A Novel Hypokalemic-Alkalotic Salt-Losing Tubulopathy in Patients with CLDN10 Mutations

CLINICAL RESEARCH www.jasn.org

† ‡ † Ernie M.H.F. Bongers,* Luke M. Shelton, Susanne Milatz, Sjoerd Verkaart, | ‡ Anneke P. Bech,§ Jeroen Schoots,* Elisabeth A.M. Cornelissen, Markus Bleich, † Joost G.J. Hoenderop, Jack F.M. Wetzels,§ Dorien Lugtenberg,* and Tom Nijenhuis§

Departments of *Human Genetics, †Physiology, §Nephrology, and |Pediatric Nephrology, Radboud University Medical Center, Nijmegen, The Netherlands; and ‡Institute of Physiology, Christian Albrechts University Kiel, Kiel, Germany

ABSTRACT Mice lacking distal tubular expression of CLDN10, the gene encoding the tight junction protein Claudin-10, show enhanced paracellular magnesium and calcium permeability and reduced sodium permeability in the thick ascending limb (TAL), leading to a urine concentrating defect. However, the function of renal Claudin-10 in humans remains undetermined. We identified and characterized CLDN10 mutations in two patients with a hypokalemic- alkalotic salt-losing nephropathy. The first patient was diagnosed with Bartter syndrome (BS) .30 years ago. At re- evaluation, we observed hypocalciuria and hypercalcemia, suggesting Gitelman syndrome (GS). However, serum magnesium was in the upper normal to hypermagnesemic range, thiazide responsiveness was not blunted, and genetic analyses did not show mutations in genes associated with GS or BS. Whole-exome sequencing revealed compound heterozygous CLDN10 sequence variants [c.446C.G (p.Pro149Arg) and c.465–1G.A (p.Glu157_Tyr192del)]. The patient had reduced urinary concentrating ability, with a preserved aquaporin-2 response to desmopressin and an intact response to furosemide. These findings were not in line with any other known salt-losing nephropathy. Subsequently, we identified a second unrelated patient showing a similar phenotype, in whom we detected compound heterozygous CLDN10 sequence variants [c.446C.G (p.(Pro149Arg) and c.217G.A (p.Asp73Asn)]. Cell surface biotinylation and immunofluorescence experiments in cells expressing the encoded mutants showed that only one mutation caused significant differences in Claudin-10 membrane localization and tight junction strand formation, indicating that these alterations do not fully explain the phenotype. These data suggest that pathogenic CLDN10 mutations affect TAL paracellular ion transport and cause a novel tight junction disease characterized by a non-BS, non-GS autosomal recessive hypokalemic-alkalotic salt-losing phenotype.

J Am Soc Nephrol 28: 3118–3128, 2017. doi: https://doi.org/10.1681/ASN.2016080881

Hypokalemic alkalosis is often due to increased wa- cause Liddle syndrome, presenting with a hypoka- ter and sodium delivery to the collecting duct with lemic alkalosis and hypertension at young age.1,2 concomitant enhanced aldosterone action due to More proximal defects in sodium reabsorption, volume contraction. Both increase sodium reabsorption, which creates a lumen-negative potential that eventually promotes excretion of potassium Received August 16, 2016. Accepted May 11, 2017. and hydrogen ions. After the exclusion of acquired E.M.H.F.B., L.M.S., and S.M. contributed equally to this work. and/or drug-induced causes of hypokalemic alka- Published online ahead of print. Publication date available at losis, the differential diagnosis includes several rare www.jasn.org. (genetic) renal tubular disorders. The most impor- Correspondence: Dr. Tom Nijenhuis, Department of Nephrol- tant molecular player in the pathogenesis of hypo- ogy, Radboud University Medical Center, 464, PO Box 9101, kalemic alkalosis is the apically located epithelial 6500 HB Nijmegen, The Netherlands. Email: Tom.Nijenhuis@ sodium channel (ENaC). Gain of function muta- Radboudumc.nl tions in the genes encoding ENaC b-andg-subunits Copyright © 2017 by the American Society of Nephrology

3118 ISSN : 1046-6673/2810-3118 JAmSocNephrol28: 3118–3128, 2017 www.jasn.org CLINICAL RESEARCH so-called salt-losing nephropathies, can also induce a hypoka- distal from PT show enhanced TAL paracellular magnesium lemic alkalosis by increasing distal tubular flow and sodium and calcium permeability and reduced paracellular sodium delivery. Several molecular players in these reabsorption permeability, leading to a urine concentrating defect. The processes were identified by studying the rare genetic tubular functional significance of renal Claudin-10 expression in hu- salt-losing disorders Bartter syndrome (BS) and Gitelman mans has remained elusive. syndrome (GS). In BS, transcellular sodium-chloride reab- In this study, we report the molecular identification of a sorption in the thick ascending limb of Henle loop (TAL) is novel non-Bartter, non-Gitelman hereditary salt-losing ne- disrupted due to mutations in transporters and channels in- phropathy and describe the clinical phenotype in two unrelated volved in this process.3–7 GS is caused by mutations affecting patients, in whom we identified compound heterozygous var- distal convoluted tubular (DCT) transcellular sodium-chlo- iants in the CLDN10 gene encoding Claudin-10. This is the ride reabsorption.8 first description of a hypokalemic-alkalotic salt-losing ne- Salt-losing disorders distal from the TAL (e.g., GS) generally phropathy putatively on the basis of a primary defect of para- lead to hypocalciuria due to either increased proximal tubular cellular ion transport in TAL. paracellular transport or increased transcellular calcium reabsorption in the DCT.9–11 In contrast, salt-losing disorders in the TAL (e.g., BS) are frequently accompanied by hypercalciu- RESULTS ria due to reduced passive paracellular reabsorption of calcium (and magnesium) in the TAL. In this tubular segment, about Patient 1 60%–70% of filtered magnesium and 10%–20% of calcium This woman was referred to the endocrinologist in 1980 at the are reabsorbed, driven by the lumen-positive transepithelial age of 21 years old because of hypokalemia detected at cardio- gradient that is maintained by transcellular sodium, chloride, logic evaluation for atypical chest pains. Detailed examination and potassium transport processes.12 The lumen-positive gra- revealed a hypokalemic alkalosis with mild renal dient permits the passive paracellular transport of cations like insufficiency, a polyuria of 3–5 L/d, and a reduced urine con- calcium and magnesium but also, sodium from the prourine centrating ability (Tables 1 and 2). Her BP was in the lower through tight junctions and back to the interstitium and vas- normal range without orthostatic hypotension. Serum mag- cular compartment.12,13 The main constituent of the size- and nesium was reported once, which was in the normal range charge-selective tight junction is the family of claudins, en- (0.98 mmol/L). At that time, in the absence of genetic screen- compassing at least 24 members in mammals.14 Claudin pro- ing tests, a presumptive diagnosis of BS was made. teins consist of four transmembrane segments, intracellular N In 2012, she was re-evaluated at our nephrology outpatient and C termini, and two extracellular loops that extend into the clinic. Her renal insufficiency had only mildly progressed, but a intercellular cleft between adjacent cells. They interact with persistent hypocalciuria with serum calcium levels in the upper other claudins within the same cell membrane (in cis)and normal to high range was noted, which in retrospect, had been claudins in the membrane of the adjacent cell (in trans). present since diagnosis in 1980 (Tables 1 and 2). A hypokale- Trans-interaction via the extracellular loops bridges the cleft mic alkalosis with hypocalciuria is more in line with GS, which between neighboring cells and results in the formation of se- is classically accompanied by hypomagnesemia. However, lective paracellular pores. The sum of multiple claudin inter- serum magnesium levels were persistently normal to even in- actions leads to the establishment of a complex tight junction creased (up to 1.22 mmol/L) at several occasions, and a chal- strand meshwork. lenge with hydrochlorothiazide showed an exaggerated rather The paracellular calcium and magnesium reabsorption in than the generally blunted response expected in GS (Tables 1 the TAL closely depends on the expression of Claudin-16 and and 2). Moreover, a challenge with furosemide, which can be -19, and mutations in the corresponding genes CLDN16 and blunted in patients with BS, also resulted in an exaggerated CLDN19 cause familial hypomagnesemia with hypercalciuria response. Mutations in SLC12A3 and CLCNKB were not and nephrocalcinosis, in which renal calcium and magnesium detected by Sanger sequencing. Recently, we showed that pa- wasting occurs.15–18 Claudin-14 was shown to be a negative tients with ADTKD-HNF1b can also show a hypo- or normo- regulator of paracellular calcium reabsorption, probably by magnesemic Gitelman-like phenotype with either preserved modifying the permeability properties of Claudin-16.19,20 or blunted response to thiazides.24,25 Because of a small right Claudin-10 exists in two major isoforms, Claudin-10a and kidney with a single cyst, the presence of an HNF1B mutation -10b, due to alternative splicing.21 Claudin-10a and -10b differ or deletion was also excluded by Sanger sequencing and Mul- only in their first transmembrane segment and first extracel- tiplex Ligation–Dependent Probe Amplification (MLPA). Re- lular loop. Claudin-10a is present in the proximal renal tubule nal ultrasound and abdominal CT scanning did not show any (PT) and thought to form an anion-selective paracellular pore. other structural renal abnormalities apart from the small right In contrast, Claudin-10b is a component of the paracellular kidney and the normal-sized left kidney or signs of nephro- pathway in the TAL and confers permeability to small cations, calcinosis. like sodium, when overexpressed in cell culture.21–23 Mice Subsequently, she was referred to our multidisciplinary with a conditional knockout of Cldn10 in nephron segments nephrogenetic outpatient clinic, and whole-exome sequencing

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was performed, inwhich the data analysis was initially confined fi 2.3 4.1 6.4 3.9 3.6 3.8 3.9 6.4 3.7 5.1 to a set of 177 genes con rmed to be associated with isolated or complex kidney diseases in humans. Open exome analysis thereafter identified two heterozygous sequence variants in the CLDN10 gene [c.446C.G (p.(Pro149Arg)) and c.465– 7 7 9 7 1G.A (p.(Glu157_Tyr192del))]. The family pedigree is depicted in Figure 1A. The presence of one CLDN10 variant in her mother (I:2), the other CLDN10 variant detected in two sisters (II:3 and II:4), and both her children (III:1 and III:2) 8.1 17 7.8 20 7.5 14 7.5 10 4.7 5.7 4.8 5.6 14.1 36 inheriting different variants are compatible with compound TTKG, % FE K, % FE Mg, % heterozygosity in the patient. One CLDN10 variant results in a single-amino acid substitution [p.(Pro149Arg)] in an evolutionary conserved amino acid in the second extracellular loop, 90 90 90 90 90 62 78 88 71 . . . . .

90/149 whereas the other was predicted to disrupt the consensus 33/37 19.9 90 . 90 ml/min

eGFR/ECC, > splice site [p.(Glu157_Tyr192del)], leading to loss of exon 4 and deletion of the fourth transmembrane segment in the Claudin-10 protein (Figure 2). According to their localization, M 83 70 90 89 90 85 86 88 66 69 the mutations found in patient 1 affect both major Claudin-10 m 144 isoforms, Claudin-10a and -10b. Creatinine,

Patient 2 ﬁ

7.41 After the identi cation of CLDN10 sequence variants as a pos- – pH, 7.43 7.39 7.34 7.57 7.38 7.38 7.41 7.42 7.39 sible cause of the hypokalemic alkalosis with hypocalciuria 7.31 and the tendency toward hypermagnesemia in patient 1, we recognized a similar phenotype in a second unrelated patient. He presented to the emergency department after a (near) col- , 3 lapse at age 15 years old in 2013, and a hypokalemia (2.6 28.0 mM 29.8 30.7 25.8 30.1 32.6 25.2 27.0 24.1 26.0 28.7 24.5 – HCO , bicarbonate; ECC, endogenous creatinine clearance rate; TTKG, transtubular potassium gradient; FE, mmol/L) and metabolic alkalosis were shown (Tables 1 and 3

22.0 2). In retrospect, hypokalemia was already present at a pre- vious emergency department visit at age 8 years old. There had

Serum been recurrent episodes of collapse or falls with minor injuries without speciﬁc cause. The BP was normal, with no ortho- Ca, 2.65 mM 2.61 2.44 2.22 2.59 2.38 2.34 2.29 2.37 2.47 2.59 2.45

– static hypertension tested. At adult age, his BP was rather low, withameanof105/55mmHg.Therewasahypocalciuriawith 2.20 serum calcium ranging from 2.54 to 2.59 mmol/L. Hypokale- mic alkalosis with hypocalciuria had prompted the suspicion of GS, but hypomagnesemia was absent with a serum magne-

1.1 mM – Mg, 1.19 0.86 0.83 1.03 0.96 0.90 0.80 0.90 0.98 0.90 0.90 sium in the normal range (0.98 1.03 mmol/L), and no se- –

0.7 quence variants in SLC12A3 and CLCNKB were detected by Sanger sequencing. After genetic counseling, the now adult patient opted for whole-exome sequencing, in which data

b analyses for known kidney disease–associated genes excluded K, 4.7 mM 3.6 3.6 3.8 2.8 4.0 3.8 3.6 2.7 3.9 3.8 – pathogenic mutations. Because the phenotype resembled that

3.4 of patient 1, we also tested the CLDN10 gene in this analysis, and two CLDN10 variants were identiﬁed. These included the a same c.446C.G (p.(Pro149Arg)) variant as detected in pa- yr 18

Age, tient 1 as well as a novel c.217G.A (p.(Asp73Asn)) variant (Figure 2). The latter mutation affects exon 1b, which is only M 24 M 15 M 48 M 21 W 58 WW 45 26 W 46 W 14 present in Claudin-10b but not present in Claudin-10a, and affects the ﬁrst extracellular loop of the protein. Segregation analysis by Sanger sequencing in the parents of patient 2 con- Clinical data of patients and family members ﬁrmed compound heterozygosity for the CLDN10 variants in this patient. The pedigree of this family is depicted in Figure

Subject Sex 1B. Thiazide responsiveness testing showed an exaggerated II:3 II:4 III:1 III:2 II:2II.2 W 21 53 2.1 4.1 0.98 2.66 31.7 109 49 II.2 II.2 I:2 I:1 II:1 II:3 Serum potassium while on potassium supplementation. Age at examination. Table 1. a b fractional excretion; W, woman; M, man. Family members Patient 1 Patient 2 Family members Individual subject numbers correspond with Figure 1. K, potassium; Mg, magnesium; Ca, calcium; HCO response to thiazide administration, consistent with patient

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1. Renal imaging showed kidneys without structural abnormalities or nephrocalcinosis. Family members of both patients were screened for electrolyte disturbances. The full hypokalemic-alkalotic, hypo- Gene magnesuric, hypocalciuric salt-losing phenotype was clearly Consequence

p.Glu157-Tyr192del restricted to the compound heterozygous patients. Interest- p.Pro149Arg p.Pro149Arg p.Pro149Arg p.Pro149Arg p.Asp73Asn p.Pro149Arg CLDN10 ingly, family members heterozygous for one CLDN10 variant showed serum potassium levels in the lower one half of the T p.Pro149Arg, A p.Pro149Arg, p.Asp73Asn . , below detection limit (urine . reference interval (Tables 1 and 2), and an occasional mild , 1G T p.Glu157-T yr192del – A G G G G G

. hypokalemia was reported in the daughter (III:1) of patient ...... 1G Genetic Analysis – 1 and the father (I:1) and sister (II:3) of patient 2. Some het- G, c.217G None G, c.465 . . c.217G c.446C c.446C c.446C c.446C c.446C erozygous family members also showed high serum bicarbon- c.465

gested to be abnormal and suggestive of GS. ate levels (Tables 1 and 2). Nucleotide Change c.446C The identiﬁed CLDN10 sequence variants were not reported in the Exome Aggregation Consortium database (Cambridge, a MA), a large database collecting next generation sequencing 15% – D FE Cl, variants in over 60,000 exomes as proxy for variant allele fre- 26.0 c.446C

8% quencies in the general population. In addition, these sequence Furosemide Test; variants were not reported in other databases, like the Genome of the Netherlands Consortium and the Human Genetic Varia- 800

> tion Database (Kyoto, Japan). 825 1048 1179

546/603 Urine Concentrating Defect mosmol/kg 2.3% in thiazide responsiveness testing sug Maximum Urine Osmolality,

, Because the conditional knockout of Cldn10 in nephron segments distal from PT in mice reduced calcium and magnesium DDAVP Test FE Cl

D excretion but increased sodium loss with a urine concentrat- 26 210 294 136 ing defect, we tested urinary concentrating ability in patient 34/39 D Urine mosmol/kg Osmolality, 1. This showed a blunted urine concentrating response to thirsting and desmopressin acetate (DDAVP) on two separate a occasions. Heterozygous family members with only one of 4.9% – 5.6 either CLDN10 variants showed normal responses to thirsting 12.9

Thiazide and/or DDAVP (Tables 1 and 2). A reduced response to 2.3% Test; FE Cl, DDAVP can result from both the inability of the collecting duct principal cells to react to the synthetic vasopressin analog and insufﬁcient buildup of the interstitial tonicity (e.g.,by

0.21 dysfunction of sodium reabsorption in the TAL). Determina- mM/mM tion of aquaporin-2 expression in urinary exosomes before Mg/Creatinine, and after DDAVP administration showed the expected increase in aquaporin-2 expression in the family members as well as the patient, consistent with an appropriate collecting mM 0.32 0.28 0.23 0.46 0.32 2.0 0.23 0.26 0.27 0.61 24 h Mg

Excretion, duct response to DDAVP (Figure 3). Chronic hypokalemia is also suggested to affect urine concentrating ability directly. Urine However, urine concentration was determined during potassium supplementation therapy, with a serum potassium in the 0.28 0.05 0.15 0.43 0.58 0.21 0.03 0.21 0.06 range of 3.8–4.0 mmol/L (normal range of 3.5–4.7 mmol/L). mM/mM Ca/Creatinine, Subcellular Localization of Claudin-10b Variants Because the renal phenotype of our patients resembled that of 1.4 – ,, ,,

mM mice with a distal tubular Claudin-10 deletion (Claudin-10b), 0.6 24-h Ca Excretion, we analyzed the subcellular localization of the three Claudin- 10b variants in cell culture models. To study whether the phe- Clinical data of patients and family members notype in these patients might be due to a reduction or lack of 0.1 mmol/L).

, Claudin-10b expression at the cell membrane, cell surface bio-

Subject tinylation experiments were performed with the Claudin-10b Maximum change of FE Cl as the main outcome measure iniazide th and furosemide responsiveness testing, with a Patient 1 II:2 II:2 Family members II:3 I:2 II:4 II.2 III:1 III:2 Patient 2 II.2 Family members I:1 II:1 II:3 calcium Table 2. Individual subject numbers correspond with Figure 1. DDAVPa was the synthetic arganine vasopressin analog. Ca, calcium; Mg,wild-type magnesium; FE, fractional excretion; Cl, chloride; and mutated proteins expressed in HEK293 cells

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Figure 1. Pedigrees of the families of patients 1 and 2 show that the clinical phenotype co-segregates with compound heterozygous CLDN10 variants. Arrows indicate index patients in (A) family 1 (subject II:2 or patient 1) and (B) family 2 (subject II:2 or patient 2). Affected individuals are indicated in black. The speciﬁc CLDN10 sequence variants found in the index patients and their family members and the predicted effects on protein level are depicted.

(Figure 4A). The wild-type Claudin-10b protein was detected at expression and localization of Claudin-10b wild type or var- the cell surface. The Pro149Arg Claudin-10b immunoreactive iants were further studied by immunostaining and confocal bands were detected at a similar density at the cell surface com- laser-scanning microscopy. Claudin-10b wild type, Asp73Asn, pared with the wild-type protein, with green fluorescent protein and Pro149Arg localized to the cell membrane (Figure 4C). immunoblotting serving to show equal transfection efficiency Moreover, they were enriched at cell-cell contacts between and loading, indicating correct expression and cellular traffick- transfected cells compared with cell membranes without con- ing of this mutant protein. However, total as well as cell surface tact to a transfected cell. Claudin contact enrichment in expression of the smaller Glu157_Tyr192del Claudin-10b vari- HEK293 cells is a clear indicator for tight junction strand ant predicted to lead to loss of exon 4 (DE4) were reduced (Fig- formation27,28 on the basis of both cis-andtrans-interaction of ure 4B). In contrast, cell surface expression of the Asp73Asn heterologous claudins. In sharp contrast to wild-type Claudin-10b Claudin-10b variant was significantly increased. Cotransfection and the other two variants, Claudin-10b DE4 was not able to of the respective variants, reconstituting the patient genotypes, localize to the cell membrane and form tight junction strands also showed correct cellular trafficking with reduced overall but was retained in the cytosol. Claudin-10 cell surface expression in the presence of the DE4 To study whether Claudin-10b Asp73Asn and Pro149Arg variant and increased Claudin-10 cell surface expression in the integrate into already existing cell junctions as previously presence of the Asp73Asn variant. shown for the wild-type protein,22 both variants were ex- To investigate whether the Claudin-10b variants were ca- pressed in the MDCK-C7 epithelial kidney cell line. This cell pableofformingtightjunctionstrandsinHEK293cells, line differentiates into an epithelial monolayer with well

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Figure 2. Sequence analyses and evolutionary conservation of the CLDN10 gene and the identiﬁed CLDN10 variants. (A) Genomic structure of the human CLDN10 gene located at chromosome 13. (B) Schematic representation of the CLDN10 cDNA on the basis of the longest protein coding sequence (NM_006984.4). CLDN10 variants are depicted by vertical lines indicating the cDNA position and describing the effect on protein level. (C) Chromatograms for the different mutations conﬁrmed by Sanger sequencing. (D) Evolutionary conservation of Asp73 and Pro149. The third CLDN10 variant is predicted to induce an alternative splice site leading to loss of glutamic acid at position 157 to tyrosine at position 192 and therefore, not depicted. (E) Predicted topology of the Claudin-10 protein with location of the different mutations and deletion indicated. TM, transmembrane region.

equipped tight junctions, comparatively high transepithelial patients’ phenotype consisted of a hypokalemic alkalosis to- resistance, low permeability to ions, and no endogenous ex- gether with reduced renal calcium and magnesium excretion, pression of Claudin-10.22,29,30 Both the Claudin-10b As- which resulted in occasional hypercalcemia and hypermagne- p73Asn and Pro149Arg variants localized to the cell junctions, semia. In addition, we report a urine concentrating defect thus forming tight junction strands in a differentiated epithe- with a preserved aquaporin-2 response to DDAVP, thus likely lial monolayer (Figure 4D). Claudin-10b DE4 was only weakly caused by a reduced medullary osmolality. The renal tubular expressed after several days of culture and never localized to role of Claudin-10 was previously studied in mice with con- cell membranes in MDCK-C7 cells. ditional knockout of Cldn10 in nephron segments distal from the PT, resulting in absence of Claudin-10b from the TAL.26 Similar to our patients, these mice showed reduced paracellu- DISCUSSION lar sodium permeability resulting in a urine concentrating defect accompanied by an enhanced TAL paracellular magne- We identiﬁed a new hypokalemic-alkalotic salt-losing sium and calcium reabsorptive capacity.26 Therefore, we nephropathy in two unrelated patients with compound het- propose that CLDN10 is a candidate gene in patients with a erozygous mutations in CLDN10. Using whole-exome se- non-Bartter, non-Gitelman hypokalemic-alkalotic salt-losing quencing, we detected three different CLDN10 variants in nephropathy with presumed autosomal recessive inheritance these two patients. Given their absence in large exome and hypothesize that this phenotype results from a primary sequencing databases, these are very rare variants virtually defect of TAL paracellular ion transport. absent in the normal population. This is the ﬁrst report on Importantly, distal tubular Cldn10 knockout mice showed a putatively pathogenic CLDN10 variants in humans. The reduced TAL sodium permeability. The exaggerated response

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hypokalemia when maintained on normal chow.35 Only when put on a stringent low-potassium diet do Slc12a3 knockouts show significantly lower serum potassium levels compared with their wild-type littermates. Conditional Cldn10 knockout mice showed hypermagnesemia combined with hypomagnesuria and increased TAL magnesium reabsorptive capacity. Patients only occasionally showed increased serum magnesium levels, such as was the case for calcium. Mice showed hypocalciuria and increased TAL calcium reabsorption, but serum calcium was not ele- Figure 3. Both patient 1 and her family members showed a normal vated, possibly due to effects mediated by the hypermagnese- physiological AQP2 response to DDAVP. Immunoblotting for AQP2 mia on calcium-sensing receptor signaling and ensuing lower was performed on urinary exosomes derived from urine collected parathyroid hormone levels. Cldn10 knockout mice showed before (2) and after (+) administration of DDAVP. Murine immor- nephrocalcinosis, whereas the patients did not. Apart from talized cortical collecting duct cells cultured in the presence or species differences and different (tubular) compensatory absence of DDAVP served as controls. Representative blots show- mechanisms in mice and humans, the renal tubular Claudin-10 ing AQP2 response to DDAVP administration in a heterozygous expression pattern should be considered. Claudin-10 is expressed family member as well as patient 1 are shown. WT, wild type. along the renal tubule, with the anion-selective Claudin-10a isoform predominantly expressed in the PTand the relatively cation- selective Claudin-10b isoform predominantly in the TAL.13,22,23 of both patients to hydrochlorothiazide in the context of a Two of the CLDN10 mutations (Pro149Arg and DE4) found hypokalemic alkalosis and enhanced tubular aldosterone in our patients affect both Claudin-10 isoforms, and only one activity is consistent with reduced sodium reabsorption prox- of them (Asp73Asn) is specific for Claudin-10b. According to imal to the DCT, leading to compensatory enhanced thiazide- its effects in cell culture models, Claudin-10a is suggested to sensitive sodium reabsorption in DCT as well as increased facilitate paracellular chloride reabsorption in the PT; how- ENaC-mediated sodium reabsorption more distally leading ever, data on its specific function are not available. Possible to the hypokalemic alkalosis. Together with the response of defects in PT ion transport could be compensated in down- patient 1 to furosemide, suggesting intact or even enhanced stream nephron segments, and thus, the patients’ phenotype transcellular sodium reabsorption in TAL, this would be con- may be primarily on the basis of Claudin-10b defects. How- sistent with a defect in paracellular sodium reabsorption. ever, the influence of Claudin-10a mutations on tubular func- Cldn10 knockout mice also showed a urine concentration tion remains elusive. Which Claudin-10 isoform is expressed defect with reduced urine osmolality and increased urine in the thin limb and its tubular transport role there are also volumes as well as a blunted response to water deprivation unresolved. compared with wild-type littermates. In patient 1, there was In the TAL, Claudin-10b exhibits a characteristic expression polyuria and reduced renal concentrating response to thirsting pattern along the corticomedullary axis. In the TAL of the inner at presentation. Recent DDAVP response testing showed a stripe of outer medulla, Claudin-10b is the onlyclaudin present blunted response to thirsting and DDAVP but a sustained and allows paracellular reabsorption of sodium along the elec- AQP2 response, in line with a disturbed concentrating ability trical gradient.26,36 In the outer stripe of outer medulla and due to insufficient buildup of the interstitial osmolality be- cortical TAL, claudins are arranged in a mosaic expression cause of TAL dysfunction. Claudin-10 expression in rodent pattern, with Claudin-10b alone and Claudin-3, -16, and nephron segments distal from the TAL was reported in some -19 in a complex.37 Although Claudin-10b paracellular chan- studies21,22,31 but was not reported in others.32–34 Therefore, nels have a preference for monovalents and primarily conduct we cannot exclude that direct effects of Claudin-10(b) loss in, sodium, tight junctions dominated by Claudin-16/19 com- for example, collecting duct mediate the disturbed concentrat- plexes prefer magnesium over sodium. The conditional ing ability. Apart from similarities between distal tubular knockout of Cldn10 led to not only an increase in Claudin- Cldn10 knockouts and our patients, supporting that the 14, -16, and -19 expression but also, a broad expansion of the CLDN10 sequence variants are involved in these patients’ tu- other TAL claudins over all tight junctions, tentatively in line bulopathy, there are also differences. Most strikingly, the hy- with the reported increased TAL calcium and magnesium re- pokalemia, which was the presenting feature in both patients, absorptive capacity.26 In contrast to mere absence of Claudin- is absent in the mouse model.26 This is remarkable, because 10b in these mice, the patients express different mutant increased distal tubular sodium delivery expected to accom- variants of Claudin-10b, of which the DE4 variant failed to pany the reduced TAL sodium reabsorption should enhance insert into the cell membrane, in line with former studies potassium excretion in the collecting duct. This discrep- showing that lack of the fourth transmembrane segment pre- ancy could be due to species differences e.g., Slc12a3 knock- vents membrane localization and leads to complete loss of out mice as a model for GS also lack the characteristic function.22 Both patients carry the Pro149Arg variant,

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Figure 4. Variable response of Claudin-10b variants on cell surface expression and tight junction strand formation when expressed in HEK293 and MDCK-C7 cells. (A) Representative immunoblots of Claudin-10b variant–transfected HEK293 cell surface and total lysates probed for the hemagglutinin (HA) tag or green fluorescent protein (GFP) expressed with the Claudin-10b variant. (B) Densitometry of immunoreactive bands quantified from three independent experiments expressed relative to GFP as an indication of successful transfection. Statistical analysis was performed with an unpaired t test. *P#0.05; **P#0.01. (C) Immunohistochemistry of HEK293 cells lacking endogenous claudin expression and transiently transfected with Claudin-10b wild type, Asp73Asn, and Pro149Arg (red) enriched at cell-cell contacts (arrows) compared with cell membranes without contact to a transfected cell (arrowheads), indicating autonomous tight junction formation. In contrast, Claudin-10b DE4 was not localized within the cell membrane and thus, could not form tight junction strands. GFP is shown in green. (D) Immunohistochemistry of MDCK subclone C7 cells with endogenous claudin expression and tight junction formation transiently transfected with Claudin-10b variants, Claudin-10b wild type, Asp73Asn, and Pro149Arg (red) integrated in the cell membrane. Claudin-10b DE4 was only weakly expressed and did not localize to the cell membrane. GFP is shown in green. affecting a residue that is conserved in the majority of claudins. loop of a cation-selective claudin is considered important, Pro149 is suggested to stabilize the conformation of the second but charged residues in similar regions of Claudin-2 or -10a extracellular loop and play a crucial role in correct tight junc- showed only small or no effect on charge selectivity.21,38 Even tion strand arrangement.28 A substitution of the respective when Asp73 is not directly involved in cation pore lining, an residueinmurineClaudin-5(Pro150Ala)sustainedtrans- indirect effect on pore formation is possible. Pore-specific and cis-interaction capability but showed a reduced surface mutations of the Claudin-16 protein have been shown to affect biotinylation and increased ER localization.28 Whereas the ca- ion selectivity,17 with Claudin-16 proteins reaching the cell pability of Claudin-10b Pro149Arg to form autonomous tight surface exhibiting either partial or complete loss of function. junction strands is in accordance with these findings, a re- Whereas defects of all three Claudin-10b variants in forming a duced membrane localization of Claudin-10b Pro149Arg functional cation pore in the TAL could explain the salt-losing was not observed in biotinylation assays. Claudin-10b As- phenotype with urine concentrating defect and secondary p73Asn affects the first extracellular loop, responsible for hypokalemia, a certain residual protein function of Asp73Asn pore selectivity.21,22,31 Asp73 is not conserved among clau- and Pro149Arg thus has to be considered. The basal capability dins, and the significance of this residue for pore selectivity for tight junction formation of variants Asp73Asn and has not been determined.14 Exchange of negatively charged Pro149Arg could be an explanation for the relatively mild residues by a hydrophilic residue in the first extracellular calcium and magnesium disturbances in patients compared

J Am Soc Nephrol 28: 3118–3128, 2017 CLDN10-Associated Tubulopathy 3125 CLINICAL RESEARCH www.jasn.org with Cldn10 knockout mice. Because these two variants form using a Life Technologies 5500XL machine or an Illumina HisEquation autonomous Claudin-10b tight junctions, Claudin-16/19 is 2000. Read mapping and variant calling were done using LifeScope TM possibly prevented from entering those junctions and thus, for the 5500XL data or BWA (mapping) and GATK (calling) for the greatly enhancing calcium and magnesium reabsorption as Illumina data. A filter for a renal disorders gene panel was applied, observed in the mouse model. This could tentatively explain consisting of approximately 200 genes implicated in hereditary kid- why one patient (patient 1) seems to have developed a more neydisorders. Genes and coverage statistics can be found at www. severe phenotype, because she carries the DE4 mutation with a genomediagnosticsnijmegen.nl/exome. Mutations were prioritized complete loss of protein function, whereas patient 2 expresses on the basis of the following criteria: frequency, nucleotide and amino Asp73Asn with a putative residual protein function. acid conservation, relation of the gene to disease (per family), and in- A limitation of our study is that we identifiedonlyone family heritance pattern. Reported variants were confirmed by Sanger se- member with biallelic CLDN10 variants in each family, which quencing. Mutation nomenclature is according to HGVS guidelines limits the value of cosegregation analysis. Furthermore, it is (www.hgvs.org). As reference sequence, the longest protein coding tran- difficult to ascertain which clinical signs and symptoms are script was used on the basis of NM_006984.4. directly caused by the CLDN10 mutations (e.g., the renal insufficiency in patient 1 does not seem to be present in patient 2 Thiazide and Furosemide Responsiveness Testing as yet), and because Claudin-10 has a broad (extrarenal) tissue The thiazide and furosemide tests were performed on the basis of the expression, there might also be additional extrarenal effects. protocols described by Colussi et al.40 and Nozu et al.41 Awashout In conclusion, the salt-losing hypokalemic-alkalotic phe- period of 7 days for therapy that could interfere with the test (i.e., notype in our two unrelated patients seems to be due to the diuretics, NSAIDs) was required, but potassium salts were allowed. presence of compound heterozygous CLDN10 mutations. Patients visited the clinic in the morning after an overnight fast. They None of these variants were found in large exome sequencing were instructed to drink 10 ml/kg tap water (t=0). At t=30 and t=90 databases as proxy for variant allele frequencies in the general minutes, patients were asked to void, but urine was discarded. At population, the particular variants are theoretically in line t=120 and t=150 minutes, basal urine samples were taken. At with functionally relevant alterations in a protein suggested t=150 minutes, basal blood samples were taken, and subsequently, to be involved in relevant renal tubular electrolyte transport 50 mg hydrochlorothiazide or 40 mg furosemide was administered processes, and the phenotype resembles mice with renal tu- orally. Urine samples were taken every 30 minutes until t=330 min- bular loss of the candidate protein. However, more research is utes. Another blood sample was drawn at t=270 minutes. As reference needed to detail the exact mechanism of disease, and identi- and cutoff values, the data derived from the papers from Colussi fication of more patients with CLDN10 mutations and sup- et al.40 and Nozu et al.41 were used. portive data from generating knock-in animals and/or diet studies in Cldn10 knockout mice should further confirm cau- DDAVP Responsiveness Testing sality and pathogenesis. On the basis of our patients’ pheno- The DDAVP test was performed after an overnight thirst starting at type, CLDN10 mutations should be considered in patients 10:00 p.m. the day before the test. At t=0 (baseline) as well as at t=30, with a hypokalemic non-Bartter, non-Gitelman salt-losing t=60, t=90, t=120, t=150, and t=180 minutes after the subcutaneous nephropathy and a renal concentration defect with low urine administration of 4 mg DDAVP, urine samples were taken to deter- calcium excretion and unexpectedly normal to high serum mine urine osmolality. In addition, serum samples were taken at magnesium levels. Because this phenotypical description is t=60, t=120, and t=180 minutes after DDAVP administration to de- on the basis of only two patients, it seems justified to sequence termine serum osmolality. CLDN10 in all incident and prevalent patients with a hypokalemic- alkalotic salt-losing nephropathy, in whom mutations in the cur- Urinary Exosome AQP2 Analyses rently known causative genes have been excluded. Patient urine was collected before and after administration of DDAVP and treated with protease inhibitors before urinary exosome isolation, which was performed as described previously.42 For immunoblotting, CONCISE METHODS exosome samples were loaded according to urinary creatinine and separated by SDS-PAGE as previously described.42 Murine immor- Genetic Analyses talized cortical collecting duct cells cultured in the presence or ab- Initial genetic testing to detect or exclude specific mutations in the sence of DDAVP served as controls. AQP2 antibody (KP9201; Merck known salt-losing nephropathy genes SLC12A3, CLCKNB,orHNF1b Milipore, Amsterdam, The Netherlands) was used at a concentration was done by Sanger sequencing and MLPA on genomic DNA derived of 1:5000. from peripheral blood cells. In Vitro CLDN10 Expression Analyses Whole-Exome Sequencing Constructs were generated using human influenza hemagglutinin- Whole-exome sequencing was essentially performed as previously tagged human Claudin-10b, and mutant Pro149Arg, p.Glu157_Tyr192del, described.39 Capture of exons was done using an Agilent SureSe- and p.Asp73Asn were inserted into a pcNeo IRES vector and lectXT Human All Exon 50-Mb Kit. Sequencing was performed transiently expressed in HEK293 cells. Subsequently, cell surface

3126 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 3118–3128, 2017 www.jasn.org CLINICAL RESEARCH biotinylationwas performed to determine expression and trafficking of 4. Vargas-Poussou R, Huang C, Hulin P, Houillier P, Jeunemaître X, wild-type Claudin-10 and -10 sequence variants.43 Resultant HEK293 Paillard M, Planelles G, Déchaux M, Miller RT, Antignac C: Functional lysate immunoreactive band volumes were quantified using Image- characterization of a calcium-sensing receptor mutation in severe autosomal dominant hypocalcemia with a Bartter-like syndrome. JAm Studio Lite (Licor) by normalizing hemagglutinin tag (Antibody 6E2; Soc Nephrol 13: 2259–2266, 2002 fl 1:5000; Bioké, Leiden, The Netherlands) expression to green uores- 5. Simon DB, Karet FE, Rodriguez-Soriano J, Hamdan JH, DiPietro A, cent protein (1:5000; G1544; Sigma-Aldrich, St. Louis, MO) and de- Trachtman H, Sanjad SA, Lifton RP: Genetic heterogeneity of Bartter’s picted as the mean6SEM. syndrome revealed by mutations in the K+ channel, ROMK. Nat Genet – To further analyze subcellular localization and tight junction 14: 152 156, 1996 6. Simon DB, Karet FE, Hamdan JM, DiPietro A, Sanjad SA, Lifton RP: strand formation of Claudin-10b wild type or variants, HEK293 cells Bartter’s syndrome, hypokalaemic alkalosis with hypercalciuria, is were grown on coverslips and transfected with the respective con- caused by mutations in the Na-K-2Cl cotransporter NKCC2. Nat Genet structs. One day post-transfection, cells were fixed with 4% PFA, 13: 183–188, 1996 blocked with 5% BSA/0.5% Triton X, and exposed to anti–Claudin- 7. Simon DB, Bindra RS, Mansfield TA, Nelson-Williams C, Mendonca E, 10 mAb (Thermo Fisher Scientific) followed by incubation with a Stone R, Schurman S, Nayir A, Alpay H, Bakkaloglu A, Rodriguez- secondary antibody (Anti-Mouse DyLight 633; Thermo Fisher Scien- Soriano J, Morales JM, Sanjad SA, Taylor CM, Pilz D, Brem A, fi Trachtman H, Griswold W, Richard GA, John E, Lifton RP: Mutations in ti c). Images were taken using a confocal laser-scanning microscope the chloride channel gene, CLCNKB, cause Bartter’ssyndrometypeIII. 510 (Zeiss). MDCK cells, subclone C7, were obtained from Albrecht Nat Genet 17: 171–178, 1997 Schwab (University of Münster, Muenster, Germany). Transfection 8. Simon DB, Nelson-Williams C, Bia MJ, Ellison D, Karet FE, Molina AM, and immunostaining procedures were carried out as described for Vaara I, Iwata F, Cushner HM, Koolen M, Gainza FJ, Gitleman HJ, Lifton ’ ’ HEK293 cells, except that MDCK-C7 cells were fixed 4 days post- RP: Gitelman s variant of Bartter s syndrome, inherited hypokalaemic alkalosis, is caused by mutations in the thiazide-sensitive Na-Cl cotransfection to allow differentiation into a functional epithelial transporter. Nat Genet 12: 24–30, 1996 monolayer. 9. Nijenhuis T, Vallon V, van der Kemp AW, Loffing J, Hoenderop JG, Bindels RJ: Enhanced passive Ca2+ reabsorption and reduced Mg2+ channel abundance explains thiazide-induced hypocalciuria and hypomagnesemia. J Clin Invest 115: 1651–1658, 2005 ACKNOWLEDGMENTS 10. Nijenhuis T, Hoenderop JG, Loffing J, van der Kemp AW, van Os CH, Bindels RJ: Thiazide-induced hypocalciuria is accompanied by a de- creased expression of Ca2+ transport proteins in kidney. Kidney Int 64: We thank the patients and their family members for participating in 555–564, 2003 additional studies and clinical characterization. We thank Albrecht 11. Loffing J, Vallon V, Loffing-Cueni D, Aregger F, Richter K, Pietri L, Schwab (University of Münster, Muenster, Germany) for the gift of Bloch-Faure M, Hoenderop JG, Shull GE, Meneton P, Kaissling B: Al- MDCK-C7 cells. tered renal distal tubule structure and renal Na(+) and Ca(2+) handling This research was partly supported by the EURenOmics project in a mouse model for Gitelman’s syndrome. JAmSocNephrol15: – from European Union Seventh Framework Programme FP7/2007– 2276 2288, 2004 12. Alexander RT, Hoenderop JG, Bindels RJ: Molecular determinants of 2013 agreement 305608 (to L.M.S. and J.G.J.H.), grants from the magnesium homeostasis: Insights from human disease. JAmSoc Innovation Fund of the Dutch health insurance companies (to A.P.B. Nephrol 19: 1451–1458, 2008 and T.N.), and The Netherlands Organization for Scientific Research 13. Yu AS: Claudins and the kidney. J Am Soc Nephrol 26: 11–19, 2015 grant VICI 016.130.668 (to J.G.J.H.). 14. Milatz S, Breiderhoff T: One gene, two paracellular ion channels- claudin-10 in the kidney. Pflugers Arch 469: 115–121, 2017 15. Konrad M, Schaller A, Seelow D, Pandey AV, Waldegger S, Lesslauer A, Vitzthum H, Suzuki Y, Luk JM, Becker C, Schlingmann KP, Schmid M, DISCLOSURES Rodriguez-Soriano J, Ariceta G, Cano F, Enriquez R, Juppner H, None. Bakkaloglu SA, Hediger MA, Gallati S, Neuhauss SC, Nurnberg P, Weber S: Mutations in the tight-junction gene claudin 19 (CLDN19) are associated with renal magnesium wasting, renal failure, and severe ocular involvement. Am J Hum Genet 79: 949–957, 2006 REFERENCES 16. Kausalya PJ, Amasheh S, Günzel D, Wurps H, Müller D, Fromm M, Hunziker W: Disease-associated mutations affect intracellular traffic 1. 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