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

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

The sequence, expression, and chromosomal localization of a novel polycystic kidney disease 1-like gene, PKD1L1, in human

Polycystin-1 and polycystin-2 are the products of PKD1 and PKD2, genes that are mutated in most cases of autosomal dominant polycystic kidney disease. Since the first two polycystins were cloned, three new members, polycystin-L, -2L2, and -REJ, have been identified. In this study, we describe a sixth member of the family, polycystin-1L1, encoded by PKD1L1 in human. The full-length cDNA sequence of PKD1L1, determined from human testis cDNA, encodes a 2849-amino-acid protein and 58 exons in a 187-kb genomic region. The deduced amino acid sequence of polycystin-1L1 has significant homology with all known polycystins, but the longest stretches of homology were found with polycystin-1 and -REJ over the 1453- and 932-amino-acid residues, respectively. Polycystin-1L1 is predicted to have two Ig-like PKD, a REJ, a GPS, a LH2/PLAT, a coiled-coil, and 11 putative transmembrane domains. Several rhodopsin-like G-protein-coupled receptor (GPCR) signatures are also found in polycystin-1L1. Dot-blot analysis and RT-PCR revealed that human PKD1L1 is expressed in testis and in fetal and adult heart. In situ hybridization analysis showed that the most abundant and specific expression of Pkd1l1 was found in Leydig cells, a known source of testosterone production, in mouse testis. We have assigned PKD1L1 to the short arm of human chromosome 7 in bands p12--p13 and Pkd1l1 to mouse chromosome 11 in band A2 by fluorescence in situ hybridization. We hypothesize a role for polycystin-1L1 in the heart and in the male reproductive system.     

A flagellar polycystin-2 homolog required for male fertility in Drosophila

A common inherited cause of renal failure, autosomal dominant polycystic kidney disease results from mutations in either of two genes, PKD1 and PKD2, which encode polycystin-1 and polycystin-2, respectively. Polycystin-2 has distant homology to TRP cation channels and associates directly with polycystin-1. The normal functions of polycystins are poorly understood, although recent studies indicate that they are concentrated in the primary cilia of a variety of cell types. In this report we identified a polycystin-2 homolog in Drosophila melanogaster; this homolog localized to the distal tip of the sperm flagella. A targeted mutation in this gene, almost there (amo), caused nearly complete male sterility. The amo males produced and transferred normal amounts of motile sperm to females, but mutant sperm failed to enter the female sperm storage organs, a prerequisite for fertilization. The finding that Amo functions in sperm flagella supports a common and evolutionarily conserved role for polycystin-2 proteins in both motile and nonmotile axonemal-containing structures.     

Molecular pathogenesis of autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is one of the commonest inherited human disorders yet remains relatively unknown to the wider medical, scientific and public audience. ADPKD is characterised by the development of bilateral enlarged kidneys containing multiple fluid-filled cysts and is a leading cause of end-stage renal failure (ESRF). ADPKD is caused by mutations in two genes: PKD1 and PKD2. The protein products of the PKD genes, polycystin-1 and polycystin-2, form a calcium-regulated, calcium-permeable ion channel. The polycystin complex is implicated in regulation of the cell cycle via multiple signal transduction pathways as well as the mechanosensory function of the renal primary cilium, an enigmatic cellular organelle whose role in normal physiology is still poorly understood. Defects in cilial function are now documented in several other human diseases including autosomal recessive polycystic kidney disease, nephronophthisis, Bardet-Biedl syndrome and many animal models of polycystic kidney disease. Therapeutic trials in these animal models of polycystic kidney disease have identified several promising drugs that ameliorate disease severity. However, elucidation of the function of the polycystins and the primary cilium will have a major impact on our understanding of renal cystic diseases and will create exciting new opportunities for the design of disease-specific therapies.     

Identification of two novel polycystic kidney disease-1-like genes in human and mouse genomes

Mutations to the prototypical members of the two general classes of polycystins, polycystin-1 encoded by PKD1 and polycystin-2 encoded by PKD2, underlie autosomal-dominant polycystic kidney disease. Here we report the identification of a pair of genes homologous to PKD1 from both the human and mouse genomes. PKD1L2 and PKD1L3 are located on human chromosome 16q22-q23 and mouse chromosome 8 and are alternatively spliced. The human and mouse forms of PKD1L2 are highly conserved, with each one consisting of 43 exons and approximately 2,460 codons. PKD1L3 shows regional sequence divergence, with the mouse form having two additional exons and a much larger exon 5. The predicted protein products of PKD1L2 and PKD1L3 contain the combination of GPS and PLAT/LH2 domains that uniquely define them as polycystin-1 family members. They are predicted to have 11 membrane-spanning regions with a large extracellular domain consistent with the proposed receptor function of this protein family. PKD1L2 and PKD1L3 contain strong ion channel signature motifs that suggest their possible function as components of cation channel pores. Polycystin-1-related proteins may not only regulate channels, but may actually be part of the pore-forming unit.     

Polycystin-1: Function as a mechanosensor

Polycystin-1 (PC1), encoded by the Pkd1 gene, is a large transmembrane protein whose mutation is involved in autosomal dominant polycystic kidney disease. When expressed, PC1 activates a G-protein signaling pathway that subsequently modulates Ca²⁺ channels. PC1 is highly expressed in developing tissue and via its C-terminus tail forms a complex with polycystin-2; this complex, found to be located at the primary cilia, seems to act as a mechanosensor that could affect proliferation, differentiation and apoptosis of cells. Also, loss of polycystins correlates with disruption of flow-dependent and steady-state intracellular Ca²⁺ signaling. Despite the lack of clarity on the role of the polycystins as mechanosensor molecules, a new interest in this PCs/primary cilium complex is providing continuously new insights. In this review, some of the known features of PC1 such as structure, function, signaling pathways involved and its role as a possible therapeutic target are being discussed.     

Structure and function of polycystin channels in primary cilia

Variants in genes which encode for polycystin-1 and polycystin-2 cause most forms of autosomal dominant polycystic disease (ADPKD). Despite our strong understanding of the genetic determinants of ADPKD, we do not understand the structural features which govern the function of polycystins at the molecular level, nor do we understand the impact of most disease-causing variants on the conformational state of these proteins. These questions have remained elusive because polycystins localize to several organelle membranes, including the primary cilia. Primary cilia are microtubule based organelles which function as cellular antennae. Polycystin-2 and related polycystin-2 L1 are members of the transient receptor potential (TRP) ion channel family, and form distinct ion channels in the primary cilia of disparate cell types which can be directly measured. Polycystin-1 has both ion channel and adhesion G-protein coupled receptor (GPCR) features—but its role in forming a channel complex or as a channel subunit chaperone is undetermined. Nonetheless, recent polycystin structural determination by cryo-EM has provided a molecular template to understand their biophysical regulation and the impact of disease-causing variants. We will review these advances and discuss hypotheses regarding the regulation of polycystin channel opening by their structural domains within the context of the primary cilia.     

Polycystin-1 and polycystin-2 are both required to amplify inositol-trisphosphate-induced Ca2+ release

Autosomal dominant polycystic kidney disease is caused by loss-of-function mutations in the PKD1 or PKD2 genes encoding respectively polycystin-1 and polycystin-2. Polycystin-2 stimulates the inositol trisphosphate (IP(3)) receptor (IP(3)R), a Ca(2+)-release channel in the endoplasmic reticulum (ER). The effect of ER-located polycystin-1 is less clear. Polycystin-1 has been reported both to stimulate and to inhibit the IP(3)R. We now studied the effect of polycystin-1 and of polycystin-2 on the IP(3)R activity under conditions where the cytosolic Ca(2+) concentration was kept constant and the reuptake of released Ca(2+) was prevented. We also studied the interdependence of the interaction of polycystin-1 and polycystin-2 with the IP(3)R. The experiments were done in conditionally immortalized human proximal-tubule epithelial cells in which one or both polycystins were knocked down using lentiviral vectors containing miRNA-based short hairpins. The Ca(2+) release was induced in plasma membrane-permeabilized cells by various IP(3) concentrations at a fixed Ca(2+) concentration under unidirectional (45)Ca(2+)-efflux conditions. We now report that knock down of polycystin-1 or of polycystin-2 inhibited the IP(3)-induced Ca(2+) release. The simultaneous presence of the two polycystins was required to fully amplify the IP(3)-induced Ca(2+) release, since the presence of polycystin-1 alone or of polycystin-2 alone did not result in an increased Ca(2+) release. These novel findings indicate that ER-located polycystin-1 and polycystin-2 operate as a functional complex. They are compatible with the view that loss-of-function mutations in PKD1 and in PKD2 both cause autosomal dominant polycystic kidney disease.     

Genomic organization and functional analysis of murine PKD2L1

Mutations in genes that encode polycystins 1 or 2 cause polycystic kidney disease (PKD). Here, we report the genomic organization and functional expression of murine orthologue of human polycystin-2L1 (PKD2L1). The murine PKD2L1 gene comprises 15 exons in chromosome 19C3. Coexpression of PKD2L1 together with polycystin-1 (PKD1) resulted in the expression of PKD2L1 channels on the cell surface, whereas PKD2L1 expressed alone was retained within the endoplasmic reticulum (ER). This suggested that interaction between PKD1 and PKD2L1 is essential for PKD2L1 trafficking and channel formation. Deletion analysis at the cytoplasmic tail of PKD2L1 revealed that the coiled-coil domain was important for trafficking by PKD1. Mutagenesis within two newly identified ER retention signal-like amino acid sequences caused PKD2L1 to be expressed at the cell surface. This indicated that the coiled-coil domain was responsible for retaining PKD2L1 within the ER. Functional analysis of murine PKD2L1 expressed in HEK 293 cells was undertaken using calcium imaging. Coexpression of PKD1 and PKD2L1 resulted in the formation of functional cation channels that were opened by hypo-osmotic stimulation, whereas neither molecule formed functional channels when expressed alone. We conclude that PKD2L1 forms functional cation channels on the plasma membrane by interacting with PKD1. These findings raise the possibility that PKD2L1 represents the third genetic locus that is responsible for PKD.     

Polycystin-1 distribution is modulated by polycystin-2 expression in mammalian cells

Mutations in PKD1 and PKD2, the genes that encode polycystin-1 and polycystin-2 respectively, account for almost all cases of autosomal dominant polycystic kidney disease. Although the polycystins are believed to interact in vivo, the two proteins often display dissimilar patterns and gradients of expression during development. In an effort to understand this apparent discrepancy, we investigated how changes in polycystin-2 expression can affect the subcellular localization of polycystin-1. We show that, when polycystin-1 is expressed alone in a PKD2 null cell line, it localizes to the cell surface, as well as to the endoplasmic reticulum. When co-expressed with polycystin-2, however, polycystin-1 is not seen at the cell surface and co-localizes completely with polycystin-2 in the endoplasmic reticulum. The localization of a polycystin-1 fusion protein was similarly affected by changes in its level of expression relative to that of polycystin-2. This phenomenon was observed in populations as well as in individual COS-7 cells. Our data suggest that the localization of polycystin-1 can be regulated via the relative expression level of polycystin-2 in mammalian cells. This mechanism may help to explain the divergent patterns and levels of expression observed for the polycystins, and may provide clues as to how the function of these two proteins are regulated during development.     

Trafficking of TRPP2 by PACS proteins represents a novel mechanism of ion channel regulation

The trafficking of ion channels to the plasma membrane is tightly controlled to ensure the proper regulation of intracellular ion homeostasis and signal transduction. Mutations of polycystin-2, a member of the TRP family of cation channels, cause autosomal dominant polycystic kidney disease, a disorder characterized by renal cysts and progressive renal failure. Polycystin-2 functions as a calcium-permeable nonselective cation channel; however, it is disputed whether polycystin-2 resides and acts at the plasma membrane or endoplasmic reticulum (ER). We show that the subcellular localization and function of polycystin-2 are directed by phosphofurin acidic cluster sorting protein (PACS)-1 and PACS-2, two adaptor proteins that recognize an acidic cluster in the carboxy-terminal domain of polycystin-2. Binding to these adaptor proteins is regulated by the phosphorylation of polycystin-2 by the protein kinase casein kinase 2, required for the routing of polycystin-2 between ER, Golgi and plasma membrane compartments. Our paradigm that polycystin-2 is sorted to and active at both ER and plasma membrane reconciles the previously incongruent views of its localization and function. Furthermore, PACS proteins may represent a novel molecular mechanism for ion channel trafficking, directing acidic cluster-containing ion channels to distinct subcellular compartments.     

The exocyst protein Sec10 interacts with Polycystin-2 and knockdown causes PKD-phenotypes

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by formation of renal cysts that destroy the kidney. Mutations in PKD1 and PKD2, encoding polycystins-1 and -2, cause ADPKD. Polycystins are thought to function in primary cilia, but it is not well understood how these and other proteins are targeted to cilia. Here, we provide the first genetic and biochemical link between polycystins and the exocyst, a highly-conserved eight-protein membrane trafficking complex. We show that knockdown of exocyst component Sec10 yields cellular phenotypes associated with ADPKD, including loss of flow-generated calcium increases, hyperproliferation, and abnormal activation of MAPK. Sec10 knockdown in zebrafish phenocopies many aspects of polycystin-2 knockdown-including curly tail up, left-right patterning defects, glomerular expansion, and MAPK activation-suggesting that the exocyst is required for pkd2 function in vivo. We observe a synergistic genetic interaction between zebrafish sec10 and pkd2 for many of these cilia-related phenotypes. Importantly, we demonstrate a biochemical interaction between Sec10 and the ciliary proteins polycystin-2, IFT88, and IFT20 and co-localization of the exocyst and polycystin-2 at the primary cilium. Our work supports a model in which the exocyst is required for the ciliary localization of polycystin-2, thus allowing for polycystin-2 function in cellular processes.     

An ever-expanding story of cyst formation

Autosomal-dominant polycystic kidney disease represents one of the most common monogenetic human disorders. The cloning of the PKD1 and PKD2 genes, which are mutated in far more than 90% of the patients affected by this disease, has generated high hopes for a quick understanding of the pathogenesis of cyst formation. However, these expectations have not yet been fulfilled, since the function of both polycystin-1 and polycystin-2, the two proteins encoded by PKD1 and PKD2, still remains a puzzle. In this review, we will highlight some of the characteristics of polycystic kidney disease, briefly touch on polycystin-1, and then go on to describe recent results of experiments with polycystin-2, since the latter is the major focus of our work. We will discuss new evidence which suggests that autosomal-dominant polycystic kidney disease actually behaves recessively on a cellular level. Finally, a model will be presented that tries to explain the available data.     

Identification, characterization, and localization of a novel kidney polycystin-1-polycystin-2 complex

The functions of the two proteins defective in autosomal dominant polycystic kidney disease, polycystin-1 and polycystin-2, have not been fully clarified, but it has been hypothesized that they may heterodimerize to form a "polycystin complex" involved in cell adhesion. In this paper, we demonstrate for the first time the existence of a native polycystin complex in mouse kidney tubular cells transgenic for PKD1, non-transgenic kidney cells, and normal adult human kidney. Polycystin-1 is heavily N-glycosylated, and several glycosylated forms of polycystin-1 differing in their sensitivity to endoglycosidase H (Endo H) were found; in contrast, native polycystin-2 was fully Endo H-sensitive. Using highly specific antibodies to both proteins, we show that polycystin-2 associates selectively with two species of full-length polycystin-1, one Endo H-sensitive and the other Endo H-resistant; importantly, the latter could be further enriched in plasma membrane fractions and co-immunoprecipitated with polycystin-2. Finally, a subpopulation of this complex co-localized to the lateral cell borders of PKD1 transgenic kidney cells. These results demonstrate that polycystin-1 and polycystin-2 interact in vivo to form a stable heterodimeric complex and suggest that disruption of this complex is likely to be of primary relevance to the pathogenesis of cyst formation in autosomal dominant polycystic kidney disease.     

A spectrum of mutations in the second gene for autosomal dominant polycystic kidney disease (PKD2).

Recently the second gene for autosomal dominant polycystic kidney disease (ADPKD), located on chromosome 4q21-q22, has been cloned and characterized. The gene encodes an integral membrane protein, polycystin-2, that shows amino acid similarity to the PKD1 gene product and to the family of voltage-activated calcium (and sodium) channels. We have systematically screened the gene for mutations by single-strand conformation-polymorphism analysis in 35 families with the second type of ADPKD and have identified 20 mutations. So far, most mutations found seem to be unique and occur throughout the gene, without any evidence of clustering. In addition to small deletions, insertions, and substitutions leading to premature translation stops, one amino acid substitution and five possible splice-site mutations have been found. These findings suggest that the first step toward cyst formation in PKD2 patients is the loss of one functional copy of polycystin-2.     

Annexin A5 interacts with polycystin-1 and interferes with the polycystin-1 stimulated recruitment of E-cadherin into adherens junctions

Polycystin-1 is the gene product of PKD1, the first gene identified to be causative for the condition of autosomal dominant polycystic kidney disease (ADPKD). Mutations in PKD1 are responsible for the majority of ADPKD cases worldwide. Polycystin-1 is a protein of the transient receptor potential channels superfamily, with 11 transmembrane spans and an extracellular N-terminal region of approximately 3109 amino acid residues, harboring multiple putative ligand binding domains. We demonstrate here that annexin A5 (ANXA5), a Ca(2+) and phospholipid binding protein, interacts with the N-terminal leucine-rich repeats of polycystin-1, in vitro and in a cell culture model. This interaction is direct and specific and involves a conserved sequence of the ANXA5 N-terminal domain. Using Madin-Darby canine kidney cells expressing polycystin-1 in an inducible manner we also show that polycystin-1 colocalizes with E-cadherin at cell-cell contacts and accelerates the recruitment of intracellular E-cadherin to reforming junctions. This polycystin-1 stimulated recruitment is significantly delayed by extracellular annexin A5.     

Localization of transient receptor potential ion channels in primary and motile cilia of the female murine reproductive organs

We have examined the subcellular localization of transient receptor potential (TRP) ion channels and the potential sensory role of cilia in murine female reproductive organs using confocal laser scanning microscopy analysis on ovary and oviduct tissue sections as well as on primary cultures of follicular granulosa cells. We show that the Ca2+ permeable cation channel, polycystin-2, as well as polycystin-1, a receptor that forms a functional protein complex with polycystin 2, distinctively localize to primary cilia emerging from granulosa cells of antral follicles in vivo and in vitro. Both polycystins are localized to motile oviduct cilia and this localization is greatly increased upon ovulatory gonadotropic stimulation. Further, the Ca2+ permeable cation channel, TRP vaniloid 4 (TRPV4), localizes to a sub-population of motile cilia on the epithelial cells of the ampulla and isthmus with high intensity in proximal invaginations of the epithelial folds. These observations are the first to demonstrate ciliary localization of TRP ion channels and their possible receptor function in the female reproductive organs. We suggest that polycystins 1 and 2 play an important role in granulosa cell differentiation and in development and maturation of ovarian follicles. In the oviduct both TRPV4 and polycystins could be important in relaying physiochemical changes in the oviduct upon ovulation.     

The ion channel polycystin-2 is required for left-right axis determination in mice

Generation of laterality depends on a pathway which involves the asymmetrically expressed genes nodal, Ebaf, Leftb, and Pitx2. In mouse, node monocilia are required upstream of the nodal cascade. In chick and frog, gap junctions are essential prior to node/organizer formation. It was hypothesized that differential activity of ion channels gives rise to unidirectional transfer through gap junctions, resulting in asymmetric gene expression. PKD2, which if mutated causes autosomal dominant polycystic kidney disease (ADPKD) in humans, encodes the calcium release channel polycystin-2. We have generated a knockout allele of Pkd2 in mouse. In addition to malformations described previously, homozygous mutant embryos showed right pulmonary isomerism, randomization of embryonic turning, heart looping, and abdominal situs. Leftb and nodal were not expressed in the left lateral plate mesoderm (LPM), and Ebaf was absent from floorplate. Pitx2 was bilaterally expressed in posterior LPM but absent anteriorly. Pkd2 was ubiquitously expressed at headfold and early somite stages, with higher levels in floorplate and notochord. The embryonic midline, however, was present, and normal levels of Foxa2 and shh were expressed, suggesting that polycystin-2 acts downstream or in parallel to shh and upstream of the nodal cascade.     

Canine PKD1 is a single-copy gene: genomic organization and comparative analysis

Most cases of autosomal dominant polycystic kidney disease are caused by mutations in the gene PKD1, encoding polycystin-1. To gain insight into the role of polycystin-1 in tubulogenesis and cystogenesis using the well-characterized canine kidney epithelial cell line MDCK, we have now cloned and characterized the exon/intron structure of the canine gene PKD1. FISH analysis showed that the dog genome lacks the multiple PKD1 homologs present in human. Intron 21 of dog PKD1 lacked the polypyrimidine tract characteristic of the human gene, whereas pyrimidine-rich elements were identified in canine intron 30. Canine polycystin-1 showed a higher degree of homology with the human counterpart and lower homology with mouse and rat. A striking degree of conservation (97% identity) was determined for the leucine-rich repeat domain between dog and human. Also, the homology analysis indicated that 4 of 16 Ig-like repeats (IgIII, IgVII, IgX, and IgXV) are likely to be functionally significant. This is particularly important in light of our recent findings demonstrating that Iglike domains form strong homophilic interactions and can mediate cell-cell adhesion. These data enable detailed analysis of the role of polycystin-1 in cystogenesis and tubulogenesis using the canine MDCK cell line.