Polycystic Kidney Disease Gene Research Articles

Human cilia proteome contains homolog of zebrafish polycystic kidney disease gene qilin

Abstract Cilia are found on most cells of the body and are thought to play important roles in physiology and development. Not surprisingly, it is now understood that a very wide range of human diseases and developmental defects results from defects in ciliary assembly or function [1]. The steadily increasing number of disease genes related to cilia suggests that identification of ciliary proteins is a productive way to identify new candidate disease genes. Recent bioinformatics studies have followed this strategy by using comparative genomics to identify likely ciliary genes found only in organisms that have cilia and flagella. This ingenious approach has turned out to be highly productive, most notably by revealing the identity of the Bardet-Biedl Syndrome BBS5 gene [2]. In a recent Current Biology Dispatch [3], Greg Pazour presented a carefully thought-out analysis of these approaches, and after discussing several potential limitations, concluded that there is an urgent need for proteomic analysis to determine directly which proteins are contained in cilia and flagella.

Polyductin, the PKHD1 gene product, comprises isoforms expressed in plasma membrane, primary cilium, and cytoplasm

PKHD1, the autosomal-recessive polycystic kidney disease (ARPKD) gene, encodes multiple alternatively spliced transcripts predicted to generate membrane-bound and secreted proteins. The longest open reading frame encodes polyductin (fibrocystin), a putative 4074 amino acid protein with a single transmembrane domain and an intracellular C-terminus. To characterize the PKHD1 products and their expression profile, we raised polyclonal antibodies against different portions of polyductin and analyzed different organs using various methods. Western blot analyses demonstrated specific bands of >440 kD in human adult kidney, liver, and pancreas and approximately 230 kD in kidney and liver, predominantly observed in membrane fractions. The >440-kD putative membrane protein was immunoprecipitated from kidney and subsequently detected by Western blotting using two distinct antisera. An additional product of approximately 140 kD was specifically recognized by affinity-purified antisera predominantly in soluble fractions. Immunohistochemistry studies revealed specific staining in cortical and medullary collecting ducts and thick ascending limbs of Henle (TALH). Serial sections were stained with antibodies against aquaporin-2 and Tamm-Horsfall protein to confirm the nephron segment localization. Positive staining was also detected in biliary and pancreatic duct epithelia. Analyses of mouse developing tissues showed specific staining in the ureteric bud branches, intra- and extrahepatic biliary ducts, pancreatic ducts, and salivary glands. Immunofluorescence studies in inner medullary collecting duct cultured cells and immunoelectron microscopy analysis of medullary collecting ducts demonstrated that the protein localizes to the primary cilium. Positive signal was also detected in the apical membrane and in cytoplasm. The results indicate that polyductin is part of the group of polycystic kidney disease (PKD)-related proteins expressed in primary apical cilia. Our data also suggest that, in addition to its likely involvement in cilia function, polyductin probably serves in other subcellular functional roles. The detection of three different products using two antisera, with evidence for distinct subcellular localizations, suggests that PKHD1 encodes membrane-bound and soluble isoforms.

Intraflagellar transport and cilia-dependent renal disease: the ciliary hypothesis of polycystic kidney disease.

Epithelial cells that line mammalian kidney nephrons have solitary nonmotile primary cilium projecting from their surface into the lumens of the ducts and tubules. Mutations that block the assembly of these cilia cause cystic kidney disease. The products of human autosomal dominant and recessive polycystic kidney disease genes and products of the nephronophthisis disease genes are at least partially localized to primary cilia. This suggests that the cilium serves as an organizing center for the early steps of the signal transduction pathway that is responsible for monitoring the integrity of the kidney nephron and controlling cell proliferation and differentiation.

A genetic screen in zebrafish identifies cilia genes as a principal cause of cystic kidney.

Polycystic kidney disease (PKD) is a common human genetic illness. It is characterized by the formation of multiple kidney cysts that are thought to result from over-proliferation of epithelial cells. Zebrafish larvae can also develop kidney cysts. In an insertional mutagenesis screen in zebrafish, we identified 12 genes that can cause cysts in the glomerular-tubular region when mutated and we cloned 10 of these genes. Two of these genes, vhnf1 (tcf2) and pkd2, are already associated with human cystic kidney diseases. Recently, defects in primary cilia have been linked to PKD. Strikingly, three out of the 10 genes cloned in this screen are homologues of Chlamydomonas genes that encode components of intraflagellar transport (IFT) particles involved in cilia formation. Mutation in a fourth blocks ciliary assembly by an unknown mechanism. These results provide compelling support for the connection between cilia and cystogenesis. Our results also suggest that lesions in genes involved in cilia formation and function are the predominant cause of cystic kidney disease, and that the genes identified here are excellent candidates for novel human PKD genes.

Depletion of PKD1 by an antisense oligodeoxynucleotide induces premature G1/S-phase transition

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the growth of epithelial cells and the influx of cyst fluid. The 14-kb mRNA of the polycystic kidney disease gene, PKD1, encodes the polycystin-1 protein, whose function remains unknown. In this study, we observed that polycystin-1 localized in epithelial cell-cell contacts of 293 cells. We found, by bromodeoxyuridine (BrdU) incorporation experiments and Western blot analysis of S-phase-specific cyclins, that the depletion of polycystin-1 led to an increased cell proliferation rate and caused a premature G1/S-phase transition. In addition, we showed that the depletion of polycystin-1 reduced the amount of p53 in 293 cells irradiated by UV light, suggesting that polycystin-1 acts as a regulator of G1 checkpoint, which controls entry into the S phase and prevents the replication of damaged DNA. Our results might provide an insight into the formation and progression of ADPKD cysts.

PKHD1 protein encoded by the gene for autosomal recessive polycystic kidney disease associates with basal bodies and primary cilia in renal epithelial cells

Mutations of the polycystic kidney and hepatic disease 1 (PKHD1) gene have been shown to cause autosomal recessive polycystic kidney disease (ARPKD), but the cellular functions of the gene product (PKHD1) remain uncharacterized. To illuminate its properties, the spatial and temporal expression patterns of PKHD1 were determined in mouse, rat, and human tissues by using polyclonal Abs and mAbs recognizing various specific regions of the gene product. During embryogenesis, PKHD1 is widely expressed in epithelial derivatives, including neural tubules, gut, pulmonary bronchi, and hepatic cells. In the kidneys of the pck rats, the rat model of which is genetically homologous to human ARPKD, the level of PKHD1 was significantly reduced but not completely absent. In cultured renal cells, the PKHD1 gene product colocalized with polycystin-2, the gene product of autosomal dominant polycystic disease type 2, at the basal bodies of primary cilia. Immunoreactive PKHD1 localized predominantly at the apical domain of polarized epithelial cells, suggesting it may be involved in the tubulogenesis and/or maintenance of duct-lumen architecture. Reduced PKHD1 levels in pck rat kidneys and its colocalization with polycystins may underlie the pathogenic basis for cystogenesis in polycystic kidney diseases.

Design, targeting, and initial screening of sTRSV-derived hairpin ribozymes for optimum helix 1 length and catalytic efficiency in vitro.

Hairpin ribozymes derived from the negative strand of satellite RNAs from the tobacco ringspot virus (sTRSV) can be engineered to target and cleave a variety of heterologous RNAs from both cellular and viral transcripts. Attention to design and targeting rules and optimization of helix 1 length and catalytic efficiency in vitro may increase the efficacy of hairpin ribozymes in reducing the expression of targeted transcripts. Here, principles for the design and targeting of sTRSV-derived hairpin ribozymes are described, as well as methods and materials for optimizing helix 1 length, and for conducting an initial screen of catalytic efficiency to identify promising candidates for further evaluation. Examples are provided for hairpin ribozymes that target human and mouse transforming growth-factor beta (TGF-beta), as well as human polycystic kidney disease gene 1 (PKD1) and JC virus large T-antigen. The tetraloop modification of the sTRSV hairpin ribozyme is considered superior to designs based on the native sTRSV hairpin ribozyme, given its potential to yield considerable improvements in stability and catalytic efficiency.

Murine models of polycystic kidney disease: molecular and therapeutic insights

Numerous murine (mouse and rat) models of polycystic kidney disease (PKD) have been described in which the mutant phenotype results from a spontaneous mutation or engineering via chemical mutagenesis, transgenic technologies, or gene-specific targeting in mouse orthologs of human PKD genes. These murine phenotypes closely resemble human PKD, with common abnormalities observed in tubular epithelia, the interstitial compartment, and the extracellular matrix of cystic kidneys. In both human and murine PKD, genetic background appears to modulate the renal cystic phenotype. In murine models, these putative modifying effects have been dissected into discrete factors called quantitative trait loci and genetically mapped. Several lines of experimental evidence support the hypothesis that PKD genes and their modifiers may define pathways involved in cystogenesis and PKD progression. Among the various pathway abnormalities described in murine PKD, recent provocative data indicate that structural and/or functional defects in the primary apical cilia of tubular epithelia may play a key role in PKD pathogenesis. This review describes the most widely studied murine models; highlights the data regarding specific gene defects and genetic modifiers; summarizes the data from these models that have advanced our understanding of PKD pathogenesis; and examines the effect of various therapeutic interventions in murine PKD.

PKD2 Cation Channel Is Required for Directional Sperm Movement and Male Fertility

Sperm of both mammals and invertebrates move toward specific sites in the female reproductive tract. However, molecular mechanisms for sperm to follow directional cues are unknown. Here, we report genetic analysis of Drosophila Pkd2 at 33E3 ( Pkd2, CG6504), which encodes a Ca 2+-activated, nonselective cation channel homologous to the human Pkd2 autosomal dominant polycystic kidney disease (ADPKD) gene [1]. The PKD2 family of genes has been implicated in sensory responses through protein localization on primary cilia of epithelia and neurons [2–4]. In renal tubules, cilium-associated PKD2 appears to mediate Ca 2+ influx in response to fluid flow, and the loss of fluid sensation probably contributes to cyst growth and ADPKD [4]. Sperm tails or flagella are specialized cilia essential for movement. Drosophila Pkd2 is abundantly associated with the tail and the acrosome-containing head region of mature sperm. Targeted disruption of Pkd2 results in male sterility without affecting spermatogenesis. The mutant sperm are motile but fail to swim into the storage organs in the female. Rare mutant sperm that reach the storage organs are able to fertilize the egg and produce viable progeny. Our data demonstrate that the Drosophila PKD2 cation channel operates in sperm for directional movement inside the female reproductive tract.

No disease-associated mutations found in the coding sequence of the canine polycystic kidney disease gene 1 in Bull Terriers with polycystic kidney disease.

The aim of this study was to identify possible disease-associated mutations in the canine homologue of the polycystic kidney disease gene 1 (PKD1) in Bull Terriers with autosomal dominant polycystic kidney disease. Messenger RNA was obtained from the blood or renal tissue of five Bull Terriers with the disease and four close relatives without the disease. Reverse transcription, PCR and 3' rapid amplification of cDNA ends were used to amplify the coding and 3' untranslated regions of this transcript. Comparison of PKD1 sequence between the affected and unaffected Bull Terriers, revealed six polymorphisms, but no disease-associated mutations.

The isolated polycystin-1 cytoplasmic COOH terminus prolongs ATP-stimulated Cl- conductance through increased Ca2+ entry.

The precise steps leading from mutation of the polycystic kidney disease (PKD1) gene to the autosomal dominant polycystic kidney disease (ADPKD) phenotype remain to be established. Fluid accumulation is a requirement for cyst expansion in ADPKD, suggesting that abnormal fluid secretion into the cyst lumen might play a role in disease. In this study, we sought to establish a link between polycystin-1 (the PKD1 gene product) and ATP-stimulated Cl- secretion in renal tubule cells. To do this, we performed a whole cell patch-clamp analysis of the effects of expression of the isolated cytoplasmic COOH-terminus of polycystin-1 in stably transfected mouse cortical collecting duct cells. The truncated polycystin-1 fusion protein prolonged the duration of ATP-stimulated Cl- conductance and intracellular Ca2+ responses. Both effects were dependent on extracellular Ca2+. It was determined that expression of the truncated polycystin-1 fusion protein introduced, or activated, an ATP-induced Ca2+ entry pathway that was undetectable in transfection control cell lines. Our findings are concordant with increasing evidence for a role of polycystin-1 in cell Ca2+ homeostasis and indicate that dysregulated Ca2+ entry might promote Cl- secretion and cyst expansion in ADPKD.

Association of mutation position in polycystic kidney disease 1 ( PKD1) gene and development of a vascular phenotype

Patients with autosomal dominant polycystic kidney disease (ADPKD) are at risk of developing intracranial aneurysms, and subarachnoid haemorrhage is a major cause of death and disability. Familial clustering of intracranial aneurysms suggests that genetic factors are important in the aetiology. We tested whether the germline mutation predisposes to this vascular phenotype. DNA samples from patients with ADPKD and vascular complications were screened for mutations throughout the PKD1 and PKD2 genes. Comparisons were made between the PKD1 and PKD2 populations and with a control PKD1 cohort (without the vascular phenotype). Mutations were characterised in 58 ADPKD families with vascular complications; 51 were PKD1 (88%) and seven PKD2 (12%). The median position of the PKD1 mutation was significantly further 59 in the vascular population than in the 87 control pedigrees (aminoacid position 2163 vs 2773, p=0.0034). Subsets of the vascular population with aneurysmal rupture, early rupture, or families with more than one vascular case had median mutation locations further 59 (aminoacid position 1811, p=0.0018; 1671, p=0.0052; and 1587, p=0.0003). Patients with PKD2, as well as those with PKD1, are at risk of intracranial aneurysm. The position of the mutation in PKD1 is predictive for development of intracranial aneurysms (59 mutations are more commonly associated with vascular disease) and is therefore of prognostic importance. Since the PKD1 phenotype is associated with mutation position, the disease is not simply due to loss of all disease allele products.

PKHDL1, a homolog of the autosomal recessive polycystic kidney disease gene, encodes a receptor with inducible T lymphocyte expression

Autosomal-recessive polycystic kidney disease (ARPKD) is caused by mutation to a large gene, PKHD1, encoding a putative receptor protein, fibrocystin. We have identified, through analysis of human genomic sequence, a PKHD1 homolog, PKHDL1, in chromosome region 8q23. The PKHDL1 transcript of 13081 bp was amplified as 16 fragments and sequenced; the sequence of the murine ortholog, Pkhdl1 (chromosome region 15B3) was also determined. PKHDL1 contains 78 exons, covers a genomic region of approximately 168 kb and encodes a large protein, fibrocystin-L. Screening PKHDL1 in ARPKD patients with no PKHD1 mutations revealed several sequence variants but no clear mutations, making it unlikely that it is ARPKD-associated. Human fibrocystin-L is predicted to be a large receptor protein (4243 aa; 466 kDa) with a signal peptide, single transmembrane domain and short cytoplasmic tail. Fibrocystin-L is homologous to fibrocystin throughout most of the extracellular region with overall identity of 25.0% and similarity of 41.5%. Fibrocystin-L has extracellular domains similar to fibrocystin with 14 copies of the TIG domain and two regions of significant homology to the protein TMEM2. Genomic sequence analysis identified no other full-length fibrocystin homologs in humans, mice or other sequenced organisms. The Fugu fish has a fibrocystin-L ortholog but no fibrocystin, suggesting that the newly identified protein may be the ancestral form. PKHDL1 and Pkhdl1 are widely expressed at a low level in most tissues but only detected in blood-derived cell-lines. Low level expression was detected in many primary immune cell subtypes but up-regulated specifically in T lymphocytes, following activation signals, suggesting a role in cellular immunity.

PKHDL1, a homolog of the autosomal recessive polycystic kidney disease gene, encodes a receptor with inducible T lymphocyte expression

Autosomal-recessive polycystic kidney disease (ARPKD) is caused by mutation to a large gene, PKHD1, encoding a putative receptor protein, fibrocystin. We have identified, through analysis of human genomic sequence, a PKHD1 homolog, PKHDL1, in chromosome region 8q23. The PKHDL1 transcript of 13081 bp was amplified as 16 fragments and sequenced; the sequence of the murine ortholog, Pkhdl1 (chromosome region 15B3) was also determined. PKHDL1 contains 78 exons, covers a genomic region of ∼168 kb and encodes a large protein, fibrocystin-L. Screening PKHDL1 in ARPKD patients with no PKHD1 mutations revealed several sequence variants but no clear mutations, making it unlikely that it is ARPKD-associated. Human fibrocystin-L is predicted to be a large receptor protein (4243 aa; 466 kDa) with a signal peptide, single transmembrane domain and short cytoplasmic tail. Fibrocystin-L is homologous to fibrocystin throughout most of the extracellular region with overall identity of 25.0% and similarity of 41.5%. Fibrocystin-L has extracellular domains similar to fibrocystin with 14 copies of the TIG domain and two regions of significant homology to the protein TMEM2. Genomic sequence analysis identified no other full-length fibrocystin homologs in humans, mice or other sequenced organisms. The Fugu fish has a fibrocystin-L ortholog but no fibrocystin, suggesting that the newly identified protein may be the ancestral form. PKHDL1 and Pkhdl1 are widely expressed at a low level in most tissues but only detected in blood-derived cell-lines. Low level expression was detected in many primary immune cell subtypes but up-regulated specifically in T lymphocytes, following activation signals, suggesting a role in cellular immunity.

Large deletions in the polycystic kidney disease 1 (PKD1) gene.

Since identification of the genes mutated in patients with Autosomal Dominant Polycystic Kidney Disease, PKD1 and PKD2, a large number of different germ line mutations in both genes have been found by conventional PCR-based mutation detection methods. Nevertheless, in approximately 40% of the PKD1 families the disease-causing mutation remains to be elucidated. Complex germ line rearrangements are often not detectable by these standard diagnostic techniques. To detect large deletions in the PKD1 gene we performed Field Inversion Gel Electrophoresis (FIGE) followed by Southern blot analysis with probes selected in the unique and in the reiterated region of this gene. Our analysis revealed 4 deletions in 125 patients, indicating that large deletions in PKD1 are rare. Likely, patients with a deletion that also affects the neighbouring Tuberous Sclerosis Complex 2 (TSC2) gene will be diagnosed as patients with tuberous sclerosis. It was speculated that the exceptional polypyrimidine tract located in intron 21 and the small tract in intron 22, might play a role in the pathogenesis of ADPKD. Since this region is extremely difficult to amplify by PCR, we analysed the 5.8 kb BamHI fragment that contains the polypyrimidine tracts. We did not observe a disease-linked alteration although we detected two different rare variants either in PKD1 or in one of its homologues.

Spectrum of mutations in the gene for autosomal recessive polycystic kidney disease (ARPKD/PKHD1).

Autosomal recessive polycystic kidney disease (ARPKD/PKHD1) is an important cause of renal-related and liver-related morbidity and mortality in childhood. Recently mutations in the PKHD1 gene on chromosome 6p21.1-p12 have been identified as the molecular cause of ARPKD. The longest continuous open reading frame (ORF) is encoded by a 67-exon transcript and predicted to yield a 4074-amino acid protein ("polyductin") of thus far unknown function. By now, a total of 29 different PKHD1 mutations have been described. This study reports mutation screening in 90 ARPKD patients and identifies mutations in 110 alleles making up a detection rate of 61%. Thirty-four of the detected mutations have not been reported previously. Two underlying mutations in 40 patients and one mutation in 30 cases are disclosed, and no mutation was detected on the remaining chromosomes. Mutations were found to be scattered throughout the gene without evidence of clustering at specific sites. About 45% of the changes were predicted to truncate the protein. All missense mutations were nonconservative, with the affected amino acid residues found to be conserved in the murine polyductin orthologue. One recurrent missense mutation (T36M) likely represents a mutational hotspot and occurs in a variety of populations. Two founder mutations (R496X and V3471G) make up about 60% of PKHD1 mutations in the Finnish population. Preliminary genotype-phenotype correlations could be established for the type of mutation rather than for the site of the individual mutation. All patients carrying two truncating mutations displayed a severe phenotype with perinatal or neonatal demise. PKHD1 mutation analysis is a powerful tool to establish the molecular cause of ARPKD in a given family. Direct identification of mutations allows an unequivocal diagnosis and accurate genetic counseling even in families displaying diagnostic challenges.

Sticky DNA: Effect of the Polypurine·Polypyrimidine Sequence

The polypurine.polypyrimidine sequence requirements for the formation of sticky DNA were evaluated in Escherichia coli plasmid systems to determine the potential occurrence of this conformation throughout biological systems. A mirror repeat, dinucleotide tract of (GA.TC)(37), which is ubiquitous in eukaryotes, formed sticky DNA, but shorter sequences of 10 or 20 repeats were inert. (GGA.TCC)(n) inserts (where n = 126, 159, and 222 bp) also formed sticky DNA. As shown previously, the control sequence (GAA.TTC)(150) (450 bp) readily adopted the X-shaped sticky structure; however, this structure has never been found for the nonpathogenic (GAAGGA.TCCTTC)(65) of the same approximate length (390 bp). A sequence that is replete with polypurine.polypyrimidine tracts that can form triplexes and slipped structures but lacks long repeating motifs (the 2.5-kbp intron 21 sequence from the polycystic kidney disease gene 1) was also inert. Interestingly, tracts of (GAA.TTC)(n) (where n = 176 or 80) readily formed sticky DNA with (GAAGGA.TCCTTC)(65) cloned into the same plasmid when the pair of inserts was in the direct, but not in the indirect (inverted), orientation. The stabilities of the triple base (Watson-Crick and Hoogsteen) interactions in the DNA/DNA associated triplex region of the sticky conformations account for these observations. Our results have significant chemical and biological implications for the structure and function of this unusual DNA conformation in Friedreich's ataxia.

Mutation detection in the duplicated region of the polycystic kidney disease 1 (PKD1) gene in PKD1-linked Australian families

Screening for disease-causing mutations in the duplicated region of the PKD1 gene was performed in 17 unrelated Australian individuals with PKD1-linked autosomal dominant polycystic kidney disease. Exons 2-21 and 23-34 were assayed using PKD1-specific PCR amplification and direct sequencing. We have identified 12 novel probably pathogenic DNA variants, including five truncating mutations (Q563X, c.5105delAT, c.5159delG, S2269X, c.9847delC), two in-frame deletions (c.7472del3, c.9292del39), and two splice-site mutations (IVS14+1G>C, IVS16+1G>T). Three of the mutations (G381C, Y2185D, G2785D) were predicted to lead to the replacement of conserved amino acid residues, with ensuing changes in protein conformation. Defects in the duplicated region of PKD1 thus account for 63% of our patients. Together with the previously detected mutations (Q4041X, R4227P) in the 3 region of the gene, the study has achieved an overall mutation detection rate of 74%. In addition, we have detected 31 variants (nine novel and 22 previously published) that did not segregate with the disease and were considered to be neutral polymorphisms. Three of the nine novel polymorphisms were missense mutations with a predicted effect on protein conformation, emphasizing the problems of interpretation in PKD1 mutation screening.

Genotypes of autosomal dominant polycystic kidney disease in Japanese.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common hereditary disorders. The prevalence of the ADPKD genotype in the Caucasian and Latin populations has been reported. Here, we used linkage analysis to demonstrate the prevalence of the genotype and the correlation between phenotypes and genotypes among 21 Japanese ADPKD families consisting of 96 individuals and including 57 affected members. Six polymorphic markers, each linked to either the polycystic kidney disease 1 (PKD1) or polycystic kidney disease 2 (PKD2) gene, were used for polymerase chain reaction analysis. Seventeen families (81%) showed linkage to PKD1, two families (10%) showed linkage to PKD2, and two families did not show linkage to either PKD1 or PKD2. One of the PKD1-linked families was indicated to have different mutations of PKD1 gene in the same family. PKD2-linked families did not have milder symptoms than PKD1-linked families.

Characterization of the region containing the jcpk PKD gene on mouse Chromosome 10

The jcpk gene on mouse Chromosome 10 causes a severe, early onset form of polycystic kidney disease (PKD) when inherited in an autosomal recessive manner. In order to positionally clone this gene, high resolution genetic and radiation hybrid maps were generated along with a detailed physical map of the approximately 500-kb region containing the jcpk gene. Additionally, sixty-nine kidney-specific ESTs were evaluated as candidates for jcpk and subsequently localized throughout the mouse genome by radiation hybrid mapping analysis. Previous studies indicating non-complementation of the jcpk mutation and 67Gso, a new PKD translocation mutant had suggested that 67Gso represents a new allele of jcpk. Fluoresence in situ hybridization (FISH) analysis using key bacterial artificial chromosome clones from the jcpk critical region, refined the 67Gso breakpoint and provided support for the allelism of jcpk and 67Gso.