The Role of Tight Junctions in Paracellular Ion Transport in the Renal Tubule: Lessons Learned from a Rare Inherited Tubular Disorder

In Translation

The Role of Tight Junctions in Paracellular Ion Transport in the Renal Tubule: Lessons Learned From a Rare Inherited Tubular Disorder

Lea Haisch, MD,1 Jorge Reis Almeida, MD,2 Paulo Roberto Abreu da Silva, MD,2 Karl Peter Schlingmann, MD,1 and Martin Konrad, MD1

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is an autosomal recessive renal tubular disorder that typically presents with disturbances in magnesium and calcium homeostasis, recurrent urinary tract infections, and polyuria and/or polydipsia. Patients with FHHNC have high risk of the development of chronic kidney disease and end-stage renal disease in early adolescence. Multiple distinct mutations in the CLDN16 gene, which encodes a tight junction protein, have been found responsible for this disorder. In addition, mutations in another member of the claudin family, CLDN19, were identified in a subset of patients with FHHNC with visual impairment. The claudins belong to the family of tight junction proteins that define the intercellular space between adjacent endo- and epithelial cells. Claudins are especially important for the regulation of paracellular ion permeability. We describe a Brazilian family with 2 affected siblings presenting with the typical FHHNC phenotype with ocular anomalies. The clinical diagnosis of FHHNC was confirmed using mutational analysis of the CLDN19 gene, which showed 2 compound heterozygous mutations. In the context of the case vignette, we summarize the clinical presentation, diagnostic criteria, and therapeutic options for patients with FHHNC. We also review recent advances in understanding the electrophysiologic function of claudin-16 and -19 in the thick ascending limb of the loop of Henle and implications for ion homeostasis in the human body. Am J Kidney Dis. 57(2):320-330. © 2011 by the National Kidney Foundation, Inc.

INDEX WORDS: Hypomagnesemia; nephrocalcinosis; claudin; tight junction; CLDN16; CLDN19.

BACKGROUND The importance of paracellular ion transport processes for electrolyte reabsorption in the kidney was Ion transport processes along the kidney tubule emphasized by the discovery of mutations in 2 mem- have a pivotal role in the homeostasis of serum bers of the claudin multigene family, CLDN16 and electrolytes and maintenance of blood pressure in the CLDN19, in patients with hereditary renal calcium human body. Transport modalities vary substantially and magnesium wasting because the claudin proteins between different nephron segments. Reabsorption of have been shown to confer ion selectivity to tight ions and water can be carried out by either active junctions. transport through an epithelial cell or passively through the paracellular pathway. Paracellular transport de- CASE VIGNETTE pends on specific characteristics of an epithelial cell A 19-year-old woman was first admitted to a primary care layer in which adjacent cells are connected by special- hospital for uremic symptoms. She was referred to the Pediatric ized cell-to-cell contacts, the so-called tight junctions. Nephrology Department for further investigations and initiation of These tight junctions may either seal the intercellular renal replacement therapy. She had an extensive medical history of recurrent urinary tract infections, and retinitis pigmentosa was space or allow selective transport of charged solutes diagnosed at 2 years of age. Physical examination showed visual and water. impairment and horizontal nystagmus, and ophthalmologic examination showed severe bilateral retinopathy with macular coloboma. Preliminary laboratory investigations confirmed end-stage renal failure (estimated glomerular filtration rate [GFR], 4 mL/min/1.73 m2 From the 1Department of General Pediatrics, Pediatric Nephrol- [0.07 mL/s/1.73 m2] calculated using the Schwartz equation)1 and ogy, University Children’s Hospital Muenster, Münster, Germany; minor electrolyte disturbances: serum calcium level was within the and 2Nephrology Unit, Hospital dos Servidores do Estado, Brazil- reference range at 9.0 mg/dL (2.25 mmol/L), but in light of advanced ian Health Ministry, Rio de Janeiro, Brazil. chronic kidney disease (CKD), serum magnesium level was low at Received May 19, 2010. Accepted in revised form August 30, 1.38 mEq/L (0.69 mmol/L). Plain radiographs of the abdomen 2010. Originally published online December 27, 2010. showed bilateral renal calcifications and nephrolithiasis (Fig Address correspondence to Martin Konrad, MD, University 1A). Renal ultrasound showed bilateral medullary nephrocalci- Children’s Hospital Münster, Department of General Pediatrics, nosis and small kidneys (Fig 1B). Additional clinical and Pediatric Nephrology, Waldeyerstr 22, D-49149 Münster, Ger- biochemical data are listed in Table 1. many. E-mail: [email protected] Assessment of family history showed that retinitis pigmen- © 2011 by the National Kidney Foundation, Inc. tosa also was diagnosed in the patient’s 13-year-old sister in 0272-6386/$36.00 early childhood, and she was almost blind. Funduscopy showed doi:10.1053/j.ajkd.2010.08.038 severe macular coloboma (Fig 1C and D). In addition, repeated

320 Am J Kidney Dis. 2011;57(2):320-330 Claudin 16 and 19 Function in the Thick Ascending Limb of the Loop of Henle

Figure 1. (A) Plain radiograph of the abdomen in the index patient shows bilateral calcifications. (B) Renal ultrasound in the same patient shows a small kidney with medullary nephrocalcinosis. (C, D) Funduscopy of the left and right eye in the 13-year-old sister shows severe macular coloboma. episodes of cerebral convulsions of unknown origin were noted ing (Michelis-Castrillo syndrome). In addition, the in the past. Renal ultrasound also showed severe medullary patients had medullary nephrocalcinosis and devel- nephrocalcinosis, and laboratory investigations showed persis- oped CKD. Subsequent case reports and small series tent hypomagnesemia caused by renal magnesium wasting (serum magnesium, 0.5 mEq/L [0.25 mmol/L]), hyperuricemia, of patients enabled phenotypic characterization of the and hypercalciuria (Table 1). As in the index case, urine entire clinical spectrum of FHHNC, allowing differen- 3-7 sediment was compatible with a chronic urinary tract infection. tiation from other hypomagnesemic disease states. Given an estimated GFR of 45 mL/min/1.73 m2 [0.75 mL/s/1.73 The most frequent clinical symptoms at presentation 2 m ], her level of kidney function was classified as CKD stage 3. are recurrent urinary tract infections and polyuria Clinical workup of the parents and the 16-year-old brother and/or polydipsia, which mostly occur between early showed unremarkable findings. 8 Based on the key clinical and laboratory findings in the 2 sisters childhood and school age. Other leading symptoms (hypomagnesemia, hypercalciuria, nephrocalcinosis, and kidney at presentation include kidney stone formation, failure failure), the diagnosis of familial hypomagnesemia with hypercal- to thrive, vomiting, abdominal pain, tetanic episodes, ciuria and nephrocalcinosis (FHHNC; with eye involvement) was or generalized seizures. In addition to hypomag- made. Genetic testing was initiated to confirm the clinical diagno- ϳ sis in this family (discussed later). nesemia with a magnesium level of 1 mEq/L (0.5 mmol/L), biochemical abnormalities include renal PATHOGENESIS magnesium and calcium wasting; kidney function often is already impaired at the age of diagnosis. Familial Hypomagnesemia With Hypercalciuria and Serum intact parathyroid hormone levels tend to be Nephrocalcinosis increased before the stage of advanced CKD. Other The first clinical description of FHHNC dates back laboratory findings may include incomplete distal to 1972 when Michelis et al2 first described a family renal tubular acidosis, hypocitraturia, and hyperurice- affected with a hypomagnesemic disorder character- mia. Extrarenal manifestations, mainly eye involve- ized by excessive renal magnesium and calcium wast- ment, have been reported repeatedly. Visual impair-

Am J Kidney Dis. 2011;57(2):320-330 321 Haisch et al

Table 1. Clinical and Biochemical Findings in the 2 Sisters

Parameter Index Case (II.1) Sister (II.3) Reference Ranges

Age (y) 19 13 Serum values Magnesium (mEq/L) 1.38 1.0 1.46-2.1 Calcium (mg/dL) 9.0 9.0 8.9-10.3 Inorganic phosphorus (mg/dL) 9.4 4.9 2.3-4.7 Sodium (mEq/L) 145 147 136-144 Potassium (mEq/L) 4.9 4.5 3.6-5.1 Bicarbonate (mEq/L) 20 24 18-23 Creatinine (mg/dL) 12.6 1.2 0.4-1 eGFR (mL/min/1.73 m2) 4 45 80-120 Urea nitrogen (mg/dL) 320 27 17-43 Uric acid (mg/dL) 6.3 8.2 2.6-6 PTH (pg/mL) 434 80 10-55

Urine values 24-h volume (mL) 2,380 3,260 Calcium excretion (mg/kg/d) 3.25 8.31 Ͻ4 Ͻ FEMg (%) ND 15.13 4 Ͻ FENa (%) ND 2.58 1 Citrate (mg/d) 305 336 90-934

Urine sediment Leukocyturia Leukocyturia Urine culture E coli E coli Renal ultrasound Nephrocalcinosis Nephrocalcinosis Plain radiograph Multiple calciﬁcations Multiple calciﬁcations Eye examination Macular coloboma, retinopathy, myopia, Macular coloboma, retinopathy, myopia nystagmus Note: Conversion factors for units: serum creatinine in mg/dL to ␮mol/L, ϫ88.4; serum urea nitrogen in mg/dL to mmol/L, ϫ0.357; uric acid in mg/dL to ␮mol/L, ϫ59.48; serum magnesium in mEq/L to mmol/L, ϫ0.5; serum calcium in mg/dL to mmol/L, ϫ0.2495; inorganic phosphorus in mg/dL to mmol/L, ϫ0.3229; eGFR in mL/min/1.73 m2 to mL/s/1.73 m2, ϫ0.01667. No conversion necessary for serum potassium, serum sodium, and bicarbonate in mEq/L and mmol/L and for PTH in pg/mL and ng/L.

Abbreviations: eGFR, estimated glomerular filtration rate; FEMg, fractional excretion of magnesium; FENa, fractional excretion of sodium; ND, not determined; PTH, parathyroid hormone. ment may be very disabling in patients with FHHNC As expected, recurrence of FHHNC after kidney and is due to severe myopia, progressive retinopathy transplant has never been observed because the pri- with the development of macular coloboma, and nys- mary defect resides in the kidney. tagmus.4,6,7 From the disease phenotype and clearance studies One third of the patients with FHHNC develop in patients with FHHNC, Rodriguez-Soriano et al4 kidney failure before adolescence.8 Because a close postulated that the primary defect must be related to correlation between progression of kidney failure and disturbances in paracellular magnesium and calcium degree of nephrocalcinosis has been reported, thera- reabsorption in the loop of Henle. Because no good peutic approaches aim at decreasing urinary calcium candidate genes were available at that time, it was by using a pure positional cloning approach that Simon et excretion.6 A short-term clinical study proved that al10 identified mutations in a novel gene (CLDN16 treatment with thiazides decreases urinary calcium 9 [formerly referred to as PCLN1]) on chromosome 3 as excretion in patients with FHHNC. However, to date, the underlying cause of FHHNC. CLDN16 encodes it is still unclear whether thiazides truly have the claudin 16 (paracellin 1), a tight junction protein of potential to attenuate the progression of GFR loss in the claudin multigene family. To date, more than 40 patients with FHHNC. different CLDN16 mutations have been identified in A second therapeutic approach aims at preventing patients with FHHNC,8,10-16 and genotype-phenotype the secondary effects of hypomagnesemia using mag- correlation suggests that the CLDN16 genotype may nesium supplementation. As with progressive CKD, help predict the clinical course in affected individu- less magnesium can be filtered and thus excreted; als.14 hence, magnesium levels tend to increase with de- There is evidence from family studies that carriers creased kidney function. of heterozygous CLDN16 mutations also may present

322 Am J Kidney Dis. 2011;57(2):320-330 Claudin 16 and 19 Function in the Thick Ascending Limb of the Loop of Henle

Figure 2. (A) Family pedigree. Two heterozygous missense mutations in CLDN19 were identified in the 2 sisters (II.1 and II.3). Black bars indicate the G20D (glycine to aspartate substitution at amino acid 20) mutant allele; grey bars, the P28L (proline to leucine change at position 28) mutant allele; and white bars, the wild-type allele (WT). (B) Schematic model of claudin 19. Arrows indicate amino acid changes caused by the different mutations. Q57E, glutamine to gluatmate substitution at amino acid 57; L90P, leucine to proline substitution at amino acid 90; G123R, glycine to arginine substitution at amino acid 123. with clinical symptoms. Two studies described a high Consequently, to characterize the molecular defect incidence of hypercalciuria, nephrolithiasis, and/or in the family described in this review (see case vi- nephrocalcinosis in first-degree relatives of patients gnette), direct sequencing of CLDN19 was performed. with FHHNC,6,8 and a subsequent smaller study re- In both affected sisters, 2 heterozygous missense ported a tendency toward mild hypomagnesemia.13 mutations in CLDN19 were identified: a glycine to Also, analysis of a Cldn16 knockout mouse model aspartate substitution at amino acid 20 and a proline to showed that heterozygous Cldn16ϩ/Ϫ mice exhibited leucine substitution at residue 28 (G20D and P28L, a tendency toward hypercalciuria and increased 1,25- respectively; Fig 2A). The mother, who originated dihydroxyvitamin D3 levels, indicating the presence from Portugal, has inherited the G20D mutation, of an intermediate phenotype, as observed in heterozy- previously described as a Hispanic founder muta- gous CLDN16 mutation carriers.17 tion.19 The father (mixed African-Brazilian ethnicity) Homozygous or compound heterozygous muta- carries the P28L mutation. This new missense mutations in CLDN16 do not uniformly result in FHHNC. tion is located in the first transmembrane domain of Muller et al18 described a homozygous threonine to claudin 19 (Fig 2B). arginine substitution at amino acid 303 (T303R; lo- In the past, the deterioration in kidney function in cated in the carboxy-terminal PDZ binding motif [the patients with FHHNC associated with progressive PDZ stands for 3 proteins in which it initially was tubulointerstitial nephropathy and end-stage renal dis- described, ie, postsynaptic density 95, discs large, and ease in early adolescence was attributed to hypercalci- zonula occludens-1 (ZO-1)]) in 2 families with iso- uria and nephrocalcinosis.6 However, other inherited lated hypercalciuria and nephrocalcinosis without evi- tubular disorders associated with severe medullary dence for a disturbance in renal magnesium handling. nephrocalcinosis do not uniformly lead to progressive Interestingly, hypercalciuria disappeared during fol- CKD. For example, in antenatal Bartter syndrome, low-up, and urinary calcium excretion normalized GFR is stable during the first 2 decades of life.20 This after puberty.18 discrepancy indicates that severe nephrocalcinosis FHHNC is a genetically heterogenous disorder be- alone is not sufficient to explain the high rate of cause mutations in CLDN19, another member of the kidney failure in patients with FHHNC. From a clini- claudin gene family, also have been identified.19 The cal point of view, the repeated episodes of febrile renal phenotype of patients with FHHNC with muta- urinary tract infections may be an additional risk tions in either CLDN16 or CLDN19 is virtually undis- factor for CKD in patients with FHHNC. As an tinguishable. However, clinical presentations differ in alternative explanation, loss of kidney function might terms of the ocular phenotype because most patients be related to the gene defect itself. This hypothesis is with CLDN19 defects show severe visual impairment, supported by the naturally occurring bovine claudin whereas patients with CLDN16 mutations have no or 16 knockout phenotype (complete deletion of Cldn16), only minor ocular abnormalities. which is characterized by early onset of end-stage

Am J Kidney Dis. 2011;57(2):320-330 323 Haisch et al renal disease associated with interstitial nephritis and of Henle is the most important segment, in which diffuse fibrosis.21 The established genotype-pheno- ϳ70% of the filtered magnesium is reabsorbed. How- type correlation for CLDN16 also is consistent with ever, the final fine-tuning of magnesium excretion is this explanation, given that a more rapid decrease in carried out in the distal convoluted tubule, where GFR is seen in patients affected by severe mutations different hormonal controls act.22 Magnesium trans- that lead to complete loss of function compared with port in the thick ascending limb occurs mainly through those with mutations in claudin 16 that preserve the paracellular pathway, with the lumen-positive po- residual function.14 tential within the thick ascending limb as the driving force. Magnesium reabsorption in the distal convo- Physiologic Principles: Ion Handling in the Thick luted tubule occurs actively through the transcellular Ascending Limb pathway.22 MagnesiumHomeostasis Magnesium has an important role in the regula- ParacellularTransportintheThickAscendingLimb tion of membrane transporters, signal transduction To understand the pathophysiologic process of and as a cofactor for a great variety of enzymes. FHHNC, it is important to appreciate the principles Under physiologic conditions, serum magnesium of ion transport in the thick ascending limb (Fig 3). levels are maintained at almost constant values by The thick ascending limb has 2 main functions: (1) up- or downregulation of intestinal absorption and absorption of sodium chloride, magnesium, and renal excretion. calcium; and (2) generation of an osmotic gradient Approximately 80% of total serum magnesium is as a prerequisite for water reabsorption in the filtered in glomeruli. More than 95% of the filtrated following nephron segments. Sodium reabsorption magnesium then is reabsorbed along the different occurs through 2 different pathways: an active nephron segments, and only 3%-5% is excreted in transcellular transport and a passive paracellular urine. Whereas the proximal tubule accounts for only route. Transcellular transport is mediated by the a small percentage of magnesium reabsorption, in sodium-potassium-chloride (Naϩ-Kϩ-2Cl–) cotrans- terms of quantity, the thick ascending limb of the loop porter 2 (NKCC2; encoded by the SLC12A1 gene)

Figure 3. Schematic model of ion transport in the thick ascending limb. The lumen-positive potential is indicated by the circled plus. In patients with FHHNC (familial hypomagnesemia with hypercalciuria and nephrocalcinosis), the mutations in CLDN16/19 abolish the cation selectivity of the thick ascending limb by decreasing the lumen-positive potential, thus diminishing the driving force for the absorption of magnesium and calcium. Abbreviations: ClC-Kb, chloride channel; NKCC2, sodium-potassium-chloride (Naϩ-Kϩ-2Cl–) cotransporter; ROMK, renal outer medullary Kϩ channel.

324 Am J Kidney Dis. 2011;57(2):320-330 Claudin 16 and 19 Function in the Thick Ascending Limb of the Loop of Henle in the apical membrane of thick ascending limb lin,28 and the protein family of claudins.29 According cells and the basolateral adenosine triphosphatase to the requirements of different epithelia, a specific ϩ ϩ sodium-potassium pump (Na -K -ATPase). At the composition of tight junction proteins can be found.25 same time, passive paracellular sodium reabsorption occurs through specialized tight junctions driven TheClaudinGeneFamily by the lumen-positive potential. Active sodium Since the discovery in 1998 of claudin 1 and 2 by transport also provides the driving force for potas- Furuse et al,29 22 additional members of this multi- sium and chloride uptake through the apical NKCC2. gene family have been identified in humans. The Whereas chloride ions leave the cell through chlo- molecular weight of the encoded proteins varies from ride channels (ClC-Ka and ClC-Kb) at the basolat- 20-27 kDa.29,30 All claudins consist of 4 transmem- eral side into the interstitium, only a small amount brane helices, 2 extracellular loops, and intracellular of the potassium eventually is reabsorbed through amino and carboxy termini. Although the first loop is potassium channels at the basolateral membrane. believed to influence paracellular charge selectivity, Instead, most potassium is recycled back into urine the second loop probably is responsible for the interac- through specific potassium channels (ROMK [renal ϩ tion between opposing claudins. outer medullary K channel]; encoded by the The carboxy terminus of most claudins contains a KCNJ1 gene) in the apical membrane of thick sequence known as the PDZ-binding motif.31 Given that ascending limb cells (Fig 3). This recycling en- the PDZ domain is found in the ZO-1 protein, claudins forces the lumen-positive potential within the thick are capable of interacting with this most important mem- ascending limb and thus the driving force for para- ber of the ZO proteins, thus anchoring with the actin cellular reabsorption of other cations, especially 31 magnesium and calcium. cytoskeleton of the cell. All claudins also share the As shown here, paracellular transport of cations in highly conserved amino acid sequence motif W-GLW- the thick ascending limb is dependent on paracellular C-C (ie, tryptophan-glycine-leucine-tryptophan-cysteine- cation selectivity and driving force. The former is cysteine) in the first extracellular loop. The extracellular loops are essential contact points provided by a specific composition of tight junction 32 strands in the thick ascending limb involving expres- for the formation of tight junction strands. Several sion of different claudins. claudins cannot only provide homotypic interaction Along the axis of the thick ascending limb, the with identical claudins, but also can establish hetero- lumen-positive potential increases constantly. At the typic interactions with different claudins. However, 33 end of the thick ascending limb, the produced sodium not every theoretical combination is possible. gradient leads to paracellular backflow of sodium The nomenclature claudin (from the Latin clau- through the tight junction network. This in turn leads dere, “to close”) refers to the first detected claudins, 29 to a further increment in lumen-positive potential and which show a sealing function. Further investiga- therefore provides an additional driving force for the tions showed that the paracellular permeability of exclusive paracellular transport of magnesium and different epithelia is modulated not only by up- or calcium.23,24 downregulation of the expressed sealing claudins,34 but also by alteration in the expression of pore- 35 RECENT ADVANCES forming claudins. Some claudins show a broad expression pattern, Claudin Physiology and Pathophysiology of FHHNC whereas others are expressed specifically in a certain TightJunction subset of tissues. In the kidney, there is a segment- The tight junctions are located in the apical part of specific claudin expression profile along the nephron, the lateral cell membrane of epithelial and endothelial according to the different functional requirements of cells, where they embrace the cells like a belt. The belt each nephron segment. In a murine kidney extract, all of one cell adjoins the belts of adjacent cells and the claudins except 6, 9, 13, and 14 can be detected using proteins interact with each other. Tight junctions serve Northern blot probing of messenger RNA expres- as diffusion barriers for the transport of water and sion.36 Claudins 1 and 2 could be identified in the electrolytes and at the same time are able to induce Bowman capsule; claudins 2, 10, and 11, in the specific paracellular permeability for ions or other proximal tubule; and claudins 3, 7, 8, and 16, in solutes.25 In addition, tight junctions also ensure cell the distal tubule. Claudins 2, 3, 4, and 8 were found in polarity. the thin ascending limb of the loop of Henle; claudins Important components of the multiprotein tight 3, 10, 11, 16, and 19, in the thick ascending limb of junction complexes are the transmembrane proteins the loop of Henle; and claudins 1, 3, 4, 8, and 10, in occludin,26 junctional adhesion molecules,27 tricellu- the collecting duct.10,19,36-38

Am J Kidney Dis. 2011;57(2):320-330 325 Haisch et al

Biology and Physiology of Claudin 16 a moderate increase in magnesium permeability.40 Claudin 16 is expressed mainly in thick ascending The increase in paracellular cation permeability seems limb cells. The gene encoding human claudin 16 has 2 to be an important physiologic function of claudin 16 in-frame start codons; the first produces a 305–amino because it could not be detected in all claudin 16 40 acid protein, and the second, which occurs at the mutants responsible for FHHNC investigated and methionine at position 71, corresponds to the start connects claudin 16 directly to the development of the codon of mouse and rat Cldn16.39 The downstream electrical transepithelial gradient that serves as the sequence of these 2 orthologues is highly conserved. driving force for the reabsorption of cations, espe- Using immunofluorescence imaging of mammalian cially magnesium and calcium, in the thick ascending cell lines expressing human CLDN16 complementary limb. DNA, Hou et al40 showed that the short claudin 16 Taken together, all published investigations concern- variant concentrated at the tight junction, whereas the ing the electrophysiologic function of claudin 16 in long claudin 16 variant was restrained in endosomes different cell types could detect only a marginal in- or lysosomes. The physiologic relevance of the sec- crease, if any, in paracellular magnesium transport ond translation start site also is supported by the and calcium permeability after overexpression of clau- observation that genetic analysis of patients with din 16. Interestingly, none of the detected mutations to FHHNC has uncovered a point mutation leading to date concern the negatively charged amino acids in the substitution of methionine by threonine at amino the first extracellular loop challenging the mentioned acid 71 (ie, a mutation that disrupts the start codon) hypothesis. that is linked to the disease and causes mistargeting of 14 claudin 16 to lysosomes. In line with these results, a Claudin16MouseModels common insertion-deletion polymorphism at position To date, 2 different mouse models are available. 55 of the long isoform can be identified in healthy 8 The claudin 16 knockdown mouse generated by RNA humans. This variant leads to a frame shift and interference (small interfering RNA [siRNA]) technol- truncation of the long isoform of the protein at a ogy shows hypomagnesemia and significantly in- premature stop codon at position 90; however, be- creased urinary excretion of magnesium and calcium. cause it occurs in the untranslated region, it does not Renal histologic examination showed calcium depos- affect the short isoform. its along the basement membranes of medullary tu- As mentioned, mutations in the human CLDN16 bules, as well as in the interstitium, similar to findings gene cause a selective disturbance in renal magne- 42 sium and calcium reabsorption in the thick ascending observed in humans with FHHNC. Studies concern- limb.13 The current hypothesis is based on the idea ing electrophysiologic function in the thick ascending that the unusually high number of negatively charged limb by perfusion of isolated thick ascending limb amino acids in the first extracellular loop may allow segments showed a decrease in transepithelial poten- the formation of a cation-selective filter and thereby tial. This decrease consequently leads to an inability to reabsorb cations through the paracellular pathway; enable the absorption of divalent cations. Most muta- 42 tions are located within the first extracellular loop. thus, urinary excretion of these cations is increased. Recently, a Cldn16 knockout mouse model was ElectrophysiologicFunctionofClaudin16 generated. In accordance with the human phenotype, Ϫ/Ϫ Different electrophysiologic studies have investi- knockout mice (Cldn16 ) show hypomagnesemia gated the underlying mechanism of the paracellular and hypercalciuria, as well as compensatory path- transport of ions through a tight junction formed by ways, such as increased parathyroid hormone and 41 claudin 16. Ikari et al transfected claudin 16 in 1,25 dihydroxyvitamin D3, similar to observations in 17 low-resistance MDCK (Madin-Darby canine kidney) patients with FHHNC. Interestingly, juvenile Cldn16 cell monolayers and reported increased calcium flux knockout mice had normal urinary excretion values 17 from the apical to basolateral direction, but not the for magnesium, sodium, and potassium. other way around. In contrast, magnesium flux re- In contrast to claudin 16 knockdown mice and the mained equal to control cells. However, overexpres- human disease phenotype, Cldn16 knockout animals sion of claudin 16 transfected in high-resistance did not develop renal calcifications. The investigators MDCK cells was able to increase paracellular magne- proposed the presence of a specific compensatory sium permeability, although this increase turned out to mechanism in knockout mice preventing renal calcifi- be moderate.11 cation.17 Interestingly, transcriptional upregulation of Finally, transfection of claudin 16 in anion-selec- other transport systems for divalent cations, including tive LLC-PK1 cells, a porcine kidney line, showed a Trpv5, Trpm6, and Calbindin-D9k, was observed.17 significant increase in sodium permeability with only Both animal models did not develop CKD.

326 Am J Kidney Dis. 2011;57(2):320-330 Claudin 16 and 19 Function in the Thick Ascending Limb of the Loop of Henle

Electrophysiologic studies of perfused thick ascend- could be found on permeability for magnesium and ing limb tubules of 9- to 10-week-old Cldn16Ϫ/Ϫ sodium.23 knockout mice showed no alteration in paracellular Angelow et al44 analyzed MDCK cells trans- permeability for monovalent cations, such as sodium fected with claudin 19 and found a significant or lithium. However, for magnesium and calcium, a decrease in paracellular permeability for monova- significant decrease in paracellular permeability could lent and divalent cations. be detected.17 All theses observations together indicate a complex In conclusion, the available data indicate that claudin function of claudin 19, rather than simply the forma- 16 confers nonselective paracellular cation permeability tion of paracellular pores for divalent cations, as to tight junction strands of the thick ascending limb. initially assumed. However, it has to be kept in mind that data for claudin 19 and 16 function were gener- Biology and Physiology of Claudin 19 ated mainly in overexpression systems disturbing the Claudin 19 is expressed in various tissues, with the physiologic composition of tight junction strands in highest expression in the kidney and eye. In the polarized cell lines. Moreover, different cell lines peripheral nervous system, claudin 19 forms tight differ greatly in background levels of claudin expres- junctions resembling structures in Swann cells.43 sion. In kidney tubules, claudin 16 and 19 share similar Claudin19MouseModel expression patterns: reverse-transcription polymerase chain reaction assays in microdissected mouse nephron On account of the presence of claudin 19 in Swann segments could detect expression of both claudins in cells, Cldn19 knockout mice show abnormal behavior medullary and cortical thick ascending limbs, as well and peripheral neuropathy, which correlated with dys- as in the distal convoluted tubule.19 This nephron functional tight junctions in Swann cells. Unfortu- segment–specific colocalization implies that claudin nately, no data were published concerning the renal phenotype of Cldn19 knockout mice.43 16 and 19 share a similar function. 45 Patients presenting with typical FHHNC symp- Recently, Hou et al created a claudin 19 knock- toms, but without a detectable mutation in the CLDN16 down mouse by using siRNA. These claudin 19 knock- gene, therefore were tested for mutations in CLDN19. down mice show a phenotype similar to the claudin 16 In addition to the known phenotype, they showed knockdown mice and patients with FHHNC. Bio- severe ocular involvement with macula coloboma, chemical analysis showed decreased magnesium and myopia, and horizontal nystagmus, as mentioned.19 In calcium plasma levels and increased urinary excretion the eye, claudin 19 is expressed specifically in the of these divalent cations. No changes in plasma or retina, with strong expression in the retinal pigment urinary sodium levels could be detected. In addition, epithelium, inner plexiform, and inner nuclear layer.19 these mice showed increased aldosterone plasma lev- Notably, claudin 16 is not expressed in the retina els, which presumably prevent sodium loss by activat- body, which may explain the lack of ocular defects in ing the renin-angiotensin-aldosterone system and thus patients with FHHNC with CLDN16 mutations. A increasing sodium reabsorption in distal nephron seg- proper tight junction formation is required for cell ments. This hypothesis is supported by the observa- polarity and epithelial barrier function. Thus, loss of tion of decreased potassium levels in plasma and correct tight junction formation can result in mistarget- increased urinary potassium excretion. In summary, ing of factors influencing retinal cell division, leading the claudin 19 knockdown mice showed renal magne- 19 sium and calcium wasting due to defective paracellu- to the observed ocular phenotype. 45 To date, 5 missense mutations affecting different lar cation permeability. parts of the claudin 19 protein have been published (Fig 2B), all of which involve the transmembrane Interaction Between Claudin 16 and Claudin 19 domains or the first extracellular loop.19,23 As mentioned, claudins can interact with each other in 2 ways: within the cell membrane of the same cell (also ElectrophysiologicFunctionofClaudin19 called cis-interaction) or between the cell membranes of Based on transfection experiments in LLC-PK1 2 adjacent cells (trans-interaction)33,46 (Fig 4). If mouse cells reported by Hou et al,23 claudin 19 leads to a L fibroblasts, which normally do not form tight junc- significant decrease in chloride permeability without tions, are transfected with both claudins, well-developed affecting sodium permeability. All CLDN19 muta- tight junction strands with claudin 16 and 19 can be tions causing FHHNC in humans (G20D, Q57E [glu- observed. Different missense mutations in either CLDN19 tamine to glumate change], L90P [leucine to proline], (L90P and G123R) or CLDN16 (L145P [leucine to and G123R [glycine to arginine]) resulted in loss of proline], L151F [leucine to phenylalanine], G191R [gly- this function. Interestingly, no effect of claudin 19 cine to arginine], A209T [alanine to threonine], and

Am J Kidney Dis. 2011;57(2):320-330 327 Haisch et al

Figure 4. Schematic model of claudin (Cldn) 16 and 19 interactions. (A) The term cis-interaction describes the interaction between claudins in the same cell membrane, whereas trans-interaction stands for the interaction between claudins located in adjacent cells. Claudin 19 is capable of interacting in a cis-ortrans- manner with itself (homomeric interaction); homomeric interaction has not been observed for claudin 16. (B) There is no evidence of a direct interaction between claudin 16 and 19 in the trans-formation. (C) Consequently, a 3-stage hypothesis of tight junction assembly arises: (i) claudin 16 cis-interacts with claudin 19 (heteromeric) in one cell membrane, (ii) claudin 19 trans-interacts with itself (homomeric) between the cell membranes of adjacent cells, and (iii) claudin 16 and 19 again cis-interact in the adjacent cell membrane, ﬁnally creating the observed tight junction strands. Data source: Hou et al23 and Hou et al.45

F232C [phenylalanine to cysteine]) that cause FHHNC sium wasting. Affected individuals have a high risk of lead to a loss of interaction between claudin 16 and the development of CKD, and extrarenal symptoms claudin 19 (heteromeric interaction), thereby disrupting include severe visual impairment in a subset of pa- the synergistic effect.23,45 tients. FHHNC is caused by mutations in 2 genes As published recently, analysis of claudin 16 and coding for tight junction proteins of the claudin multi- 19 knockdown mice (using siRNA) showed that loss gene family, claudins 16 and 19. In the kidney, both of claudin 16 or 19 expression prevents the respective proteins are expressed predominantly in the thick interacting claudin from reaching the tight junction.45 ascending limb of the loop of Henle, the major site of This observation again emphasizes the strong connec- passive paracellular reabsorption of divalent cations. tion between these 2 claudins. Of interest, in that Functional data from overexpression studies in epithe- analysis of Cldn16 knockout mice, significantly lower lial cell monolayers point to an important function of claudin 19 expression was observed.17 Expression claudin 16 in conferring paracellular cation permeabil- decreases with age and was decreased by 30% in adult ity, whereas claudin 19 prevents the paracellular flux compared with neonatal mice. Maybe the late-onset of of chloride. Genetic analyses and subsequent func- hypomagnesemia in the juvenile Cldn16 knockout tional studies of mutated claudin 16 and 19 in this rare animals is triggered by the decrease in claudin 19 disorder show the highly specialized tissue-specific levels during development,17 which supports the im- nature of tight junction composition and emphasize portance of the claudin 16 and claudin 19 interaction. the critical role of claudins in selectively permitting or inhibiting paracellular ion flux along the kidney tu- SUMMARY bule. In the future, claudins and tight junctions eventu- FHHNC is an autosomal recessive renal tubular ally may provide targets for pharmacologic interven- disorder characterized by urinary calcium and magne- tion. However, our present knowledge of the tight

328 Am J Kidney Dis. 2011;57(2):320-330 Claudin 16 and 19 Function in the Thick Ascending Limb of the Loop of Henle junction physiologic process is still incomplete, and 16. Hampson G, Konrad MA, Scoble J. Familial hypomagne- much work remains to be done before we could even saemia with hypercalciuria and nephrocalcinosis (FHHNC): com- think about successful therapeutic strategies for FH- pound heterozygous mutation in the claudin 16 (CLDN16) gene. BMC Nephrol. 2008;9:12. HNC. 17. Will C, Breiderhoff T, Thumfart J, et al. Targeted deletion of murine CLDN16 identifies extra- and intrarenal compensatory ACKNOWLEDGEMENTS ϩ ϩ mechanisms of Ca2 and Mg2 wasting. Am J Physiol Renal Support: Dr Konrad receives support from the Peter Foundation Physiol. 2010;298:F1152-1161. for Nephrology. 18. Muller D, Kausalya PJ, Claverie-Martin F, et al. 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