Mutations in PCBD1 Cause Hypomagnesemia and Renal Magnesium Wasting

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† ‡ 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 Children’s Hospital, Calgary, Alberta, Canada; ‡Städtisches Klinikum Karlsruhe, Klinik für Kinder und Jugendmedizin, Karlsruhe, Germany; and §Sophia Children’s 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 Mg2+ loss, and two patients developed diabetes with characteristics of maturity onset diabetes of the young (MODY), regardless of serum Mg2+ levels. Our results suggest that these clinical findings 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 Mg2+-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 Mg2+ reabsorption in the DCT. Of seven PCBD1 mutations previously reported in HPABH4D patients, five mutations caused proteolytic instability, leading to reduced FXYD2 promoter activity. Furthermore, cytosolic localization of PCBD1 increased when coexpressed with HNF1B mutants. Overall, our findings establish PCBD1 as a coactivator of the HNF1B-mediated transcription necessary for fine 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: 574–586, 2014. doi: 10.1681/ASN.2013040337

Hypomagnesemia is a common clinical manifesta- In adult kidney, HNF1B is expressed in epithelial tion in patients with mutations in the transcription cells along all segments of the nephron. The role of factor hepatocyte nuclear factor 1 homeobox B HNF1B in renal Mg2+ handling was, however, (HNF1B [Mendelian Inheritance in Man (MIM) 189907]).1 Mutations in HNF1B are associated with an autosomal dominant syndrome characterized by Received April 3, 2013. Accepted September 14, 2013. renal malformations with or without cysts, liver and S.F. and J.H.F.d.B. contributed equally to this work. genital tract abnormalities, gout, and maturity- Published online ahead of print. Publication date available at onset diabetes of the young type 5 (MODY5; renal www.jasn.org. cysts and diabetes syndrome [MIM 137920]).1,2 Hy- Correspondence: Dr. René J.M. Bindels, Radboud University Medi- 2+ pomagnesemia (plasma Mg levels,0.7 mmol/L) cal Center, Department of Physiology 286, PO Box 9101, 6500 HB, with hypermagnesuria affects up to 50% of the Nijmegen, The Netherlands. Email: [email protected] HNF1B patients.1,3 Copyright © 2014 by the American Society of Nephrology

574 ISSN : 1046-6673/2503-574 J Am Soc Nephrol 25: 574–586, 2014 www.jasn.org BASIC RESEARCH pinpointed to the distal convoluted tubule (DCT), where the final might be related to hypomagnesemia, including fatigue, mus- urinary Mg2+ excretion is determined.1,4 In DCT, the Na+/K+- cular pain, weakness and cramps in arms, numbness, difficulty ATPase provides the necessary driving force for active Mg2+ with memory, chest pains, and blurred vision, with some im- reabsorption from the prourine into the blood.4 Heterozygous provement after Mg2+ supplementation. An abdominal ultra- mutations in the FXYD2 gene, encoding the g-subunit of the sound showed slightly increased echogenicity of both liver and Na+/K+-ATPase, result in an autosomal dominant renal hypo- kidney of uncertain cause; renal size was normal, with no cysts magnesemia with hypocalciuria (MIM 154020).5 More recently, present (Supplemental Figure 2, A and B). Renal function was functional HNF1B binding sites were identified in the promoter normal (GFR=128 ml/min per 1.73 m2, N$90). Laboratory region of FXYD2, suggesting that impaired transcription of investigations of patient 2 revealed a relatively high 24-hour FXYD2 by HNF1B results in renal Mg2+ wasting.1,6 Additional urinary Mg2+ excretion (5.25 mmol/24 h, N=2–8) (Table 1) in HNF1B target genes in kidney include renal cystic genes7–9 as the presence of hypomagnesemia (0.65 mmol/L), with no sec- well as genes involved in tubular electrolyte transport.10,11 ondary symptoms. Serum and 24-hour urinary Ca2+ excretion HNF1B forms heterotetrameric complexes with the protein were within the normal range as was creatinine clearance pterin-4a-carbinolamine dehydratase/dimerization cofactor (126 ml/min, N=89–143). Ultrasound examinations excluded of hepatocyte nuclear factor 1 homeobox A (PCBD1 [MIM the presence of renal hypoplasia or cysts (Supplemental Figure 126090]).12 PCBD1 is a protein of 12 kDa with two distinct 2C). Patient 3 showed borderline serum Mg2+ levels biologic functions: transcriptional coactivation of HNF1A- (0.74 mmol/L) but displayed normal urinary Mg2+ excretion mediated transcription within the nucleus and PCBD1 (EC (FEMg=2.5%). 4.2.1.96) in the cell cytosol.12,13 The enzymatic activity of PCBD1, together with dihydropteridine reductase, regenerates MODY in Two Patients with PCBD1 Mutations tetrahydrobiopterin (BH4), which is the cofactor for phenylal- In addition to hypomagnesemia, patient 1 was diagnosed with anine hydroxylase and other aromatic amino acid hydrolases.14 diabetes. Type 1 autoimmune diabetes was unlikely, because The crystal structure of PCBD1 revealed that the protein forms a the patient lacked islet cell antibodies and showed normal tetramer of identical subunits comprising two saddle-like shaped serum C-peptide levels (0.69 nmol/L, N=0.3–1.32 nmol/L). dimers.15,16 HNF1 binding sites are located at the same surface Because MODY patients are generally highly sensitive to sul- that mediates interaction of the PCBD1 homodimers on the op- phonylureas,22 he was treated with Gliclazide 80 mg two times posite side of the catalytic domain.17 PCBD1 knockout mice dis- per day, to which he responded well, allowing his previous play hyperphenylalaninemia, predisposition to cataracts, and mild insulin treatment of 20–30 units/d to be discontinued. Addi- glucose intolerance.18 Homozygous or compound heterozygous tional extrarenal manifestations in HNF1B disease include PCBD1 mutations in humans are associated with transient neo- liver test abnormalities.23,24 Liver function tests in patient 1 natal hyperphenyalaninemia and high urinary levels of primap- revealed that the plasma levels of high-sensitivity C-reactive terin (HPABH4D; or primapterinuria [MIM 264070]).19–21 To protein (hs-CRP) were significantly low at ,0.1 mg/L (N,8 date, there have been no reports of late complications or possible mg/L) (Table 2). IgG was slightly low at 5.66 g/L (N=6.94– phenotypic consequences of impaired stimulation of the HNF1 16.18 g/L), whereas the remaining liver function tests were all transcription factors. within the normal range. Based on his glycosylated hemoglo- In our study, the occurrence of hypomagnesemia and MODY bin (HbA1c) levels, we concluded that patient 2 did not diabetes was investigated in three patients carrying PCBD1 mu- develop diabetes (HbA1c=4.95%, N=4.3%–6.1%) (Table 1). tations. We evaluated whether PCBD1 plays a role in renal Mg2+ Abdominal ultrasound analysis showed that the pancreas and reabsorption by directly affecting HNF1B-regulated FXYD2 liver were normal (Supplemental Figure 2, D and E). Patient 3 transcription to gain new insight into the molecular basis of was also diagnosed with diabetes of no autoimmune origin the PCBD1–HNF1B interaction. because of the absence of islet cell antibodies. HbA1c levels were 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- RESULTS tations in HNF4A, GCK,andHNF1A, respectively, were excluded by DNA analysis. Presently, the patient is not receiving Homozygous PCBD1 Mutations Are Associated with medications or insulin treatment. Hypomagnesemia and Renal Mg2+ Wasting We diagnosed hypomagnesemia and hypermagnesuria in two Pcbd1 Expression in DCT Is Modulated by Dietary patients carrying mutations on both alleles in the PCBD1 gene Mg2+ Content (Table 1). In patient 1, hypomagnesemia was partially correc- We examined Pcbd1 mRNA expression levels in a mouse tissue tedwithoralMg2+ supplements at a dose of 500 mg/d (0.64– panel using real-time RT-PCR. Highest expression was mea- 0.76 mmol/L after first supplementation, 0.66–0.69 mmol/L sured in kidney and liver (Figure 1A). Furthermore, immu- after second supplementation), although at the expense of nohistochemical analysis of mouse pancreas sections revealed increased magnesuria (fractional excretion of Mg2+ [FEMg]; that Pcbd1 is expressed in pancreatic cells (Figure 1B). Mouse 4.6%–7.8%, N,4%). He had nonspecificsymptomsthat kidney sections were stained for Pcbd1 to evaluate whether

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Table 1. Pertinent laboratory investigations for three patients with PCBD1 mutations Patient 1 (BIODEF 272) Patient 2 (BIODEF 329) Patient 3 (BIODEF 319) Parameter Reference Range Baseline 500 mg/d Mg2+ Baseline Baseline Serum indices Na+ (mmol/L) 141 — 138 139 135–145 K+ (mmol/L) 4.0 — 4.5 4.0 3.5–5.0 Mg2+ (mmol/L) 0.64a 0.76 0.65a 0.74 0.7–1.1 Ca2+ (mmol/L) 2.41 2.55 2.25 2.43 2.20–2.65 Pi (mmol/L) 1.13 1.16 — 1.15 0.8–1.4 Creatinine (mmol/L) 0.069 0.066 0.062 0.056 0.045–0.110 HbA1c (%) 7.2a — 4.95 6.5a 4.3–6.1 Uric acid (mmol/L) 310 —— —135–510 Urinary indices Mg2+ (mmol/L) 6.16 3.96 — 2.6 — Mg2+ (mmol/24 h) —— 5.25 — 2–8 FEMg (%) 6.6a 11.1a — 3.5 ,4 Ca2+ (mmol/L) 4.47 3.04 — 2.99 — Ca2+ (mmol/24 h) —— 5.61 — ,7.5 FeCa (%) 0.9 1.8 — 0.86 .1 Pi (mmol/L) 40.29 13.61 — 18.2 — FePi (%) 17 17.6 ——5–20 Creatinine (mmol/L) 14.29 4.40 — 8.0 — Creatinine (mmol/24 h) —— 14 — 9–18 GFR (ml/min per 1.73 m2) 128 135 — 124 $90 CCr (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 Disease formula. FeCa, fractional excretion of Ca2+; FePi, fractional excretion of phosphate (Pi); CCr, creatinine clearance. aAbnormal value.

isolated DCT fragments. Pcbd1 expression was significantly Table 2. Liver function tests in patient 1 (BIODEF 272) higher in DCT compared with whole kidney (Figure 1H). Hnf1b transcript was not enriched in the DCT fraction. In- Parameter Patient 1 (BIODEF 272) Reference Range terestingly, Pcbd1 expression in the DCT was significantly up- Albumin (g/L) 41 38–50 regulated when mice were fed a low Mg2+-containing diet Prealbumin (g/L) 0.381 0.1–0.4 2+ a compared with a high Mg -containing diet (Figure 1I). hs-CRP (mg/L) ,0.1 ,8 2+ fi fi – Mice on a Mg -de cient diet displayed a signi cantly lower a-1 antitrypsin (g/L) 1.4 0.9 2.6 2+ – 24-hour Mg excretion (Supplemental Figure 1B) in response C3 (g/L) 1.17 0.8 2.1 – C4 (g/L) 0.18 0.15–0.5 to the hypomagnesemia (1.1 versus 2.2 mmol/L, N=1.2 1.5 25 IgG (g/L) 5.66a 6.94–16.18 mmol/L ) (Supplemental Figure 1A). Differences were not IgM (g/L) 0.75 0.6–3.0 observed in the serum and urinary levels of phenylalanine IgA (g/L) 1.99 0.7–4.0 between the experimental groups (Supplemental Figure 1, C ALT (U/L) 20a 21–72 and D). AST (U/L) 26 15–46 – ALP (U/L) 108 30 130 PCBD1 Enhances FXYD2 and PKHD1 Promoter C3, complement component 3; C4, complement component 4; ALT, alanine Activation by HNF1B transaminase; AST, aspartate transaminase; ALP, alkaline phosphatase. aAbnormal value. To 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 structural Pcbd1 locates to the sites of renal Mg2+ reabsorption (Figure 1, homology model of the PCBD1–HNF1B dimerization domain 2 C–G). Pcbd1 staining colocalized with Na+/Cl cotransporter (HNF1B–D) complex using the structure of the PCBD1–HNF1A (NCC) in DCT and in part, Tamm Horsfall and calbindin-D28K dimerization domain tetramer (Figure 2, A and B). The p.Glu26* expression in the cortical thick ascending limb of Henle’s mutation leads to a major protein truncation and complete loss of loop (TAL) and connecting tubule, respectively. No colocali- the interaction domain with HNF1B (Figure 2B). Although zation of Pcbd1 was detected with markers of the proximal p.Gln97* does not directly affect the HNF1 binding domain of tubule or collecting duct, which are Bcrp and Aqp2, respec- PCBD1, the mutation results in a truncation of the central a-helix tively. The expression of Pcbd1 in DCT was confirmed in of the protein. Consequently, the interaction domain might be

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destabilized, explaining the PCBD1 dysfunc- tion.Similarly,thenegativelychargedGlu96 residue could be important for the stabiliza- tion of the interaction domain, which may be hampered by the p.Glu96Lys mutation. To test the effects of the mutations on PCBD1 function, we studied all previously described patient mutations in their ability to coactivate FXYD2 transcription in a luciferase assay (Figure 2C).19–21 Expression of wild-type PCBD1 did not change the luciferase activity compared with mock- transfected cells, whereas HNF1B signiﬁ- cantly enhanced the FXYD2 promoter activity (Figure 2D). Interestingly, coexpression of PCBD1 with HNF1B further increased FXYD2 promoter activation by ;1.5 fold (Figure 2D). Of all PCBD1 mutants, only PCBD1 p.Arg87Gln and p.Cys81Arg maintained their coactivator properties (Figure 2D). The transcription of another established target gene of HNF1B in the kidney, PKHD1,8 was

tissue expression. (B) Immunohistochemical analysis of Pcbd1 in mouse pancreas tissue. Scale bar, 20 mm. (C–G) Mouse kidney sections were 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-diamidino-2-phenylindole staining (blue). TH, Tamm Horsfall; 28K, calbindin-D28K. Scale bar, 20 mm. (H) Kidney expression pattern of Pcbd1 shows the highest expression in DCT. The mRNA expression levels of Pcbd1 and Hnf1b in (black bars) COPAS-selected mouse DCT and (white bars) control (nonselected) kidneys were measured by quantitative RT-PCR and normalized for Gapdh expression. Data represent the mean of three individual experiments6SEM and are expressed as fold difference compared with the expression in nonselected tubules. *P,0.05 versus total kidney. (I) DCT expression of Pcbd1 is regulated by dietary Mg2+ intake. The mRNA expression levels of Pcbd1 and Hnf1b in COPAS- selected mouse DCT kidney tubules from mice fed with (white bars) low and (black bars) high Mg2+-containing diets were measured by real- time RT-PCR and normalized for Gapdh expression. Data represent the mean of four individual experiments6SEM and are expressed as fold Figure 1. Pcbd1 is expressed in the DCT of the kidney. (A) Tissue expression pattern of the difference compared with the expression in high Pcbd1 transcript. Pcbd1 mRNA expression level was measured in a panel of mouse tissues Mg2+-containing diets. *P,0.05 versus high Mg2+. by quantitative RT-PCR and normalized for Gapdh1 expression. Data represent the mean Aqp2,aquaporin2;Bcrp,breastcancerre- of three individual experiments6SEM and are expressed as the percentage of the total sistance protein.

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Figure 2. PCBD1 coactivates HNF1B-induced FXYD2 and PKHD1 promoter activity. (A) Homology model of the PCBD1–HNF1B dimerization 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 helix sequences. The HNF1A–D monomer is shown in yellow. Residues in the PCBD1 protein that were found mutates in patients affected by

578 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 574–586, 2014 www.jasn.org BASIC RESEARCH assessed in the presence of PCBD1. PKHD1 promoter activity assays showed that only HNF1B p.His324Ser325fsdelCA and showed a ;1.5-fold increase expression levels when HNF1B p.Tyr352fsinsA, which retained partial transcriptional activity, and PCBD1 were coexpressed (Figure 2E). PCBD1 p.Arg87Gln, respond to the coactivation by PCBD1 to the same extent as but not p.Cys81Arg, was the only PCBD1 mutant that retained HNF1B wild type (;1.5-fold) (Figure 4C). Importantly, im- its coactivator activity (Figure 2E). munocytochemical analysis revealed that coexpression of PCBD1 with the HNF1B mutants p.Gln253Pro and p.His324- Mutations Detected in HPABH4D Patients Cause Ser325fsdelCA causes a predominant cytosolic localization of Protein Degradation of PCBD1 PCBD1 compared with the nuclear translocation observed on To explain why the PCBD1 mutants are not capable of en- coexpression with HNF1B wild type (Figure 4, D and E). hancing HNF1B-induced transcription, we examined their subcellular localization and their capacity to bind HNF1B. Immunostaining for PCBD1 in transiently transfected human DISCUSSION embryonic kidney (HEK293) cells revealed that wild-type PCBD1 translocates to the nucleus on coexpression with Mutations in PCBD1 have been shown to cause a transient and HNF1B compared with mock DNA (Figure 3A). Furthermore, benign form of neonatal HPABH4D.19–21 Here, we present the PCBD1 p.Glu26*, p.Glu86*, p.Glu96Lys, and p.Gln97* were first follow-up study of HPABH4D patients reporting the on- not expressed, whereas p.Thr78Ile and p.Cys81Arg were set of late complications linked to the deficient activity of detected significantly less than the wild-type protein. PCBD1 as a transcriptional coactivator of HNF1B. Our results PCBD1 p.Arg87Gln was the only mutant showing a compa- suggest that PCBD1 acts as an important transcriptional reg- rable expression level with wild-type PCBD1 (Figure 3A). ulator of FXYD2, contributing to renal Mg2+ reabsorption in Coimmunoprecipitation studies confirmed that only DCT. Our observations are based on the following results: (1) PCBD1 p.Arg87Gln, p.Thr78Ile, and p.Cys81Arg were ex- hypomagnesemia with renal Mg2+ wasting was reported in pressed and able to bind HNF1B (Figure 3B, top and bottom two of three patients carrying mutations in the PCBD1 gene; panels). Importantly HNF1B was equally expressed in all con- (2) two patients were diagnosed with MODY; (3)PCBD1is ditions (Figure 3B, middle panel). To examine whether mu- present in the pancreas and kidney, predominantly in DCT, tated PCBD1 proteins are degraded by the proteasomal path- where its expression is sensitive to dietary Mg2+ content; (4) in way for misfolded proteins, PCBD1-expressing HEK293 cells vitro data showed that PCBD1 binds HNF1B to costimulate were treated for 24 hours with 10 nM proteasome inhibitor the FXYD2 promoter, and its activity contributes to Mg2+ re- MG-132 (N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)- absorption in DCT; and (5) PCBD1 mutations reported in 1-formyl-3-methylbutyl]-L-leucinamide). Protein expression HPABH4D patients caused proteolytic instability, leading to was restored for all of the PCBD1 mutants, with the only ex- degradation through the proteasomal pathway. ception being p.Glu26* (Figure 3C). To our knowledge, we are the first to report that PCBD1 mutations associate with hypomagnesemia because of renal HNF1B Mutations Affect the Subcellular Localization of Mg2+ wasting as well as MODY. In the kidney, the key sites for PCBD1 Mg2+ reabsorption are TAL and DCT. Although defects in the We evaluated five HNF1B mutations (p.Lys156Glu, p.Gln253Pro, molecular pathway for Mg2+ handling in TAL lead to a con- p.Arg276Gly, p.His324Ser325fsdelCA, and p.Tyr352fsinsA) for comitant waste of Ca2+, shortcomings in DCT cause hyper- their ability to bind and functionally respond to PCBD1 (Figure magnesuria associated with hypo- or normocalciuria. Based 4A).1,3 All HNF1B mutants, except HNF1B D2–30, bound on the serum Ca2+ levels and urinary Ca2+ excretion values, it PCBD1 in coimmunoprecipitation studies in transiently trans- is likely that, in our patients, the DCT is primarily affected. fected HEK293 cells (Figure 4B, top panel). PCBD1 and Fluorescence-based sorting of DCT-enhanced green fluores- HNF1B were expressed in all conditions tested (Figure 4B, cent protein (eGFP) tubules using a Complex Object Para- middle and bottom panels). FXYD2 promoter luciferase 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–D monomer is shown in yellow. The residues that differ between HNF1B–D and HNF1A–D are visualized in grey. (C) Linear representation of the secondary structure elements of the human PCBD1 protein. Red arrowheads indicate the positions of the patient mutations described in the literature. Green balls indicate the histidine residues involved in the dehydratase active site (His61, His62, and His79). A luciferase assay was performed in HEK293 cells transiently cotransfected with a (D) Firefly luciferase FXYD2 and (E) PKHD1 promoter construct and HNF1B or mock DNA in the presence of wild-type or mutant PCBD1. A Renilla luciferase construct was cotransfected to correct for transfection efficiency. Firefly/Renilla luciferase ratios were determined as a measure of promoter activity. Results are depicted as percentage 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 of Pcbd1 expression in DCT tubules compared with other nephron segments. Pcbd1 expression in DCT was increased in mice fed with a low Mg2+ diet, suggesting that Pcbd1 is important for renal Mg2+ reabsorption in DCT. Serum and urinary phenylalanine levels were stable in the mice, and thus, they did not modulate Pcbd1 expression. Immunohistochemical analysis showed that Pcbd1 expression is not limited to DCT but partially extended to cortical TAL and connecting tubule. Overall, these findings confirm previous immunohistochemical studies showing Pcbd1 expression in the cortex of the kidney.26 Because Pcbd1 was present in the cytosol of renal cells, we hypothesized that the relative abundance of PCBD1 and HNF1B in the kidney may favor the cytosolic localization. Nevertheless, a nuclear localization of PCBD1 in the kidney could occur according to tissue-specific dynamics of the PCBD1–HNF1B complex. Our results showed that PCBD1 enhances the FXYD2 promoter activation by HNF1B in vitro. The relevance of FXYD2 activity in Mg2+ handling in DCT has been suggested previously, because both patients with FXYD2 mutations and patients carrying HNF1B mutations may present with hypomagnesemia.1,5 It was shown that HNF1B binds the FXYD2 promoter to specifically regulate the transcription of the g-subunit of the Na+/K+-ATPase isoform- a.1,6 ga-Subunit is mainly expressed in proximal tubule and medullary TAL, but it is also expressed in DCT (Supplemental Figure 3),6,27 where PCBD1 is expressed. To date, the exact molecular mechanism by which the g-subunit regulates Mg2+ handling in DCT remains elusive.4 Figure 3. Several PCBD1 mutations lead to protein degradation HEK293 cells. (A) In the kidney, HNF1B is ubiquitously Immunocytochemistry analysis of the subcellular localization of HA-tagged PCDB1 wild type or HA-tagged PCDB1 mutants when coexpressed in a 1:1 ratio with FLAG-tagged expressed, where it regulates the transcrip- 9 HNF1B or mock DNA in HEK293 cells. Red signal represents immunodetected HA tion of several cystic genes, like PKHD1. epitopes. Nuclei stained with 49,6-diamidino-2-phenylindole are shown in blue. Scale Mutations in PKHD1 are causative for au- bar, 10 mm. Representation immunocytochemical images are shown. (B) HA-tagged tosomal recessive polycystic kidney disease PCDB1 wild-type, PCDB1 mutant, or mock DNA were transiently expressed in HEK293 (MIM 263200).8 Similar to the FXYD2 cells with or without FLAG-tagged HNF1B. Immunoprecipitations on nuclear extracts using an anti-FLAG antibody were separated by SDS-PAGE, and Western blots were probed with (top) anti-HA and (middle) anti-FLAG antibodies. (Bottom) HA- PCDB1 input (25%) expression was also included in the analysis. The immunoblots immunoprecipitation; Mock, mock DNA; WB, shown are representative of three independent experiments. (C) Western blot analysis Western blot; WT, wild-type; 26, p.Glu26+;78, of HA-tagged PCDB1 mutants expressed in HEK293 cells treated with (+) or without p.Thr78lle; 81, p.Cys81Arg; 86, p.Glu86*; 87, (2) 10 nM MG-132 for 24 hours. A representative immunoblot is shown. IP, 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, transcription coactivation, and subcellular localization. (A) Linear representation of the human HNF1B protein. Red arrowheads indicate patient mutations 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-FLAG antibody 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 three independent experiments. IP, immunoprecipitation. (C) A luciferase assay was performed in HEK293 cells transiently cotransfected with aFirefly luciferase FXYD2 promoter construct and each of the HNF1B variants (black bars) with and (white bars) without PCDB1. A Renilla luciferase construct was cotransfected to correct for transfection efficiency. Firefly/Renilla luciferase ratios were determined as a measure of promoter activity. Results are depicted as percentage compared with HNF1B/Mock transfected cells. *P,0.05 compared with the HNF1B/Mock condition (n=9). (D) Immunocytochemistry analysis of the subcellular localization of FLAG-tagged PCDB1 when coexpressed in a 1:1 ratio with HA-tagged HNF1B WTs, HA-tagged HNF1B mutants, or mock DNA HEK293 cells. Red signal represents immunodetected FLAG epitopes. Scale bar, 20 mm. The immunocytochemical images shown are representative for three independent experiments. (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 with Mock. #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.

J Am Soc Nephrol 25: 574–586, 2014 PCBD1 and Hypomagnesemia 581 BASIC RESEARCH www.jasn.org promoter, a PKHD1 reporter construct showed increased ex- PCBD1 monomers are small molecules that can passively pression levels when HNF1B and PCBD1 were cotransfected diffuse from the cytosol into the nucleus.36 It was suggested in HEK293 cells. However, the renal expression of PKHD1 in that, by assembling through the same interface, PCBD1 ho- vivo is restricted to the loops of Henle and collecting duct, motetramer and PCBD1–HNF1 complexes are mutually ex- where PCBD1 expression is negligible.28 Thus, under physi- clusive.17 In our immunocytochemical analysis, the HNF1B ologic conditions, renal PKHD1 gene transcription seems to mutants p.Gln253Pro and p.His324Ser325fsdelCA signifi- not be affected by PCBD1. This finding is supported by the cantly stimulated a cytosolic localization of PCBD1 compared absence of kidney abnormalities in patients with PCBD1 with the nuclear PCBD1 localization observed in the presence mutations. of wild-type HNF1B. This finding suggests that HNF1B mu- Additional evidence for an impaired HNF1-mediated tations may disturb the stability of the PCBD1–HNF1 com- transcription in HPABH4D patients was provided by the di- plexes in the nucleus and therefore, favor the formation of agnosis of MODY in patients 1 and 3. MODY is a monogenic PCBD1 homotetramers in the cytosol of the cell.1,3 The re- form of autosomal dominant type 2 diabetes characterized duced nuclear localization of PCBD1 will indirectly result in a by age of onset often below 25 years and negative pancreatic decreased coactivation of HNF1B and could contribute to the autoantibodies. Heterozygous mutations in HNF1B and its hypomagnesemia observed in some HNF1B patients. Because homolog HNF1A associate with MODY types 5 and 3, respec- the presentation and development of HNF1B disease is di- tively.29 We showed that Pcbd1 is localized in the nuclei of verse, variations in HNF1B interacting proteins may be re- pancreatic cells. Knowing that PCBD1 acts as transcriptional sponsible for the phenotypic heterogeneity. Screening of coactivator of both HNF1B and HNF1A, a dysregulation of HNF1B patients for polymorphisms in PCBD1 should, there- HNF1B and/or HNF1A is potentially responsible for the fore, be considered. MODY diagnosed in patients 1 and 3. Interestingly, the low Inconclusion, weidentified PCBD1 as a new molecular player plasma hs-CRP levels in patient 1 are in line with two recent in renal Mg2+ handling. Our results suggest that PCBD1 regu- studies showing lower hs-CRP levels in MODY3 patients than lates the HNF1B-mediated FXYD2 transcription, influencing individuals with other forms of diabetes and nondiabetic con- active renal Mg2+ reabsorption in DCT. So far, HPABH4D trols.30,31 This evidence favors the diagnosis of MODY3-like caused by PCBD1 mutations has been considered a transient, diabetes in patient 1. Patient 2 was not diagnosed with diabe- benign condition, primarily related to impaired BH4 regenerates, but he will be monitored for later onset. tion. To date, 23 patients with PCBD1 mutations linked to In this study, we showed that the HPABH4D patient mu- HPABH4D are listed in the International Database of Tetrahy- tations reported in the literature lead to protein degradation of drobiopterin Deficiencies (BIODEF database; http://www.bio- PCBD1 through the proteasome pathway. Our data, together pku.org/biodef/).37 Here, we suggest that patients affected by with previous studies on proteolytic instability for the same HPABH4D should be monitored for late complications related mutants in both mammalian and bacterial expression systems, to the interactions with HNF1 transcription factors, including support the hypothesis that this degradation process may also hypomagnesemia and MODY. occur in HPABH4D patients.19,20,32 Considering the inheritance of the disease, HNF1-mediated transcription seems to be sensitive to changes in PCBD1 quantity only when both PCBD1 al- CONCISE METHODS leles are affected, suggesting that PCBD1 probably belongs to an ancillary regulatory mechanism to which other HNF1B partners Patients may participate.33 In vitro data in HEK293 cells revealed that The three patients reported in this study were ascertained by PCBD1 p.Arg87Gln was the only mutant showing an expression contacting the authors of papers related to hyperphenylalaninemia, level comparable with the wild-type protein. Nevertheless, in tetrahydrobiopterin-deficient HPABH4D (MIM 264070).19–21 Of 23 patient 2, p.Arg87Gln is homozygously present on both alleles, patients listed in the International Database of Tetrahydrobiopterin with a p.Glu26* mutation that leads to a significant decrease in Deficiencies (BIODEF database; http://www.biopku.org/biodef/),37 the cellular content of PCBD1.20 PCBD1 p.Glu26* degradation BIODEF 272, 329, and 319 were assessed by P.F. (Canada), R.G. could not be rescued by proteasome inhibition, suggesting that (Germany), and J.B.C.d.K. (The Netherlands), respectively. The re- itstranscriptisdegradedbymRNAsurveillancemecha- sults are included in this study. Informed consent to participate in this nisms.34,35 PCBD1 p.Thr78Ile and p.Cys81Arg were signifi- study was obtained, and the procedures followed were in accordance cantly less expressed than the wild-type protein. Both mutants with the standards of the medical ethics committee of each partici- showed binding to HNF1B, but only PCBD1 p.Cys81Arg co- pating institution. Patients were not treated with any medication that activated the FXYD2 promoter. PCBD1 p.Cys81Arg did not could affect serum levels of Mg2+ (e.g., diuretics, calcineurin inhib- coactivate the PKHD1 promoter, suggesting insufficient ex- itors, and corticosteroids). pression to enhance the transcription of PKHD1. In the near Patient 1 (BIODEF 272) was reported in detail previously.19 In future, it would be of interest to screen patients with PCBD1 summary, he was found to have borderline hyperphenylalaninemia p.Thr78Ile and p.Cys81Arg mutations for complications re- on newborn screening, but this markedly increased to a peak of lated to the HNF1B disease. 2589 mmol/L at 3.5 weeks. The diagnosis of HPABH4D was suspected

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because of primapterinuria (7-biopterin) as well as a marked re- 5 mmol/L KCl, 1 mmol/L NaH2PO4, 2.5 mmol/L CaCl2,1.8mmol/L sponse to BH4. There was no parental consanguinity; molecular anal- MgSO4, 10 mmol/L glucose, and 10 mmol/L Hepes/NaOH (pH 7.4). ysis confirmed the diagnosis, showing that he was homozygous for a Kidneys were harvested and digested in buffer of 145 mmol/L NaCl, c.312C.T (p.Gln97*) mutation in the PCBD1 gene. He was treated 5 mmol/L KCl, 1 mmol/L NaH2PO4, 2.5 mmol/L CaCl2,1.8mmol/L with phenylalanine restriction and BH4 until 4 months of age, at which MgSO4, 10 mmol/L glucose, and 10 mmol/L Hepes/NaOH (pH 7.4) time his phenylalanine levels normalized without additional treatment containing 1 mg/ml collagenase (Worthington, Lakewood, NJ) and on a normal diet. Intermittent follow-up into adolescence revealed 2000 units/ml hyaluronidase (Sigma-Aldrich, Houten, The Nether- normal health, growth, and cognitive development. He was recon- lands) for three cycles of a maximum of 15 minutes each at 37°C. tacted for this study at the age of 19 years; at that time, it was noticed Subsequently, the kidney tubules between 40 and 100 mm were col- that he had become an insulin-dependent diabetic about 6 months lected by filtration. Tubules collected from the three digestions were previously. ice cooled and sorted by the COPAS (Union Biometrica, Holliston, In patient 2 (BIODEF 329), initial neonatal screening showed MA). Sorted tubules were directly collected in 1% (vol/vol) b-mer- hyperphenylalaninemia with high urinary levels of primapterin captoethanol containing RNA extraction buffer that was supplied by 19,20 (7-biopterin), which resolved after daily treatment with BH4. the RNeasy RNA Extraction Kit (Qiagen, Venlo, The Netherlands). There was parental consanguinity, and molecular studies showed Per mouse, 4000 eGFP-fluorescent tubules were collected; 4000 tu- that he was homozygote for two separate mutations in the PCBD1 bules were pooled on a microcolumn for RNA extraction according to gene: c.99G.T (p.Glu26*) and 283G.A (p.Arg87Gln). He was fol- the manufacturer’s protocol. Subsequently, reverse transcription of lowed by pediatricians at the Klinik für Kinder und Jugendmedizin. the RNA by M-MLV reverse transcription (Invitrogen) was per- Patient 3 (BIODEF 319) is the first child of nonconsanguineous formed for 1 hour at 37°C. Gene expression levels were determined parents. A previous sibling of the patient died neonatally, whereas a by quantitative real-time PCR on a BioRad Analyzer and normalized younger brother was born healthy. The patient was diagnosed with for Gapdh expression levels. Real-time PCR primers (Supplemental hyperphenylalaninemia and primapterinuria at the age of 1 month.20 Table1)weredesignedusingtheonlinecomputerNCBI/Primer- Molecular analysis revealed compound heterozygous c.309G. BLAST software. A/c.312C.T (p.Glu96Lys/p.Gln97*) mutations in the PCBD1 gene. She needed cofactor BH4 replacement therapy on a normal diet to Phenylalanine Measurements maintain phenylalanine levels within the reference range. At the mo- Phenylalanine in plasma and urine samples was measured by liquid ment of recruitment for this study, she was 17 years old. At that time, chromatography–tandem mass spectrometry (MS) by a standardized she had stopped taking BH4 around 1 year earlier and had slightly method, in which the MS was operated in positive mode. For these elevated phenylalanine levels of 120 mmol/L (n,60 mmol/L). It was MS measurements, a Waters Premier triple quadrupole mass spec- also noticed that, just before turning 16 years old, she was diagnosed trometer (tandem MS) interfaced with an electrospray ionization with diabetes. MODY diabetes types 1, 2, and 3 were excluded by source and equipped with an Alliance ultra-performance liquid chro- DNA analysis. The patient is presently healthy and developing nor- matography (Waters, Etten-Leur, The Netherlands) was used. Briefly, 13 mally. to a 10-ml plasma or urine sample, C6-labeled phenylalanine was added as an internal control to correct for possible ion suppression. Calculation of FEMg Samples were deproteinized with methanol, diluted with water, and The FEMg was calculated using the following formula: FEMg= injected on an RP C18 column (Waters Atlantis T3; 3 mm, 2.13100

(UMg3PCr)/[(0.73PMg)3UCr3100]. U and P refer to the urine and mm). Samples were eluted in 17.5% methanol/0.1 M formic acid. 2+ + 13 serum concentrations, respectively of Mg (Mg) and creatinine (Cr). Positively charged [M-H] phenylalanine (m/z=166) and C6-phe- Serum Mg2+ concentration was multiplied by 0.7, because only ap- nylalanine (m/z=172) were selected as parent ions. After collision- 2+ proximately 70% of the circulating Mg was unbound by albumin induced dissociation, the losses of -NH3 and -COOH groups were and therefore, able to be ﬁltered across the glomerulus. 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- Animal Study spectively) were chosen as qualiﬁer ions. In Multiple Reaction Mon- All the experimental procedures are in compliance with the animal itoring mode, the transitions m/z=166→120 and m/z=166→103 (for 13 ethics board of the Radboud University Nijmegen. The transgenic phenylalanine) and m/z=172→126 and m/z=172→109 (for C6- parvalbumin-eGFP mice were a gift from Dr. Hannah Monyer.38 In phenylalanine) were measured. Phenylalanine concentrations were the Mg2+ diet experiment, the mice were fed low (0.02% wt/wt) or calculated using a calibration curve. high (0.48% wt/wt) Mg2+-containing diets for 15 days (SSNIFF Spe- zialdiäten GmbH, Soest, Germany). During the last 48 hours of the DNA Constructs experiment, the mice were housed in metabolic cages for urine col- Human HNF1B wild types and mutants were cloned into the pCINeo lection (24 hours of adaptation and 24 hours of sampling). Blood hemagglutinin (HA)internalribosome entry site(IRES) GFPvectoras 6 samples were taken at the start of the experiment and just before described previously. HNF1B was FLAG-tagged at the NH2 terminal euthanasia. Parvalbumin-eGFP–positive tubules were isolated as de- by Phusion PCR using NheIandAgeI restriction sites. To obtain an scribed previously.39 In short, mice ages 4–6 weeks were anesthetized HNF1B lacking the dimerization domain spanning residues 2–30 and perfused transcardially with ice cold buffer of 145 mmol/L NaCl, (D2–30), HNF1B was ampliﬁed using a Phusion polymerase

J Am Soc Nephrol 25: 574–586, 2014 PCBD1 and Hypomagnesemia 583 BASIC RESEARCH www.jasn.org

(Finnzymes, Vantaa, Finland) with speciﬁc primers covering from goat serum, 0.3% [vol/vol] Triton X-100, and 0.3 M NaCl in PBS). residue p.Glu31. The HNF1B-D2–30 was ligated into pCINeo IRES After incubation overnight at 4°C with a rabbit anti-FLAG M2 (F7425, GFP using AscI/AgeI restriction sites. The open reading frame of hu- 1:100; Sigma-Aldrich) or a mouse anti-HA (6E2, 1:100; Cell Signaling man PCBD1 was ampliﬁed using Phusion polymerase from PCBD1 Technology), cells were washed three times with Tris-buffered NaCl pCMV-SPORT6 (Genbank accession number BC006324; ImaGenes) Tween-20 (150 mmol/L NaCl, 0.1 mol/LTris/HCl [pH 7.5], and 0.05% and subcloned into the pCINeo HA IRES GFP vector using AgeI/ [vol/vol] Tween-20) and subsequently incubated with a secondary EcoRI restriction sites. Wild-type PCBD1 was FLAG-tagged at the goat anti-rabbit antibody (A4914, 1:300; Sigma-Aldrich) or a sheep

NH2 terminal by PCR using NheI/XhoI restriction sites. PCBD1 mu- anti-mouse (1:300; Jackson Immunoresearch) coupled to AlexaFluor tations were inserted in the construct using the QuikChange site- 594 for 45 minutes at room temperature. After incubation with directed mutagenesis kit (Stratagene, La Jolla, CA) according to the 49,6-diamidino-2-phenylindole for 30 minutes at room temperature, manufacturer’s protocol. The luciferase reporter plasmid containing cells were washed three times with Tris-buffered NaCl Tween-20 and the FXYD2 promoter linked to the coding region of firefly luciferase finally mounted with Fluoromount-G (SouthernBiotech). Photo- was produced as previously described.6 Briefly, the human FXYD2 graphs were taken using a Zeiss Axio Imager 1 microscope (Oberkochen, promoter region that controls transcription of the g-subunit iso- Germany) equipped with an HXP120 Kubler Codix fluorescence lamp form-a (23229/+91 bp from the transcription start site) was ampli- and a Zeiss Axiocam MRm digital camera. Images were analyzed by use fied from genomic DNA using Phusion polymerase (Finnzymes) and of the software ImageJ.40 cloned into pGL3-Basic (Promega) using KpnI/BglII restriction sites. The human PKHD1 promoter region extending from exon 1 to 1.5 kb Immunohistochemistry upstream to the transcription start site was amplified from genomic Staining was performed on 5-mmsectionsofPLPfixed frozen mouse DNA and cloned into pGL3-Basic using KpnI/HindIII sites. The pRL- kidney or pancreas samples. Subsequently, sections were washed CMV vector encoding Renilla luciferase under control of a CMV three times with buffer of 0.15 mol/L NaCl and 0.1 mol/L Tris/HCl promoter was commercially available (Promega, Fitchburg, WI). (pH 7.6). Antigen retrieval was done with 10 mmol/L sodium citrate Primer sequences used for cloning or mutagenesis PCR are reported (pH 6.0), and nonspecific binding was blocked by incubation in inSupplementalTable2.Allconstructswereverified by sequence blocking buffer for 1 hour. Sections were stained overnight at 4°C analysis. with a polyclonal rabbit anti-PCBD1 antibody (F3862, 1:5000; kind gift from Prof. Beat Thöny).26 The following day, sections were Cell Culture washed and incubated with a goat anti-rabbit secondary antibody HEK293 cells were grown in DMEM (Bio Whittaker-Europe, Verviers, coupled to Alexa Fluor 488 (A11008, 1:300; Invitrogen) for 1 hour Belgium) containing 10% (vol/vol) FCS (Thermo Fisher HyClone), at room temperature. Kidney sections were costained for Bcrp, Tamm

10 ml/ml nonessential amino acids, and 2 mmol/L L-glutamine at 37°C Horsfall, NCC, calbindin-D28k (D28K), or Aqp2. Briefly, sections in a humidity-controlled incubator with 5% (vol/vol) CO2. The cells were washed and incubated for 2 hours at room temperature with were transiently transfected with the respective constructs using poly- rat anti-Bcrp (Clone BXP-9, MC-980, 1:200; Kamya Biomedical ethylenimine cationic polymer (Polysciences, Inc.) at a 1:6 DNA to Company), sheep anti-Tamm Horsfall (8595–0054, 1:1500; AbD Se- polyethylenimine cationic polymer ratio for 48 hours unless otherwise rotec), guinea pig anti-NCC (1:50; gift from Jan Loffing), mouse anti- stated. 28K (C-9848, 1:1500; Sigma-Aldrich), or guinea pig anti-Aqp2 (1:1000; gift from Peter Deen). Secondary antibodies were coupled Western Blotting with Alexa Fluor 495 (1:300; Invitrogen) and incubated for 45 minutes HEK293 cells were transfected for 24 hours with HA-PCBD1 mutants at room temperature. Subsequently, sections were washed, incubated and treated at the same time with 10 mmol/L MG-132. Protein lysates for 10 minutes with 49,6-diamidino-2-phenylindole, and mounted were denatured in Laemmli containing 100 mmol/L dithiothreitol for with Mowiol. Photographs were taken using a Zeiss Axio Imager 1 30 minutes at 37°C and subsequently subjected to SDS-PAGE. Then, microscope equipped with an HXP120 Kubler Codix fluorescence immunoblots were incubated with a mouse anti-HA (high-affinity lamp and a Zeiss Axiocam MRm digital camera. 3F10, 1:5000; Roche) primary antibody and peroxidase-conjugated sheep anti-mouse secondary antibodies (1:10,000; Jackson Immu- Coimmunoprecipitation noresearch). HEK293 cells were seeded on 55-mm Petri dishes and cotransfected with 5 mg PCBD1 constructs and 5 mg HNF1B constructs or an empty Immunocytochemistry pCINeo IRES GFP vector (mock DNA); 48 hours after transfection, HEK293 cells were seeded in 12-well plates on glass coverslips and nuclear and cytosolic fractions were prepared using the NE-PER Nu- cotransfected with 400 ng PCBD1 constructs and 400 ng HNF1B clear Protein Extraction Kit (Pierce). The next incubation steps were constructs or empty pCINeo IRES GFP vector (mock DNA). After 48 all done under rotary agitation at 4°C; 35 ml protein A-agarose beads hours, HEK293 cells were fixed with 4% (wt/vol) paraformaldehyde (Santa Cruz Biotechnology) were previously incubated overnight at for 30 minutes at 4°C followed by the subsequent steps at room 4°C with 2.5 mg rabbit anti-FLAG antibody (F7425; Sigma) and temperature: permeabilization for 15 minutes with 0.3% (vol/vol) washed with IPP500 (500 mmol/L NaCl, 10 mmol/L Tris adjusted Triton X-100 in PBS, incubation for 15 minutes with 50 mmol/L to pH 8.0 with HCl, 0.1% [vol/vol] NP-40, 0.1% [vol/vol] Tween-20,

NH4Cl, and ﬁnally, incubation in blocking buffer (16% [vol/vol] and 0.1% [wt/vol] BSA, and protein inhibitors) four times. After sampling

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60 ml as the input control, the remaining 180 ml lysate sample was added REFERENCES 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 1. Adalat S, Woolf AS, Johnstone KA, Wirsing A, Harries LW, Long DA, four times with lysis buffer. The proteins were separated from the beads by Hennekam RC, Ledermann SE, Rees L, van’t Hoff W, Marks SD, incubation for 30 minutes at 37°C in 13 Laemmli sample buffer supple- Trompeter RS, Tullus K, Winyard PJ, Cansick J, Mushtaq I, Dhillon HK, Bingham C, Edghill EL, Shroff R, Stanescu H, Ryffel GU, Ellard S, mented with 100 mmol/L dithiothreitol and detected by immunoblotting Bockenhauer D: HNF1B mutations associate with hypomagnesemia using a mouse anti-HA (high-affinity 3F10, 1:5000; Roche) or a mouse and renal magnesium wasting. J Am Soc Nephrol 20: 1123–1131, 2009 anti-FLAG M2 (F3165, 1:5000; Sigma-Aldrich) primary antibodies and 2. 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Meij IC, Koenderink JB, van Bokhoven H, Assink KF, Groenestege WT, 10 ng pRL-CMV was used as a reference. FireflyandRenilla luciferase de Pont JJ, Bindels RJ, Monnens LA, van den Heuvel LP, Knoers NV: activities were measured by use of a Dual-Luciferase Reporter Assay Dominant isolated renal magnesium loss is caused by misrouting of the – (Promega) 48 hours after transfection. Na(+),K(+)-ATPase gamma-subunit. Nat Genet 26: 265 266, 2000 6. Ferrè S, Veenstra GJ, Bouwmeester R, Hoenderop JG, Bindels RJ: HNF- 1B specifically regulates the transcription of the ga-subunit of the Na – Homology Modeling +/K+-ATPase. Biochem Biophys Res Commun 404: 284 290, 2011 A homology model was built using the modeling script in the WHAT 7. Gong Y, Ma Z, Patel V, Fischer E, Hiesberger T, Pontoglio M, Igarashi P: HNF-1beta regulates transcription of the PKD modifier gene Kif12. 41,42 IF & YASARATwinset with standard parameters. 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Statistical analyses were con- mouse urate transporter 1 gene by hepatocyte nuclear factor 1 alpha/ ducted by unpaired t test when comparing two experimental condi- beta and DNA methylation. Mol Pharmacol 72: 1619–1625, 2007 tions and one-way ANOVA with Bonferroni test when comparing 11. Kikuchi R, Kusuhara H, Hattori N, Shiota K, Kim I, Gonzalez FJ, more conditions. P values ,0.05 were considered significant. Sugiyama Y: Regulation of the expression of human organic anion transporter 3 by hepatocyte nuclear factor 1alpha/beta and DNA methylation. Mol Pharmacol 70: 887–896, 2006 12. Mendel DB, Khavari PA, Conley PB, Graves MK, Hansen LP, Admon A, Crabtree GR: Characterization of a cofactor that regulates dimerization ACKNOWLEDGMENTS of a mammalian homeodomain protein. Science 254: 1762–1767, 1991 13. Citron BA, Davis MD, Milstien S, Gutierrez J, Mendel DB, Crabtree GR, The authors are grateful to the patients for their participation in this KaufmanS:Identityof4a-carbinolaminedehydratase,acomponentofthe study. We appreciate the help of Drs. N. Blau and B. Thöny in the phenylalanine hydroxylation system, and DCoH, a transregulator of homeodomain proteins. Proc Natl Acad Sci U S A 89: 11891– 11894, 1992 recruitment of the patients and provision of the PCBD1 antibody. We 14. Werner ER, Blau N, Thöny B: Tetrahydrobiopterin: Biochemistry and also thank Lonneke Duijkers and Annelies van Angelen for their ex- pathophysiology. Biochem J 438: 397–414, 2011 cellent technical support. 15. Endrizzi JA, Cronk JD, Wang W, Crabtree GR, Alber T: Crystal structure This work was supported by The Netherlands Organization for of DCoH, a bifunctional, protein-binding transcriptional coactivator. – ScientificResearch (NWO)Grants ZonMw 9120.8026 and NWOALW Science 268: 556 559, 1995 16. 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