Roles of HNF-1β in kidney development and congenital cystic diseases

Roles of HNF-1β in kidney development and congenital cystic diseases. Hepatocyte nuclear factor-1β (HNF-1β) is a Pit-1/Oct-1/Unc-86 (POU)/homeodomain-containing transcription factor that regulates tissue-specific gene expression in the kidney, liver, pancreas, and other epithelial organs. Mutations of HNF-1β produce maturity-onset diabetes of the young type 5 (MODY5) and are associated with congenital cystic abnormalities of the kidney. Transgenic mice expressing mutant HNF-1β under the control of a kidney-specific promoter develop kidney cysts and renal failure, which is similar to the phenotype of humans with MODY5. Similarly, kidney-specific deletion of HNF-1β using Cre/loxP recombination results in renal cyst formation. HNF-1β directly regulates the Pkhd1 promoter. HNF-1β mutant mice show decreased expression of Pkhd1, the gene that is mutated in humans with autosomal-recessive polycystic kidney disease (ARPKD). These studies demonstrate that HNF-1β is required for the development of the mammalian kidney. They establish a previously unrecognized link between two renal cystic diseases, MODY5 and ARPKD, and suggest that the mechanism of cyst formation in humans with mutations of HNF-1β involves down-regulation of PKHD1 gene transcription. Roles of HNF-1β in kidney development and congenital cystic diseases. Hepatocyte nuclear factor-1β (HNF-1β) is a Pit-1/Oct-1/Unc-86 (POU)/homeodomain-containing transcription factor that regulates tissue-specific gene expression in the kidney, liver, pancreas, and other epithelial organs. Mutations of HNF-1β produce maturity-onset diabetes of the young type 5 (MODY5) and are associated with congenital cystic abnormalities of the kidney. Transgenic mice expressing mutant HNF-1β under the control of a kidney-specific promoter develop kidney cysts and renal failure, which is similar to the phenotype of humans with MODY5. Similarly, kidney-specific deletion of HNF-1β using Cre/loxP recombination results in renal cyst formation. HNF-1β directly regulates the Pkhd1 promoter. HNF-1β mutant mice show decreased expression of Pkhd1, the gene that is mutated in humans with autosomal-recessive polycystic kidney disease (ARPKD). These studies demonstrate that HNF-1β is required for the development of the mammalian kidney. They establish a previously unrecognized link between two renal cystic diseases, MODY5 and ARPKD, and suggest that the mechanism of cyst formation in humans with mutations of HNF-1β involves down-regulation of PKHD1 gene transcription. Hepatocyte nuclear factor-1β (HNF-1β, vHNF1) is a transcription factor that regulates tissue-specific gene expression in the kidney, liver, pancreas, and other organs[1.Mendel D.B. Hansen L.P. Graves M.K. et al.HNF-1α and HNF-1β (vHNF-1) share dimerization and homeo domains, but not activation domains, and form heterodimers in vitro.Genes Dev. 1991; 5: 1042-1056Crossref PubMed Scopus (241) Google Scholar],[2.Rey-Campos J. Chouard T. Yaniv M. et al.vHNF1 is a homeoprotein that activates transcription and forms heterodimers with HNF1.Embo J. 1991; 10: 1445-1457Crossref PubMed Scopus (236) Google Scholar]. HNF-1β and the related family member, HNF-1α, have a similar structure consisting of an N-terminal dimerization domain, a Pit-1/Oct-1/Unc-86 (POU) domain and homeodomain that mediate DNA binding, and a C-terminal transcriptional activation domain Figure 1. HNF-1α and HNF-1β recognize the consensus sequence 5′-GTTAATNATTAAC-3′ and bind to DNA as homodimers or heterodimers. In general, HNF-1α and HNF-1β bind to the promoters of target genes and activate transcription, although transcriptional repression has also been reported. HNF-1α and HNF-1β were first discovered because of their roles in liver-specific gene regulation, but both proteins are also expressed in other epithelial organs, including the kidney. HNF-1α is expressed in renal proximal tubules, whereas HNF-1β is expressed in tubular epithelial cells in all segments of the nephron and in renal collecting ducts[3.Pontoglio M. Barra J. Hadchouel M. et al.Hepatocyte nuclear factor 1 inactivation results in hepatic dysfunction, phenylketonuria, and renal Fanconi syndrome.Cell. 1996; 84: 575-585Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar],[4.Coffinier C. Barra J. Babinet C. et al.Expression of the vHNF1/HNF1β homeoprotein gene during mouse organogenesis.Mech Dev. 1999; 89: 211-213Crossref PubMed Scopus (170) Google Scholar]. Neither transcription factor is expressed in glomerular tufts, blood vessels, or interstitial cells. During embryonic development, HNF-1β is expressed in the branching ureteric bud that will give rise to the renal collecting system and ureter as well as in comma- and S-shaped bodies that will form the nephrons proper. HNF-1α is restricted to late S-shaped bodies and developing proximal tubules. HNF-1β is also expressed in the mesonephric (Wolffian) duct, which is the anlage of the vas deferens and epididymis in males, and the Müllerian duct, which is the anlage of the oviduct, uterus, and cervix in females. HNF-1α regulates the tissue-specific expression of genes that encode transport proteins in the renal proximal tubule. Knockout mice that lack HNF-1α have morphologically normal kidneys but develop glucosuria due to decreased expression of the sodium/glucose cotransporter, Sglt2[3.Pontoglio M. Barra J. Hadchouel M. et al.Hepatocyte nuclear factor 1 inactivation results in hepatic dysfunction, phenylketonuria, and renal Fanconi syndrome.Cell. 1996; 84: 575-585Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar],[5.Pontoglio M. Prie D. Cheret C. et al.HNF1α controls renal glucose reabsorption in mouse and man.EMBO Rep. 2000; 11: 359-365Crossref Scopus (164) Google Scholar]. Defects in the renal reabsorption of glucose are also seen in humans with mutations of HNF-1α. HNF-1α knockout mice also exhibit phosphaturia, which has been attributed to decreased expression of Npt1 and Npt4 in the proximal tubule[6.Cheret C. Doyen A. Yaniv M. et al.Hepatocyte nuclear factor 1α controls renal expression of the Npt1-Npt4 anionic transporter locus.J Mol Biol. 2002; 322: 929-941Crossref PubMed Scopus (41) Google Scholar]. In contrast to HNF-1α, the functions of HNF-1β in the kidney have been poorly understood. HNF-1β recognizes the same DNA sequence as HNF-1α and could also be involved in kidney-specific gene regulation. For example, HNF-1β binding sites have been identified in the promoters of the genes encoding kidney-specific Ksp-cadherin (Cdh16) and Na-K-Cl cotransporter (Nkcc2)[7.Bai Y. Pontoglio M. Hiesberger T. et al.Regulation of kidney-specific Ksp-cadherin gene promoter by hepatocyte nuclear factor-1β.Am J Physiol. 2002; 283: F839-F851Crossref PubMed Scopus (43) Google Scholar] [abstract; Whyte DA, et al, J Am Soc Nephrol 6:713A, 1995]. Studies in lower vertebrates suggest that HNF-1β plays an important role in nephrogenesis. Transgenic expression of mutant HNF-1β in Xenopus embryos leads to abnormal development of the pronephros[8.Wild W. Von STRANDMANN E.P. Nastos A. et al.The mutated human gene encoding hepatocyte nuclear factor 1β inhibits kidney formation in developing Xenopus embryos.Proc Natl Acad Sci USA. 2000; 97: 4695-4700Crossref PubMed Scopus (65) Google Scholar],[9.Bohn S. Thomas H. Turan G. et al.Distinct molecular and morphogenetic properties of mutations in the human HNF1β gene that lead to defective kidney development.J Am Soc Nephrol. 2003; 14: 2033-2041Crossref PubMed Scopus (59) Google Scholar], and mutation of zebrafish HNF-1β produces cysts in the pronephric tubules[10.Sun Z. Hopkins N. vhnf1, the MODY5 and familial GCKD-associated gene, regulates regional specification of the zebrafish gut, pronephros, and hindbrain.Genes Dev. 2001; 15: 3217-3229Crossref PubMed Scopus (170) Google Scholar]. However, the roles of HNF-1β in the development of the mammalian kidney have not been apparent because HNF-1β knockout mice die prior to renal organogenesis[11.Barbacci E. Reber M. Ott M-O. et al.Variant hepatocyte nuclear factor 1 is required for visceral endoderm specification.Development. 1999; 126: 4795-4805Crossref PubMed Google Scholar],[12.Coffinier C. Thepot D. Babinet C. et al.Essential role for the homeoprotein vHNF1/HNF1β in visceral endoderm differentiation.Development. 1999; 126: 4785-4794PubMed Google Scholar]. Mutations of HNF-1β in humans produce maturity-onset diabetes of the young, type 5 (MODY5). MODY5 is a rare familial form of type 2 diabetes mellitus that is characterized by autosomal-dominant inheritance, early age of onset, impaired insulin secretion, and absence of obesity[13.Fajans S.S. Bell G.I. Polonsky K.S. Molecular mechanisms and clinical pathophysiology of maturity-onset diabetes of the young.N Engl J Med. 2001; 345: 971-980Crossref PubMed Scopus (812) Google Scholar]. MODY5 is frequently, perhaps invariably, associated with congenital abnormalities of the kidney and genitourinary tract[14.Bingham C. Hattersley A.T. Renal cysts and diabetes syndrome resulting from mutations in hepatocyte nuclear factor-1β.Nephrol Dial Transplant. 2004; 19: 2703-2708Crossref PubMed Scopus (147) Google Scholar],[15.Bellanne-Chantelot C. Chauveau D. Gautier J-F. et al.Clinical spectrum associated with hepatocyte nuclear factor-1β mutations.Ann Intern Med. 2004; 140: 510-517Crossref PubMed Scopus (268) Google Scholar]. The most common kidney abnormalities involve the formation of cysts in the renal tubules, and the acronym RCAD (renal cysts and diabetes) has been coined to describe this syndrome. The spectrum of congenital renal cystic abnormalities includes simple cysts, multicystic renal dysplasia, and hypoplastic glomerulocystic kidney disease. Renal involvement is often severe and can lead to kidney failure. In addition to cysts, other kidney abnormalities that have been observed in MODY5 include oligomeganephronia, familial juvenile hyperuricemic nephropathy, and renal agenesis or hypoplasia[16.Bingham C. Ellard S. Van'T Hoff W.G. et al.Atypical familial juvenile hyperuricemic nephropathy associated with a hepatocyte nuclear factor-1β gene mutation.Kidney Int. 2003; 63: 1645-1651Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar]. Abnormalities of the male and female genital tract are also seen in some patients. To date, more than 30 different mutations of HNF-1β have been identified in humans with MODY5, some of which are shown in Figure 1. The molecular mechanism by which mutation of one allele of HNF-1β produces disease has been controversial. The A263fsinsGG mutation affects the DNA-binding domain and has been shown to produce a dominant-negative mutant[17.Tomura H. Nishigori H. Sho K. et al.Loss-of-function and dominant-negative mechanisms associated with hepatocyte nuclear factor-1β mutations in familial type 2 diabetes mellitus.J Biol Chem. 1999; 274: 12975-12978Crossref PubMed Scopus (40) Google Scholar], whereas the P328L329fsdelCCTCT mutation deletes the C-terminal domain and has been reported to produce a gain-of-function mutant[8.Wild W. Von STRANDMANN E.P. Nastos A. et al.The mutated human gene encoding hepatocyte nuclear factor 1β inhibits kidney formation in developing Xenopus embryos.Proc Natl Acad Sci USA. 2000; 97: 4695-4700Crossref PubMed Scopus (65) Google Scholar]. Although MODY5 is a rare familial disorder, mutations of HNF-1β may also be responsible for a subset of the sporadic congenital renal abnormalities that occur frequently in humans. A recent survey of 105 subjects with sporadic renal hypoplasia/dysplasia and chronic kidney disease found mutations of HNF-1β in three individuals [abstract; Salomon R, et al, J Am Soc Nephrol 15:662A, 2004]. These findings suggest that mutations of HNF-1β may be a more common cause of congenital renal abnormalities than is currently appreciated. HNF-1β may also be involved in the pathogenesis of renal cancer, since biallelic inactivation of the gene has been identified in chromophobe renal cell carcinomas[18.Rebouissou S. Vasiliu V. Thomas C. et al.Germline hepatocyte nuclear factor 1α and 1β mutations in renal cell carcinomas.Hum Mol Genet. 2005; 14: 603-614Crossref PubMed Scopus (93) Google Scholar]. To create an animal model of the renal abnormalities in MODY5, we generated transgenic mice expressing a dominant-negative HNF-1β mutant under the control of a kidney-specific promoter[19.Hiesberger T. Bai Y. Shao X. et al.Mutation of hepatocyte nuclear factor-1β inhibits Pkhd1 gene expression and produces renal cysts in mice.J Clin Invest. 2004; 113: 814-825Crossref PubMed Scopus (136) Google Scholar]. A tissue-specific promoter was required for these experiments, because constitutive inhibition of HNF-1β produces embryonic lethality. Transgenic mice expressing dominant-negative HNF-1β are viable at birth but develop kidney cysts and renal failure. Cysts are observed in the renal tubules and collecting ducts as well as in Bowman's capsules. The severity of cyst formation is directly correlated with the level of expression of the mutant transgene. These results indicate that kidney-specific expression of a dominant-negative HNF-1β mutant produces congenital cystic kidney disease in mice, similar to the phenotype of humans with MODY5. To determine how mutations of HNF-1β produce kidney cysts, we measured the expression of other cystic disease genes in the kidneys from the transgenic mice. A striking abnormality was a marked reduction in the expression of the autosomal-recessive polycystic kidney disease (ARPKD) gene, Pkhd1. Real-time reverse transcription-polymerase chain reaction (RT-PCR) showed that Pkhd1 expression is reduced by up to 70% in the cystic kidneys compared with nontransgenic controls. In situ hybridization showed that Pkhd1 mRNA transcripts are absent in the cells lining the cysts but can be detected in the surrounding noncystic tubules. In contrast, the expression of the ADPKD genes (Pkd1 and Pkd2) is not affected in this model. ARPKD is a congenital cystic disorder that is characterized by dilatation of the collecting ducts, bilateral kidney enlargement, and intrauterine renal failure[20.Igarashi P. Somlo S. Genetics and pathogenesis of polycystic kidney disease.J Am Soc Nephrol. 2002; 13: 2384-2398Crossref PubMed Scopus (422) Google Scholar]. The cystic collecting ducts are invariably associated with biliary dysgenesis, a ductal plate malformation that produces aberrant intrahepatic bile ducts and portal fibrosis. ARPKD is caused by mutations of Pkhd1, which encodes a membrane protein of unknown function that has been localized to primary cilia[21.Ward C.J. Yuan D. Masyuk T.V. et al.Cellular and subcellular localization of the ARPKD protein, fibrocystin is expressed on primary cilia.Hum Mol Genet. 2003; 12: 2703-2710Crossref PubMed Scopus (248) Google Scholar]. The expression of Pkhd1 is tissue-specific and overlaps with the expression of HNF-1β[22.Nagasawa Y. Matthiesen S. Onuchic L.F. et al.Identification and characterization of Pkhd1, the mouse orthologue of the human ARPKD gene.J Am Soc Nephrol. 2002; 13: 2246-2258Crossref PubMed Scopus (80) Google Scholar]. Pkhd1 is highly expressed in renal collecting ducts with lower levels of expression in proximal and distal tubules. In the developing metanephros, Pkhd1 is expressed in the branching ureteric bud and developing tubules. In the developing liver Pkhd1 is expressed in epithelial cells of the ductal plate, and expression continues in the intra- and extrahepatic bile ducts in the mature liver. Additional sites of overlapping expression include the mesonephros, Wolffian duct, pancreatic ducts, and gastrointestinal tract. Liver-specific inactivation of HNF-1β produces a ductal plate malformation that resembles the biliary dysgenesis seen in humans with ARPKD[23.Coffinier C. Gresh L. Fiette L. et al.Bile system morphogenesis defects and liver dysfunction upon targeted deletion of HNF1β.Development. 2002; 129: 1829-1838Crossref PubMed Google Scholar]. The preceding results suggested that HNF-1β might directly regulate Pkhd1 transcription. To test this hypothesis, we cloned and characterized the mouse Pkhd1 promoter[19.Hiesberger T. Bai Y. Shao X. et al.Mutation of hepatocyte nuclear factor-1β inhibits Pkhd1 gene expression and produces renal cysts in mice.J Clin Invest. 2004; 113: 814-825Crossref PubMed Scopus (136) Google Scholar]. A genomic fragment containing the first two exons and 9 kb of 5′ flanking sequence of Pkhd1 was obtained from a BAC clone, and the major transcription initiation site was mapped by primer extension. The Pkhd1 promoter was linked to a luciferase reporter gene and transfected into cultured cells. Transfection into renal epithelial cells produced high levels of luciferase, whereas no significant activity was produced in fibroblasts, which indicated that the activity of the promoter was cell-specific. Truncation of the promoter from 6.6 kb to 1.8 kb does not significantly affect its activity in kidney cells, whereas further deletions decrease promoter activity. The proximal Pkhd1 promoter contains a DNase I hypersensitive site that is present in renal epithelial cells but not in fibroblasts, which suggests that activation of the promoter is associated with local alterations in chromatin structure. An evolutionarily conserved binding site for HNF-1 is located 60 bp upstream to the transcription initiation site near the region of DNase I hypersensitivity. HNF-1β binds specifically to the -60 bp site and stimulates Pkhd1 promoter activity, whereas mutations of the HNF-1 site or expression of a dominant-negative HNF-1β mutant inhibit promoter activity. Collectively, these findings demonstrate that HNF-1β directly regulates the Pkhd1 promoter. To confirm the role of HNF-1β in the pathogenesis of renal cystic diseases, we collaborated with Dr. Marco Pontoglio at the Pasteur Institute to conditionally inactivate the HNF-1β gene using kidney-specific Cre/loxP recombination[24.Gresh L. Fischer E. Reimann A. et al.A transcriptional network in polycystic kidney disease.Embo J. 2004; 23: 1657-1668Crossref PubMed Scopus (252) Google Scholar]. Mice expressing Cre recombinase under the control of the kidney-specific Ksp-cadherin promoter were crossed with mice carrying a floxed HNF-1β gene to produce progeny in which HNF-1β was deleted specifically in the kidney and genitourinary tract. Kidney-specific inactivation of HNF-1β leads to postnatal lethality and renal failure. The mutant mice develop hydroureter and enlarged kidneys that contain multiple cysts which are lined by a hyperplastic epithelium. Similar to the findings in the dominant-negative HNF-1β transgenic mice, the expression of Pkhd1 is markedly decreased in the cystic kidneys of HNF-1β knockout mice. In addition, there is a reduction in the expression of Umod, the gene that is mutated in familial juvenile hyperuricemic nephropathy and medullary cystic kidney disease type 2, and a smaller decrease in the expression of the ADPKD gene, Pkd2. These results uncovered a transcriptional hierarchy involving HNF-1β and several cystic disease genes. In vivo chromatin immunoprecipitation experiments showed that HNF-1β binds to several DNA elements in the 5′ flanking regions of the Umod, Pkhd1, and Pkd2 genes. In addition to the site at -60 bp, the Pkhd1 gene contains HNF-1β binding sites at -200 bp, -35 kb, and within intron 5. To understand how HNF-1β activates the Pkdh1 promoter, we first examined the role of the C-terminal domain[25.Hiesberger T. Shao X. Gourley E. et al.Role of the hepatocyte nuclear factor-1β (HNF-1β) C-terminal domain in Pkhd1 (ARPKD) gene transcription and renal cystogenesis.J Biol Chem. 2005; 280: 10578-10586Crossref PubMed Scopus (63) Google Scholar]. Removal of 236 amino acids from the C-terminus of HNF-1β does not interfere with dimerization or DNA-binding but produces a dominant-negative inhibitor of a chromosomally integrated Pkhd1 promoter. The C-terminal domain of HNF-1β contains a transcriptional activation domain that remains functional when fused to a heterologous DNA-binding domain. Deletion analysis showed that the activation domain extends from amino acids 352-483. The activation of the Pkhd1 promoter by wild-type HNF-1β is stimulated by CBP [cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB) binding protein] and P/CAF (p300/CBP-associated factor), which are transcriptional coactivators that have intrinsic histone acetyl transferase activity. Sodium butyrate, an inhibitor of histone deacetylase (HDAC), also stimulates promoter activity, whereas an inhibitor of DNA methylation has no effect. CBP and P/CAF directly interact with the C-terminal domain of HNF-1β, and deletion of the C-terminal domain prevents the coactivators from activating the Pkhd1 promoter. These results indicate that HNF-1β activates Pkhd1 transcription by recruiting coactivators that promote histone acetylation and chromatin remodeling at the promoter. Deletion mutants lacking the C-terminal domain function as dominant-negative mutants, possibly by preventing the recruitment of coactivators. To test whether the C-terminal domain is required for the function of HNF-1β in vivo, we expressed the C-terminal deletion mutant in transgenic mice using a kidney-specific promoter[25.Hiesberger T. Shao X. Gourley E. et al.Role of the hepatocyte nuclear factor-1β (HNF-1β) C-terminal domain in Pkhd1 (ARPKD) gene transcription and renal cystogenesis.J Biol Chem. 2005; 280: 10578-10586Crossref PubMed Scopus (63) Google Scholar]. Expression of the C-terminal deletion mutant produces renal cysts, increased cell proliferation, and dilatation of the ureter, which is similar to the phenotype of mice with kidney-specific inactivation of HNF-1β. The expression of Pkhd1 is inhibited in cystic collecting ducts but not in noncystic proximal tubules, despite expression of the HNF-1β deletion mutant in both nephron segments. These results verify that deletion of the C-terminal domain of HNF-1β, as results from some disease-causing MODY5 mutations, produces a dominant-negative mutation. Cyst formation in the deletion mutants correlates with inhibition of Pkhd1 expression rather than transgene expression per se, which further argues that mutations of HNF-1β produce kidney cysts by down-regulating Pkhd1. The expression of HNF-1α in proximal tubules may protect against cystogenesis in this nephron segment. In summary, our studies confirm that HNF-1β plays a central role in the development of the mammalian kidney. The identification of Pkhd1 as an HNF-1β gene target establishes a previously unrecognized link between two congenital renal cystic diseases, MODY5 and ARPKD, and suggests that cyst formation in humans with mutations of HNF-1β results from inhibition of PKHD1 gene transcription. Further studies of the transcriptional hierarchy involving HNF-1β and cystic disease genes may clarify the molecular mechanisms of renal epithelial differentiation and the pathogenesis of congenital renal cystic diseases. Work from the authors' laboratory is supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases, Texas Advanced Technology Program, and PKD Foundation.