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BASIC RESEARCH www.jasn.org Mutations in PCBD1 Cause Hypomagnesemia and Renal Magnesium Wasting Silvia Ferrè,* Jeroen H.F. de Baaij,* Patrick Ferreira, Roger Germann, Johannis B.C. de Klerk, § Marla Lavrijsen,* Femke van Zeeland,* Hanka Venselaar, | Leo A.J. Kluijtmans, Joost G.J. Hoenderop,* and René J.M. Bindels* *Department of Physiology, Nijmegen Centre for Molecular Life Sciences, | Centre for Molecular and Biomolecular Informatics, and Department of Laboratory Medicine, Laboratory of Genetic, Endocrine and Metabolic Diseases, Radboud University Medical Center, Nijmegen, The Netherlands; Division of Medical Genetics, Alberta Childrens Hospital, Calgary, Alberta, Canada; Städtisches Klinikum Karlsruhe, Klinik für Kinder und Jugendmedizin, Karlsruhe, Germany; and § Sophia Childrens Hospital, Erasmus University, Rotterdam, The Netherlands ABSTRACT Mutations in PCBD1 are causative for transient neonatal hyperphenylalaninemia and primapterinuria (HPABH4D). Until now, HPABH4D has been regarded as a transient and benign neonatal syndrome without complications in adulthood. In our study of three adult patients with homozygous mutations in the PCBD1 gene, two patients were diagnosed with hypomagnesemia and renal Mg 2+ loss, and two patients developed diabetes with characteristics of maturity onset diabetes of the young (MODY), regardless of serum Mg 2+ levels. Our results suggest that these clinical ndings are related to the function of PCBD1 as a dimerization cofactor for the transcription factor HNF1B. Mutations in the HNF1B gene have been shown to cause renal malformations, hypomagnesemia, and MODY. Gene expression studies combined with immunohistochemical analysis in the kidney showed that Pcbd1 is expressed in the distal convoluted tubule (DCT), where Pcbd1 transcript levels are upregulated by a low Mg 2+ -containing diet. Overexpression in a human kidney cell line showed that wild-type PCBD1 binds HNF1B to costimulate the FXYD2 promoter, the activity of which is instrumental in Mg 2+ reab- sorption in the DCT. Of seven PCBD1 mutations previously reported in HPABH4D patients, ve mutations caused proteolytic instability, leading to reduced FXYD2 promoter activity. Furthermore, cytosolic localization of PCBD1 increased when coexpressed with HNF1B mutants. Overall, our ndings establish PCBD1 as a coac- tivator of the HNF1B-mediated transcription necessary for ne tuning FXYD2 transcription in the DCT and suggest that patients with HPABH4D should be monitored for previously unrecognized late complications, such as hypomagnesemia and MODY diabetes. J Am Soc Nephrol 25: 574586, 2014. doi: 10.1681/ASN.2013040337 Hypomagnesemia is a common clinical manifesta- tion in patients with mutations in the transcription factor hepatocyte nuclear factor 1 homeobox B (HNF1B [Mendelian Inheritance in Man (MIM) 189907]). 1 Mutations in HNF1B are associated with an autosomal dominant syndrome characterized by renal malformations with or without cysts, liver and genital tract abnormalities, gout, and maturity- onset diabetes of the young type 5 (MODY5; renal cysts and diabetes syndrome [MIM 137920]). 1,2 Hy- pomagnesemia (plasma Mg 2+ levels,0.7 mmol/L) with hypermagnesuria affects up to 50% of the HNF1B patients. 1,3 In adult kidney, HNF1B is expressed in epithelial cells along all segments of the nephron. The role of HNF1B in renal Mg 2+ handling was, however, Received April 3, 2013. Accepted September 14, 2013. S.F. and J.H.F.d.B. contributed equally to this work. Published online ahead of print. Publication date available at www.jasn.org. Correspondence: Dr. René J.M. Bindels, Radboud University Medi- cal Center, Department of Physiology 286, PO Box 9101, 6500 HB, Nijmegen, The Netherlands. Email: [email protected] Copyright © 2014 by the American Society of Nephrology 574 ISSN : 1046-6673/2503-574 J Am Soc Nephrol 25: 574586, 2014

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Page 1: Mutations in PCBD1 Cause Hypomagnesemia and Renal ...jasn.asnjournals.org/content/25/3/574.full.pdf · BASIC RESEARCH Mutations in PCBD1 Cause Hypomagnesemia and Renal Magnesium Wasting

BASIC RESEARCH www.jasn.org

Mutations in PCBD1 Cause Hypomagnesemia andRenal Magnesium Wasting

Silvia Ferrè,* Jeroen H.F. de Baaij,* Patrick Ferreira,† Roger Germann,‡ Johannis B.C. deKlerk,§ Marla Lavrijsen,* Femke van Zeeland,* Hanka Venselaar,| Leo A.J. Kluijtmans,¶

Joost G.J. Hoenderop,* and René J.M. Bindels*

*Department of Physiology, Nijmegen Centre for Molecular Life Sciences, |Centre for Molecular and BiomolecularInformatics, and ¶Department of Laboratory Medicine, Laboratory of Genetic, Endocrine and Metabolic Diseases,Radboud University Medical Center, Nijmegen, The Netherlands; †Division of Medical Genetics, Alberta Children’sHospital, Calgary, Alberta, Canada; ‡Städtisches Klinikum Karlsruhe, Klinik für Kinder und Jugendmedizin, Karlsruhe,Germany; and §Sophia Children’s Hospital, Erasmus University, Rotterdam, The Netherlands

ABSTRACTMutations in PCBD1 are causative for transient neonatal hyperphenylalaninemia and primapterinuria(HPABH4D). Until now, HPABH4D has been regarded as a transient and benign neonatal syndrome withoutcomplications in adulthood. In our studyof three adult patientswith homozygousmutations in thePCBD1gene,two patients were diagnosedwith hypomagnesemia and renalMg2+ loss, and two patients developed diabeteswith characteristics of maturity onset diabetes of the young (MODY), regardless of serum Mg2+ levels. Ourresults suggest that these clinical findings are related to the function of PCBD1 as a dimerization cofactor for thetranscription factor HNF1B. Mutations in the HNF1B gene have been shown to cause renal malformations,hypomagnesemia, and MODY. Gene expression studies combined with immunohistochemical analysis in thekidney showed that Pcbd1 is expressed in the distal convoluted tubule (DCT), wherePcbd1 transcript levels areupregulated by a low Mg2+-containing diet. Overexpression in a human kidney cell line showed that wild-typePCBD1 binds HNF1B to costimulate the FXYD2 promoter, the activity of which is instrumental in Mg2+ reab-sorption in the DCT. Of seven PCBD1 mutations previously reported in HPABH4D patients, five mutationscaused proteolytic instability, leading to reduced FXYD2 promoter activity. Furthermore, cytosolic localizationof PCBD1 increased when coexpressed with HNF1B mutants. Overall, our findings establish PCBD1 as a coac-tivator of the HNF1B-mediated transcription necessary for fine tuning FXYD2 transcription in the DCT andsuggest that patientswithHPABH4Dshould bemonitored for previously unrecognized late complications, suchas hypomagnesemia and MODY diabetes.

J Am Soc Nephrol 25: 574–586, 2014. doi: 10.1681/ASN.2013040337

Hypomagnesemia is a common clinical manifesta-tion in patients with mutations in the transcriptionfactor hepatocyte nuclear factor 1 homeobox B(HNF1B [Mendelian Inheritance in Man (MIM)189907]).1 Mutations inHNF1B are associated withan autosomal dominant syndrome characterized byrenal malformations with or without cysts, liver andgenital tract abnormalities, gout, and maturity-onset diabetes of the young type 5 (MODY5; renalcysts and diabetes syndrome [MIM 137920]).1,2 Hy-pomagnesemia (plasma Mg2+ levels,0.7 mmol/L)with hypermagnesuria affects up to 50% of theHNF1B patients.1,3

In adult kidney, HNF1B is expressed in epithelialcells along all segments of the nephron. The role ofHNF1B in renal Mg2+ handling was, however,

Received April 3, 2013. Accepted September 14, 2013.

S.F. and J.H.F.d.B. contributed equally to this work.

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Dr. René J.M. Bindels, Radboud University Medi-cal Center, Department of Physiology 286, PO Box 9101, 6500 HB,Nijmegen, The Netherlands. Email: [email protected]

Copyright © 2014 by the American Society of Nephrology

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pinpointed to the distal convoluted tubule (DCT), where the finalurinary Mg2+ excretion is determined.1,4 In DCT, the Na+/K+-ATPase provides the necessary driving force for active Mg2+

reabsorption from the prourine into the blood.4 Heterozygousmutations in the FXYD2 gene, encoding the g-subunit of theNa+/K+-ATPase, result in an autosomal dominant renal hypo-magnesemia with hypocalciuria (MIM 154020).5 More recently,functional HNF1B binding sites were identified in the promoterregion of FXYD2, suggesting that impaired transcription ofFXYD2 by HNF1B results in renal Mg2+ wasting.1,6 AdditionalHNF1B target genes in kidney include renal cystic genes7–9 aswell as genes involved in tubular electrolyte transport.10,11

HNF1B forms heterotetrameric complexes with the proteinpterin-4a-carbinolamine dehydratase/dimerization cofactorof hepatocyte nuclear factor 1 homeobox A (PCBD1 [MIM126090]).12 PCBD1 is a protein of 12 kDa with two distinctbiologic functions: transcriptional coactivation of HNF1A-mediated transcription within the nucleus and PCBD1 (EC4.2.1.96) in the cell cytosol.12,13 The enzymatic activity ofPCBD1, together with dihydropteridine reductase, regeneratestetrahydrobiopterin (BH4), which is the cofactor for phenylal-anine hydroxylase and other aromatic amino acid hydrolases.14

The crystal structure of PCBD1 revealed that the protein forms atetramer of identical subunits comprising two saddle-like shapeddimers.15,16 HNF1 binding sites are located at the same surfacethat mediates interaction of the PCBD1 homodimers on the op-posite side of the catalytic domain.17 PCBD1 knockout mice dis-play hyperphenylalaninemia, predisposition to cataracts, andmildglucose intolerance.18 Homozygous or compound heterozygousPCBD1 mutations in humans are associated with transient neo-natal hyperphenyalaninemia and high urinary levels of primap-terin (HPABH4D; or primapterinuria [MIM 264070]).19–21 Todate, there have been no reports of late complications or possiblephenotypic consequences of impaired stimulation of the HNF1transcription factors.

In our study, the occurrence of hypomagnesemia andMODYdiabetes was investigated in three patients carrying PCBD1mu-tations. We evaluated whether PCBD1 plays a role in renal Mg2+

reabsorption by directly affecting HNF1B-regulated FXYD2transcription to gain new insight into the molecular basis ofthe PCBD1–HNF1B interaction.

RESULTS

Homozygous PCBD1 Mutations Are Associated withHypomagnesemia and Renal Mg2+ WastingWe diagnosed hypomagnesemia and hypermagnesuria in twopatients carrying mutations on both alleles in the PCBD1 gene(Table 1). In patient 1, hypomagnesemia was partially correc-ted with oral Mg2+ supplements at a dose of 500 mg/d (0.64–0.76 mmol/L after first supplementation, 0.66–0.69 mmol/Lafter second supplementation), although at the expense ofincreased magnesuria (fractional excretion of Mg2+ [FEMg];4.6%–7.8%, N,4%). He had nonspecific symptoms that

might be related to hypomagnesemia, including fatigue, mus-cular pain, weakness and cramps in arms, numbness, difficultywith memory, chest pains, and blurred vision, with some im-provement after Mg2+ supplementation. An abdominal ultra-sound showed slightly increased echogenicity of both liver andkidney of uncertain cause; renal size was normal, with no cystspresent (Supplemental Figure 2, A and B). Renal function wasnormal (GFR=128 ml/min per 1.73 m2, N$90). Laboratoryinvestigations of patient 2 revealed a relatively high 24-hoururinaryMg2+ excretion (5.25 mmol/24 h,N=2–8) (Table 1) inthe presence of hypomagnesemia (0.65 mmol/L), with no sec-ondary symptoms. Serum and 24-hour urinary Ca2+ excretionwere within the normal range as was creatinine clearance(126 ml/min,N=89–143). Ultrasound examinations excludedthe presence of renal hypoplasia or cysts (Supplemental Figure2C). Patient 3 showed borderline serum Mg2+ levels(0.74 mmol/L) but displayed normal urinary Mg2+ excretion(FEMg=2.5%).

MODY in Two Patients with PCBD1 MutationsIn addition to hypomagnesemia, patient 1 was diagnosed withdiabetes. Type 1 autoimmune diabetes was unlikely, becausethe patient lacked islet cell antibodies and showed normalserum C-peptide levels (0.69 nmol/L, N=0.3–1.32 nmol/L).Because MODY patients are generally highly sensitive to sul-phonylureas,22 he was treated with Gliclazide 80 mg two timesper day, to which he responded well, allowing his previousinsulin treatment of 20–30 units/d to be discontinued. Addi-tional extrarenal manifestations in HNF1B disease includeliver test abnormalities.23,24 Liver function tests in patient 1revealed that the plasma levels of high-sensitivity C-reactiveprotein (hs-CRP) were significantly low at ,0.1 mg/L (N,8mg/L) (Table 2). IgG was slightly low at 5.66 g/L (N=6.94–16.18 g/L), whereas the remaining liver function tests were allwithin the normal range. Based on his glycosylated hemoglo-bin (HbA1c) levels, we concluded that patient 2 did notdevelop diabetes (HbA1c=4.95%, N=4.3%–6.1%) (Table 1).Abdominal ultrasound analysis showed that the pancreas andliver were normal (Supplemental Figure 2, D and E). Patient 3was also diagnosed with diabetes of no autoimmune originbecause of the absence of islet cell antibodies. HbA1c levelswere 6.5% (N=4.3%–6.1%), and glucose was 6.4 mmol/L(N=4.4–6.1 mmol/L). MODY types 1, 2, and 3 caused by mu-tations in HNF4A, GCK, and HNF1A, respectively, were ex-cluded by DNA analysis. Presently, the patient is not receivingmedications or insulin treatment.

Pcbd1 Expression in DCT Is Modulated by DietaryMg2+ ContentWe examined Pcbd1mRNA expression levels in a mouse tissuepanel using real-time RT-PCR. Highest expression was mea-sured in kidney and liver (Figure 1A). Furthermore, immu-nohistochemical analysis of mouse pancreas sections revealedthat Pcbd1 is expressed in pancreatic cells (Figure 1B). Mousekidney sections were stained for Pcbd1 to evaluate whether

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Pcbd1 locates to the sites of renal Mg2+ reabsorption (Figure 1,C–G). Pcbd1 staining colocalized with Na+/Cl2 cotransporter(NCC) in DCTand in part, Tamm Horsfall and calbindin-D28K

expression in the cortical thick ascending limb of Henle’sloop (TAL) and connecting tubule, respectively. No colocali-zation of Pcbd1 was detected with markers of the proximaltubule or collecting duct, which are Bcrp and Aqp2, respec-tively. The expression of Pcbd1 in DCT was confirmed in

isolated DCT fragments. Pcbd1 expression was significantlyhigher in DCT compared with whole kidney (Figure 1H).Hnf1b transcript was not enriched in the DCT fraction. In-terestingly, Pcbd1 expression in the DCTwas significantly up-regulated when mice were fed a low Mg2+-containing dietcompared with a high Mg2+-containing diet (Figure 1I).Mice on a Mg2+-deficient diet displayed a significantly lower24-hourMg2+ excretion (Supplemental Figure 1B) in responseto the hypomagnesemia (1.1 versus 2.2 mmol/L, N=1.2–1.5mmol/L25) (Supplemental Figure 1A). Differences were notobserved in the serum and urinary levels of phenylalaninebetween the experimental groups (Supplemental Figure 1, Cand D).

PCBD1 Enhances FXYD2 and PKHD1 PromoterActivation by HNF1BTo investigate whether the PCBD1 p.Glu26*, p.Arg87Gln,p.Glu96Lys, and p.Gln97* mutations can lead to an impair-ment of the interaction with HNF1B, we generated a structuralhomology model of the PCBD1–HNF1B dimerization domain(HNF1B–D) complex using the structure of the PCBD1–HNF1Adimerization domain tetramer (Figure 2, A and B). The p.Glu26*mutation leads to amajor protein truncation and complete loss ofthe interaction domain with HNF1B (Figure 2B). Althoughp.Gln97* does not directly affect the HNF1 binding domain ofPCBD1, themutation results in a truncation of the centrala-helixof the protein. Consequently, the interaction domain might be

Table 1. Pertinent laboratory investigations for three patients with PCBD1 mutations

ParameterPatient 1 (BIODEF 272) Patient 2 (BIODEF 329)

BaselinePatient 3 (BIODEF 319)

BaselineReference Range

Baseline 500 mg/d Mg2+

Serum indicesNa+ (mmol/L) 141 — 138 139 135–145K+ (mmol/L) 4.0 — 4.5 4.0 3.5–5.0Mg2+ (mmol/L) 0.64a 0.76 0.65a 0.74 0.7–1.1Ca2+ (mmol/L) 2.41 2.55 2.25 2.43 2.20–2.65Pi (mmol/L) 1.13 1.16 — 1.15 0.8–1.4Creatinine (mmol/L) 0.069 0.066 0.062 0.056 0.045–0.110HbA1c (%) 7.2a — 4.95 6.5a 4.3–6.1Uric acid (mmol/L) 310 — — — 135–510

Urinary indicesMg2+ (mmol/L) 6.16 3.96 — 2.6 —

Mg2+ (mmol/24 h) — — 5.25 — 2–8FEMg (%) 6.6a 11.1a — 3.5 ,4Ca2+ (mmol/L) 4.47 3.04 — 2.99 —

Ca2+ (mmol/24 h) — — 5.61 — ,7.5FeCa (%) 0.9 1.8 — 0.86 .1Pi (mmol/L) 40.29 13.61 — 18.2 —

FePi (%) 17 17.6 — — 5–20Creatinine (mmol/L) 14.29 4.40 — 8.0 —

Creatinine (mmol/24 h) — — 14 — 9–18GFR (ml/min per 1.73 m2) 128 135 — 124 $90CCr (ml/min) — — 126 — 89–143

FEMg was calculated using the following formula: FEMg=(UMg3PCr)/[(0.73PMg)3UCr3100]. GFR was calculated by the Modification of Diet in Renal Diseaseformula. FeCa, fractional excretion of Ca2+; FePi, fractional excretion of phosphate (Pi); CCr, creatinine clearance.aAbnormal value.

Table 2. Liver function tests in patient 1 (BIODEF 272)

Parameter Patient 1 (BIODEF 272) Reference Range

Albumin (g/L) 41 38–50Prealbumin (g/L) 0.381 0.1–0.4hs-CRP (mg/L) ,0.1a ,8a-1 antitrypsin (g/L) 1.4 0.9–2.6C3 (g/L) 1.17 0.8–2.1C4 (g/L) 0.18 0.15–0.5IgG (g/L) 5.66a 6.94–16.18IgM (g/L) 0.75 0.6–3.0IgA (g/L) 1.99 0.7–4.0ALT (U/L) 20a 21–72AST (U/L) 26 15–46ALP (U/L) 108 30–130

C3, complement component 3; C4, complement component 4; ALT, alaninetransaminase; AST, aspartate transaminase; ALP, alkaline phosphatase.aAbnormal value.

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destabilized, explaining the PCBD1 dysfunc-tion. Similarly, the negatively charged Glu96residue could be important for the stabiliza-tion of the interaction domain, whichmay behampered by the p.Glu96Lys mutation.

To test the effects of the mutations onPCBD1 function, we studied all previouslydescribed patient mutations in their abilityto coactivate FXYD2 transcription in a lu-ciferase assay (Figure 2C).19–21 Expressionof wild-type PCBD1 did not change theluciferase activity compared with mock-transfected cells, whereas HNF1B signifi-cantly enhanced the FXYD2 promoteractivity (Figure 2D). Interestingly, coex-pression of PCBD1 with HNF1B furtherincreased FXYD2 promoter activation by;1.5 fold (Figure 2D). Of all PCBD1mutants, only PCBD1 p.Arg87Gln andp.Cys81Arg maintained their coactivatorproperties (Figure 2D). The transcriptionof another established target gene ofHNF1B in the kidney, PKHD1,8 was

Figure 1. Pcbd1 is expressed in theDCTof the kidney. (A) Tissue expression pattern of thePcbd1 transcript. Pcbd1mRNA expression level was measured in a panel of mouse tissuesby quantitative RT-PCR and normalized for Gapdh1 expression. Data represent the meanof three individual experiments6SEM and are expressed as the percentage of the total

tissue expression. (B) Immunohistochemicalanalysis of Pcbd1 in mouse pancreas tissue.Scale bar, 20 mm. (C–G) Mouse kidney sectionswere costained for Pcdb1 (green) and (C) Bcrp(red), (D) TH (red), (E) NCC (red), (F) 28K (red), or(G) Aqp2 (red). Nuclei are shown by 49,6-dia-midino-2-phenylindole staining (blue). TH,Tamm Horsfall; 28K, calbindin-D28K. Scale bar,20 mm. (H) Kidney expression pattern of Pcbd1shows the highest expression inDCT. ThemRNAexpression levels of Pcbd1 and Hnf1b in (blackbars) COPAS-selected mouse DCT and (whitebars) control (nonselected) kidneys were mea-sured by quantitative RT-PCR and normalized forGapdh expression. Data represent the mean ofthree individual experiments6SEM and are ex-pressed as fold difference compared with theexpression in nonselected tubules. *P,0.05versus total kidney. (I) DCT expression of Pcbd1is regulated by dietary Mg2+ intake. The mRNAexpression levels ofPcbd1 andHnf1b inCOPAS-selected mouse DCT kidney tubules from micefed with (white bars) low and (black bars) highMg2+-containing diets were measured by real-time RT-PCR and normalized for Gapdh expres-sion. Data represent the mean of four individualexperiments6SEM and are expressed as folddifference compared with the expression in highMg2+-containingdiets. *P,0.05versushighMg2+.Aqp2, aquaporin 2; Bcrp, breast cancer re-sistance protein.

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Figure 2. PCBD1 coactivates HNF1B-induced FXYD2 and PKHD1 promoter activity. (A) Homology model of the PCBD1–HNF1B di-merization domain (HNF1B–D) tetramer modeled using the structure of the PCBD1–HNF1A dimerization domain (HNF1A–D) complex(Protein Data Bank ID code 1F93). (Light blue and grey) The PCBD1 dimer binds the (orange and grey) HNF1B dimer through helixsequences. The HNF1A–D monomer is shown in yellow. Residues in the PCBD1 protein that were found mutates in patients affected by

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assessed in the presence of PCBD1. PKHD1 promoter activityshowed a ;1.5-fold increase expression levels when HNF1Band PCBD1 were coexpressed (Figure 2E). PCBD1 p.Arg87Gln,but not p.Cys81Arg, was the only PCBD1 mutant that retainedits coactivator activity (Figure 2E).

Mutations Detected in HPABH4D Patients CauseProtein Degradation of PCBD1To explain why the PCBD1 mutants are not capable of en-hancing HNF1B-induced transcription, we examined theirsubcellular localization and their capacity to bind HNF1B.Immunostaining for PCBD1 in transiently transfected humanembryonic kidney (HEK293) cells revealed that wild-typePCBD1 translocates to the nucleus on coexpression withHNF1B compared with mock DNA (Figure 3A). Furthermore,PCBD1 p.Glu26*, p.Glu86*, p.Glu96Lys, and p.Gln97* werenot expressed, whereas p.Thr78Ile and p.Cys81Arg weredetected significantly less than the wild-type protein.PCBD1 p.Arg87Gln was the only mutant showing a compa-rable expression level with wild-type PCBD1 (Figure 3A).Coimmunoprecipitation studies confirmed that onlyPCBD1 p.Arg87Gln, p.Thr78Ile, and p.Cys81Arg were ex-pressed and able to bind HNF1B (Figure 3B, top and bottompanels). Importantly HNF1B was equally expressed in all con-ditions (Figure 3B, middle panel). To examine whether mu-tated PCBD1 proteins are degraded by the proteasomal path-way for misfolded proteins, PCBD1-expressing HEK293 cellswere treated for 24 hours with 10 nM proteasome inhibitorMG-132 (N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide). Protein expressionwas restored for all of the PCBD1 mutants, with the only ex-ception being p.Glu26* (Figure 3C).

HNF1B Mutations Affect the Subcellular Localization ofPCBD1We evaluated fiveHNF1Bmutations (p.Lys156Glu, p.Gln253Pro,p.Arg276Gly, p.His324Ser325fsdelCA, and p.Tyr352fsinsA) fortheir ability to bind and functionally respond to PCBD1 (Figure4A).1,3 All HNF1B mutants, except HNF1B D2–30, boundPCBD1 in coimmunoprecipitation studies in transiently trans-fected HEK293 cells (Figure 4B, top panel). PCBD1 andHNF1B were expressed in all conditions tested (Figure 4B,middle and bottom panels). FXYD2 promoter luciferase

assays showed that only HNF1B p.His324Ser325fsdelCA andp.Tyr352fsinsA, which retained partial transcriptional activity,respond to the coactivation by PCBD1 to the same extent asHNF1B wild type (;1.5-fold) (Figure 4C). Importantly, im-munocytochemical analysis revealed that coexpression ofPCBD1 with the HNF1B mutants p.Gln253Pro and p.His324-Ser325fsdelCA causes a predominant cytosolic localization ofPCBD1 compared with the nuclear translocation observed oncoexpression with HNF1B wild type (Figure 4, D and E).

DISCUSSION

Mutations in PCBD1 have been shown to cause a transient andbenign form of neonatal HPABH4D.19–21 Here, we present thefirst follow-up study of HPABH4D patients reporting the on-set of late complications linked to the deficient activity ofPCBD1 as a transcriptional coactivator of HNF1B. Our resultssuggest that PCBD1 acts as an important transcriptional reg-ulator of FXYD2, contributing to renal Mg2+ reabsorption inDCT. Our observations are based on the following results: (1)hypomagnesemia with renal Mg2+ wasting was reported intwo of three patients carrying mutations in the PCBD1 gene;(2) two patients were diagnosed with MODY; (3) PCBD1 ispresent in the pancreas and kidney, predominantly in DCT,where its expression is sensitive to dietaryMg2+ content; (4) invitro data showed that PCBD1 binds HNF1B to costimulatethe FXYD2 promoter, and its activity contributes to Mg2+ re-absorption in DCT; and (5) PCBD1 mutations reported inHPABH4D patients caused proteolytic instability, leading todegradation through the proteasomal pathway.

To our knowledge, we are the first to report that PCBD1mutations associate with hypomagnesemia because of renalMg2+ wasting as well as MODY. In the kidney, the key sites forMg2+ reabsorption are TAL and DCT. Although defects in themolecular pathway for Mg2+ handling in TAL lead to a con-comitant waste of Ca2+, shortcomings in DCT cause hyper-magnesuria associated with hypo- or normocalciuria. Basedon the serum Ca2+ levels and urinary Ca2+ excretion values, itis likely that, in our patients, the DCT is primarily affected.Fluorescence-based sorting of DCT-enhanced green fluores-cent protein (eGFP) tubules using a Complex Object Para-meric Analyzer and Sorter (COPAS) coupled with real-time

hyperphenylalaninemia are depicted in red. (B) Homology model of the interaction site within the PCBD1–HNF1B dimerization domain(HNF1A–D) complex. (Orange) The bound HNF1B monomer forms a helix bundle with the (light blue) PCBD1 monomer. The HNF1A–Dmonomer is shown in yellow. The residues that differ between HNF1B–D andHNF1A–D are visualized in grey. (C) Linear representation ofthe secondary structure elements of the human PCBD1 protein. Red arrowheads indicate the positions of the patient mutations describedin the literature. Green balls indicate the histidine residues involved in the dehydratase active site (His61, His62, and His79). A luciferaseassay was performed in HEK293 cells transiently cotransfected with a (D) Firefly luciferase FXYD2 and (E) PKHD1 promoter construct andHNF1B or mock DNA in the presence of wild-type or mutant PCBD1. A Renilla luciferase construct was cotransfected to correct fortransfection efficiency. Firefly/Renilla luciferase ratios were determined as a measure of promoter activity. Results are depicted as per-centage compared with HNF1B-Mock transfected cells. *P,0.05 versus HNF1B/Mock (n=0). Mock, mock DNA; WT, wild type; 26,p.Glu26+; 78, p.Thr78lle; 81, p.Cys81Arg; 86, p.Glu86*; 87, Arg87Gln; 96, p.Glu96Lys; 97, p.Gln97*.

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PCR analysis confirmed an enrichment ofPcbd1 expression in DCT tubules com-pared with other nephron segments.Pcbd1 expression in DCTwas increased inmice fed with a low Mg2+ diet, suggestingthat Pcbd1 is important for renal Mg2+ re-absorption in DCT. Serum and urinaryphenylalanine levels were stable in themice, and thus, they did not modulatePcbd1 expression. Immunohistochemicalanalysis showed that Pcbd1 expression isnot limited to DCT but partially extendedto cortical TAL and connecting tubule.Overall, these findings confirm previousimmunohistochemical studies showingPcbd1 expression in the cortex of the kid-ney.26 Because Pcbd1 was present in thecytosol of renal cells, we hypothesizedthat the relative abundance of PCBD1 andHNF1B in the kidney may favor the cyto-solic localization. Nevertheless, a nuclear lo-calization of PCBD1 in the kidney couldoccur according to tissue-specific dynamicsof the PCBD1–HNF1B complex.

Our results showed that PCBD1 enhan-ces the FXYD2 promoter activation byHNF1B in vitro. The relevance of FXYD2activity in Mg2+ handling in DCT has beensuggested previously, because both patientswith FXYD2mutations and patients carry-ing HNF1B mutations may present withhypomagnesemia.1,5 It was shown thatHNF1B binds the FXYD2 promoter to spe-cifically regulate the transcription of theg-subunit of the Na+/K+-ATPase isoform-a.1,6 ga-Subunit is mainly expressed inproximal tubule and medullary TAL, butit is also expressed in DCT (SupplementalFigure 3),6,27 where PCBD1 is expressed.To date, the exact molecular mechanismby which the g-subunit regulates Mg2+

handling in DCT remains elusive.4

In the kidney, HNF1B is ubiquitouslyexpressed, where it regulates the transcrip-tion of several cystic genes,9 like PKHD1.Mutations in PKHD1 are causative for au-tosomal recessive polycystic kidney disease(MIM 263200).8 Similar to the FXYD2

Figure 3. Several PCBD1 mutations lead to protein degradation HEK293 cells. (A)Immunocytochemistry analysis of the subcellular localization of HA-tagged PCDB1 wildtype or HA-tagged PCDB1 mutants when coexpressed in a 1:1 ratio with FLAG-taggedHNF1B or mock DNA in HEK293 cells. Red signal represents immunodetected HAepitopes. Nuclei stained with 49,6-diamidino-2-phenylindole are shown in blue. Scalebar, 10 mm. Representation immunocytochemical images are shown. (B) HA-taggedPCDB1 wild-type, PCDB1 mutant, or mock DNA were transiently expressed in HEK293cells with or without FLAG-tagged HNF1B. Immunoprecipitations on nuclear extractsusing an anti-FLAG antibody were separated by SDS-PAGE, and Western blots wereprobed with (top) anti-HA and (middle) anti-FLAG antibodies. (Bottom) HA-PCDB1 input (25%) expression was also included in the analysis. The immunoblotsshown are representative of three independent experiments. (C) Western blot analysisof HA-tagged PCDB1 mutants expressed in HEK293 cells treated with (+) or without(2 ) 10 nM MG-132 for 24 hours. A representative immunoblot is shown. IP,

immunoprecipitation; Mock, mock DNA; WB,Western blot;WT, wild-type; 26, p.Glu26+; 78,p.Thr78lle; 81, p.Cys81Arg; 86, p.Glu86*; 87,Arg87Gln; 96, p.Glu96Lys; 97, p.Gln97*.

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Figure 4. HNF1B mutations result in cytosolic localization of PCBD1. Effect of HNF1B mutations on PCDB1 binding, transcriptioncoactivation, and subcellular localization. (A) Linear representation of the human HNF1B protein. Red arrowheads indicate patientmutations that were tested in this study. D, dimerization domain; NLS, nuclear localization signal; POUH, atypical POU homeodomain;POUS, POU-specific domain. (B) HA-tagged HNF1B wild-type (WT), HNF1B mutants, or HNF1B lacking the extracts using an anti-FLAGantibody were separated by SDS-PAGE, and Western blots (WBs) were probed with (top) anti-HA or (middle) anti-FLAG antibodies.(Lower) HA-HNF1B input (25%) expression was also included in the analysis. The immunoblots shown are representative for threeindependent experiments. IP, immunoprecipitation. (C) A luciferase assay was performed in HEK293 cells transiently cotransfected witha Firefly luciferase FXYD2 promoter construct and each of the HNF1B variants (black bars) with and (white bars) without PCDB1. ARenilla luciferase construct was cotransfected to correct for transfection efficiency. Firefly/Renilla luciferase ratios were determined asa measure of promoter activity. Results are depicted as percentage compared with HNF1B/Mock transfected cells. *P,0.05 comparedwith the HNF1B/Mock condition (n=9). (D) Immunocytochemistry analysis of the subcellular localization of FLAG-tagged PCDB1 whencoexpressed in a 1:1 ratio with HA-tagged HNF1B WTs, HA-tagged HNF1B mutants, or mock DNA HEK293 cells. Red signal representsimmunodetected FLAG epitopes. Scale bar, 20 mm. The immunocytochemical images shown are representative for three independentexperiments. (E) Quantification of the nuclear versus cytosolic localization of PCDB1 when coexpressed in a 1:1 ratio with mock DNA(n=24), HNF1B D1–32 (n=33), WT (n=41), 156 (n=38), 253 (n=35), 276 (n=33), 324_325 (n=37), or 352 (n=36). *P,0.05 compared withMock. #P,0.05 compared with WT. Mock, mock DNA; D1–32, HNF1B lacking the dimerization domain; 156, p.Lys156Glu; 253,p.Gln253Pro; 276, p.Arg276Gly; 324_325, His324Ser325fsdelCA; 352, p.Tyr352fsinsA.

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promoter, a PKHD1 reporter construct showed increased ex-pression levels when HNF1B and PCBD1 were cotransfectedin HEK293 cells. However, the renal expression of PKHD1 invivo is restricted to the loops of Henle and collecting duct,where PCBD1 expression is negligible.28 Thus, under physi-ologic conditions, renal PKHD1 gene transcription seems tonot be affected by PCBD1. This finding is supported by theabsence of kidney abnormalities in patients with PCBD1mutations.

Additional evidence for an impaired HNF1-mediatedtranscription in HPABH4D patients was provided by the di-agnosis of MODY in patients 1 and 3. MODY is a monogenicform of autosomal dominant type 2 diabetes characterizedby age of onset often below 25 years and negative pancreaticautoantibodies. Heterozygous mutations in HNF1B and itshomolog HNF1A associate with MODY types 5 and 3, respec-tively.29 We showed that Pcbd1 is localized in the nuclei ofpancreatic cells. Knowing that PCBD1 acts as transcriptionalcoactivator of both HNF1B and HNF1A, a dysregulation ofHNF1B and/or HNF1A is potentially responsible for theMODY diagnosed in patients 1 and 3. Interestingly, the lowplasma hs-CRP levels in patient 1 are in line with two recentstudies showing lower hs-CRP levels in MODY3 patients thanindividuals with other forms of diabetes and nondiabetic con-trols.30,31 This evidence favors the diagnosis of MODY3-likediabetes in patient 1. Patient 2 was not diagnosed with diabe-tes, but he will be monitored for later onset.

In this study, we showed that the HPABH4D patient mu-tations reported in the literature lead to protein degradation ofPCBD1 through the proteasome pathway. Our data, togetherwith previous studies on proteolytic instability for the samemutants in both mammalian and bacterial expression systems,support the hypothesis that this degradation process may alsooccur inHPABH4D patients.19,20,32 Considering the inheritanceof the disease, HNF1-mediated transcription seems to be sensi-tive to changes in PCBD1 quantity only when both PCBD1 al-leles are affected, suggesting that PCBD1 probably belongs to anancillary regulatorymechanism towhich other HNF1B partnersmay participate.33 In vitro data in HEK293 cells revealed thatPCBD1 p.Arg87Glnwas the onlymutant showing an expressionlevel comparable with the wild-type protein. Nevertheless, inpatient 2, p.Arg87Gln is homozygously present on both alleles,with a p.Glu26* mutation that leads to a significant decrease inthe cellular content of PCBD1.20 PCBD1 p.Glu26* degradationcould not be rescued by proteasome inhibition, suggesting thatits transcript is degraded by mRNA surveillance mecha-nisms.34,35 PCBD1 p.Thr78Ile and p.Cys81Arg were signifi-cantly less expressed than the wild-type protein. Both mutantsshowed binding to HNF1B, but only PCBD1 p.Cys81Arg co-activated the FXYD2 promoter. PCBD1 p.Cys81Arg did notcoactivate the PKHD1 promoter, suggesting insufficient ex-pression to enhance the transcription of PKHD1. In the nearfuture, it would be of interest to screen patients with PCBD1p.Thr78Ile and p.Cys81Arg mutations for complications re-lated to the HNF1B disease.

PCBD1 monomers are small molecules that can passivelydiffuse from the cytosol into the nucleus.36 It was suggestedthat, by assembling through the same interface, PCBD1 ho-motetramer and PCBD1–HNF1 complexes are mutually ex-clusive.17 In our immunocytochemical analysis, the HNF1Bmutants p.Gln253Pro and p.His324Ser325fsdelCA signifi-cantly stimulated a cytosolic localization of PCBD1 comparedwith the nuclear PCBD1 localization observed in the presenceof wild-type HNF1B. This finding suggests that HNF1B mu-tations may disturb the stability of the PCBD1–HNF1 com-plexes in the nucleus and therefore, favor the formation ofPCBD1 homotetramers in the cytosol of the cell.1,3 The re-duced nuclear localization of PCBD1 will indirectly result in adecreased coactivation of HNF1B and could contribute to thehypomagnesemia observed in some HNF1B patients. Becausethe presentation and development of HNF1B disease is di-verse, variations in HNF1B interacting proteins may be re-sponsible for the phenotypic heterogeneity. Screening ofHNF1B patients for polymorphisms in PCBD1 should, there-fore, be considered.

Inconclusion,we identifiedPCBD1as anewmolecular playerin renal Mg2+ handling. Our results suggest that PCBD1 regu-lates the HNF1B-mediated FXYD2 transcription, influencingactive renal Mg2+ reabsorption in DCT. So far, HPABH4Dcaused by PCBD1 mutations has been considered a transient,benign condition, primarily related to impaired BH4 regenera-tion. To date, 23 patients with PCBD1 mutations linked toHPABH4D are listed in the International Database of Tetrahy-drobiopterin Deficiencies (BIODEF database; http://www.bio-pku.org/biodef/).37 Here, we suggest that patients affected byHPABH4D should be monitored for late complications relatedto the interactions with HNF1 transcription factors, includinghypomagnesemia and MODY.

CONCISE METHODS

PatientsThe three patients reported in this study were ascertained by

contacting the authors of papers related to hyperphenylalaninemia,

tetrahydrobiopterin-deficient HPABH4D (MIM 264070).19–21 Of 23

patients listed in the International Database of Tetrahydrobiopterin

Deficiencies (BIODEF database; http://www.biopku.org/biodef/),37

BIODEF 272, 329, and 319 were assessed by P.F. (Canada), R.G.

(Germany), and J.B.C.d.K. (The Netherlands), respectively. The re-

sults are included in this study. Informed consent to participate in this

study was obtained, and the procedures followed were in accordance

with the standards of the medical ethics committee of each partici-

pating institution. Patients were not treated with any medication that

could affect serum levels of Mg2+ (e.g., diuretics, calcineurin inhib-

itors, and corticosteroids).

Patient 1 (BIODEF 272) was reported in detail previously.19 In

summary, he was found to have borderline hyperphenylalaninemia

on newborn screening, but this markedly increased to a peak of

2589mmol/L at 3.5 weeks. The diagnosis of HPABH4Dwas suspected

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because of primapterinuria (7-biopterin) as well as a marked re-

sponse to BH4. There was no parental consanguinity; molecular anal-

ysis confirmed the diagnosis, showing that he was homozygous for a

c.312C.T (p.Gln97*) mutation in the PCBD1 gene. He was treated

with phenylalanine restriction and BH4 until 4months of age, at which

time his phenylalanine levels normalizedwithout additional treatment

on a normal diet. Intermittent follow-up into adolescence revealed

normal health, growth, and cognitive development. He was recon-

tacted for this study at the age of 19 years; at that time, it was noticed

that he had become an insulin-dependent diabetic about 6 months

previously.

In patient 2 (BIODEF 329), initial neonatal screening showed

hyperphenylalaninemia with high urinary levels of primapterin

(7-biopterin),19,20 which resolved after daily treatment with BH4.

There was parental consanguinity, and molecular studies showed

that he was homozygote for two separate mutations in the PCBD1

gene: c.99G.T (p.Glu26*) and 283G.A (p.Arg87Gln). He was fol-

lowed by pediatricians at the Klinik für Kinder und Jugendmedizin.

Patient 3 (BIODEF 319) is the first child of nonconsanguineous

parents. A previous sibling of the patient died neonatally, whereas a

younger brother was born healthy. The patient was diagnosed with

hyperphenylalaninemia and primapterinuria at the age of 1 month.20

Molecular analysis revealed compound heterozygous c.309G.

A/c.312C.T (p.Glu96Lys/p.Gln97*) mutations in the PCBD1 gene.

She needed cofactor BH4 replacement therapy on a normal diet to

maintain phenylalanine levels within the reference range. At the mo-

ment of recruitment for this study, she was 17 years old. At that time,

she had stopped taking BH4 around 1 year earlier and had slightly

elevated phenylalanine levels of 120 mmol/L (n,60 mmol/L). It was

also noticed that, just before turning 16 years old, she was diagnosed

with diabetes. MODY diabetes types 1, 2, and 3 were excluded by

DNA analysis. The patient is presently healthy and developing nor-

mally.

Calculation of FEMgThe FEMg was calculated using the following formula: FEMg=

(UMg3PCr)/[(0.73PMg)3UCr3100]. U and P refer to the urine and

serum concentrations, respectively ofMg2+ (Mg) and creatinine (Cr).

Serum Mg2+ concentration was multiplied by 0.7, because only ap-

proximately 70% of the circulating Mg2+ was unbound by albumin

and therefore, able to be filtered across the glomerulus.

Animal StudyAll the experimental procedures are in compliance with the animal

ethics board of the Radboud University Nijmegen. The transgenic

parvalbumin-eGFP mice were a gift from Dr. Hannah Monyer.38 In

the Mg2+ diet experiment, the mice were fed low (0.02% wt/wt) or

high (0.48% wt/wt) Mg2+-containing diets for 15 days (SSNIFF Spe-

zialdiäten GmbH, Soest, Germany). During the last 48 hours of the

experiment, the mice were housed in metabolic cages for urine col-

lection (24 hours of adaptation and 24 hours of sampling). Blood

samples were taken at the start of the experiment and just before

euthanasia. Parvalbumin-eGFP–positive tubules were isolated as de-

scribed previously.39 In short, mice ages 4–6 weeks were anesthetized

and perfused transcardially with ice cold buffer of 145 mmol/L NaCl,

5 mmol/L KCl, 1 mmol/L NaH2PO4, 2.5 mmol/L CaCl2, 1.8 mmol/L

MgSO4, 10 mmol/L glucose, and 10 mmol/L Hepes/NaOH (pH 7.4).

Kidneys were harvested and digested in buffer of 145 mmol/L NaCl,

5 mmol/L KCl, 1 mmol/L NaH2PO4, 2.5 mmol/L CaCl2, 1.8 mmol/L

MgSO4, 10 mmol/L glucose, and 10 mmol/L Hepes/NaOH (pH 7.4)

containing 1 mg/ml collagenase (Worthington, Lakewood, NJ) and

2000 units/ml hyaluronidase (Sigma-Aldrich, Houten, The Nether-

lands) for three cycles of a maximum of 15 minutes each at 37°C.

Subsequently, the kidney tubules between 40 and 100 mm were col-

lected by filtration. Tubules collected from the three digestions were

ice cooled and sorted by the COPAS (Union Biometrica, Holliston,

MA). Sorted tubules were directly collected in 1% (vol/vol) b-mer-

captoethanol containing RNA extraction buffer that was supplied by

the RNeasy RNA Extraction Kit (Qiagen, Venlo, The Netherlands).

Per mouse, 4000 eGFP-fluorescent tubules were collected; 4000 tu-

bules were pooled on amicrocolumn for RNAextraction according to

the manufacturer’s protocol. Subsequently, reverse transcription of

the RNA by M-MLV reverse transcription (Invitrogen) was per-

formed for 1 hour at 37°C. Gene expression levels were determined

by quantitative real-time PCR on a BioRad Analyzer and normalized

for Gapdh expression levels. Real-time PCR primers (Supplemental

Table 1) were designed using the online computer NCBI/Primer-

BLAST software.

Phenylalanine MeasurementsPhenylalanine in plasma and urine samples was measured by liquid

chromatography–tandemmass spectrometry (MS) by a standardized

method, in which the MS was operated in positive mode. For these

MS measurements, a Waters Premier triple quadrupole mass spec-

trometer (tandem MS) interfaced with an electrospray ionization

source and equipped with an Alliance ultra-performance liquid chro-

matography (Waters, Etten-Leur, The Netherlands) was used. Briefly,

to a 10-ml plasma or urine sample, 13C6-labeled phenylalanine was

added as an internal control to correct for possible ion suppression.

Samples were deproteinized with methanol, diluted with water, and

injected on an RP C18 column (Waters Atlantis T3; 3 mm, 2.13100

mm). Samples were eluted in 17.5% methanol/0.1 M formic acid.

Positively charged [M-H]+ phenylalanine (m/z=166) and 13C6-phe-

nylalanine (m/z=172) were selected as parent ions. After collision-

induced dissociation, the losses of -NH3 and -COOH groups were

chosen as quantifier ions (m/z=120 and =126, respectively), and the

losses of -NH3, -COOH, and -OH2 groups (m/z=103 and 109, re-

spectively) were chosen as qualifier ions. In Multiple Reaction Mon-

itoring mode, the transitionsm/z=166→120 and m/z=166→103 (for

phenylalanine) and m/z=172→126 and m/z=172→109 (for 13C6-

phenylalanine) were measured. Phenylalanine concentrations were

calculated using a calibration curve.

DNA ConstructsHumanHNF1B wild types andmutants were cloned into the pCINeo

hemagglutinin (HA) internal ribosomeentry site (IRES)GFPvector as

described previously.6 HNF1B was FLAG-tagged at the NH2 terminal

by Phusion PCR using NheI and AgeI restriction sites. To obtain an

HNF1B lacking the dimerization domain spanning residues 2–30

(D2–30), HNF1B was amplified using a Phusion polymerase

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(Finnzymes, Vantaa, Finland) with specific primers covering from

residue p.Glu31. The HNF1B-D2–30 was ligated into pCINeo IRES

GFP using AscI/AgeI restriction sites. The open reading frame of hu-

man PCBD1 was amplified using Phusion polymerase from PCBD1

pCMV-SPORT6 (Genbank accession number BC006324; ImaGenes)

and subcloned into the pCINeo HA IRES GFP vector using AgeI/

EcoRI restriction sites. Wild-type PCBD1 was FLAG-tagged at the

NH2 terminal by PCR using NheI/XhoI restriction sites. PCBD1 mu-

tations were inserted in the construct using the QuikChange site-

directed mutagenesis kit (Stratagene, La Jolla, CA) according to the

manufacturer’s protocol. The luciferase reporter plasmid containing

the FXYD2 promoter linked to the coding region of firefly luciferase

was produced as previously described.6 Briefly, the human FXYD2

promoter region that controls transcription of the g-subunit iso-

form-a (23229/+91 bp from the transcription start site) was ampli-

fied from genomic DNAusing Phusion polymerase (Finnzymes) and

cloned into pGL3-Basic (Promega) using KpnI/BglII restriction sites.

The human PKHD1 promoter region extending from exon 1 to 1.5 kb

upstream to the transcription start site was amplified from genomic

DNA and cloned into pGL3-Basic usingKpnI/HindIII sites. The pRL-

CMV vector encoding Renilla luciferase under control of a CMV

promoter was commercially available (Promega, Fitchburg, WI).

Primer sequences used for cloning or mutagenesis PCR are reported

in Supplemental Table 2. All constructs were verified by sequence

analysis.

Cell CultureHEK293 cellswere grown inDMEM(BioWhittaker-Europe,Verviers,

Belgium) containing 10% (vol/vol) FCS (Thermo Fisher HyClone),

10 ml/ml nonessential amino acids, and 2 mmol/L L-glutamine at 37°C

in a humidity-controlled incubator with 5% (vol/vol) CO2. The cells

were transiently transfected with the respective constructs using poly-

ethylenimine cationic polymer (Polysciences, Inc.) at a 1:6 DNA to

polyethylenimine cationic polymer ratio for 48 hours unless otherwise

stated.

Western BlottingHEK293 cells were transfected for 24 hours withHA-PCBD1mutants

and treated at the same time with 10mmol/LMG-132. Protein lysates

were denatured in Laemmli containing 100mmol/L dithiothreitol for

30 minutes at 37°C and subsequently subjected to SDS-PAGE. Then,

immunoblots were incubated with a mouse anti-HA (high-affinity

3F10, 1:5000; Roche) primary antibody and peroxidase-conjugated

sheep anti-mouse secondary antibodies (1:10,000; Jackson Immu-

noresearch).

ImmunocytochemistryHEK293 cells were seeded in 12-well plates on glass coverslips and

cotransfected with 400 ng PCBD1 constructs and 400 ng HNF1B

constructs or empty pCINeo IRES GFP vector (mock DNA). After 48

hours, HEK293 cells were fixed with 4% (wt/vol) paraformaldehyde

for 30 minutes at 4°C followed by the subsequent steps at room

temperature: permeabilization for 15 minutes with 0.3% (vol/vol)

Triton X-100 in PBS, incubation for 15 minutes with 50 mmol/L

NH4Cl, and finally, incubation in blocking buffer (16% [vol/vol]

goat serum, 0.3% [vol/vol] Triton X-100, and 0.3 M NaCl in PBS).

After incubation overnight at 4°Cwith a rabbit anti-FLAGM2 (F7425,

1:100; Sigma-Aldrich) or a mouse anti-HA (6E2, 1:100; Cell Signaling

Technology), cells were washed three times with Tris-buffered NaCl

Tween-20 (150mmol/LNaCl, 0.1mol/LTris/HCl [pH 7.5], and 0.05%

[vol/vol] Tween-20) and subsequently incubated with a secondary

goat anti-rabbit antibody (A4914, 1:300; Sigma-Aldrich) or a sheep

anti-mouse (1:300; Jackson Immunoresearch) coupled to AlexaFluor

594 for 45 minutes at room temperature. After incubation with

49,6-diamidino-2-phenylindole for 30 minutes at room temperature,

cells were washed three times with Tris-buffered NaCl Tween-20 and

finally mounted with Fluoromount-G (SouthernBiotech). Photo-

graphswere taken using a Zeiss Axio Imager 1microscope (Oberkochen,

Germany) equipped with an HXP120 Kubler Codix fluorescence lamp

and a Zeiss AxiocamMRm digital camera. Images were analyzed by use

of the software ImageJ.40

ImmunohistochemistryStaining was performed on 5-mm sections of PLP fixed frozen mouse

kidney or pancreas samples. Subsequently, sections were washed

three times with buffer of 0.15 mol/L NaCl and 0.1 mol/L Tris/HCl

(pH 7.6). Antigen retrieval was done with 10 mmol/L sodium citrate

(pH 6.0), and nonspecific binding was blocked by incubation in

blocking buffer for 1 hour. Sections were stained overnight at 4°C

with a polyclonal rabbit anti-PCBD1 antibody (F3862, 1:5000; kind

gift from Prof. Beat Thöny).26 The following day, sections were

washed and incubated with a goat anti-rabbit secondary antibody

coupled to Alexa Fluor 488 (A11008, 1:300; Invitrogen) for 1 hour

at room temperature. Kidney sections were costained for Bcrp, Tamm

Horsfall, NCC, calbindin-D28k (D28K), or Aqp2. Briefly, sections

were washed and incubated for 2 hours at room temperature with

rat anti-Bcrp (Clone BXP-9, MC-980, 1:200; Kamya Biomedical

Company), sheep anti-Tamm Horsfall (8595–0054, 1:1500; AbD Se-

rotec), guinea pig anti-NCC (1:50; gift from Jan Loffing), mouse anti-

28K (C-9848, 1:1500; Sigma-Aldrich), or guinea pig anti-Aqp2

(1:1000; gift from Peter Deen). Secondary antibodies were coupled

with Alexa Fluor 495 (1:300; Invitrogen) and incubated for 45minutes

at room temperature. Subsequently, sections were washed, incubated

for 10 minutes with 49,6-diamidino-2-phenylindole, and mounted

with Mowiol. Photographs were taken using a Zeiss Axio Imager 1

microscope equipped with an HXP120 Kubler Codix fluorescence

lamp and a Zeiss Axiocam MRm digital camera.

CoimmunoprecipitationHEK293 cells were seeded on 55-mm Petri dishes and cotransfected

with 5mg PCBD1 constructs and 5mgHNF1B constructs or an empty

pCINeo IRES GFP vector (mock DNA); 48 hours after transfection,

nuclear and cytosolic fractions were prepared using the NE-PER Nu-

clear Protein Extraction Kit (Pierce). The next incubation steps were

all done under rotary agitation at 4°C; 35 ml protein A-agarose beads

(Santa Cruz Biotechnology) were previously incubated overnight at

4°C with 2.5 mg rabbit anti-FLAG antibody (F7425; Sigma) and

washed with IPP500 (500 mmol/L NaCl, 10 mmol/L Tris adjusted

to pH 8.0 with HCl, 0.1% [vol/vol] NP-40, 0.1% [vol/vol] Tween-20,

and0.1%[wt/vol] BSA, andprotein inhibitors) four times.After sampling

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60ml as the input control, the remaining 180 ml lysate sample was added

to the antibody beads mixture overnight at 4°C. Then, the beads were

collected by centrifugation at 2000 rpm for 2 minutes at 4°C and washed

four timeswith lysis buffer. Theproteinswere separated from the beads by

incubation for 30 minutes at 37°C in 13 Laemmli sample buffer supple-

mentedwith 100mmol/L dithiothreitol and detected by immunoblotting

using a mouse anti-HA (high-affinity 3F10, 1:5000; Roche) or a mouse

anti-FLAG M2 (F3165, 1:5000; Sigma-Aldrich) primary antibodies and

peroxidase-conjugated sheep anti-mouse secondary antibodies (1:10,000;

Jackson Immunoresearch).

Luciferase Reporter AssayHEK293 cells were seeded on 12-well plates and transfected with the

followingDNA amounts: 700 ng FXYD2 promoter luciferase construct,

50ngPCBD1constructs, and50ngHNF1Bconstructs oremptypCINeo

IRES GFP vector (mock DNA). To correct for transfection efficiency,

10 ng pRL-CMV was used as a reference. Firefly and Renilla luciferase

activities were measured by use of a Dual-Luciferase Reporter Assay

(Promega) 48 hours after transfection.

Homology ModelingA homology model was built using the modeling script in the WHAT

IF & YASARATwinset with standard parameters.41,42 The structure of

the PCBD1 dimer in complex with the HNF1A dimerization domain

dimer was used as a template for modeling of HNF1B (Protein Data

Bank ID code 1f93). Our model contains only the dimerization do-

main (31 amino acids) of HNF1B, and it has 68% sequence identity

with the dimerization domain of HNF1A.

Statistical AnalysesAll results are depicted as mean6SEM. Statistical analyses were con-

ducted by unpaired t test when comparing two experimental condi-

tions and one-way ANOVA with Bonferroni test when comparing

more conditions. P values ,0.05 were considered significant.

ACKNOWLEDGMENTS

The authors are grateful to the patients for their participation in this

study. We appreciate the help of Drs. N. Blau and B. Thöny in the

recruitment of the patients and provision of the PCBD1 antibody.We

also thank Lonneke Duijkers and Annelies van Angelen for their ex-

cellent technical support.

This work was supported by The Netherlands Organization for

ScientificResearch (NWO)GrantsZonMw9120.8026andNWOALW

818.02.001, a European Young Investigator Award 2006, and Vici Grant

NWO-ZonMw016.130.668(toJ.G.J.H.)aswell asEURenOmics funding

from the European Union Seventh Framework Programme (FP7/2007-

2013, Agreement 305608).

DISCLOSURESNone.

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