Autosomal recessive polycystic kidney disease (ARPKD) is a severe form of polycystic kidney disease that presents primarily in infancy and childhood and that is characterized by enlarged kidneys and congenital hepatic fibrosis. We have identified PKHD1, the gene mutated in ARPKD. PKHD1 extends over > or =469 kb, is primarily expressed in human fetal and adult kidney, and includes a minimum of 86 exons that are variably assembled into a number of alternatively spliced transcripts. The longest continuous open reading frame encodes a 4,074-amino-acid protein, polyductin, that is predicted to have a single transmembrane (TM)-spanning domain near its carboxyl terminus, immunoglobulin-like plexin-transcription-factor domains, and parallel beta-helix 1 repeats in its amino terminus. Several transcripts encode truncated products that lack the TM and that may be secreted if translated. The PKHD1-gene products are members of a novel class of proteins that share structural features with hepatocyte growth-factor receptor and plexins and that belong to a superfamily of proteins involved in regulation of cell proliferation and of cellular adhesion and repulsion.

Autosomal dominant polycystic kidney disease (ADPKD) is a common disease and an important cause of renal failure. It is characterized by considerable intrafamilial phenotypic variation and focal cyst formation. To elucidate the molecular basis for these observations, we have developed a novel method for isolating renal cystic epithelia from single cysts and have used it to show that individual renal cysts in ADPKD are monoclonal. Loss of heterozygosity was discovered within a subset of cysts for two closely linked polymorphic markers located within the PKD1 gene. Genetic analysis revealed that it was the normal haplotype that was lost. This study provides a molecular explanation for the focal nature of cyst formation and a probable mechanism whereby mutations cause disease. The high rate at which "second hits" must occur to account for the large number of cysts observed suggests that unique structural features of the PKD1 gene may be responsible for its mutability.

Abstract. 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. cbergmann@ukaachen.de

The major form of autosomal dominant polycystic kidney disease (ADPKD) results from mutation of a gene (PKD1) of unknown function that is essential for the later stages of renal tubular differentiation. In this report, we describe a novel cell culture system for studying how PKD1 regulates this process. We show that expression of human PKD1 in MDCK cells slows their growth and protects them from programmed cell death. MDCK cells expressing PKD1 also spontaneously form branching tubules while control cells form simple cysts. Increased cell proliferation and apoptosis have been implicated in the pathogenesis of cystic diseases. Our study suggests that PKD1 may function to regulate both pathways, allowing cells to enter a differentiation pathway that results in tubule formation.

A novel gene, designated ML-1, was identified from a human genomic DNA clone and human T cell cDNA sequences. The second exon of ML-1 gene shares significant sequence identity with the gene encoding IL-17 (IL-17). ML-1 gene expression was up-regulated in activated PBMCs, CD4+ T cells, allergen-specific Th0, Th1, and Th2 clones, activated basophils, and mast cells. Increased expression of the ML-1 gene, but not IL-17, was seen following allergen challenge in four asthmatic subjects, suggesting its role in allergic inflammatory responses. ML-1 from transiently transfected COS-7 cells was able to induce gene expression and protein production for IL-6 and IL-8 (at 10 ng/ml of ML-1: for IL-6, 599.6 ± 19.1 pg/ml; for IL-8, 1724.2 ± 132.9 pg/ml; and at 100 ng/ml of ML-1: for IL-6, 1005.3 ± 55.6 pg/ml; for IL-8, 4371.4 ± 280.5 pg/ml; p < 0.05 for both doses vs baseline) in primary bronchial epithelial (PBE) cells. Furthermore, increased expression of ICAM-1 was found in ML-1-stimulated PBE cells (mean fluorescence intensity (MFI) = 31.42 ± 4.39 vs baseline, MFI = 12.26 ± 1.77, p < 0.05), a functional feature distinct from IL-17 (MFI = 11.07 ± 1.22). This effect was not inhibited by a saturating amount of IL-17. These findings demonstrate that ML-1 is a novel cytokine with a distinct function, and suggest a different receptor for ML-1 on PBE cells.

The primary structure of polycystin predicts a large integral membrane protein with multiple cell recognition motifs, but its function remains unknown. Insight into polycystin's normal function and its role in the development of autosomal dominant polycystic kidney disease (PKD1) requires the assembly of an extensive collection of molecular reagents to examine its expression and create model systems for functional studies. Development of these crucial reagents has been complicated due to the presence of transcriptionally active homologous loci. We have assembled the authentic full-length PKD1 cDNA and demonstrated expression of polycystin in vitro. Polyclonal antibodies directed against distinct extra-and intracellular domains specifically immunoprecipitated in vitro translated polycystin. The panel of antibodies was used to determine localization of polycystin in renal epithelial and endothelial cell lines and tissues of fetal, adult, and cystic origins. In normal adult kidney and maturing fetal nephrons, polycystin expression was confined to epithelial cells of the distal nephron and vascular endothelial cells. Expression in the proximal nephron was only observed after injury-induced cell proliferation. Polycystin expression was confined to ductal epithelium in liver, pancreas, and breast, and restricted to astrocytes in normal brain. We report clear evidence for the membrane localization of polycystin by both tissue sections and by confocal microscopy in cultured renal and endothelial cells. Interestingly, when cultured cells made cell-cell contact, polycystin was localized to the lateral membranes of cells in contact. These data suggest that polycystin is likely to have a widespread role in epithelial cell differentiation and maturation and in cell-cell interactions.

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.

Polycystic kidney disease (PKD) describes a heterogeneous collection of disorders that differ significantly with respect to their etiology and clinical presentation. They share, however, abnormal tubular morphology as a common feature, leading to the hypothesis that their respective gene products may function cooperatively in a common pathway to maintain tubular integrity. To study the pathobiology of one major form of human PKD, we generated a mouse line with a floxed allele of Pkhd1, the orthologue of the gene mutated in human autosomal recessive PKD. Cre-mediated excision of exons 3-4 results in a probable hypomorphic allele. Pkhd1(del3-4/del3-4) developed a range of phenotypes that recapitulate key features of the human disease. Like in humans, abnormalities of the biliary tract were an invariant finding. Most mice 6 months or older also developed renal cysts. Subsets of animals presented with either perinatal respiratory failure or exhibited growth retardation that was not due to the renal disease. We then tested for genetic interaction between Pkhd1 and Pkd1, the mouse orthologue of the gene most commonly linked to human autosomal dominant PKD. Pkd1(+/-); Pkhd1(del3-4/del3-4) mice had markedly more severe disease than Pkd1(+/+); Pkhd1(del3-4/del3-4) littermates. These studies are the first to show genetic interaction between the major loci responsible for human renal cystic disease in a common PKD pathway.