aneurysm-related gene changes effected by AngII were modulated following the addition of pioglitazone. Among those genes, polycystic kidney disease 1 (PKD1) was significantly up-regulated (multiple testing corrected p < 0.05). The analysis of the PKD1 proximal promoter revealed a putative early growth response 1 (EGR1) binding site, which was confirmed by chromatin immunoprecipitation (ChIP) and quantitative PCR. Further analysis of publicly available ChIP-sequencing data revealed that this putative binding site overlapped with a conserved EGR1 binding peak present in 5 other cell lines. Quantitative real-time PCR showed that EGR1 suppressed PKD1, while AngII significantly up-regulated PKD1, an effect counteracted by pioglitazone. Conversely, in EGR1 short hairpin RNA lentivirally transduced THP-1 cells, reduced EGR1 led to a significant up-regulation of PKD1, especially after treatment with pioglitazone. In vivo, deficiency of Egr1 in the haematopoietic compartment of mice completely abolished the incidence of CaCl2-induced aneurysm formation. © 2015 S. Karger AG, Basel.
Breast epithelial cells differentiate into tubules when cultured in floating three-dimensional (3D) collagen gels, but not when the cells are cultured in the same collagen matrix that is attached to the culture dish. These observations suggest that the biophysical properties of collagenous matrices regulate epithelial differentiation, but the mechanism by which this occurs is unknown. Tubulogenesis required the contraction of floating collagen gels through Rho and ROCK-mediated contractility. ROCK-mediated contractility diminished Rho activity in a floating 3D collagen gel, and corresponded to a loss of FAK phosphorylated at Y397 localized to 3D matrix adhesions. Increasing the density of floating 3D collagen gels also disrupted tubulogenesis, promoted FAK phosphorylation, and sustained high Rho activity. These data demonstrate the novel finding that breast epithelial cells sense the rigidity or density of their environment via ROCK-mediated contractility and a subsequent down-regulation of Rho and FAK function, which is necessary for breast epithelial tubulogenesis to occur.
Tensin is a focal adhesion phosphoprotein that binds to F-actin and contains a functional Src homology 2 domain. To explore the biological functions of tensin, we cloned the mouse tensin gene, determined its program of expression, and used gene targeting to generate mice lacking tensin. Even though tensin is expressed in many different tissues during embryogenesis, tensin null mice developed normally and appeared healthy postnatally for at least several months. Over time, −/− mice became frail because of abnormalities in their kidneys, an organ that expresses high levels of tensin. Mice with overt signs of weakness exhibited signs of renal failure and possessed multiple large cysts in the proximal kidney tubules, but even in tensin null mice with normal blood analysis, cysts were prevalent. Ultrastructurally, noncystic areas showed typical cell– matrix junctions that readily labeled with antibodies against other focal adhesion molecules. In abnormal regions, cell–matrix junctions were disrupted and tubule cells lacked polarity. Taken together, our data imply that, in the kidney, loss of tensin leads to a weakening, rather than a severing, of focal adhesion. All other tissues appeared normal, suggesting that, in most cases, tensin's diverse functions are redundant and may be compensated for by other focal adhesion proteins.
The aetiology and pathogenesis of abdominal wall hernia formation is complex. Optimal treatment of hernias depends on a full understanding of the pathophysiological mechanisms involved in their formation. The aim of this study was to review the literature on specific collagen alterations in abdominal wall hernia formation.
A computer-assisted search of the medical databases PubMed and Embase was performed, together with a cross-reference search of eligible papers.
Fifty-two papers were included. Collagen alteration depended on the type of hernia; there were more pronounced changes in patients with a direct inguinal hernia than in those with an indirect inguinal hernia, recurrent inguinal hernia or incisional hernia. A consistent finding was a significant increase in immature type III collagen relative to the stronger type I collagen in patients with a hernia. This resulted in thinner collagen fibres with a correspondingly diminished biomechanical strength. It has been suggested that these alterations are due to variation in the synthesis, maturation or degradation of collagen by matrix metalloproteinases, in combination or alone.
Hernia formation and recurrence is associated with altered collagen metabolism manifested by a decreased type I:III collagen ratio.
Polycystin-1 (PC1) is a large membrane protein that is expressed along the renal tubule and exposed to a wide range of concentrations of urea. Urea is known as a common denaturing osmolyte that affects protein function by destabilizing their structure. However, it is known that the native conformation of proteins can be stabilized by protecting osmolytes that are found in the mammalian kidney. PC1 has an unusually long ectodomain with a multimodular structure including 16 Ig-like polycystic kidney disease (PKD) domains. Here, we used single-molecule force spectroscopy to study directly the effects of several naturally occurring osmolytes on the mechanical properties of PKD domains. This experimental approach more closely mimics the conditions found in vivo. We show that upon increasing the concentration of urea there is a remarkable decrease in the mechanical stability of human PKD domains. We found that protecting osmolytes such as sorbitol and trimethylamine N-oxide can counteract the denaturing effect of urea. Moreover, we found that the refolding rate of a structurally homologous archaeal PKD domain is significantly slowed down in urea, and this effect was counteracted by sorbitol. Our results demonstrate that naturally occurring osmolytes can have profound effects on the mechanical unfolding and refolding pathways of PKD domains. Based on these findings, we hypothesize that osmolytes such as urea or sorbitol may modulate PC1 mechanical properties and may lead to changes in the activation of the associated polycystin-2 channel or other intracellular events mediated by PC1.
Polycystic Kidney Disease is characterized by the formation of large fluid-filled cysts that eventually destroy the renal parenchyma leading to end-stage renal failure. Although remarkable progress has been made in understanding the pathologic mechanism of the disease, the precise orchestration of the early events leading to cyst formation is still unclear. Abnormal cellular proliferation was traditionally considered to be one of the primary irregularities leading to cyst initiation and growth. Consequently, many therapeutic interventions have focused on targeting this abnormal proliferation, and some have even progressed to clinical trials. However, the role of proliferation in cyst development was primarily examined at stages where cysts are already visible in the kidneys and therefore at later stages of disease development.
In this study we focused on the cystic phenotype since birth in an attempt to clarify the temporal contribution of cellular proliferation in cyst development. Using a PKD2 transgenic rat model (PKD2 (1-703)) of different ages (0-60 days after birth) we performed gene expression profiling and phenotype analysis by measuring various kidney parameters.
Phenotype analysis demonstrated that renal cysts appear immediately after birth in the PKD2 transgenic rat model (PKD2 (1-703)). On the other hand, abnormal proliferation occurs at later stages of the disease as identified by gene expression profiling. Interestingly, other pathways appear to be deregulated at early stages of the disease in this PKD model. Specifically, gene expression analysis demonstrated that at day 0 the RAS system is involved. This is altered at day 6, when Wnt signaling and focal adhesion pathways are affected. However, at and after 24 days, proliferation, apoptosis, altered ECM signaling and many other factors become involved.
Our data suggest that cystogenesis precedes deregulation of proliferation-related pathways, suggesting that proliferation abnormalities may contribute in cyst growth rather than cyst formation.
Deseasin MCP-01 is a bacterial collagenolytic serine protease. Its catalytic domain alone can degrade collagen, and its C-terminal PKD domain is a collagen-binding domain (CBD) that can improve the collagenolytic efficiency of the catalytic domain by an unknown mechanism. Here, scanning electron microscopy (SEM), atomic force microscopy (AFM), zeta potential, and circular dichroism spectroscopy were used to clarify the functional mechanism of the PKD domain in MCP-01 collagenolysis. The PKD domain observably swelled insoluble collagen. Its collagen-swelling ability and its improvement to the collagenolysis of the catalytic domain are both temperature-dependent. SEM observation showed the PKD domain swelled collagen fascicles with an increase of their diameter from 5.3 mum to 8.8 mum after 1 h of treatment, and the fibrils forming the fascicles were dispersed. AFM observation directly showed that the PKD domain bound collagen, swelled the microfibrils, and exposed the monomers. The PKD mutant W36A neither bound collagen nor disturbed its structure. Zeta potential results demonstrated that PKD treatment increased the net positive charges of the collagen surface. PKD treatment caused no change in the content or the thermostability of the collagen triple helix. Furthermore, the PKD-treated collagen could not be degraded by gelatinase. Therefore, though the triple helix monomers were exposed, the PKD domain could not unwind the collagen triple helix. Our study reveals the functional mechanism of the PKD domain of the collagenolytic serine protease MCP-01 in collagen degradation, which is distinct from that of the CBDs of mammalian matrix metalloproteases.
Thoracic aortic aneurysms (TAA) increase the risk of aortic dissection or rupture and represent an important source of morbidity and mortality. Inherited forms of the disease, including Marfan syndrome, have been recognized for a long time but were considered degenerative diseases characterized by cystic medial necrosis of the aortic wall. Improved definition of the structure and function of the normal aortic wall, coupled with the discovery of genetic mutations in key regulatory molecules, have contributed to a more detailed understanding of the pathophysiology of syndromic, familial and sporadic TAAs. We here review the cellular and molecular mechanisms involved in TAA formation and outline areas for future research.
Mutations in polycystin-1 (PC1) can cause autosomal dominant polycystic kidney disease, which is a leading cause of renal
failure. The available evidence suggests that PC1 acts as a mechanosensor, receiving signals from the primary cilia, neighboring
cells, and extracellular matrix. PC1 is a large membrane protein that has a long N-terminal extracellular region (about 3000
amino acids) with a multimodular structure including 16 Ig-like polycystic kidney disease (PKD) domains, which are targeted
by many naturally occurring missense mutations. Nothing is known about the effects of these mutations on the biophysical properties
of PKD domains. Here we investigate the effects of several naturally occurring mutations on the mechanical stability of the
first PKD domain of human PC1 (HuPKDd1). We found that several missense mutations alter the mechanical unfolding pathways
of HuPKDd1, resulting in distinct mechanical phenotypes. Moreover, we found that these mutations also alter the thermodynamic
stability of a structurally homologous archaeal PKD domain. Based on these findings, we hypothesize that missense mutations
may cause autosomal dominant polycystic kidney disease by altering the stability of the PC1 ectodomain, thereby perturbing
its ability to sense mechanical signals.
Autosomal dominant polycystic kidney disease (ADPKD), often caused by mutations in the PKD1 gene, is associated with life-threatening vascular abnormalities that are commonly attributed to the frequent occurrence of hypertension. A previously reported targeted mutation of the mouse homologue of PKD1 was not associated with vascular fragility, leading to the suggestion that the vascular lesion may be of a secondary nature. Here we demonstrate a primary role of PKD1 mutations in vascular fragility. Mouse embryos homozygous for the mutant allele (Pkd1L) exhibit s.c. edema, vascular leaks, and rupture of blood vessels, culminating in embryonic lethality at embryonic day 15.5. Kidney and pancreatic ductal cysts are present. The Pkd1-encoded protein, mouse polycystin 1, was detected in normal endothelium and the surrounding vascular smooth muscle cells. These data reveal a requisite role for polycystin 1 in maintaining the structural integrity of the vasculature as well as epithelium and suggest that the nature of the PKD1 mutation contributes to the phenotypic variance in ADPKD.
An increase in pressure inside most small arteries unexpectedly results in a vasoconstriction. This phenomenon, known as the Bayliss effect, involves the stretch-induced activation of non-selective cation channels in the vascular smooth muscle cells. Recent work by Mederos y Schnitzler et al. now demonstrates that the stretch-induced channel activity originates from a fascinating interplay between a TRP cation channel and the angiotensin receptor. © 2009 European Molecular Biology Organization | Some Rights Reserved.
Collagens are the most abundant proteins in marine animals and their degradation is important for the recycling of marine nitrogen. However, it is rather unclear how marine collagens are degraded because few marine collagenolytic proteases are studied in detail. Deseasins are a new type of multidomain subtilases. Here, the collagenolytic activity of deseasin MCP-01, the type example of deseasins, was studied. MCP-01 had broad substrate specificity to various type collagens from terrestrial and marine animals. It completely decomposed insoluble collagen into soluble peptides and amino acids, and was more prone to degrade marine collagen than terrestrial collagen. Thirty-seven cleavage sites of MCP-01 on bovine collagen chains were elucidated, showing the cleavage is various but specific. As the main extracellular cold-adapted protease from deep-sea bacterium Pseudoalteromonas sp. SM9913, MCP-01 displayed high activity at low temperature and alkaline range. Our data also showed that the C-terminal polycystic kidney disease (PKD) domain of MCP-01 was able to bind insoluble collagen and facilitate the insoluble collagen digestion by MCP-01. Site-directed mutagenesis demonstrated that Trp-36 of the PKD domain played a key role in its binding to insoluble collagen. It is the first time that the structure and function of a marine collagenolytic protease, deseasin MCP-01, has been studied in detail. Moreover, the PKD domain was experimentally proven to bind to insoluble protein for the first time. These results imply that MCP-01 would play an important role in the degradation of deep-sea sedimentary particulate organic nitrogen.
Progressive renal enlargement due to the growth of innumerable fluid-filled cysts is a central pathophysiological feature of autosomal dominant polycystic kidney disease (ADPKD). These epithelial neoplasms enlarge slowly and damage noncystic parenchyma by mechanisms that have not been clearly defined. In a microarray analysis of cultured human ADPKD cyst epithelial cells, periostin mRNA was overexpressed 15-fold compared with normal human kidney (NHK) cells. Periostin, initially identified in osteoblasts, is not expressed in normal adult kidneys but is expressed transiently during renal development. We found periostin in cyst-lining cells in situ in the extracellular matrix adjacent to the cysts and within cyst fluid. ADPKD cells secreted periostin across luminal and basolateral plasma membranes. Periostin increased proliferation of cyst epithelial cells 27.9 +/- 3.1% (P < 0.001) above baseline and augmented in vitro cyst growth but did not affect proliferation of normal renal cells. Expression of alphaV-integrin, a periostin receptor, was ninefold higher in ADPKD cells compared with NHK cells, and antibodies that block alphaV-integrin inhibited periostin-induced cell proliferation. We conclude that periostin is a novel autocrine mitogen secreted by mural epithelial cells with the potential to accelerate cyst growth and promote interstitial remodeling in ADPKD.
Tensin is a focal adhesion phosphoprotein that binds to F-actin and contains a functional Src homology 2 domain. To explore the biological functions of tensin, we cloned the mouse tensin gene, determined its program of expression, and used gene targeting to generate mice lacking tensin. Even though tensin is expressed in many different tissues during embryogenesis, tensin null mice developed normally and appeared healthy postnatally for at least several months. Over time, -/- mice became frail because of abnormalities in their kidneys, an organ that expresses high levels of tensin. Mice with overt signs of weakness exhibited signs of renal failure and possessed multiple large cysts in the proximal kidney tubules, but even in tensin null mice with normal blood analysis, cysts were prevalent. Ultrastructurally, noncystic areas showed typical cell-matrix junctions that readily labeled with antibodies against other focal adhesion molecules. In abnormal regions, cell-matrix junctions were disrupted and tubule cells lacked polarity. Taken together, our data imply that, in the kidney, loss of tensin leads to a weakening, rather than a severing, of focal adhesion. All other tissues appeared normal, suggesting that, in most cases, tensin's diverse functions are redundant and may be compensated for by other focal adhesion proteins.
Most cases of autosomal dominant polycystic kidney disease (ADPKD) are the result of mutations in the PKD1 gene. The PKD1 gene codes for a large cell-surface glycoprotein, polycystin-1, of unknown function, which, based on its predicted domain structure, may be involved in protein-protein and protein-carbohydrate interactions. Approximately 30% of polycystin-1 consists of 16 copies of a novel protein module called the PKD domain. Here we show that this domain has a beta-sandwich fold. Although this fold is common to a number of cell-surface modules, the PKD domain represents a distinct protein family. The tenth PKD domain of human and Fugu polycystin-1 show extensive conservation of surface residues suggesting that this region could be a ligand-binding site. This structure will allow the likely effects of missense mutations in a large part of the PKD1 gene to be determined.
Polycystin-1, the protein defective in a majority of patients with autosomal dominant polycystic kidney disease, is a ubiquitously
expressed multi-span transmembrane protein of unknown function. Subcellular localization studies found this protein to be
a component of various cell junctional complexes and to be associated with the cytoskeleton, but the specificity and nature
of such associations are not known. To identify proteins that interact with the polycystin-1 C-tail (P1CT), this segment was
used as bait in a yeast two-hybrid screening of a kidney epithelial cell library. The intermediate filament (IF) protein vimentin
was identified as a strong polycystin-1-interacting partner. Cytokeratins K8 and K18 and desmin were also found to interact
with P1CT. These interactions were mediated by coiled-coil motifs in polycystin-1 and IF proteins. Vimentin, cytokeratins
K8 and K18, and desmin also bound directly to P1CT in GST pull-down and in in vitro filament assembly assays. Two observations confirmed these interactions in vivo: (i) a cell membrane-anchored form of recombinant P1CT decorated the IF network and was found to associate with the cytoskeleton
in detergent-solubilized cells and (ii) endogenous polycystin-1 distributed with IF at desmosomal junctions. Polycystin-1
may utilize this association for structural, storage, or signaling functions.
Polycystin-1, the product of the PKD1 gene, is a membrane-bound multidomain protein with a unique structure and a molecular weight of approximately 460 kD. The purpose of this study is to investigate the binding of the cystein-flanked leucine-rich repeats (LRR) of polycystin-1 to extracellular matrix (ECM) components. These interactions may play a role in normal renal development as well as the pathogenesis of autosomal-dominant polycystic kidney disease (ADPKD). In vitro assays were used to assess the binding of a fusion protein containing the LRR of polycystin-1 and that of affinity purified polycystin-1 to a number of ECM components. The results showed that the LRR modulate the binding of polycystin-1 to collagen I, fibronectin, laminin, and cyst fluid-derived laminin fragments. The addition of the LRR fusion protein to cells in culture resulted in a significant dose-dependent reduction in the rate of proliferation. Cyst fluid-derived laminin fragments had a stimulatory effect on cell proliferation, which was reversed by the LRR fusion protein. These results suggest that the LRR of polycystin-1 act as mediators of the polycystin-1 interaction with the ECM. The observed suppression effect of the LRR on cell proliferation suggests a functional role of the LRR-mediated polycystin-1 involvement in cell-matrix and cell-cell interactions. These interactions may result in the enhanced cell proliferation that is a characteristic feature of ADPKD.
Polycystin-2, the product of the gene mutated in type 2 autosomal dominant polycystic kidney disease (ADPKD), is the prototypical member of a subfamily of the transient receptor potential (TRP) channel superfamily, which is expressed abundantly in the endoplasmic reticulum (ER) membrane. Here, we show by single channel studies that polycystin-2 behaves as a calcium-activated, high conductance ER channel that is permeable to divalent cations. Epithelial cells overexpressing polycystin-2 show markedly augmented intracellular calcium release signals that are lost after carboxy-terminal truncation or by the introduction of a disease-causing missense mutation. These data suggest that polycystin-2 functions as a calcium-activated intracellular calcium release channel in vivo and that polycystic kidney disease results from the loss of a regulated intracellular calcium release signalling mechanism.
Evidence is emerging that mechanical stretching can alter the functional states of proteins. Fibronectin (Fn) is a large, extracellular matrix protein that is assembled by cells into elastic fibrils and subjected to contractile forces. Assembly into fibrils coincides with expression of biological recognition sites that are buried in Fn's soluble state. To investigate how supramolecular assembly of Fn into fibrillar matrix enables cells to mechanically regulate its structure, we used fluorescence resonance energy transfer (FRET) as an indicator of Fn conformation in the fibrillar matrix of NIH 3T3 fibroblasts. Fn was randomly labeled on amine residues with donor fluorophores and site-specifically labeled on cysteine residues in modules FnIII(7) and FnIII(15) with acceptor fluorophores. Intramolecular FRET was correlated with known structural changes of Fn in denaturing solution, then applied in cell culture as an indicator of Fn conformation within the matrix fibrils of NIH 3T3 fibroblasts. Based on the level of FRET, Fn in many fibrils was stretched by cells so that its dimer arms were extended and at least one FnIII module unfolded. When cytoskeletal tension was disrupted using cytochalasin D, FRET increased, indicating refolding of Fn within fibrils. These results suggest that cell-generated force is required to maintain Fn in partially unfolded conformations. The results support a model of Fn fibril elasticity based on unraveling and refolding of FnIII modules. We also observed variation of FRET between and along single fibrils, indicating variation in the degree of unfolding of Fn in fibrils. Molecular mechanisms by which mechanical force can alter the structure of Fn, converting tensile forces into biochemical cues, are discussed.
Polycystin-1 is a novel protein predicted to be a large membrane-spanning glycoprotein with an extracellular N-terminus and an intracellular C-terminus, harboring several structural motifs. To study the subcellular localization, antibodies raised against various domains of polycystin-1 and against specific adhesion complex proteins were used for two-color immunofluorescence staining. In Madine Darby canine kidney (MDCK) cells, polycystin-1 was detected in the cytoplasm as well as co-localizing with desmosomes, but not with tight or adherens junctions. Using confocal laser scanning and immunoelectron microscopy we confirmed the desmosomal localization. By performing a calcium switch experiment, we demonstrated the sequential reassembly of tight junctions, subsequently adherens junctions and finally desmosomes. Polycystin-1 only stained the membrane after incorporation of desmoplakin into the desmosomes, suggesting that membrane-bound polycystin-1 may be important for cellular signaling or cell adhesion, but not for the assembly of adhesion complexes.
The human kidney is composed of roughly 1.2-million renal tubules that must maintain their tubular structure to function properly. In autosomal dominant polycystic kidney disease (ADPKD) cysts develop from renal tubules and enlarge independently, in a process that ultimately causes renal failure in 50% of affected individuals1,2. Mutations in either PKCD1 or PKD2 are associated with ADPKD but the function of these genes is unknown. PKD1 is thought to encode a membrane protein, polycystin-1, involved in cell-cell or cell-matrix interactions3-5, whereas the PKD2 gene product, polycystin-2, is thought to be a channel protein6. Here we show that polycystin-1 and -2 interact to produce new calcium-permeable non-selective cation currents. Neither polycystin- 1 nor-2 alone is capable of producing currents. Moreover, disease-associated mutant forms of either potycystin protein that are incapable of heterodimerization do not result in new channel activity. We also show that polycystin-2 is localized in the cell in the absence of polycystin-1, but is translocated to the plasma membrane in its presence. Thus, polycystin-1 and -2 co-assemble at the plasma membrane to produce a new channel and to regulate renal tubular morphology and function.
Extracellular matrix alterations have been suggested to be part of the early events occurring in Autosomal Dominant Polycystic Kidney Disease (ADPKD), a disease characterized by formation of renal cysts and progressive renal failure. Here we report that cDNA array analysis identified beta(4) integrin aberrant expression in ADPKD cells. Furthermore, laminin 5 (Ln-5), the main alpha(6)beta(4) integrin ligand, was also found to be abnormally expressed in ADPKD. Studies performed with ADPKD cyst-lining epithelial cells (CC) by comparison with normal tubular cells indicate that integrin alpha(6)beta(4)-Ln-5 interactions are involved in cellular events of potential importance for cystogenesis: 1) laminin 5 is a preferential adhesion substrate for CC, mainly through alpha(6)beta(4) interaction, 2) CC increased haptotactic and chemotactic motility depends on the presence of Ln-5 and requires integrin alpha(3)beta(1) cooperation, and 3) CC haptotactic or chemotactic migration is specifically increased by mAb-mediated beta(4) integrin ligation, through an alpha(3)beta(1) integrin-dependent and independent pathway, respectively. These results highlight the role of Ln-5 and alpha(6)beta(4) integrin in adhesive and motility properties of cyst-lining epithelial cells, and further suggest that integrins and extracellular matrix modifications may be of general relevance to kidney epithelial cell cyst formation.
Mutations in polycystin1 (PKD1) account for the majority of autosomal dominant polycystic kidney disease (ADPKD). PKD1 mutations are also associated with vascular aneurysm and abdominal wall hernia, suggesting a role for polycystin1 in extracellular matrix (ECM) integrity. In zebrafish, combined knockdown of the PKD1 paralogs pkd1a and pkd1b resulted in dorsal axis curvature, hydrocephalus, cartilage and craniofacial defects, and pronephric cyst formation at low frequency (10-15%). Dorsal axis curvature was identical to the axis defects observed in pkd2 knockdown embryos. Combined pkd1a/b, pkd2 knockdown demonstrated that these genes interact in axial morphogenesis. Dorsal axis curvature was linked to notochord collagen overexpression and could be reversed by knockdown of col2a1 mRNA or chemical inhibition of collagen crosslinking. pkd1a/b- and pkd2-deficient embryos exhibited ectopic, persistent expression of multiple collagen mRNAs, suggesting a loss of negative feedback signaling that normally limits collagen gene expression. Knockdown of pkd1a/b also dramatically sensitized embryos to low doses of collagen-crosslinking inhibitors, implicating polycystins directly in the modulation of collagen expression or assembly. Embryos treated with wortmannin or LY-29400 also exhibited dysregulation of col2a1 expression, implicating phosphoinositide 3-kinase (PI3K) in the negative feedback signaling pathway controlling matrix gene expression. Our results suggest that pkd1a/b and pkd2 interact to regulate ECM secretion or assembly, and that altered matrix integrity may be a primary defect underlying ADPKD tissue pathologies.
Autosomal-dominant polycystic kidney disease, the most frequent monogenic cause of kidney failure, is induced by mutations in the PKD1 or PKD2 genes, encoding polycystins TRPP1 and TRPP2, respectively. Polycystins are proposed to form a flow-sensitive ion channel complex in the primary cilium of both epithelial and endothelial cells. However, how polycystins contribute to cellular mechanosensitivity remains obscure. Here, we show that TRPP2 inhibits stretch-activated ion channels (SACs). This specific effect is reversed by coexpression with TRPP1, indicating that the TRPP1/TRPP2 ratio regulates pressure sensing. Moreover, deletion of TRPP1 in smooth muscle cells reduces SAC activity and the arterial myogenic tone. Inversely, depletion of TRPP2 in TRPP1-deficient arteries rescues both SAC opening and the myogenic response. Finally, we show that TRPP2 interacts with filamin A and demonstrate that this actin crosslinking protein is critical for SAC regulation. This work uncovers a role for polycystins in regulating pressure sensing.
Mechanotransduction research has focused historically on how externally applied forces can affect cell signalling and function. A growing body of evidence suggests that contractile forces that are generated internally by the actomyosin cytoskeleton are also important in regulating cell behaviour, and suggest a broader role for mechanotransduction in biology. Although the molecular basis for these cellular forces in mechanotransduction is being pursued in cell culture, researchers are also beginning to appreciate their contribution to in vivo developmental processes. Here, we examine the role for mechanical forces and contractility in regulating cell and tissue structure and function during development.
A number of inherited disorders result in renal cyst development. The most common form, autosomal dominant polycystic kidney disease (ADPKD), is a disorder most often diagnosed in adults and caused by mutation in PKD1 or PKD2. The PKD1 protein, polycystin-1, is a large receptor-like protein, whereas polycystin-2 is a transient receptor potential channel. The polycystin complex localizes to primary cilia and may act as a mechanosensor essential for maintaining the differentiated state of epithelia lining tubules in the kidney and biliary tract. Elucidation of defective cellular processes has highlighted potential therapies, some of which are now being tested in clinical trials. ARPKD is the neonatal form of PKD and is associated with enlarged kidneys and biliary dysgenesis. The disease phenotype is highly variable, ranging from neonatal death to later presentation with minimal kidney disease. ARPKD is caused by mutation in PKHD1, and two truncating mutations are associated with neonatal lethality. The ARPKD protein, fibrocystin, is localized to cilia/basal body and complexes with polycystin-2. Rare, syndromic forms of PKD also include defects of the eye, central nervous system, digits, and/or neural tube and highlight the role of cilia and pathways such as Wnt and Hh in their pathogenesis.
Substrate rigidity plays crucial roles in regulating cellular functions, such as cell spreading, traction forces, and stem cell differentiation. However, it is not clear how substrate rigidity influences early cell signaling events such as calcium in living cells. Using highly sensitive Ca(2+) biosensors based on fluorescence resonance energy transfer (FRET), we investigated the molecular mechanism by which substrate rigidity affects calcium signaling in human mesenchymal stem cells (HMSCs). Spontaneous Ca(2+) oscillations were observed inside the cytoplasm and the endoplasmic reticulum (ER) using the FRET biosensors targeted at subcellular locations in cells plated on rigid dishes. Lowering the substrate stiffness to 1 kPa significantly inhibited both the magnitudes and frequencies of the cytoplasmic Ca(2+) oscillation in comparison to stiffer or rigid substrate. This Ca(2+) oscillation was shown to be dependent on ROCK, a downstream effector molecule of RhoA, but independent of actin filaments, microtubules, myosin light chain kinase, or myosin activity. Lysophosphatidic acid, which activates RhoA, also inhibited the frequency of the Ca(2+) oscillation. Consistently, either a constitutive active mutant of RhoA (RhoA-V14) or a dominant negative mutant of RhoA (RhoA-N19) inhibited the Ca(2+) oscillation. Further experiments revealed that HMSCs cultured on gels with low elastic moduli displayed low RhoA activities. Therefore, our results demonstrate that RhoA and its downstream molecule ROCK may mediate the substrate rigidity-regulated Ca(2+) oscillation, which determines the physiological functions of HMSCs.
Autosomal dominant polycystic kidney disease, the most common monogenetic disorder, is characterized by gradual replacement of normal renal parenchyma by fluid-filled cysts. Mutations in either PKD1 or PKD2 cause autosomal dominant polycystic kidney disease. Pkd1(-/-) or Pkd2(-/-) mice develop rapid renal cystic disease and exhibit embryonic lethality; this supports the "two-hit" hypothesis, which proposes that a germline mutation in PKD1 (or PKD2) followed by a second somatic mutation later in life is responsible for the phenotype. Here, for investigation of the loss of Pkd1 at specific times of development, an inducible Pkd1-knockout mouse model was generated. Inactivation of Pkd1 in 5-wk-old mice resulted in formation of only focal renal cysts 6 to 9 wk later but in a severe polycystic phenotype nearly 1 yr later. Cysts derived from either collecting tubules or distal tubules but not from proximal tubules, which correlated with sites of Cre-mediated recombination. Inactivation of Pkd1 in 1-wk-old mice, however, resulted in massive cyst disease 6 wk later, despite a similar pattern of Cre-mediated recombination between 1- and 5-wk-old kidneys. Moreover, a germline heterozygous Pkd1 mutation facilitated cyst formation when a somatic Pkd1 mutation was induced. A marked increase in proliferating cell nuclear antigen expression was observed in cyst-lining epithelia and in normal-looking tubules adjacent to but not in those distant from cysts. These data suggest that Pkd1 inactivation is not sufficient to initiate the cell proliferation necessary for cyst formation; a paracrine mechanism may account for focal cell proliferation and regional disease progression. We propose that an additional genetic or nongenetic "third hit" may be required for rapid development of cysts in polycystic kidney disease.
Mutations in PKD1 are associated with autosomal dominant polycystic kidney disease (ADPKD), which leads to major cardiovascular complications. We used mice with a heterozygous deletion of Pkd1 (Pkd1+/-) and wild-type (Pkd1+/+) littermates to test whether Pkd1 haploinsufficiency is associated with a vascular phenotype in different age groups. Systolic blood pressure measured by the tail-cuff method was similar up to 20 weeks of age, but significantly higher in 30-week-old Pkd1+/- compared to Pkd1+/+. By contrast, similar telemetric recordings were obtained in unrestrained Pkd1+/- and Pkd1+/+ mice. The contractile responses evoked by KCl or phenylephrine were similar in young animals but increased in abdominal aortas of 30-week-old Pkd1+/- mice, and acetylcholine-evoked relaxation was depressed. Basal cytosolic calcium, KCl, and phenylephrine-evoked calcium signals were significantly lower in the Pkd1+/- aortas, whereas calcium release evoked by caffeine or thapsigargin was significantly larger. These changes were paralleled with a significant change in the mRNA expression of Pkd2, Trpc1, Orai1, and Serca2a in the aortas from Pkd1+/- vs. Pkd1+/+. These results are the first to indicate that haploinsufficiency in Pkd1 is associated with altered intracellular calcium homeostasis and increased vascular reactivity in the aorta with compensatory changes in transport proteins involved in the calcium signaling network.
Cells from the cysts of patients with autosomal dominant polycystic kidney disease (PKD) were grown in vitro under standard conditions without the aid of collagen-pretreated surfaces, and both the synthesis and composition of the extracellular matrix were investigated. At confluence, PKD cells presented the typical features of epithelial cells, but showed a different collagen composition from fibroblasts. Compared with normal tubular epithelia (NTE), PKD monolayers produced an excess of extracellular matrix, which accounted for 30% of the total incorporation of [3H] proline, although this value was considerably lower (by a factor of 10) in the case of NTE. Immunohistochemical and electrophoretic techniques revealed a complex collagen composition in the extracellular matrix which included [alpha (III)]3 and collagen IV. However, part of the collagen components remained unidentified in spite of the fact that they exhibited a typical M(r) of alpha 1(I) and alpha 2(I) in the presence of urea. Immunoprecipitation with monospecific antibodies and Northern blotting with specific probes failed to recognize alpha 1(I) and alpha 2(I), but demonstrated their presence in fibroblasts. Purification and cyanogen bromide digestion demonstrated a strong interhomology in fingerprint peptide composition among the uncharacterized collagens synthesized by PKD cells, thus suggesting a common identity. These observations document a markedly augmented production of extracellular matrix by PKD cultured cells in vitro, and show the presence of collagens which do not share homologies with the major collagen molecules. A better characterization of extracellular matrix composition is central to any comprehension of the cytogenetic mechanisms in vivo.
Human autosomal dominant polycystic kidney disease (ADPKD) epithelia were grown in primary monolayer cultures and their properties compared with intact kidney epithelial cultures derived from individually microdissected normal human kidney proximal convoluted tubules (PCT), proximal straight tubules (PST), and cortical collecting tubules (CCT). In vivo, ADPKD cyst epithelia exhibited a thickened basement membrane, and immunofluorescence demonstrated the presence of laminin, fibronectin, type IV collagen, and heparan sulfate proteoglycan in basement membranes and type I collagen in the interstitium. ADPKD epithelia grown in culture synthesized and secreted basally a unique, extracellular matrix that took the form of proteinaceous spheroids when the cells were grown on dried, type I collagen. Incorporation of H2[S35O4] into basement membrane extracts was increased more than ten-fold in ADPKD epithelia by comparison to normal PST and CCT. In addition to incorporation into the normal tubular basement membrane 220 kD band, radioactivity was also seen at 175 kD and 150 kD in ADPKD extracts. Growth in culture of cyst-lining ADPKD epithelia was more rapid than normal tubules, and was abnormal since there was no absolute requirement for added extracellular matrix. However, when ADPKD epithelia were grown on different, exogenous matrix protein components, a profound influence on both structure and epithelial cell proliferation was seen. Growth on a complete basement membrane three-dimensional gel derived from the Engelbreth-Holm-Swarm (EHS) sarcoma led to a reduction in the numbers of spheroids and increase in amorphous filaments. Incorporation of [3H]-thymidine into ADPKD epithelia was greater than into normal PCT, PST, and CCT and was also greatly modified by the type of extracellular matrix components provided. In studies using single matrix components, the strongest proliferative response was seen when ADPKD epithelia were plated on type I collagen greater than type IV collagen greater than fibronectin greater than laminin. These findings suggest that the excessive growth of cyst-lining epithelia may be, at least in part, a result of abnormal basement membrane and extracellular matrix production by ADPKD cells.
Basement membranes surround the renal tubules and have been shown to limit their distension in vitro. Therefore, it has been postulated that a defect in a basement membrane component(s) underlies the pathogenesis of polycystic kidney disease. Here we have studied a murine model of congenital polycystic kidney disease and found by immunohistology, that the components of the peri-cyst basement membrane appeared to diminish with time. We also measured mRNA levels for collagen IV and laminin, and found a different pattern than in the normal mouse kidney. In normal kidneys, mRNA levels for the B1 and B2 chains of laminin were maximal at birth, and at 1 week for the alpha 1(IV) chain of collagen IV. With all three chains, the levels then rapidly declined. In contrast, mRNA for the alpha 1(IV) chain in congenital polycystic kidneys was half normal 1 week after birth and then increased. Laminin B1 and B2 chain mRNA's were 80% of normal at 1 week but were maintained at that level. As a control, beta-actin mRNA was examined and found to remain constant in both normal and diseased kidneys. In situ hybridization of cRNA probes for the alpha 1(IV) chain confirmed that cells associated with cysts were the principal source of expression of these basement membrane mRNAs. Thus, there exists an abnormal regulation of basement membrane gene expression in congenital polycystic kidney disease. The first stage is characterized by reduced levels of expression. In the second stage, the levels are abnormally high, perhaps representing a compensatory synthesis of basement membrane as cysts enlarge.
Echocardiography, including Doppler analysis, was performed to assess the prevalence of cardiac abnormalities in 163 patients with autosomal dominant polycystic kidney disease, 130 unaffected family members, and 100 control subjects. In these three groups, the prevalence of mitral-valve prolapse was 26, 14, and 2 percent, respectively (P less than 0.0005). A higher prevalence of mitral incompetence (31, 14, and 9 percent, respectively; P less than 0.005), aortic incompetence (8, 3, and 1 percent, respectively; P less than 0.05), tricuspid incompetence (15, 7, and 4 percent, respectively; P less than 0.02), and tricuspid-valve prolapse (6, 2, and 0 percent, respectively; P less than 0.02) was also found in the patients with polycystic kidney disease. These findings reflect the systemic nature of polycystic kidney disease and support the hypothesis that the disorder involves a defect in the extracellular matrix and the cardiac abnormalities are an expression of that defect.
Twenty cysts from five patients with adult polycystic kidney disease were evaluated morphologically by electron microscopy and functionally by cyst fluid chemical analysis in order to correlate the structure with the function of the cyst wall. Thirteen proximal cysts, as defined by cyst fluid/serum sodium ratios of 0.8 to 1.2, were lined by epithelial cells with open or short closed apical junctions that appeared permeable to lanthanum. In contrast, seven distal cysts, as defined by cyst fluid/serum sodium ratios of less than 0.4, were lined by epithelial cells with long closed apical junctions that appeared impermeable to lanthanum. Cell organelles showed no distinction between proximal and distal cysts. Cyst basement membranes appeared abnormal, but there was no consistent pattern. The fluid of proximal cysts contained lower creatinine, potassium, and hydrogen ion, and higher chloride concentrations than did the distal cysts. These studies provide morphologic and chemical evidence consistent with te view that cysts originate from nephrons. Moreover, the maintenance of the epithelial lining and transmembrane solute gradients over many years extending up to and beyond the development of renal insufficiency suggests that the cysts function as nephronsthrughout the life of the patient.
The DBA/2FG-pcy mouse has a form of slowly progressive kidney disease that appears similar in many respects to that seen in the autosomal dominant form of human polycystic kidney disease. This study was designed to assess how the expression of extracellular matrix component genes is regulated in a model of murine polycystic kidney disease and control DBA/2 mice at 8, 16, and 30 weeks of age. The mRNA levels encoding for collagen IV, the B1 and B2 chains of laminin, heparan sulfate proteoglycan, fibronectin, and collagens I and III increased with the progression of cystic lesions in the kidney of DBA/2FG-pcy mice. At 30 weeks of age, mRNA levels for collagen IV, laminin B1 and B2, heparan sulfate proteoglycan, fibronectin, and collagens I and III were increased 8.1-fold, 7.0-fold, 7.0-fold, 9.8-fold, 7.0-fold, 5.5-fold, and 5.4-fold, respectively, compared to those of control DBA/2 mice. An immunofluorescence study revealed the irregular staining for collagen IV, laminin, heparan sulfate proteoglycan, and collagens I and III around the cysts. These data suggest that changes in the expression of basement membrane components and interstitial collagens are associated with the development of polycystic kidney disease.
Once viewed as hopelessly incurable disorders and the dustbin for careers in academic medicine, the polycystic kidney diseases have emerged as prime targets of pathophysiologic study and palliative and definitive treatment in the era of molecular medicine. Polycystic kidney disease (PKD) may be hereditary or acquired. The major inherited types are autosomal dominant (AD) and autosomal recessive (AR). ADPKD is caused by at least two (and possibly three) genes located on separate chromosomes, while ADPKD-1 is due to a 14 kb transcript in a duplicated region on the short arm of chromosome 16 very near the alpha-globin gene cluster and the gene for one form of tuberous sclerosis. ADPKD-2 has been assigned to the long arm of chromosome 4. ARPKD is due to a mutated gene on both copies of the long arm of chromosome 6. Cysts originate in renal tubules. Proliferation of tubule epithelial cells modulated by endocrine, paracrine, and autocrine factors is a major element in the pathogenesis of renal cystic diseases. In addition, fluid that is abnormally accumulated within the cysts is derived from glomerular filtrate and, to a greater extent, by transepithelial fluid secretion. Abnormal synthesis and degradation of matrix components associated with interstitial inflammation are additional features in the pathogenesis of renal cystic diseases. The ADPKD genotypes are characterized by bilateral kidney cysts, hypertension, hematuria, renal infection, stones, and renal insufficiency. ADPKD is a systemic disorder; cysts appear with decreasing frequency in the kidneys, liver, pancreas, brain, spleen, ovaries, and testis. Cardiac valvular disorders, abdominal and inguinal hernias, and aneurysms of cerebral and coronary arteries and aorta are also associated with ADPKD. Treatment is supportive: dietary regulation of salt and protein intake, control of hypertension and renal stones, and dialysis and transplantation at the end stage. ARPKD is a relatively rare disease that causes clinical symptoms at birth, with significant mortality in the first month of life. The cysts develop primarily in the collecting ducts because of a failure in the maturation process. Early complications include Potter's syndrome; excessive size of the kidneys, causing respiratory dysfunction; hypertension; and renal insufficiency. Hepatic fibrosis is an associated extrarenal problem that results in significant morbidity in young children and adolescents. Treatment includes supportive care, dialysis, and renal transplantation. Acquired cysts (solitary/simple) are commonplace in older persons. Multiple cysts may be seen in association with potassium deficiency, congenital disorders, metabolic diseases, and toxic renal injury.(ABSTRACT TRUNCATED AT 400 WORDS)
Characterization of the polycystic kidney disease 1 (PKD1) gene has been complicated by genomic rearrangements on chromosome 16. We have used an exon linking strategy, taking RNA from a cell line containing PKD1 but not the duplicate loci, to clone a cDNA contig of the entire transcript. The transcript consists of 14,148 bp (including a correction to the previously described C terminus), distributed among 46 exons spanning 52 kb. The predicted PKD1 protein, polycystin, is a glycoprotein with multiple transmembrane domains and a cytoplasmic C-tail. The N-terminal extracellular region of over 2,500 aa contains leucine-rich repeats, a C-type lectin, 16 immunoglobulin-like repeats and four type III fibronectin-related domains. Our results indicate that polycystin is an integral membrane protein involved in cell-cell/matrix interactions.
Changes of extracellular matrix are involved in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD). The relationship between epithelial changes and extracellular matrix production was studied in a new rat model (Han:SPRD/cy+) at an early phase of cystogenesis. Messenger RNA expression of the alpha 1(IV)-chain of collagen type IV, the main structural component of basement membrane, was localized by in situ hybridization. The presence of collagen IV-protein was shown by immunohistochemistry. At an initial stage, cysts were lined with normal-appearing epithelium except for focal zones of less differentiated cells exhibiting strong collagen alpha 1(IV) mRNA expression and a thickened basement membrane. In these zones, an increase in cell number (2.39-fold) per unit epithelial area indicated hyperplastic growth. Conspicuously, these zones were found at 'bottleneck'-like transitions from normal-size tubules to cystic expansions. Intermediate stages of cysts showed more prominent extracellular matrix deposits and an overall maximally enhanced collagen IV mRNA expression, whereas terminal stages were lined with a flat, simplified epithelium and exhibited moderate collagen IV expression. We suggest that focally enhanced expression of collagen IV in the tubular epithelium and surrounding interstitium of Han:SPRD/cy+ rat kidney is initially involved in cyst development.
The known cardiovascular manifestations in polycystic kidney include the life-threatening berry aneurysms of the cerebral circulation, as well as mitral valve prolapse, mild dilatation of the aortic root, occasional thoracic and abdominal aneurysms, and a predisposition to valvular regurgitation, which have been considered suggestive of an underlying connective tissue disorder.1,2
A second gene for autosomal dominant polycystic kidney disease was identified by positional cloning. Nonsense mutations in
this gene (PKD2) segregated with the disease in three PKD2 families. The predicted 968-amino acid sequence of the PKD2 gene product has six transmembrane spans with intracellular amino- and carboxyl-termini. The PKD2 protein has amino acid
similarity with PKD1, the Caenorhabditis elegans homolog of PKD1, and the family of voltage-activated calcium (and sodium) channels, and it contains a potential calcium-binding
domain.
Autosomal dominant polycystic kidney disease is genetically heterogenous, with at least two chromosomal loci accounting for the disease. When the mutation is located on chromosome 16 (PKD1), extra-renal manifestations such as the rupture of intracranial aneurysms are well known. In the case of localization on chromosome 4 (PKD2), in which the renal disease runs a milder course, not much is known about the incidence of extrarenal manifestations. A PKD2 family is reported in which two members had subarachnoidal bleeding due to intracranial aneurysms; there was strong clinical evidence of subarachnoidal bleeding in a third family member. This indicates that the familial clustering of intracranial aneurysms may also occur in PKD2 families. Because of the considerable mortality and morbidity of intracranial aneurysms, screening with magnetic resonance angiography in PKD2 patients with a positive family history of intracranial aneurysms is recommended.
Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of renal impairment with a number of well recognized extrarenal associations. A high incidence of abdominal wall hernia was noted in patients with ADPKD.
A retrospective review of the notes of all patients with ADPKD on the South Wales renal replacement therapy database was performed. These patients were compared with age- and sex-matched patients with renal failure but without ADPKD and with general surgical patients.
The prevalence of hernia in patients with ADPKD was 38 of 85 (45 per cent) compared with seven of 85 (8 per cent) for other forms of renal failure and three of 85 (4 per cent) for general surgical controls (P < 0.001). There were significantly greater numbers of inguinal (P < 0.001), incisional (P = 0.019) and paraumbilical (P = 0.007) hernias in patients with ADPKD compared with the other two groups.
These results show a significantly greater incidence of hernia, which could be an additional manifestation of the underlying defect in extracellular matrix production identified in patients with ADPKD.
This short review summarizes some information concerning what is known about matrix adhesion molecules, focal adhesion proteins, and cell-cell adhesion molecules in normal renal development and cystic diseases of the kidney. The focus is on human nephrogenesis and disease, but utilizes critical information gained from genetically manipulated mouse models. Interestingly, a significant role for the human PKD-1-encoded gene product, polycystin-1, has been found in cell-matrix interactions via integrins during development, and mutations lead to autosomal dominant polycystic kidney disease (ADPKD). Recent studies on human ADPKD have implicated polycystin-1 in the formation of multiprotein complexes containing focal adhesion proteins at the basal cell surface of the normal ureteric bud. Further evidence of a critical role of cell-matrix interactions via focal adhesion complex formation is provided by the development of renal cystic disease in tensin knockout mice.
Mutations in the PKD1 gene are responsible for autosomal dominant polycystic kidney disease (ADPKD). Although PKD1 has been cloned and shown to be expressed at high levels in the fetal ureteric bud and ADPKD cystic epithelia in the human kidney, the function of its encoded protein, "polycystin-1" is unknown. In this study we used primary and immortalized human renal epithelial cell lines derived from normal fetal, adult, and ADPKD kidneys, that endogenously express PKD1, to study the biologic function of the polycystin-1 protein. ADPKD renal epithelial cells expressed high levels of polycystin-1 protein and showed increased adhesion to type I collagen by comparison with normal adult human renal epithelia that expressed little polycystin. Adherent ADPKD cells also expressed high levels of alpha2beta1-integrin and their attachment was inhibited by a functional monoclonal antibody to alpha2-integrin. Double labeling and confocal microscopy as well as coimmunoprecipitation analysis showed overlapping colocalization of polycystin-1 with alpha2beta1-integrin as well as with the focal adhesion proteins vinculin and paxillin in multiprotein clusters localized to focal areas of cell membrane contact with type I collagen matrix after short periods of attachment. Immunoprecipitation and Western immunoblot studies also showed that polycystin-1 was posttranslationally modified by tyrosine phosphorylation. These studies suggest that the PKD1-encoded protein is part of a large multiprotein complex in epithelial cells that functions in the regulation of extracellular matrix interactions with the plasma membrane and cell cytoskeleton.
Directional cell locomotion is critical in many physiological processes, including morphogenesis, the immune response, and wound healing. It is well known that in these processes cell movements can be guided by gradients of various chemical signals. In this study, we demonstrate that cell movement can also be guided by purely physical interactions at the cell-substrate interface. We cultured National Institutes of Health 3T3 fibroblasts on flexible polyacrylamide sheets coated with type I collagen. A transition in rigidity was introduced in the central region of the sheet by a discontinuity in the concentration of the bis-acrylamide cross-linker. Cells approaching the transition region from the soft side could easily migrate across the boundary, with a concurrent increase in spreading area and traction forces. In contrast, cells migrating from the stiff side turned around or retracted as they reached the boundary. We call this apparent preference for a stiff substrate "durotaxis." In addition to substrate rigidity, we discovered that cell movement could also be guided by manipulating the flexible substrate to produce mechanical strains in the front or rear of a polarized cell. We conclude that changes in tissue rigidity and strain could play an important controlling role in a number of normal and pathological processes involving cell locomotion.
Polycystin-1 is a novel protein predicted to be a large membrane-spanning glycoprotein with an extracellular N-terminus and an intracellular C-terminus, harboring several structural motifs. To study the subcellular localization, antibodies raised against various domains of polycystin-1 and against specific adhesion complex proteins were used for two-color immunofluorescence staining. In Madine Darby canine kidney (MDCK) cells, polycystin-1 was detected in the cytoplasm as well as co-localizing with desmosomes, but not with tight or adherens junctions. Using confocal laser scanning and immunoelectron microscopy we confirmed the desmosomal localization. By performing a calcium switch experiment, we demonstrated the sequential reassembly of tight junctions, subsequently adherens junctions and finally desmosomes. Polycystin-1 only stained the membrane after incorporation of desmoplakin into the desmosomes, suggesting that membrane-bound polycystin-1 may be important for cellular signaling or cell adhesion, but not for the assembly of adhesion complexes.
Mutations in the PKD1 gene are responsible for >85% of autosomal dominant polycystic kidney disease (ADPKD). The protein product of PKD1, polycystin-1, is a large, modular membrane protein, with putative ligand-binding motifs in the extracelluar N-terminal portion, 9-11 transmembrane domains and an intracellular C-terminal portion with phosphorylation sites. A role for polycystin-1 as a cell surface receptor involved in cell-matrix and cell-cell interactions has been proposed. In this study, we have analyzed polycystin-1 and associated protein distribution in normal human epithelial cells and examined the role of cell-matrix versus cell-cell interactions in regulation of the assembly of polycystin-1 multiprotein complexes. Immunocytochemistry, sucrose density gradient sedimentation, co-immunoprecipitation analyses and in vitro binding assays have shown that polycystin-1 associates with the focal adhesion proteins talin, vinculin, p130Cas, FAK, alpha-actinin, paxillin and pp60c-src in subconfluent normal human fetal collecting tubule (HFCT) epithelia when cell-matrix interactions predominate. Polycystin-1 also forms higher S value complexes with the cell-cell adherens junction proteins E-cadherin, beta- and gamma-catenins in confluent cultures when cell-cell interactions are predominant. Polycystin-1 multiprotein complexes can be disrupted by cytochalasin D but not by colchicine, suggesting involvement of the actin cytoskeleton. Although inhibition of tyrosine phosphorylation by tyrphostin inhibits polycystin-1-FAK interactions, E-cadherin interactions are enhanced. High calcium treatment also increases polycystin-1-E-cadherin interactions.
Mutations in the PKD1 gene are responsible for 85% of cases of autosomal dominant polycystic kidney disease (ADPKD). This gene encodes a large membrane associated glycoprotein, polycystin-1, which is predicted to contain a number of extracellular protein motifs, including a C-type lectin domain between amino acids 403--532. We have cloned and expressed the PKD1 C-type lectin domain, and have demonstrated that it binds carbohydrate matrices in vitro, and that Ca(2+) is required for this interaction. This domain also binds to collagens type I, II and IV in vitro. This binding is greatly enhanced in the presence of Ca(2+) and can be inhibited by soluble carbohydrates such as 2-deoxyglucose and dextran. These results suggest that polycystin-1 may be involved in protein-carbohydrate interactions in vivo. The data presented indicate that there may a direct interaction between the PKD1 gene product and an ubiquitous extracellular matrix (ECM) protein.