Overexpression of PKD1 causes polycystic kidney disease

The pathogenetic mechanisms underlying autosomal dominant polycystic kidney disease (ADPKD) remain to be elucidated. While there is evidence that Pkd1 gene haploinsufficiency and loss of heterozygosity can cause cyst formation in mice, paradoxically high levels of Pkd1 expression have been detected in the kidneys of ADPKD patients. To determine whether Pkd1 gain of function can be a pathogenetic process, a Pkd1 bacterial artificial chromosome (Pkd1-BAC) was modified by homologous recombination to solely target a sustained Pkd1 expression preferentially to the adult kidney. Several transgenic lines were generated that specifically overexpressed the Pkd1 transgene in the kidneys 2- to 15-fold over Pkd1 endogenous levels. All transgenic mice reproducibly developed tubular and glomerular cysts and renal insufficiency and died of renal failure. This model demonstrates that overexpression of wild-type Pkd1 alone is sufficient to trigger cystogenesis resembling human ADPKD. Our results also uncovered a striking increased renal c-myc expression in mice from all transgenic lines, indicating that c-myc is a critical in vivo downstream effector of Pkd1 molecular pathways. This study not only produced an invaluable and first PKD model to evaluate molecular pathogenesis and therapies but also provides evidence that gain of function could be a pathogenetic mechanism in ADPKD.     

Human-mouse homologies in the region of the polycystic kidney disease gene (PKD1).

Autosomal dominant polycystic kidney disease (PKD1) is linked to the alpha-globin locus near the telomere of chromosome 16p. We established the existence of a conserved linkage group in mouse by mapping conserved sequences and cDNAs from the region surrounding the PKD1 gene in the mouse genome. Results obtained with the BXD recombinant strain system and somatic cell hybrids show the homologous region to be located on mouse chromosome 17 near the globin pseudogene Hba-ps4, an unprocessed alpha-like globin gene. The markers we mapped are widely distributed over the region known to contain the PKD1 gene, and it is therefore likely that the mouse homologue of PKD1 is also located on mouse chromosome 17.     

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.     

Novel mutations of the PKD1 gene in Korean patients with autosomal dominant polycystic kidney disease

The gene for the most common form of autosomal dominant polycystic kidney disease (ADPKD), PKD1, has recently been characterized and shown to encode an integral membrane protein, polycystin-1, which is involved in cell-cell and cell-matrix interactions. Until now, approximately 30 mutations of the 3' single copy region of the PKD1 gene have been reported in European and American populations. However, there is no report of mutations in Asian populations. Using the polymerase chain reaction and single-strand conformation polymorphism (SSCP) analysis, 91 Korean patients with ADPKD were screened for mutation in the 3' single copy region of the PKD1 gene. As a result, we have identified and characterized six mutations: three frameshift mutations (11548del8bp, 11674insG and 12722delT), a nonsense mutation (Q4010X), and two missense mutations (R3752W and D3814N). Five mutations except for Q4010X are reported here for the first time. Our findings also indicate that many different mutations are likely to be responsible for ADPKD in the Korean population. The detection of additional disease-causing PKD1 mutations will help in identifying the location of the important functional regions of polycystin-1 and help us to better understand the pathophysiology of ADPKD.     

Molecular cloning, expression and chromosomal localization of a novel human REG family gene, REG III

Regenerating gene (Reg), first isolated from a regenerating islet cDNA library, encodes a secretory protein with a growth stimulating effect on pancreatic beta cells that ameliorates the diabetes of 90% depancreatized rats and non-obese diabetic mice. Reg and Reg-related genes have been revealed to constitute a multigene family, the Reg family, which consists of four subtypes (types I, II, III, IV) based on the primary structures of the encoded proteins of the genes [Diabetes 51(Suppl. 3) (2002) S462]. Plural type III Reg genes were found in mouse and rat. On the other hand, only one type III REG gene, HIP/PAP (gene expressed in hepatocellular carcinoma-intestine-pancreas/gene encoding pancreatitis-associated protein), was found in human. In the present study, we found a novel human type III REG gene, REG III. This gene is divided into six exons spanning about 3 kilobase pairs (kb), and encodes a 175 amino acid (aa) protein with 85% homology with HIP/PAP. REG III was expressed predominantly in pancreas and testis, but not in small intestine, whereas HIP/PAP was expressed strongly in pancreas and small intestine. IL-6 responsive elements existed in the 5'-upstream region of the human REG III gene indicating that the human REG III gene might be induced during acute pancreatitis. All the human REG family genes identified so far (REG Ialpha, REG Ibeta, HIP/PAP, REG III and REG IV) have a common gene structure with 6 exons and 5 introns, and encode homologous 158-175-aa secretory proteins. By database searching and PCR analysis using a yeast artificial chromosome clone, the human REG family genes on chromosome 2, except for REG IV on chromosome 1, were mapped to a contiguous 140 kb region of the human chromosome 2p12. The gene order from centromere to telomere was 5' HIP/PAP 3'-5' RS 3'-3' REG Ialpha 5'-5' REG Ibeta 3'-3' REG III 5'. These results suggest that the human REG gene family is constituted from an ancestor gene by gene duplication and forms a gene cluster on the region.     

The human polycystic kidney disease 2-like (PKDL) gene: exon/intron structure and evidence for a novel splicing mechanism

Polycystin-L is a member of the expanding family of polycystins. Mutations in polycystin-1 or -2 cause human autosomal dominant polycystic kidney disease (ADPKD). The mouse ortholog of PKDL, Pkdl, is deleted in a mouse line with renal and retinal defects. We recently have shown that polycystin-L has calcium channel properties. In the current study, we determined the exon/intron organization of the PKDL gene and its alternative splicing. We show that PKDL has 16 exons. All splice acceptor/donor sites for these exons conform to the GT-AG rule. The positions of introns and the sizes of exons in the PKDL gene are very similar to those of PKD2, except for the last two 3′ end exons. RT-PCR demonstrates the existence of at least three polycystin-L splice variants: PKDL(Δ5), PKDL(Δ456), and PKDL(Δ15) that are expressed in a tissue-specific manner. In addition, we have localized polymorphic marker D10S603 to intron 4 and exon 5 of PKDL. Elucidation of the gene structure, exact location, and alternative splicing patterns of PKDL will facilitate its evaluation as a candidate gene in cystic or other genetic disorders. Received: 26 July 1999 / Accepted: 16 September 1999     

Detection of a novel nonsense mutation and an intragenic polymorphism in the PKD1 gene of a Cypriot family with autosomal dominant polycystic kidney disease

Mutations in the PKD1 gene on the short arm of chromosome 16 account for 85%–90% of polycystic kidney disease patients in the Caucasian population. After the recent characterization of the gene, we started a search for mutations in its 3′-end unique portion in Cypriot patients, by using the method of single-strand conformation polymorphism (SSCP). In one large family, we identified a nucleotide substitution at position 12 258 of the cDNA; this substitutes cysteine-4086 by a premature termination codon (C4086X). It has been inherited by every affected family member but not by unaffected members, nor by patients from 13 other Cypriot families. A new polymerase chain reaction (PCR) primer has been designed to engineer a novel DdeI recognition site upon PCR amplification, thereby allowing easy detection of the mutation by PCR-restriction digestion. The premature STOP codon is expected to remove 217 residues from the putative C-terminal intracellular domain of the gene product, polycystin and thus identifies this part as being critical to the production of the disease phenotype, possibly by interfering with the transmission of signals from the extracellular matrix to the cytoplasm. We also describe the identification of the first polymorphism within the encoding region of the gene. It is at alanine 4091, which is encoded by either GCA or GCG. With a heterozygosity of 35%, it should be extremely useful in informative families, especially because the gene lies in an unstable region and is prone to rearrangements. This polymorphism is readily detectable by PCR-restriction digestion with Bsp1286I. Received: 19 February 1996 / Revised: 20 April 1996     

Novel mutations in the 3' region of the polycystic kidney disease 1 (PKD1) gene

Mutation screening in 90 unrelated ADPKD1 patients was carried out on some of the exons in the single copy area (37, 38, 39, 44, 45) using genomic PCR and SSCP. Four novel mutations were found: a 15 bp in-frame deletion in exon 39 [nt11449 (del 15)], a 2 bp deletion in exon 44 [nt12252 (del 2)], a G insertion in exon 44 [nt12290 (Ins G)], and a GTT in-frame deletion in exon 45 [nt12601 (del 3)].     

Nucleotide sequence, chromosomal localization and developmental expression of the mouse int-1-related gene

cDNA clones encoding the murine int-1-related protein (m-irp) were isolated from an 8.5-day mouse embryo library. m-irp and its human counterpart, h-irp, share extensive nucleotide homology in coding (92%) and 3' untranslated (69%) regions. At the amino acid level, m-irp and h-irp share 97% of amino acids including all 24 cysteine residues, which are highly conserved among members of the int-1 family. However, in contrast to h-irp and int-1, the predicted m-irp protein sequence did not contain a signal peptide sequence. Analysis of polymerase chain reaction, amplified cDNA, and genomic sequences strongly suggests that a single-base substitution has created a new 5' splice site 17 bp 5' of a highly conserved splice site. Splicing at this new site generates a mRNA-encoding an amino-terminal truncated protein. Splicing at the conserved splice site generates a mRNA species encoding a protein with a signal peptide sequence similar to h-irp. Close linkage between m-irp and the met oncogene maps m-irp sequences to proximal mouse chromosome 6. Adult and fetal expression of m-irp was examined by RNA blot analysis. Adult expression of m-irp is restricted to lungs and heart, and fetal expression, to placental tissue and to all stages of fetal development examined. In situ hybridization localized early fetal m-irp expression to the pericardium of the heart, to the umbilicus and associated allantoic mesoderm, and to the ventral lateral mesenchyme tissue surrounding the umbilical vein in the fetus. These results suggest a role for m-irp in the development of fetal allantoic communication.     

The structure of a PKD domain from polycystin-1: implications for polycystic kidney disease.

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.     

Apoptosis and autophagy in polycystic kidney disease (PKD)

Apoptosis in the cystic epithelium is observed in most rodent models of polycystic kidney disease (PKD) and in human autosomal dominant PKD (ADPKD). Apoptosis inhibition decreases cyst growth, whereas induction of apoptosis in the kidney of Bcl-2 deficient mice increases proliferation of the tubular epithelium and subsequent cyst formation. However, alternative evidence indicates that both induction of apoptosis as well as increased overall rates of apoptosis are associated with decreased cyst growth. Autophagic flux is suppressed in cell, zebra fish and mouse models of PKD and suppressed autophagy is known to be associated with increased apoptosis. There may be a link between apoptosis and autophagy in PKD. The mammalian target of rapamycin (mTOR), B-cell lymphoma 2 (Bcl-2) and caspase pathways that are known to be dysregulated in PKD, are also known to regulate both autophagy and apoptosis. Induction of autophagy in cell and zebrafish models of PKD results in suppression of apoptosis and reduced cyst growth supporting the hypothesis autophagy induction may have a therapeutic role in decreasing cyst growth, perhaps by decreasing apoptosis and proliferation in PKD. Future research is needed to evaluate the effects of direct autophagy inducers on apoptosis in rodent PKD models, as well as the cause and effect relationship between autophagy, apoptosis and cyst growth in PKD.     

The human vigilin gene: identification,chromosomal localization and expression pattern

Chick vigilin cRNA clones were used to isolate the cognate human gene, by screening a pWE15 genomic library. Three independent cosmid clones were isolated and characterized by restriction mapping. The gene was identified by sequencing an internal EcoRI fragment containing two exons homologous to exon 24 and 25 of the chicken vigilin gene and corresponding to nucleotides 1973–2104 of the human HBP-cDNA. The homology between the chicken and human sequences was 77% and 82% at the cDNA level, and 91% and 100% at the amino acid level. In addition, the analyzed intron/exon boundaries were invariantly conserved. The 5 and 3 regions of the human gene were mapped by Southern analysis of the respective clones with synthetic oligonucleotides. The entire vigilin gene spans a region of about 50 kb and has been assigned to chromosome 2q36–q37.2 (FL-pter value of 0.96 ± 0.03) by fluorescence in situ hybridization to metaphase spreads from normal peripheral blood lymphocytes. The vigilin gene is localized in a chromosomal region comprising a cluster of collagen genes (COLIVA3, COLVIA3) and the locus of the Waardenburg syndrome I. Only one mRNA species of 4.4 kb is transcribed from the human vigilin gene. In accordance with previous observations on chicken mRNA, the expression of the human vigilin mRNA depends on the stage of cytodifferentiation both in vitro and in situ.     

Placental expression and chromosomal localization of the human Gcm 1 gene.

Although gcm was first recognized for its role in specifying glial cell fate in Drosophila melanogaster, its mammalian counterparts are expressed predominantly in non-neural tissues. Here we demonstrate expression of the mouse and human GCM 1 proteins in placenta. We have prepared a highly specific antibody that recognizes the GCM 1 protein and have used it to assess the temporal and spatial expression profile of the protein. In both mouse and human placenta, the protein is associated with cells that are involved with exchange between maternal and fetal blood supplies: the labyrinthine cells of the mouse placenta and the syncytio- and cytotrophoblasts of the human placenta. Using the full-length hGcm 1 cDNA as a probe, we have mapped the gene on human chromosome 6p12 by fluorescent in situ hybridization.     

The human glucagon receptor encoding gene: structure, cDNA sequence and chromosomal localization

Characterization of the human glucagon-receptor-encoding gene (GGR) should provide a greater understanding of blood glucose regulation and may reveal a genetic basis for the pathogenesis of diabetes. A cDNA encoding a complete functional human glucagon receptor (GGR) was isolated from a liver cDNA library by a combination of polymerase chain reaction and colony hybridization. The cDNA encodes a receptor protein with 80% identity to rat GGR that binds [¹²⁵I] glucagon and transduces a signal leading to increases in the concentration of intracellular cyclic adenosine 3′,5′-monophosphate. Southern blot analysis of human DNA reveals a hybridization pattern consistent with a single GGR locus. In situ hybridization to metaphase chromosome preparations maps the GGR locus to chromosome 17q25. Analysis of the genomic sequence shows that the coding region spans over 5.5 kb and is interrupted by 12 introns.     

Fine genetic localization of the gene for autosomal dominant polycystic kidney disease (PKD1) with respect to physically mapped markers.

PKD1, the gene for the chromosome 16-linked form of autosomal dominant polycystic kidney disease, has previously been genetically mapped to an interval bounded by the polymorphic loci Fr3-42/EKMDA2 distally and O327hb/O90a proximally. More recently, 26.6PROX was identified as the closest proximal flanking locus. We set out to refine the localization of PKD1 by identifying a series of single recombinant events between the flanking markers Fr3-42/EKMDA2 and O327hb/O90a and analyzing them with a new set of polymorphic loci that have been physically mapped within the PKD1 interval. We identified 11 such crossovers in eight families; 6 of these fell into the interval between GGG1 and 26.6PROX, a distance of less than 750 kb. Three of these crossovers placed PKD1 proximal to GGG1 and two crossovers placed PKD1 distal to 26.6PROX. Both of the latter also placed PKD1 telomeric to a locus 92.6SH1.0, which lies 200-250 kb distal to 26.6PROX. The sixth recombinant, however, placed the disease mutation proximal to the locus 92.6SH1.0. Several possible explanations for these observations are discussed. An intensive study to locate deletions, insertions, and other chromosomal rearrangements associated with PKD1 mutations failed to detect any such abnormalities. Thus we have defined, in genetic and physical terms, the segment of 16p13.3 where PKD1 resides and conclude that a gene-by-gene analysis of the region will be necessary to identify the mutation(s).     

Construction of a transgenic pig model overexpressing polycystic kidney disease 2 (PKD2) gene

Autosomal dominant polycystic kidney disease (ADPKD) is a common human genetic disease, affecting millions of people worldwide. The progressive growth of cysts in kidneys eventually leads to renal failure in 50 % of patients, and there is currently no effective treatment. Various murine models have been studied to elucidate the disease mechanisms, and much information has been acquired. However, the course of the disease cannot be fully recapitulated using these models. The pig is a suitable model for biomedical research, and pig PKD2 has high similarity to the human ortholog at the molecular level. Here, a mini-pig PKD2 transgenic model was generated, driven by a ubiquitous cytomegalovirus enhancer/promoter. Using somatic cell nuclear transfer, four transgenic pigs with approximately 10 insertion events each were generated. Quantitative real-time PCR and western blotting showed that PKD2 was more highly expressed in transgenic pigs than in wild-type counterparts. Because of the chronic nature of ADPKD, blood urea nitrogen and serum creatinine levels were continuously measured to assess the pig kidney function. The transgenic pigs continue to show no significant alteration in kidney function; it is estimated that 1–2 more years may be required for manifestation of renal cystogenesis in these pigs.