CLINICAL RESEARCH www.jasn.org A Novel Hypokalemic-Alkalotic Salt-Losing Tubulopathy in Patients with CLDN10 Mutations † ‡ † 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 ascend- ing 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 pheno- type, 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 reab- sorption, 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 reab- sorption 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