Luiz F. Onuchic

PKHD1, the Polycystic Kidney and Hepatic Disease 1 Gene, Encodes a Novel Large Protein Containing Multiple Immunoglobulin-Like Plexin-Transcription–Factor Domains and Parallel Beta-Helix 1 Repeats

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.

Polycystin-1, the gene product of PKD1, induces resistance to apoptosis and spontaneous tubulogenesis in MDCK cells.

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.

Identification of a novel cytokine, ML-1, and its expression in subjects with asthma.

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.

Identification and Characterization of Pkhd1, the Mouse Orthologue of the Human ARPKD Gene

PKHD1, the gene mutated in human autosomal recessive polycystic kidney disease has recently been identified. Its translation products are predicted to belong to a superfamily of proteins involved in the regulation of cellular adhesion and repulsion. One notable aspect of the gene is its unusually complex pattern of splicing. This study shows that mouse Pkhd1 and its translation products have very similar properties to its human orthologue. Mouse Pkhd1 extends over approximately 500 kb of genomic DNA, includes a minimum of 68 nonoverlapping exons, and exhibits a complex pattern of splicing. The longest ORF encodes a protein of 4059aa predicted to have an N-terminal signal peptide, multiple IPTs and PbH1 repeats, a single transmembrane span (TM), and a short cytoplasmic C-terminus. Although the protein sequence is generally well conserved (approximately 73% average identity), the C-termini share only 55% identity. The pattern of Pkhd1 expression by in situ hybridization was also examined in developing and adult mouse tissues over a range of ages (E12.5 to 3 mo postnatal). High levels of expression were present in renal and biliary tubular structures at all time points examined. Prominent Pkhd1 signals were also found in a number of other organs and tissues. Tissue-specific differences in transcript expression were revealed through the use of single exon probes. These data show that key features of human PKHD1 are highly conserved in the mouse and suggest that the complicated pattern of splicing is likely to be functionally important.

Milder Presentation of Recessive Polycystic Kidney Disease Requires Presence of Amino Acid Substitution Mutations

Autosomal recessive polycystic kidney disease (ARPKD; MIM 263200) is a hereditary and severe form of polycystic disease affecting the kidneys and biliary tract with an estimated incidence of 1 in 20,000 live births. The clinical spectrum is widely variable: up to 50% of affected neonates die shortly after birth, whereas others survive to adulthood. Mutations at a single locus, polycystic kidney and hepatic disease 1 (PKHD1), are responsible for all typical forms of ARPKD. Mutation detection was performed in PKHD1 by DHPLC in 85 affected, unrelated individuals. Seventy-four amplicons were amplified and analyzed from the PKHD1 genomic locus. Sequence variants were considered pathogenic when they were not observed in 160 control individuals (320 chromosomes). For purposes of genotype-phenotype comparisons, families were stratified by clinical presentation into two groups: the severe perinatal group, in which at least one affected child presented with perinatal disease and neonatal demise, and the less severe, nonperinatal group, in which none of the affected children died in the neonatal period. Forty-one mutations were found in 55 affected disease chromosomes; 32 of these mutations have not been reported previously. Mutations were distributed throughout the portions of gene encoding the predicted extracellular portion of the protein product. The most commonly encountered mutation, T36M, was found in 8 of 55 disease chromosomes. Amino acid substitutions were found to be more commonly associated with a nonlethal presentation, whereas chain terminating mutations were more commonly associated with neonatal demise (chi(2) = 11.54, P = 0.003). All patients who survive the neonatal period have at least one amino acid substitution mutation, suggesting that such substitutions produce milder disease through production of partially functional protein products. The nature of the germline mutations in ARPKD plays a significant role in determining clinical outcome.

Comprehensive genomic analysis of PKHD1 mutations in ARPKD cohorts

Autosomal recessive polycystic kidney disease (ARPKD; MIM 263200) is an important childhood nephropathy, occurring in 1 in 20 000 live births.1 The clinical phenotype is dominated by dilatation of the renal collecting ducts, biliary dysgenesis, and portal tract fibrosis. Affected children often present in utero with enlarged, echogenic kidneys, as well as oligohydramnios secondary to poor urine output. Approximately 30% of affected neonates die shortly after birth as a result of severe pulmonary hypoplasia and secondary respiratory insufficiency. Those who survive the perinatal period express widely variable disease phenotypes with systemic hypertension, renal insufficiency, and portal hypertension due to portal tract fibrosis as the most common clinical features.2 Linkage analysis indicates that mutations in a single locus on chromosome 6p12 are responsible for all typical forms of ARPKD.3,4 Two groups working independently have identified PKHD1 (MIM 606702) as the locus responsible for ARPKD and have demonstrated that this novel gene is among the largest in the human genome, extending over at least 470 kb and including a minimum of 86 exons.5,6 Both PKHD1 and its mouse orthologue ( Pkhd1 ) encode a complex and extensive array of splice variants, with most abundant transcriptional expression in fetal and adult kidney and weaker expression in other tissues including liver and pancreas.5,7 The longest PKHD1 transcript includes 67 exons with an open reading frame (ORF) composed of 66 exons that encode a 4074 amino acid protein, polyductin/fibrocystin.5,6 The full length protein is predicted to have several immunoglobulin-like, plexin, transcription factor (IPT) domains and multiple parallel beta-helix 1 (PbH1) repeats in its approximately 3860 amino acid extracellular amino terminus; a single transmembrane (TM) spanning domain; and a short, cytoplasmic carboxyl terminus with potential phosphorylation sites. Alternatively spliced transcripts are predicted to fall into two broad groups. …

Biliary and Pancreatic Dysgenesis in Mice Harboring a Mutation in Pkhd1

Autosomal recessive polycystic kidney disease is a hereditary fibrocystic disease that involves the kidneys and the biliary tract. Mutations in the PKHD1 gene are responsible for typical forms of autosomal recessive polycystic kidney disease. We have generated a mouse model with targeted mutation of Pkhd1 by disrupting exon 4, resulting in a mutant transcript with deletion of 66 codons and expression at approximately 30% of wild-type levels. Pkhd1(del4/del4) mice develop intrahepatic bile duct proliferation with progressive cyst formation and associated periportal fibrosis. In addition, these mice exhibit extrahepatic manifestations, including pancreatic cysts, splenomegaly, and common bile duct dilation. The kidneys are unaffected both histologically and functionally. Fibrocystin is expressed in the apical membranes and cilia of bile ducts and distal nephron segments but is absent from the proximal tubule. This pattern is unchanged in orthologous models of autosomal dominant polycystic kidney disease due to mutation in Pkd1 or Pkd2. Mutant fibrocystin in Pkhd1(del4/del4) mice also retains this expression pattern. The hypomorphic Pkhd1(del4/del4) mouse model provides evidence that reduced functional levels of fibrocystin are sufficient for cystogenesis and fibrosis in the liver and pancreas, but not the kidney, and supports the hypothesis of species-dependent differences in susceptibility of tissues to Pkhd1 mutations.

Pkd1 Haploinsufficiency Increases Renal Damage and Induces Microcyst Formation following Ischemia/Reperfusion

Mutations in PKD1 cause the majority of cases of autosomal dominant polycystic kidney disease (ADPKD). Because polycystin 1 modulates cell proliferation, cell differentiation, and apoptosis, its lower biologic activity observed in ADPKD might influence the degree of injury after renal ischemia/reperfusion. We induced renal ischemia/reperfusion in 10- to 12-wk-old male noncystic Pkd1(+/-) and wild-type mice. Compared with wild-type mice, heterozygous mice had higher fractional excretions of sodium and potassium and higher serum creatinine after 48 h. In addition, in heterozygous mice, also cortical damage, rates of apoptosis, and inflammatory infiltration into the interstitium at time points out to 14 d after injury all increased, as well as cell proliferation at 48 h and 7 d. The mRNA and protein expression of p21 was lower in heterozygous mice than wild-type mice at 48 h. After 6 wk, we observed dilated tubules, microcysts, and increased renal fibrosis in heterozygotes. The early mortality of heterozygotes was significantly higher than that of wild-type mice when we extended the duration of ischemia from 32 to 35 min. In conclusion, ischemia/reperfusion induces a more severe injury in kidneys of Pkd1-haploinsufficient mice, a process that apparently depends on a relative deficiency of p21 activity, tubular dilation, and microcyst formation. These data suggest the possibility that humans with ADPKD from PKD1 mutations may be at greater risk for damage from renal ischemia/reperfusion injury.

Renal cyst growth is the main determinant for hypertension and concentrating deficit in Pkd1-deficient mice

We have bred a Pkd1 floxed allele with a nestin-Cre expressing line to generate cystic mice with preserved GFR to address the pathogenesis of complex ADPKD phenotypes. Hypertension affects about 60% of these patients before loss of renal function, leading to significant morbimortality. Cystic mice were hypertensive at 5 and 13 weeks of age, a phenotype not seen in non-cystic controls and Pkd1-haploinsufficient animals, which do not develop renal cysts. Fractional sodium excretion was reduced in cystic mice at these ages. Angiotensinogen gene expression was higher in cystic than non-cystic kidneys at 18 weeks, while ACE and the AT1 receptor were expressed in renal cyst epithelia. Cystic animals displayed increased renal cAMP, cell proliferation and apoptosis. At 24 weeks mean arterial pressure and fractional sodium excretion did not significantly differ between the cystic and non-cystic groups, whereas cardiac mass increased in cystic mice. Renal concentrating deficit is also an early finding in ADPKD. Maximum urine osmolality and urine nitrite excretion were reduced in 10–13 and 24-week-old cystic mice, deficits not found in haploinsufficient and non-cystic controls. A trend of higher plasma vasopressin was observed in cystic mice. Thus, cyst growth most probably plays a central role in early-stage ADPKD-associated hypertension, with activation of the intrarenal renin-angiotensin system as a key mechanism. Cyst expansion is also likely essential for the development of the concentrating deficit in this disease. Our findings are consistent with areas of reduced perfusion in the kidneys of patients with ADPKD.

Immunohistochemical detection of polyductin and co-localization with liver progenitor cell markers during normal and abnormal development of the intrahepatic biliary system and in adult hepatobiliary carcinomas

The longest open reading frame of PKHD1 (polycystic kidney and hepatic disease 1), the autosomal recessive polycystic kidney disease (ARPKD) gene, encodes a single‐pass, integral membrane protein named polyductin or fibrocystin. A fusion protein comprising its intracellular C‐terminus, FP2, was previously used to raise a polyclonal antiserum shown to detect polyductin in several human tissues, including liver. In the current study, we aimed to investigate by immunohistochemistry the detailed polyductin localization pattern in normal (ductal plate [DP], remodelling ductal plate [RDP], remodelled bile ducts) and abnormal development of the primitive intrahepatic biliary system, known as ductal plate malformation (DPM). This work also included the characterization of polyductin expression profile in various histological forms of neonatal and infantile cholestasis, and in cholangiocellular carcinoma (CCC) and hepatocellular carcinoma (HCC). We detected polyductin expression in the intrahepatic biliary system during the DP and the RDP stages as well as in DPM. No specific staining was found at the stage of remodelled bile ducts. Polyductin was also detected in liver biopsies with neonatal cholestasis, including mainly biliary atresia and neonatal hepatitis with ductular reaction as well as congenital hepatic fibrosis. In addition, polyductin was present in CCC, whereas it was absent in HCC. Polyductin was also co‐localized in some DP cells together with oval stem cell markers. These results represent the first systematic study of polyductin expression in human pathologies associated with abnormal development of intrahepatic biliary tree, and support the following conclusions: (i) polyductin expression mirrors developmental properties of the primitive intrahepatic biliary system; (ii) polyductin is re‐expressed in pathological conditions associated with DPM and (iii) polyductin might be a potential marker to distinguish CCC from HCC.