A novel mutation of the claudin 16 gene in familial hypomagnesemia with hypercalciuria and nephrocalcinosis mimicking rickets

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is caused by a mutation in the gene CLDN16, which encodes paracellin 1 (claudin-16), atight junction protein mediating paracellular transport which is expressed in the thick ascending loop of Henle and in the distal convoluted tubule, where reabsorption of magnesium occurs. We present a 4 years old Turkish female child with a chief complaint of hypocalcemic tetany. A diagnosis of FHHNC was confirmed by genetic testing for a mutation in claudin 16 gene. Claudin 16 gene revealed homozygosity for the p.K183E(AAA>GAA) C. 547A>G indicating the diagnosis of hypomagnesemia with hypercalciuria and nephrocalcinosis. To our knowledge, this is the first case of FHHNC reported in Turkish population diagnosed at molecular level.     

Transgenic RNAi depletion of claudin-16 and the renal handling of magnesium

Tight junctions play a key role in mediating paracellular ion reabsorption in the kidney. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a human disorder caused by mutations in the tight junction protein claudin-16. However, the molecular mechanisms underlining the renal handling of magnesium and its dysfunction causing FHHNC are unknown. Here we show that claudin-16 plays a key role in maintaining the paracellular cation selectivity of the thick ascending limbs of the nephron. Using RNA interference, we have generated claudin-16-deficient mouse models. Claudin-16 knock-down (KD) mice exhibit chronic renal wasting of magnesium and calcium and develop renal nephrocalcinosis. Our data suggest that claudin-16 forms a non-selective paracellular cation channel, rather than a selective Mg(2+)/Ca(2+) channel as previously proposed. Our study highlights the pivotal importance of the tight junction in renal control of ion homeostasis and provides answer to the pathogenesis of FHHNC. We anticipate our study to be a starting point for more sophisticated in vivo analysis of tight junction proteins in renal functions. Furthermore, tight junction proteins could be major targets of drug development for electrolyte disorders.     

An unusual patient with hypercalciuria, recurrent nephrolithiasis, hypomagnesemia and G227R mutation of Paracellin-1. An unusual patient with hypercalciuria and hypomagnesemia unresponsive to thiazide diuretics

A 19-year-old female patient with hypercalciuria and recurrent nephrolithiasis/urinary tract infection unresponsive to thiazide type diuretics is presented. The patient first experienced nephrolithiasis at the age of 4 years. Afterwards, recurrent passages of stones and urinary tract infection occurred. On diagnostic evaluation at the age of 19 years, she also had hypocitraturia and hypomagnesemia. Her serum calcium concentrations were near the lower limit of normal (8.5-8.8 mg/dl; normal range: 8.5-10.5), her serum magnesium concentrations were 1.15-1.24 mg/dl (normal range: 1.4-2.5) and urinary calcium excretion was 900 mg/24 h. PTH concentrations were increased (110-156 pg/ml; normal range: 10-65). We tried to treat the patient with hydrochlorothiazide at a dose of 50 mg/day. During treatment with thiazide diuretics, PTH concentration remained high and the patient had recurrent urinary tract infections and passages of stones. Serum magnesium concentration did not normalize even under the parenteral magnesium infusion. Her mother had a history of nephrolithiasis 20 years ago. Severe hypomagnesemia in association with hypercalciuria/urinary stones is reported as a rare autosomal recessive disorder caused by impaired reabsorption of magnesium and calcium in the thick assending limp of Henle's loop. Recent studies showed that mutations in the CLDN16 gene encoding paracellin-1 cause the disorder. In exon 4, a homozygous nucleotide exchange (G679C) was identified for the patient. This results in a point mutation at position Glycine227, which is replaced by an Arginine residue (G227R). The mother was heterozygous for this mutation. G227 is located in the fourth transmembrane domain and is highly conserved in the claudin gene family. This case indicates the pathogenetic role of paracellin-1 mutation in familial hypomagnesemia with hypercalciuria and nephrocalcinosis and further underlines the risk of stone formation in heterozygous mutation carriers.     

Lecture: New light on the role of claudins in the kidney

The physiology of paracellular permeation of ions and solutes in the kidney is pivotally important but poorly understood. Claudins are the key components of the paracellular pathway. Defects in claudin function result in a broad range of renal diseases, including hypomagnesemia, hypercalciuria and nephrolithiasis. This review describes recent findings on the physiological function of claudins underlying paracellular transport mechanisms with a focus on renal Ca ( 2+) handling. We have uncovered a molecular mechanism underlying paracellular Ca ( 2+) transport in the thick ascending limb of Henle (TAL) that involves the functional interplay of three important claudin genes: claudin-14, -16 and -19, all of which are associated with human kidney diseases with hypercalciuria, nephrolithiasis and bone mineral loss. The Ca ( 2+) sensing receptor (CaSR) signaling in the kidney has long been a mystery. By analyzing small non-coding RNA molecules in the kidney, we have uncovered a novel microRNA based signaling pathway downstream of CaSR that directly regulates claudin-14 gene expression and establishes the claudin-14 molecule as a key regulator for renal Ca ( 2+) homeostasis. The molecular cascade of CaSR-microRNAs-claudins forms a regulatory loop to maintain proper Ca ( 2+) homeostasis in the kidney.     

Association of paracellin-1 with ZO-1 augments the reabsorption of divalent cations in renal epithelial cells

Paracellin-1 (PCLN-1) belongs to the claudin family of tight junction proteins and possibly plays a critical role in the reabsorption of magnesium and calcium. So far, the physiological properties of PCLN-1 have not been clarified. In the present study, we investigated whether PCLN-1 is associated with ZO-1. We also investigated whether (45)Ca(2+) transport across the paracellular barrier is affected by this association. In vitro binding analysis using glutathione S-transferase fusion protein showed that the C-terminal TRV sequence, especially Thr and Val residues, of PCLN-1 interacts with ZO-1. Next, PCLN-1 was stably expressed in Madin-Darby canine kidney cells using a FLAG tagging vector. ZO-1 was co-immunoprecipitated with the wild-type PCLN-1 and the alanine substitution (TAV) mutant. However, mutants of the deletion (Delta TRV) and the alanine substitution (ARV and TRA) inhibited the association of PCLN-1 with ZO-1. Confocal immunofluorescence demonstrated that the wild-type PCLN-1 and the TAV mutant localized in the tight junction along with ZO-1, but the Delta TRV, ARV, and TRA mutants were widely distributed in the lateral membrane including the tight junction area. Interestingly, monolayers of cells expressing the wild-type PCLN-1 and the TAV mutant showed higher activities of (45)Ca(2+) transport from apical to basal compartments, compared with those expressing the Delta TRV, ARV, and TRA mutants and the mock cells. (45)Ca(2+) transport was inhibited by increased magnesium concentration suggesting that magnesium and calcium were competitively transported by PCLN-1. It was noted that a positive electrical potential gradient enhanced (45)Ca(2+) transport from apical to basal compartments without affecting the opposite direction of transport. Thus, PCLN-1 localizes to the tight junction followed by association with ZO-1, and the PCLN-1.ZO-1 complex may play an essential role in the reabsorption of divalent cations in renal epithelial cells.     

Claudin-19 Mutations and Clinical Phenotype in Spanish Patients with Familial Hypomagnesemia with Hypercalciuria and Nephrocalcinosis

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis is an autosomal recessive tubular disorder characterized by excessive renal magnesium and calcium excretion and chronic kidney failure. This rare disease is caused by mutations in the CLDN16 and CLDN19 genes. These genes encode the tight junction proteins claudin-16 and claudin-19, respectively, which regulate the paracellular ion reabsortion in the kidney. Patients with mutations in the CLDN19 gene also present severe visual impairment. Our goals in this study were to examine the clinical characteristics of a large cohort of Spanish patients with this disorder and to identify the disease causing mutations. We included a total of 31 patients belonging to 27 unrelated families and studied renal and ocular manifestations. We then analyzed by direct DNA sequencing the coding regions of CLDN16 and CLDN19 genes in these patients. Bioinformatic tools were used to predict the consequences of mutations. Clinical evaluation showed ocular defects in 87% of patients, including mainly myopia, nystagmus and macular colobomata. Twenty two percent of patients underwent renal transplantation and impaired renal function was observed in another 61% of patients. Results of the genetic analysis revealed CLDN19 mutations in all patients confirming the clinical diagnosis. The majority of patients exhibited the previously described p.G20D mutation. Haplotype analysis using three microsatellite markers showed a founder effect for this recurrent mutation in our cohort. We also identified four new pathogenic mutations in CLDN19, p.G122R, p.I41T, p.G75C and p.G75S. A strategy based on microsequencing was designed to facilitate the genetic diagnosis of this disease. Our data indicate that patients with CLDN19 mutations have a high risk of progression to chronic renal disease.     

Analysis of CLDN14 gene in deaf Moroccan patients with non-syndromic hearing loss

Mutations in the CLDN14 gene, encoding the tight junction claudin 14 protein has been reported to date in an autosomal recessive form of isolated hearing loss DFNB29. In order to identify the contribution of CLDN14 to inherited deafness in Moroccan population, we performed a genetic analysis of this gene in 80 Moroccan familial cases. Our results show the presence of 7 mutations: 6 being conservative and one leading to a missense mutation (C11T) which was found at heterozygous and homozygous states, with a general frequency of 6.87%. The pathogenicity of the resulting T4M substitution is under discussion.     

Welcome to Organogenesis!

The physiology of paracellular permeation of ions and solutes in the kidney is pivotally important but poorly understood. Claudins are the key components of the paracellular pathway. Defects in claudin function result in a broad range of renal diseases, including hypomagnesemia, hypercalciuria and nephrolithiasis. This review describes recent findings on the physiological function of claudins underlying paracellular transport mechanisms with a focus on renal Ca²⁺ handling. We have uncovered a molecular mechanism underlying paracellular Ca²⁺ transport in the thick ascending limb of Henle (TAL) that involves the functional interplay of three important claudin genes: claudin-14, -16 and -19, all of which are associated with human kidney diseases with hypercalciuria, nephrolithiasis and bone mineral loss. The Ca²⁺ sensing receptor (CaSR) signaling in the kidney has long been a mystery. By analyzing small non-coding RNA molecules in the kidney, we have uncovered a novel microRNA based signaling pathway downstream of CaSR that directly regulates claudin-14 gene expression and establishes the claudin-14 molecule as a key regulator for renal Ca²⁺ homeostasis. The molecular cascade of CaSR-microRNAs-claudins forms a regulatory loop to maintain proper Ca²⁺ homeostasis in the kidney.     

Developmental regulation of claudin localization by fetal alveolar epithelial cells

Tight junction proteins in the claudin family regulate epithelial barrier function. We examined claudin expression by human fetal lung (HFL) alveolar epithelial cells cultured in medium containing dexamethasone, 8-bromo-cAMP, and isobutylmethylxanthanine (DCI), which promotes alveolar epithelial cell differentiation to a type II phenotype. At the protein level, HFL cells expressed claudin-1, claudin-3, claudin-4, claudin-5, claudin-7, and claudin-18, where levels of expression varied with culture conditions. DCI-treated differentiated HFL cells cultured on permeable supports formed tight transepithelial barriers, with transepithelial resistance (TER) >1,700 ohm/cm(2). In contrast, HFL cells cultured in control medium without DCI did not form tight barriers (TER <250 ohm/cm(2)). Consistent with this difference in barrier function, claudins expressed by HFL cells cultured in DCI medium were tightly localized to the plasma membrane; however, claudins expressed by HFL cells cultured in control medium accumulated in an intracellular compartment and showed discontinuities in claudin plasma membrane localization. In contrast to claudins, localization of other tight junction proteins, zonula occludens (ZO)-1, ZO-2, and occludin, was not sensitive to HFL cell phenotype. Intracellular claudins expressed by undifferentiated HFL cells were localized to a compartment containing early endosome antigen-1, and treatment of HFL cells with the endocytosis inhibitor monodansylcadaverine increased barrier function. This suggests that during differentiation to a type II cell phenotype, fetal alveolar epithelial cells use differential claudin expression and localization to the plasma membrane to help regulate tight junction permeability.     

Zonula occludens-1 function in the assembly of tight junctions in Madin-Darby canine kidney epithelial cells

Zonula occludens (ZO)-1 was the first tight junction protein to be cloned and has been implicated as an important scaffold protein. It contains multiple domains that bind a diverse set of junction proteins. However, the molecular functions of ZO-1 and related proteins such as ZO-2 and ZO-3 have remained unclear. We now show that gene silencing of ZO-1 causes a delay of approximately 3 h in tight junction formation in Madin-Darby canine kidney (MDCK) epithelial cells, but mature junctions seem functionally normal even in the continuing absence of ZO-1. Depletion of ZO-2, cingulin, or occludin, proteins that can interact with ZO-1, had no discernible effects on tight junctions. Rescue of junction assembly using murine ZO-1 mutants demonstrated that the ZO-1 C terminus is neither necessary nor sufficient for normal assembly. Moreover, mutation of the PDZ1 domain did not block rescue. However, point mutations in the Src homology 3 (SH3) domain almost completely prevented rescue. Surprisingly, the isolated SH3 domain of ZO-1 could also rescue junction assembly. These data reveal an unexpected function for the SH3 domain of ZO-1 in regulating tight junction assembly in epithelial cells and show that cingulin, occludin, or ZO-2 are not limiting for junction assembly in MDCK monolayers.     

Tight junction claudins and the kidney in sickness and in health

The epithelial cell tight junction has several functions including the control of paracellular transport between epithelial cells. Renal paracellular transport has been long recognized to exhibit unique characteristics within different segments of the nephron, functions as an important component of normal renal physiology and has been speculated to contribute to renal related pathology if functioning abnormally. The discovery of a large family of tight junction associated 4-transmembrane spanning domain proteins named claudins has advanced our understanding on how the paracellular permeability properties of tight junctions are determined. In the kidney, claudins are expressed in a nephron-specific pattern and are major determinants of the paracellular permeability of tight junctions in different nephron segments. The combination of nephron segment claudin expression patterns, inherited renal diseases, and renal epithelial cell culture models is providing important clues about how tight junction claudin molecules function in different segments of the nephron under normal and pathological conditions. This review discusses early observations of renal tubule paracellular transport and more recent information on the discovery of the claudin family of tight junction associated membrane proteins and how they relate to normal renal function as well as diseases of the human kidney.     

A deletion of the paracellin-1 gene is responsible for renal tubular dysplasia in cattle

Various hereditary diseases analogous to particular human heritable diseases have been identified in cattle. Investigation of these cattle diseases will provide useful information regarding the pathogenesis of the corresponding human diseases. Renal tubular dysplasia is an autosomal recessive disease of Japanese black cattle characterized by renal failure and growth retardation. We have previously mapped the locus responsible for the disease within a region on bovine chromosome 1. In the present study, we further typed additional markers in this region and found that a genomic segment of bovine chromosome 1 including the microsatellite marker BMS4009 was deleted in the affected animals. Construction of a physical map covering this region with BAC clones and comparison of the nucleotide sequences of this region between normal and affected animals revealed that a region of 37 kb including exons 1 to 4 of the bovine paracellin-1 gene was deleted in the affected animals. The paracellin-1 gene, which is the causative gene for human renal hypomagnesemia with hypercaciuria and nephrocalcinosis, encodes a tight junction protein of renal epithelial cells. Therefore, we concluded that deletion of the paracellin-1 gene is responsible for renal tubular dysplasia of cattle, and the cattle disease could be a good model for the human disease.     

Connexin-occludin chimeras containing the ZO-binding domain of occludin localize at MDCK tight junctions and NRK cell contacts.

Occludin is a transmembrane protein of the tight junction that functions in creating both an intercellular permeability barrier and an intramembrane diffusion barrier. Creation of the barrier requires the precise localization of occludin, and a distinct family of transmembrane proteins called claudins, into continuous linear fibrils visible by freeze-fracture microscopy. Conflicting evidence exists regarding the relative importance of the transmembrane and extracellular versus the cytoplasmic domains in localizing occludin in fibrils. To specifically address whether occludin's COOH-terminal cytoplasmic domain is sufficient to target it into tight junction fibrils, we created chimeras with the transmembrane portions of connexin 32. Despite the gap junction targeting information present in their transmembrane and extracellular domains, these connexin-occludin chimeras localized within fibrils when expressed in MDCK cells, as assessed by immunofluorescence and immunogold freeze-fracture imaging. Localization of chimeras at tight junctions depends on the COOH-terminal ZO-binding domain and not on the membrane proximal domain of occludin. Furthermore, neither endogenous occludin nor claudin is required for targeting to ZO-1-containing cell-cell contacts, since in normal rat kidney fibroblasts targeting of chimeras again required only the ZO-binding domain. These results suggest an important role for cytoplasmic proteins, presumably ZO-1, ZO-2, and ZO-3, in localizing occludin in tight junction fibrils. Such a scaffolding and cytoskeletal coupling function for ZO MAGUKs is analogous to that of other members of the MAGUK family.     

The unique-5 and -6 motifs of ZO-1 regulate tight junction strand localization and scaffolding properties

The proper cellular location and sealing of tight junctions is assumed to depend on scaffolding properties of ZO-1, a member of the MAGUK protein family. ZO-1 contains a conserved SH3-GUK module that is separated by a variable region (unique-5), which in other MAGUKs has proven regulatory functions. To identify motifs in ZO-1 critical for its putative scaffolding functions, we focused on the SH3-GUK module including unique-5 (U5) and unique-6 (U6), a motif immediately C-terminal of the GUK domain. In vitro binding studies reveal U5 is sufficient for occludin binding; U6 reduces the affinity of this binding. In cultured cells, U5 is required for targeting ZO-1 to tight junctions and removal of U6 results in ectopically displaced junction strands containing the modified ZO-1, occludin, and claudin on the lateral cell membrane. These results provide evidence that ZO-1 can control the location of tight junction transmembrane proteins and reveals complex protein binding and targeting signals within its SH3-U5-GUK-U6 region. We review these findings in the context of regulated scaffolding functions of other MAGUK proteins.     

The carboxyl terminus of zona occludens-3 binds and recruits a mammalian homologue of discs lost to tight junctions

Mammalian homologues of the Drosophila polarity proteins Stardust, Discs Lost, and Crumbs have been identified as Pals1, Pals1-associated tight junction protein (PATJ), and human Crumbs homologue 1 (CRB1), respectively. We have previously demonstrated that PATJ, Pals1, and CRB1 can form a tripartite tight junction complex in epithelial cells and that PATJ recruits Pals1 to tight junctions. Here, we observed that the Pals1/PATJ interaction was not crucial for the ultimate targeting of PATJ itself to tight junctions. This prompted us to examine if any of the 10 post-synaptic density-95/Discs Large/zona occludens-1 (PDZ) domains of PATJ could bind to the carboxyl termini of known tight junction constituents. We found that the 6th and 8th PDZ domains of PATJ can interact with the carboxyl termini of zona occludens-3 (ZO-3) and claudin 1, respectively. PATJ missing the 6th PDZ domain was found to mislocalize away from cell contacts. Surprisingly, deleting the 8th PDZ domain had little effect on PATJ localization. Finally, reciprocal co-immunoprecipitation experiments revealed that full-length ZO-3 can associate with PATJ. Hence, the PATJ/ZO-3 interaction is likely important for recruiting PATJ and its associated proteins to tight junctions.     

Functional characterization and localization of a gill-specific claudin isoform in Atlantic salmon

Claudins are the major determinants of paracellular epithelial permeability in multicellular organisms. In Atlantic salmon (Salmo salar L.), we previously found that mRNA expression of the abundant gill-specific claudin 30 decreases during seawater (SW) acclimation, suggesting that this claudin is associated with remodeling of the epithelium during salinity change. This study investigated localization, protein expression, and function of claudin 30. Confocal microscopy showed that claudin 30 protein was located at cell-cell interfaces in the gill filament in SW- and fresh water (FW)-acclimated salmon, with the same distribution, overall, as the tight junction protein ZO-1. Claudin 30 was located at the apical tight junction interface and in cell membranes deeper in the epithelia. Colocalization with the α-subunit of the Na(+)-K(+)-ATPase was negligible, suggesting limited association with mitochondria-rich cells. Immunoblotting of gill samples showed lower claudin 30 protein expression in SW than FW fish. Retroviral transduction of claudin 30 into Madin-Darby canine kidney cells resulted in a decreased conductance of 19%. The decreased conductance correlated with a decreased permeability of the cell monolayer to monovalent cations, whereas permeability to chloride was unaffected. Confocal microscopy revealed that claudin 30 was expressed in the lateral membrane, as well as in tight junctions of Madin-Darby canine kidney cells, thereby paralleling the findings in the native gill. This study suggests that claudin 30 functions as a cation barrier between pavement cells in the gill and also has a general role in cell-cell adhesion in deeper layers of the epithelium.     

Sinuous is a Drosophila claudin required for septate junction organization and epithelial tube size control

Epithelial tubes of the correct size and shape are vital for the function of the lungs, kidneys, and vascular system, yet little is known about epithelial tube size regulation. Mutations in the Drosophila gene sinuous have previously been shown to cause tracheal tubes to be elongated and have diameter increases. Our genetic analysis using a sinuous null mutation suggests that sinuous functions in the same pathway as the septate junction genes neurexin and scribble, but that nervana 2, convoluted, varicose, and cystic have functions not shared by sinuous. Our molecular analyses reveal that sinuous encodes a claudin that localizes to septate junctions and is required for septate junction organization and paracellular barrier function. These results provide important evidence that the paracellular barriers formed by arthropod septate junctions and vertebrate tight junctions have a common molecular basis despite their otherwise different molecular compositions, morphologies, and subcellular localizations.     

Renal ischemia�Creperfusion injury causes intercalated cell-specific disruption of occludin in the collecting duct

Renal ischemic events open tight junctions and disrupt epithelial polarity. The purpose of this study was to examine the effects of ischemia-reperfusion (IR) injury on expression and distribution of the tight junction proteins, occludin and ZO-1, in the rat kidney. IR injury was induced by clamping both renal pedicles for 30 min and animals were killed at 6 h after the reperfusion. IR injury decreased blood bicarbonate level, but did not persistently alter pH, Na(+), K(+), or Cl(-). In control kidneys, occludin immunoreactivity was intense in the tight junctions in the thick ascending limb, distal convoluted tubule, and collecting duct, moderate in the thin limbs of the loop of Henle, and was not detected in the proximal tubule, glomerulus, and blood vessels. ZO-1 was expressed in the same sites in which occludin was expressed, and additionally was also expressed in the proximal tubule, glomerulus, and vascular endothelial cells. IR kidneys exhibited damaged renal tubular epithelial cells in both proximal tubule and collecting duct segments in the outer medulla. In the collecting duct, the response of intercalated cells and principal cells differed. Following IR injury, intercalated cells, but not principal cells, lost their normal epithelial polarity and were frequently extruded into the tubule lumen. Occludin, instead of being localized to tight junctions, was localized diffusely in the cytoplasm in intercalated cells of IR kidneys. Principal cells, in contrast, were not detectably affected and neither occludin nor ZO-1 expression were altered in response to IR injury. The normal localization of ZO-1 expression to tight junction sites in both the proximal tubule and collecting duct was altered in response to IR, and, instead, ZO-1 expression was present diffusely in the cytoplasm. IR injury did not alter detectably either occludin or ZO-1 localization to the tight junction of the thick ascending limb cells. The abundance of total occludin protein by immunoblot analysis was not changed with IR injury. These results demonstrate that renal IR injury causes tight junction disruptions in both the proximal tubule and the collecting duct, and that altered distribution of the tight junction protein, occludin, may play a critical role in the collecting duct dysfunction which IR induces.