Kidney International, Vol. 68 (2005), pp. 1944–1947

Roles of HNF-1b in kidney development and congenitalcystic diseases

PETER IGARASHI, XINLI SHAO, BRIAN T. MCNALLY, and THOMAS HIESBERGER

Department of Internal Medicine and Division of Basic Science, University of Texas Southwestern Medical Center, Dallas, Texas

Roles of HNF-1b in kidney development and congenital cysticdiseases. Hepatocyte nuclear factor-1b (HNF-1b) is a Pit-1/Oct-1/Unc-86 (POU)/homeodomain-containing transcription factorthat regulates tissue-specific gene expression in the kidney, liver,pancreas, and other epithelial organs. Mutations of HNF-1bproduce maturity-onset diabetes of the young type 5 (MODY5)and are associated with congenital cystic abnormalities of thekidney. Transgenic mice expressing mutant HNF-1b under thecontrol of a kidney-specific promoter develop kidney cysts andrenal failure, which is similar to the phenotype of humans withMODY5. Similarly, kidney-specific deletion of HNF-1b usingCre/loxP recombination results in renal cyst formation. HNF-1b directly regulates the Pkhd1 promoter. HNF-1b mutant miceshow decreased expression of Pkhd1, the gene that is mutatedin humans with autosomal-recessive polycystic kidney disease(ARPKD). These studies demonstrate that HNF-1b is requiredfor the development of the mammalian kidney. They establish apreviously unrecognized link between two renal cystic diseases,MODY5 and ARPKD, and suggest that the mechanism of cystformation in humans with mutations of HNF-1b involves down-regulation of PKHD1 gene transcription.

Hepatocyte nuclear factor-1b (HNF-1b , vHNF1) is atranscription factor that regulates tissue-specific gene ex-pression in the kidney, liver, pancreas, and other organs[1, 2]. HNF-1b and the related family member, HNF-1a, have a similar structure consisting of an N-terminaldimerization domain, a Pit-1/Oct-1/Unc-86 (POU) do-main and homeodomain that mediate DNA binding, anda C-terminal transcriptional activation domain (Fig. 1).HNF-1a and HNF-1b recognize the consensus sequence5′-GTTAATNATTAAC-3′ and bind to DNA as homod-imers or heterodimers. In general, HNF-1a and HNF-1bbind to the promoters of target genes and activate tran-scription, although transcriptional repression has alsobeen reported. HNF-1a and HNF-1b were first dis-covered because of their roles in liver-specific generegulation, but both proteins are also expressed in otherepithelial organs, including the kidney. HNF-1a is ex-pressed in renal proximal tubules, whereas HNF-1b isexpressed in tubular epithelial cells in all segments of thenephron and in renal collecting ducts [3, 4]. Neither tran-

C© 2005 by the International Society of Nephrology

scription factor is expressed in glomerular tufts, bloodvessels, or interstitial cells. During embryonic develop-ment, HNF-1b is expressed in the branching uretericbud that will give rise to the renal collecting system andureter as well as in comma- and S-shaped bodies that willform the nephrons proper. HNF-1a is restricted to lateS-shaped bodies and developing proximal tubules. HNF-1b is also expressed in the mesonephric (Wolffian) duct,which is the anlage of the vas deferens and epididymis inmales, and the Mullerian duct, which is the anlage of theoviduct, uterus, and cervix in females.

HNF-1a regulates the tissue-specific expression ofgenes that encode transport proteins in the renal prox-imal tubule. Knockout mice that lack HNF-1a have mor-phologically normal kidneys but develop glucosuria dueto decreased expression of the sodium/glucose cotrans-porter, Sglt2 [3, 5]. Defects in the renal reabsorption ofglucose are also seen in humans with mutations of HNF-1a. HNF-1a knockout mice also exhibit phosphaturia,which has been attributed to decreased expression ofNpt1 and Npt4 in the proximal tubule [6]. In contrastto HNF-1a, the functions of HNF-1b in the kidney havebeen poorly understood. HNF-1b recognizes the sameDNA sequence as HNF-1a and could also be involvedin kidney-specific gene regulation. For example, HNF-1bbinding sites have been identified in the promoters of thegenes encoding kidney-specific Ksp-cadherin (Cdh16)and Na-K-Cl cotransporter (Nkcc2) [7] [abstract; WhyteDA, et al, J Am Soc Nephrol 6:713A, 1995]. Studies inlower vertebrates suggest that HNF-1b plays an impor-tant role in nephrogenesis. Transgenic expression of mu-tant HNF-1b in Xenopus embryos leads to abnormaldevelopment of the pronephros [8, 9], and mutation ofzebrafish HNF-1b produces cysts in the pronephrictubules [10]. However, the roles of HNF-1b in the de-velopment of the mammalian kidney have not been ap-parent because HNF-1b knockout mice die prior to renalorganogenesis [11, 12].

Mutations of HNF-1b in humans produce maturity-onset diabetes of the young, type 5 (MODY5). MODY5is a rare familial form of type 2 diabetes mellitus that ischaracterized by autosomal-dominant inheritance, early

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Igarashi et al: HNF-1b in kidney development and congenital cystic diseases 1945

HNF-1β

R177X

A263fs

R137-K161del

P328L329fs

1 32domain

Dimerization POU Homeo Activationdomain domain domain

106 178 231 310 55 Fig. 1. Structure of wild-type and mutanthepatocyte nuclear factor (HNF-1b). Upperbar depicts wild-type human HNF-1b . Boxesindicate the N-terminal dimerization domain,POU domain, homeodomain, and C-terminalactivation domain. Lower bars depict muta-tions of HNF-1b in humans with maturity-onset diabetes of the young, type 5 (MODY5)selected from ∼30 described in the litera-ture. Most mutations affect the DNA bindingdomain. The P328L329fsdelCCTCT mutationproduces a deletion of the C-terminal do-main and addition of 29 amino acids from theshifted reading frame (hatched box).

age of onset, impaired insulin secretion, and absence ofobesity [13]. MODY5 is frequently, perhaps invariably,associated with congenital abnormalities of the kidneyand genitourinary tract [14, 15]. The most common kid-ney abnormalities involve the formation of cysts in therenal tubules, and the acronym RCAD (renal cysts anddiabetes) has been coined to describe this syndrome.The spectrum of congenital renal cystic abnormalitiesincludes simple cysts, multicystic renal dysplasia, and hy-poplastic glomerulocystic kidney disease. Renal involve-ment is often severe and can lead to kidney failure. Inaddition to cysts, other kidney abnormalities that havebeen observed in MODY5 include oligomeganephronia,familial juvenile hyperuricemic nephropathy, and renalagenesis or hypoplasia [16]. Abnormalities of the maleand female genital tract are also seen in some patients.To date, more than 30 different mutations of HNF-1bhave been identified in humans with MODY5, some ofwhich are shown in Figure 1. The molecular mechanismby which mutation of one allele of HNF-1b produces dis-ease has been controversial. The A263fsinsGG mutationaffects the DNA-binding domain and has been shown toproduce a dominant-negative mutant [17], whereas theP328L329fsdelCCTCT mutation deletes the C-terminaldomain and has been reported to produce a gain-of-function mutant [8]. Although MODY5 is a rare familialdisorder, mutations of HNF-1b may also be responsiblefor a subset of the sporadic congenital renal abnormal-ities that occur frequently in humans. A recent surveyof 105 subjects with sporadic renal hypoplasia/dysplasiaand chronic kidney disease found mutations of HNF-1bin three individuals [abstract; Salomon R, et al, J AmSoc Nephrol 15:662A, 2004]. These findings suggest thatmutations of HNF-1b may be a more common cause ofcongenital renal abnormalities than is currently appreci-ated. HNF-1b may also be involved in the pathogenesisof renal cancer, since biallelic inactivation of the genehas been identified in chromophobe renal cell carcino-mas [18].

To create an animal model of the renal abnormalitiesin MODY5, we generated transgenic mice expressing a

dominant-negative HNF-1b mutant under the controlof a kidney-specific promoter [19]. A tissue-specificpromoter was required for these experiments, becauseconstitutive inhibition of HNF-1b produces embryoniclethality. Transgenic mice expressing dominant-negativeHNF-1b are viable at birth but develop kidney cysts andrenal failure. Cysts are observed in the renal tubules andcollecting ducts as well as in Bowman’s capsules. Theseverity of cyst formation is directly correlated with thelevel of expression of the mutant transgene. These resultsindicate that kidney-specific expression of a dominant-negative HNF-1b mutant produces congenital cystic kid-ney disease in mice, similar to the phenotype of humanswith MODY5.

To determine how mutations of HNF-1b produce kid-ney cysts, we measured the expression of other cys-tic disease genes in the kidneys from the transgenicmice. A striking abnormality was a marked reductionin the expression of the autosomal-recessive polycystickidney disease (ARPKD) gene, Pkhd1. Real-time re-verse transcription-polymerase chain reaction (RT-PCR)showed that Pkhd1 expression is reduced by up to 70%in the cystic kidneys compared with nontransgenic con-trols. In situ hybridization showed that Pkhd1 mRNAtranscripts are absent in the cells lining the cysts but canbe detected in the surrounding noncystic tubules. In con-trast, the expression of the ADPKD genes (Pkd1 andPkd2) is not affected in this model. ARPKD is a con-genital cystic disorder that is characterized by dilatationof the collecting ducts, bilateral kidney enlargement, andintrauterine renal failure [20]. The cystic collecting ductsare invariably associated with biliary dysgenesis, a ductalplate malformation that produces aberrant intrahepaticbile ducts and portal fibrosis. ARPKD is caused by mu-tations of Pkhd1, which encodes a membrane protein ofunknown function that has been localized to primary cilia[21]. The expression of Pkhd1 is tissue-specific and over-laps with the expression of HNF-1b [22]. Pkhd1 is highlyexpressed in renal collecting ducts with lower levels ofexpression in proximal and distal tubules. In the devel-oping metanephros, Pkhd1 is expressed in the branching

1946 Igarashi et al: HNF-1b in kidney development and congenital cystic diseases

ureteric bud and developing tubules. In the developingliver Pkhd1 is expressed in epithelial cells of the ductalplate, and expression continues in the intra- and extrahep-atic bile ducts in the mature liver. Additional sites of over-lapping expression include the mesonephros, Wolffianduct, pancreatic ducts, and gastrointestinal tract. Liver-specific inactivation of HNF-1b produces a ductal platemalformation that resembles the biliary dysgenesis seenin humans with ARPKD [23].

The preceding results suggested that HNF-1b mightdirectly regulate Pkhd1 transcription. To test this hy-pothesis, we cloned and characterized the mouse Pkhd1promoter [19]. A genomic fragment containing the firsttwo exons and 9 kb of 5′ flanking sequence of Pkhd1was obtained from a BAC clone, and the major tran-scription initiation site was mapped by primer extension.The Pkhd1 promoter was linked to a luciferase reportergene and transfected into cultured cells. Transfection intorenal epithelial cells produced high levels of luciferase,whereas no significant activity was produced in fibrob-lasts, which indicated that the activity of the promoterwas cell-specific. Truncation of the promoter from 6.6 kbto 1.8 kb does not significantly affect its activity in kidneycells, whereas further deletions decrease promoter activ-ity. The proximal Pkhd1 promoter contains a DNase Ihypersensitive site that is present in renal epithelial cellsbut not in fibroblasts, which suggests that activation ofthe promoter is associated with local alterations in chro-matin structure. An evolutionarily conserved binding sitefor HNF-1 is located 60 bp upstream to the transcriptioninitiation site near the region of DNase I hypersensitivity.HNF-1b binds specifically to the −60 bp site and stimu-lates Pkhd1 promoter activity, whereas mutations of theHNF-1 site or expression of a dominant-negative HNF-1b mutant inhibit promoter activity. Collectively, thesefindings demonstrate that HNF-1b directly regulates thePkhd1 promoter.

To confirm the role of HNF-1b in the pathogenesisof renal cystic diseases, we collaborated with Dr. MarcoPontoglio at the Pasteur Institute to conditionally inac-tivate the HNF-1b gene using kidney-specific Cre/loxPrecombination [24]. Mice expressing Cre recombinaseunder the control of the kidney-specific Ksp-cadherinpromoter were crossed with mice carrying a floxed HNF-1b gene to produce progeny in which HNF-1b wasdeleted specifically in the kidney and genitourinary tract.Kidney-specific inactivation of HNF-1b leads to postna-tal lethality and renal failure. The mutant mice develophydroureter and enlarged kidneys that contain multiplecysts which are lined by a hyperplastic epithelium. Sim-ilar to the findings in the dominant-negative HNF-1btransgenic mice, the expression of Pkhd1 is markedly de-creased in the cystic kidneys of HNF-1b knockout mice.In addition, there is a reduction in the expression ofUmod, the gene that is mutated in familial juvenile hype-

ruricemic nephropathy and medullary cystic kidney dis-ease type 2, and a smaller decrease in the expression ofthe ADPKD gene, Pkd2. These results uncovered a tran-scriptional hierarchy involving HNF-1b and several cys-tic disease genes. In vivo chromatin immunoprecipitationexperiments showed that HNF-1b binds to several DNAelements in the 5′ flanking regions of the Umod, Pkhd1,and Pkd2 genes. In addition to the site at −60 bp, thePkhd1 gene contains HNF-1b binding sites at −200 bp,−35 kb, and within intron 5.

To understand how HNF-1b activates the Pkdh1 pro-moter, we first examined the role of the C-terminaldomain [25]. Removal of 236 amino acids from the C-terminus of HNF-1b does not interfere with dimerizationor DNA-binding but produces a dominant-negative in-hibitor of a chromosomally integrated Pkhd1 promoter.The C-terminal domain of HNF-1b contains a transcrip-tional activation domain that remains functional whenfused to a heterologous DNA-binding domain. Deletionanalysis showed that the activation domain extends fromamino acids 352-483. The activation of the Pkhd1 pro-moter by wild-type HNF-1b is stimulated by CBP [cyclicadenosine monophosphate (cAMP)-responsive elementbinding protein (CREB) binding protein] and P/CAF(p300/CBP-associated factor), which are transcriptionalcoactivators that have intrinsic histone acetyl transferaseactivity. Sodium butyrate, an inhibitor of histone deacety-lase (HDAC), also stimulates promoter activity, whereasan inhibitor of DNA methylation has no effect. CBP andP/CAF directly interact with the C-terminal domain ofHNF-1b , and deletion of the C-terminal domain preventsthe coactivators from activating the Pkhd1 promoter.These results indicate that HNF-1b activates Pkhd1 tran-scription by recruiting coactivators that promote histoneacetylation and chromatin remodeling at the promoter.Deletion mutants lacking the C-terminal domain functionas dominant-negative mutants, possibly by preventing therecruitment of coactivators.

To test whether the C-terminal domain is requiredfor the function of HNF-1b in vivo, we expressed theC-terminal deletion mutant in transgenic mice usinga kidney-specific promoter [25]. Expression of the C-terminal deletion mutant produces renal cysts, increasedcell proliferation, and dilatation of the ureter, which issimilar to the phenotype of mice with kidney-specific in-activation of HNF-1b . The expression of Pkhd1 is inhib-ited in cystic collecting ducts but not in noncystic proximaltubules, despite expression of the HNF-1b deletion mu-tant in both nephron segments. These results verify thatdeletion of the C-terminal domain of HNF-1b , as re-sults from some disease-causing MODY5 mutations, pro-duces a dominant-negative mutation. Cyst formation inthe deletion mutants correlates with inhibition of Pkhd1expression rather than transgene expression per se, whichfurther argues that mutations of HNF-1b produce kidney

Igarashi et al: HNF-1b in kidney development and congenital cystic diseases 1947

cysts by down-regulating Pkhd1. The expression of HNF-1a in proximal tubules may protect against cystogenesisin this nephron segment.

In summary, our studies confirm that HNF-1b plays acentral role in the development of the mammalian kidney.The identification of Pkhd1 as an HNF-1b gene targetestablishes a previously unrecognized link between twocongenital renal cystic diseases, MODY5 and ARPKD,and suggests that cyst formation in humans with muta-tions of HNF-1b results from inhibition of PKHD1 genetranscription. Further studies of the transcriptional hi-erarchy involving HNF-1b and cystic disease genes mayclarify the molecular mechanisms of renal epithelial dif-ferentiation and the pathogenesis of congenital renal cys-tic diseases.

ACKNOWLEDGMENTS

Work from the authors’ laboratory is supported by grants from theNational Institute of Diabetes and Digestive and Kidney Diseases,Texas Advanced Technology Program, and PKD Foundation.

Reprint requests to Peter Igarashi, M.D., Division of Nephrology Uni-versity of Texas Southwestern Medical Center, 5323 Harry Hines Blvd.,MC8856, Dallas, TX 75390-8856.E-mail: peter.igarashi@utsouthwestern.edu

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