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Felix Claverie-martin - One of the best experts on this subject based on the ideXlab platform.
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Novel compound heterozygous mutations of CLDN16 in a patient with familial hypomagnesemia with hypercalciuria and nephrocalcinosis
Molecular genetics & genomic medicine, 2020Co-Authors: Alejandro García-castaño, Ana Perdomo-ramirez, Gema Ariceta, Elena Ramos-trujillo, Mònica Vall-palomar, Leire Madariaga, Felix Claverie-martinAbstract:Background Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is an autosomal recessive tubulopathy characterized by excessive urinary wasting of magnesium and calcium, bilateral nephrocalcinosis, and progressive chronic renal failure in childhood or adolescence. FHHNC is caused by mutations in CLDN16 and CLDN19, which encode the tight-junction proteins claudin-16 and claudin-19, respectively. Most of these mutations are missense mutations and large deletions are rare. Methods We examined the clinical and biochemical features of a Spanish boy with early onset of FHHNC symptoms. Exons and flanking intronic segments of CLDN16 and CLDN19 were analyzed by direct sequencing. We developed a new assay based on Quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF) to investigate large CLDN16 deletions. Results Genetic analysis revealed two novel compound heterozygous mutations of CLDN16, comprising a missense mutation, c.277G>A; p.(Ala93Thr), in one allele, and a gross deletion that lacked exons 4 and 5,c.(840+25_?)del, in the other allele. The patient inherited these variants from his mother and father, respectively. Conclusions Using direct sequencing and our QMPSF assay, we identified the genetic cause of FHHNC in our patient. This QMPSF assay should facilitate the genetic diagnosis of FHHNC. Our study provided additional data on the genotypic spectrum of the CLDN16 gene.
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Exonic CLDN16 mutations associated with familial hypomagnesemia with hypercalciuria and nephrocalcinosis can induce deleterious mRNA alterations
BMC Medical Genetics, 2019Co-Authors: Ana Perdomo-ramirez, Elena Ramos-trujillo, Marian De Armas-ortiz, Lorena Suarez-artiles, Felix Claverie-martinAbstract:Background Familial hypomagnesaemia with hypercalciuria and nephrocalcinosis type 1 is an autosomal recessive disease characterized by excessive renal magnesium and calcium excretion, bilateral nephrocalcinosis, and progressive chronic renal failure. This rare disease is caused by mutations in CLDN16 that encodes claudin-16, a tight-junction protein involved in paracellular reabsorption of magnesium and calcium in the renal tubule. Most of these variants are located in exons and have been classified as missense mutations. The functional consequences of some of these claudin-16 mutant proteins have been analysed after heterologous expression showing indeed a significant loss of function compared to the wild-type claudin-16. We hypothesize that a number of CLDN16 exonic mutations can be responsible for the disease phenotype by disrupting the pre-mRNA splicing process. Methods We selected 12 previously described presumed CLDN16 missense mutations and analysed their potential effect on pre-mRNA splicing using a minigene assay. Results Our results indicate that five of these mutations induce significant splicing alterations. Mutations c.453G > T and c.446G > T seem to inactivate exonic splicing enhancers and promote the use of an internal cryptic acceptor splice site resulting in inclusion of a truncated exon 3 in the mature mRNA. Mutation c.571G > A affects an exonic splicing enhancer resulting in partial skipping of exon 3. Mutations c.593G > C and c.593G > A disturb the acceptor splice site of intron 3 and cause complete exon 4 skipping. Conclusions To our knowledge, this is the first report of CLDN16 exonic mutations producing alterations in splicing. We suggest that in the absence of patients RNA samples, splicing functional assays with minigenes could be valuable for evaluating the effect of exonic CLDN16 mutations on pre-mRNA splicing.
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Additional file 2: of Exonic CLDN16 mutations associated with familial hypomagnesemia with hypercalciuria and nephrocalcinosis can induce deleterious mRNA alterations
2019Co-Authors: Ana Perdomo-ramirez, Elena Ramos-trujillo, Marian De Armas-ortiz, Lorena Suarez-artiles, Felix Claverie-martinAbstract:Table S2. Bioinformatics predictions of splicing defects for CLDN16 exonic mutations and comparison with experimental results. (PDF 94 kb
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Characterization of two novel mutations in the claudin-16 and claudin-19 genes that cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis.
Gene, 2018Co-Authors: Ana Perdomo-ramirez, Mireia Aguirre, Tinatin Davitaia, Gema Ariceta, Elena Ramos-trujillo, Felix Claverie-martinAbstract:Abstract Familial hypomagnesemia with hypercalciuria and nephrocalcinosis is an autosomal-recessive renal tubular disorder characterized by excessive urinary losses of magnesium and calcium, bilateral nephrocalcinosis and progressive chronic renal failure in childhood or adolescence. The disease is caused by mutations in the tight-junction proteins claudin-16 and claudin-19 that are encoded by the CLDN16 and CLDN19 genes, respectively. Patients with CLDN19 mutations also are affected with severe ocular abnormalities. The aim of our study was to identify and characterize the molecular defects causing this disease in a Georgian girl and two Spanish siblings. Clinical and biochemical parameters were studied. The CLDN16 and CLDN19 genes were analyzed by DNA sequencing. The functional consequences of the identified mutations on pre-mRNA splicing were investigated using a minigene assay. Sequence analysis revealed that the patient from Georgia was homozygous for a novel mutation, c.602G > A; p.(G201E), in exon 4 of the CLDN16 gene. The two Spanish siblings were homozygous for a new CLDN19 mutation, c.388G > T; p.(G130C), located in exon 2, and both parents were heterozygous carriers of the mutation. Bioinformatics analysis predicted that the amino acid substitutions generated by these mutations were pathogenic. Functional studies showed that mutation c.388G > T also results in partial skipping of CLDN19 exon 2, which would imply significant alterations in the claudin-19 protein structure. Conversely, CLDN16 mutation c.602G > A had no effect on pre-mRNA splicing. Our study expands the genotypic classification of this rare disease and provides the first report of a CLDN19 mutation affecting splicing.
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Familial hypomagnesaemia with hypercalciuria and nephrocalcinosis: clinical and molecular characteristics.
Clinical kidney journal, 2015Co-Authors: Felix Claverie-martinAbstract:Familial hypomagnesaemia with hypercalciuria and nephrocalcinosis (FHHNC) is an autosomal-recessive renal tubular disorder characterized by excessive urinary losses of magnesium and calcium, bilateral nephrocalcinosis and progressive chronic renal failure. Presentation with FHHNC symptoms generally occurs early in childhood or before adolescence. At present, the only therapeutic option is supportive and consists of oral magnesium supplementation and thiazide diuretics. However, neither treatment seems to have a significant effect on the levels of serum magnesium or urine calcium or on the decline of renal function. In end-stage renal disease patients, renal transplantation is the only effective approach. This rare disease is caused by mutations in the CLDN16 or CLDN19 genes. Patients with mutations in CLDN19 also present severe ocular abnormalities such as myopia, nystagmus and macular colobamata. CLDN16 and CLDN19 encode the tight-junction proteins claudin-16 and claudin-19, respectively, which are expressed in the thick ascending limb of Henle's loop and form an essential complex for the paracellular reabsorption of magnesium and calcium. Claudin-19 is also expressed in retinal epithelium and peripheral neurons. Research studies using mouse and cell models have generated significant advances on the understanding of the pathophysiology of FHHNC. A recent finding has established that another member of the claudin family, claudin-14, plays a key regulatory role in paracellular cation reabsorption by inhibiting the claudin-16-claudin-19 complex. Furthermore, several studies on the molecular and cellular consequences of disease-causing CLDN16 and CLDN19 mutations have provided critical information for the development of potential therapeutic strategies.
Martin Konrad - One of the best experts on this subject based on the ideXlab platform.
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Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC): report of three cases with a novel mutation in CLDN19 gene.
Saudi journal of kidney diseases and transplantation : an official publication of the Saudi Center for Organ Transplantation Saudi Arabia, 2013Co-Authors: Amar Al-shibli, Martin Konrad, Waleed Altay, Omar Al Masri, Lihad Al-gazali, Ibrahim Al AttrachAbstract:Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare autosomal recessive disorder that is caused by mutation in the genes coding for tight junction proteins Claudin-16 and Claudin-19. Affected individuals usually develop nephrocalcinosis and progressive renal failure; some of them may have ophthalmologic involvement as well. Phenotypic description of three affected individuals from the same Middle Eastern kindred (two sisters and their cousin) is presented. This includes both clinical and laboratory findings upon initial presentation and subsequent follow-up. Molecular analysis of the CLDN19 gene was performed on the three cases and one set of parents. A novel homozygous missense mutation in CLDN19 (c.241C>T, p.Arg81Cys) was detected in all three affected children. The parents were heterozygous. Clinical and laboratory data in the three children with renal and ocular manifestations of FHHNC are described. Genetic analysis revealed a novel mutation in the CLDN19 gene. FHHNC is a rare cause of nephrocalcinosis, and we believe that it should be considered in the presence of nephrocalcinosis with hypercalcuria and hypermagnesuria.
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Hypomagnesemia-hypercalciuria-nephrocalcinosis and ocular findings: a new claudin-19 mutation.
The Turkish journal of pediatrics, 2012Co-Authors: Zelal Ekinci, Levent Karabaş, Martin KonradAbstract:Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is an autosomal recessive syndrome that affects the tight junction proteins claudin-16 and claudin-19 in the thick ascending limb. In patients with claudin-19 mutations, additional symptoms such as visual impairment and other ophthalmologic findings are expected. In this report, we present a seven-year-old girl with polyuria and polydipsia. She was the daughter of consanguineous parents with a history of neonatal hypomagnesemic convulsion. On physical examination, bilateral horizontal nystagmus, retinitis pigmentosa and severe myopia were detected. Laboratory examination revealed hypomagnesemia, hypercalciuria and hypermagnesuria. A clinical diagnosis of FHHNC caused possibly by claudin-19 mutation was decided with the ocular findings. DNA analysis revealed a novel homozygous nonsense mutation (W169X) in the CLDN19 gene. In conclusion, in a patient with consanguineous parents, history of hypomagnesemic convulsion and disturbed organization and development of the retina, a diagnosis of FHHNC caused by claudin-19 mutation should be considered.
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The role of tight junctions in paracellular ion transport in the renal tubule: lessons learned from a rare inherited tubular disorder.
American journal of kidney diseases : the official journal of the National Kidney Foundation, 2010Co-Authors: Lea Haisch, Jorge Reis Almeida, Paulo Roberto Abreu Da Silva, Karl Peter Schlingmann, Martin KonradAbstract: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.
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Claudins and Renal Magnesium Handling
Current Topics in Membranes, 2010Co-Authors: Jianghui Hou, Martin KonradAbstract:Publisher Summary Claudins are tight junction (TJ) integral membrane proteins that are key regulators of the paracellular pathway. Defects in claudin-16 (CLDN16) and claudin-19 (CLDN19) function result in the inherited human renal disorder familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC). Significant advances have been made toward understanding the mechanisms underlying the roles of these claudins in mediating paracellular ion reabsorption in the kidney. This chapter reviews the biosynthesis, trafficking, and interaction of CLDN16 and CLDN19 molecules; the biophysical properties of CLDN16 and CLDN19 channels; and the pathogenic mechanisms for the role of mutant forms of CLDN16 and CLDN19 in the development of FHHNC. FHHNC is a genetically heterogeneous disorder. Mutations in TJ gene encoding CLDN19 are also linked to this disease. The renal tubular phenotypes are indistinguishable of patients with mutations in CLDN16 from those with CLDN19. CLDN19 mutations are invariably associated with severe ocular abnormalities. This association has been named FHHNC with severe ocular involvement.
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Claudin-16 and claudin-19 interact and form a cation-selective tight junction complex.
The Journal of clinical investigation, 2008Co-Authors: Jianghui Hou, Aparna Renigunta, Antonio S. Gomes, David L. Paul, Siegfried Waldegger, Martin Konrad, Eveline E. Schneeberger, Daniel A. GoodenoughAbstract:Tight junctions (TJs) play a key role in mediating paracellular ion reabsorption in the kidney. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is an inherited disorder caused by mutations in the genes encoding the TJ proteins claudin-16 (CLDN16) and CLDN19; however, the mechanisms underlying the roles of these claudins in mediating paracellular ion reabsorption in the kidney are not understood. Here we showed that in pig kidney epithelial cells, CLDN19 functioned as a Cl(-) blocker, whereas CLDN16 functioned as a Na(+) channel. Mutant forms of CLDN19 that are associated with FHHNC were unable to block Cl(-) permeation. Coexpression of CLDN16 and CLDN19 generated cation selectivity of the TJ in a synergistic manner, and CLDN16 and CLDN19 were observed to interact using several criteria. In addition, disruption of this interaction by introduction of FHHNC-causing mutant forms of either CLDN16 or CLDN19 abolished their synergistic effect. Our data show that CLDN16 interacts with CLDN19 and that their association confers a TJ with cation selectivity, suggesting a mechanism for the role of mutant forms of CLDN16 and CLDN19 in the development of FHHNC.
Aimee K. Ryan - One of the best experts on this subject based on the ideXlab platform.
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Functional Validation of CLDN Variants Identified in a Neural Tube Defect Cohort Demonstrates Their Contribution to Neural Tube Defects.
Frontiers in neuroscience, 2020Co-Authors: Amanda I. Baumholtz, Patrizia De Marco, Valeria Capra, Aimee K. RyanAbstract:Neural tube defects (NTDs) are severe malformations of the central nervous system that affect 1-2 individuals per 2,000 births. Their etiology is complex and involves both genetic and environmental factors. Our recent discovery that simultaneous removal of Cldn3, -4, and -8 from tight junctions results in cranial and spinal NTDs in both chick and mouse embryos suggests that claudins play a conserved role in neural tube closure in vertebrates. To determine if claudins were associated with NTDs in humans, we used a Fluidigm next generation sequencing approach to identify genetic variants in CLDN loci in 152 patients with spinal NTDs. We identified eleven rare and four novel missense mutations in ten CLDN genes. In vivo validation of variant pathogenicity using a chick embryo model system revealed that overexpression of four variants caused a significant increase in NTDs: CLDN3 A128T, CLDN8 P216L, CLDN19 I22T, and E209G. Our data implicate rare missense variants in CLDN genes as risk factors for spinal NTDs and suggest a new family of proteins involved in the pathogenesis of these malformations.
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Claudin-7, -16, and -19 during mouse kidney development.
Tissue barriers, 2014Co-Authors: Halim Khairallah, Yan-hua Chen, Jasmine El Andalousi, Annie Simard, Nicholas Haddad, Jianghui Hou, Aimee K. Ryan, Indra R. GuptaAbstract:Members of the claudin family of tight junction proteins are critical for establishing epithelial barriers and for the regulation of paracellular transport. To understand their roles during kidney development, we first performed RT-PCR analyses and determined that 23 claudin family members were expressed in embryonic day (E) 13.5 mouse kidneys. Based on their developmental expression and phenotypes in mouse models, we hypothesized that 3 claudin members could affect nephron formation during kidney development. Using whole mount in situ hybridization and immunohistochemistry, we demonstrated that Claudin-7 (Cldn7) was expressed in the nephric duct, the emerging ureteric bud, and in tubules derived from ureteric bud branching morphogenesis. In contrast, Claudin-16 (Cldn16) and Claudin-19 (CLDN19) were expressed at later stages of kidney development in immature renal tubules that become the Loop of Henle. To determine if a loss of these claudins would perturb kidney development, we examined newborn kidneys from mutant mouse models lacking Cldn7 or Cldn16. In both models, we noted no evidence for any congenital renal malformation and quantification of nephron number did not reveal a decrease in nephron number when compared to wildtype littermates. In summary, Cldn7, Cldn16, and CLDN19 are expressed in different epithelial lineages during kidney development. Mice lacking Cldn7 or Cldn16 do not have defects in de novo nephron formation, and this suggests that these claudins primarily function to regulate paracellular transport in the mature nephron.
Jianghui Hou - One of the best experts on this subject based on the ideXlab platform.
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Mosaic expression of claudins in thick ascending limbs of Henle results in spatial separation of paracellular Na+ and Mg2+ transport.
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Susanne Milatz, Jianghui Hou, Dominik N. Müller, Nina Himmerkus, Vera Christine Wulfmeyer, Hoora Drewell, Kerim Mutig, Tilman Breiderhoff, Michael Fromm, Markus BleichAbstract:The thick ascending limb (TAL) of Henle's loop drives paracellular Na+, Ca2+, and Mg2+ reabsorption via the tight junction (TJ). The TJ is composed of claudins that consist of four transmembrane segments, two extracellular segments (ECS1 and -2), and one intracellular loop. Claudins interact within the same (cis) and opposing (trans) plasma membranes. The claudins Cldn10b, -16, and -19 facilitate cation reabsorption in the TAL, and their absence leads to a severe disturbance of renal ion homeostasis. We combined electrophysiological measurements on microperfused mouse TAL segments with subsequent analysis of claudin expression by immunostaining and confocal microscopy. Claudin interaction properties were examined using heterologous expression in the TJ-free cell line HEK 293, live-cell imaging, and Forster/FRET. To reveal determinants of interaction properties, a set of TAL claudin protein chimeras was created and analyzed. Our main findings are that (i) TAL TJs show a mosaic expression pattern of either cldn10b or cldn3/cldn16/CLDN19 in a complex; (ii) TJs dominated by cldn10b prefer Na+ over Mg2+, whereas TJs dominated by cldn16 favor Mg2+ over Na+; (iii) cldn10b does not interact with other TAL claudins, whereas cldn3 and cldn16 can interact with CLDN19 to form joint strands; and (iv) further claudin segments in addition to ECS2 are crucial for trans interaction. We suggest the existence of at least two spatially distinct types of paracellular channels in TAL: a cldn10b-based channel for monovalent cations such as Na+ and a spatially distinct site for reabsorption of divalent cations such as Ca2+ and Mg2.
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Claudin-7, -16, and -19 during mouse kidney development.
Tissue barriers, 2014Co-Authors: Halim Khairallah, Yan-hua Chen, Jasmine El Andalousi, Annie Simard, Nicholas Haddad, Jianghui Hou, Aimee K. Ryan, Indra R. GuptaAbstract:Members of the claudin family of tight junction proteins are critical for establishing epithelial barriers and for the regulation of paracellular transport. To understand their roles during kidney development, we first performed RT-PCR analyses and determined that 23 claudin family members were expressed in embryonic day (E) 13.5 mouse kidneys. Based on their developmental expression and phenotypes in mouse models, we hypothesized that 3 claudin members could affect nephron formation during kidney development. Using whole mount in situ hybridization and immunohistochemistry, we demonstrated that Claudin-7 (Cldn7) was expressed in the nephric duct, the emerging ureteric bud, and in tubules derived from ureteric bud branching morphogenesis. In contrast, Claudin-16 (Cldn16) and Claudin-19 (CLDN19) were expressed at later stages of kidney development in immature renal tubules that become the Loop of Henle. To determine if a loss of these claudins would perturb kidney development, we examined newborn kidneys from mutant mouse models lacking Cldn7 or Cldn16. In both models, we noted no evidence for any congenital renal malformation and quantification of nephron number did not reveal a decrease in nephron number when compared to wildtype littermates. In summary, Cldn7, Cldn16, and CLDN19 are expressed in different epithelial lineages during kidney development. Mice lacking Cldn7 or Cldn16 do not have defects in de novo nephron formation, and this suggests that these claudins primarily function to regulate paracellular transport in the mature nephron.
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Claudins and Renal Magnesium Handling
Current Topics in Membranes, 2010Co-Authors: Jianghui Hou, Martin KonradAbstract:Publisher Summary Claudins are tight junction (TJ) integral membrane proteins that are key regulators of the paracellular pathway. Defects in claudin-16 (CLDN16) and claudin-19 (CLDN19) function result in the inherited human renal disorder familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC). Significant advances have been made toward understanding the mechanisms underlying the roles of these claudins in mediating paracellular ion reabsorption in the kidney. This chapter reviews the biosynthesis, trafficking, and interaction of CLDN16 and CLDN19 molecules; the biophysical properties of CLDN16 and CLDN19 channels; and the pathogenic mechanisms for the role of mutant forms of CLDN16 and CLDN19 in the development of FHHNC. FHHNC is a genetically heterogeneous disorder. Mutations in TJ gene encoding CLDN19 are also linked to this disease. The renal tubular phenotypes are indistinguishable of patients with mutations in CLDN16 from those with CLDN19. CLDN19 mutations are invariably associated with severe ocular abnormalities. This association has been named FHHNC with severe ocular involvement.
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Claudin-16 and claudin-19 interaction is required for their assembly into tight junctions and for renal reabsorption of magnesium
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Jianghui Hou, Aparna Renigunta, Antonio S. Gomes, Mingli Hou, David L. Paul, Siegfried Waldegger, Daniel A. GoodenoughAbstract:Claudins are tight junction integral membrane proteins that are key regulators of the paracellular pathway. Defects in claudin-16 (CLDN16) and CLDN19 function result in the inherited human renal disorder familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC). Previous studies showed that siRNA knockdown of CLDN16 in mice results in a mouse model for FHHNC. Here, we show that CLDN19-siRNA mice also developed the FHHNC symptoms of chronic renal wasting of magnesium and calcium together with defective renal salt handling. siRNA knockdown of CLDN19 caused a loss of CLDN16 from tight junctions in the thick ascending limb (TAL) without a decrease in CLDN16 expression level, whereas siRNA knockdown of CLDN16 produced a similar effect on CLDN19. In both mouse lines, CLDN10, CLDN18, occludin, and ZO-1, normal constituents of TAL tight junctions, remained correctly localized. CLDN16- and CLDN19-depleted tight junctions had normal barrier function but defective ion selectivity. These data, together with yeast two-hybrid binding studies, indicate that a heteromeric CLDN16 and CLDN19 interaction was required for assembling them into the tight junction structure and generating cation-selective paracellular channels.
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Insights into driving forces and paracellular permeability from claudin-16 knockdown mouse.
Annals of the New York Academy of Sciences, 2009Co-Authors: Qixian Shan, Jianghui Hou, Daniel A. Goodenough, Nina Himmerkus, Markus BleichAbstract:Tight junction (TJ) properties are determined by membrane protein complexes of neighboring cells that form both a barrier and a selective pathway for paracellular substrate transport. Our previous work supports the view that paracellular permeability changes in the thick ascending limb (TAL) may underlie the mechanism for familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), a rare autosomal recessive disease linked to mutations in claudin-16 (CLDN16) and claudin-19 (CLDN19). CLDN16 knockdown (KD) mice are lacking CLDN16 expression by transgenic RNA interference. We observed that the transport defect for Mg2+ and Ca2+ in this animal model is caused by a loss of paracellular cation selectivity. The permeability ratio for Na+ over Cl- in KD mice was lower by a factor of two without a change in paracellular conductance, compared to wild type (WT). This resulted in a collapse of the transepithelial voltage, which is the driving force for Mg2+ and Ca2+ absorption in TAL. Since CLDN16 KD mice revealed lower blood pressure and an increased aldosterone plasma concentration, we hypothesize that the reduction in paracellular selectivity could allow backflow of Na+ and Cl- into the lumen of the TAL, thus enhancing the distal NaCl load and challenging the organism with a latent NaCl loss.
Kursad Turksen - One of the best experts on this subject based on the ideXlab platform.
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Abstract 4200: Transgenic mice overexpressing a Claudin-6 tail deletion mutant are resistant to tumor formation
Tumor Biology, 2010Co-Authors: Kursad Turksen, Azadeh Arabzadeh, Tammy-claire TroyAbstract:Morphological and physiological observations suggest that the tight junction (TJ)-based permeability barrier is modified/disrupted during tumorigenesis. It is now also widely recognized that the Claudin (Cldn) family of four tetraspan transmembrane proteins is crucial for tight junction assembly and permeability barrier function. However, the detailed roles of the Cldn cytoplasmic tail and extracellular loop domains in these processes or their dysfunction in tumorigenesis are not yet understood. We recently demonstrated that the tail domain of Cldn6 is crucial for membrane targeting and epidermal permeability barrier (EPB) formation, by forced expression of a Cldn6 tail deletion mutant (CDelta187) via the involucrin (Inv) promoter in the suprabasal compartment of the mouse epidermis. Even though a functional barrier formed, Inv-CDelta187 mice displayed histological and biochemical abnormalities in the epidermal differentiation program leading to a thickening of the epidermis after 1 week of age that persisted throughout life. Notably, a significant amount of not only Cldn6, but also Cldn10, Cldn11, and Cldn18 remained cytoplasmically localized and the protein-unfolding pathway was activated in transgenic epidermal cells. Using a well-established two-stage chemical carcinogenesis model, we are now investigating the temporal and spatial changes in expression of these and other (Cldn1, Cldn12) Cldns that we have previously demonstrated to be important in epidermal differentiation and tumorigenesis in CDelta187 versus wild type (WT) mice. METHODS: The lower dorsal backskin of mice was treated topically with 7,12-dimethylbenz(a)anthracene (DMBA; 0.25 mg/ml in acetone) and following a 10-day incubation period, 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 25 microg/ml in acetone) was applied three times a week to the same area. Backskin samples were dissected 2, 4, 6, 8 and 12 weeks after the initiation of the experimental protocol and immunohistochemistry was performed on sections using antibodies against the following: Cldn1, Cldn6, Cldn11, Cldn12, Cldn18, Ki67 (proliferation marker) and CD3 (immune infiltration marker). Strikingly, epidermal tumor formation was inhibited in CDelta187 as compared to WT animals. This is the first demonstration that alterations in Cldn structure and homeostasis may play a protective role against DMBA/TPA-induced skin tumors. Further investigation into the molecular mechanisms underlying this protective function is underway. Supported by CIHR Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4200.
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involucrin claudin 6 tail deletion mutant cδ206 transgenic mice a model of delayed epidermal permeability barrier formation and repair
Disease Models & Mechanisms, 2010Co-Authors: Adebola Enikanolaiye, Tammy-claire Troy, Azadeh Arabzadeh, Nathalie Larivière, Elif Atasoy, Kursad TurksenAbstract:Preterm birth is a major global health problem that results in a large number of infant deaths, many of which are attributable to the complications of an immature epidermal permeability barrier (EPB), for which there is currently no effective therapeutic option. The mammalian EPB is formed during development and is essential for survival as it maintains thermoregulation and hydration, and provides a defense against infection. Using transgenic mouse technology, we have demonstrated the importance of claudin (Cldn)-containing tight junctions (TJs) in epidermal differentiation and, in particular, that epidermal suprabasal overexpression of Cldn6 results in an EPB-deficient phenotype that phenocopies the dysfunctional EPB of premature human infants. In this study, we used the same approach to target a Cldn6 tail deletion mutant to the epidermis of mice [involucrin (Inv)-Cldn6-CDelta206 transgenic mice]. The Inv-Cldn6-CDelta206 transgenic mice displayed a developmental delay in EPB formation, as shown by the expression of keratins and Cldns, and by X-Gal penetration assays. Trans-epidermal water loss measurements and immunolocalization studies indicated that the epidermal differentiation program was also perturbed in postnatal Inv-Cldn6-CDelta206 transgenic mice resulting in a delayed maturation. Notably, however, expression/localization of epidermal differentiation and maturation markers, including Cldns, indicated that the transgenic epidermis matured and normalized by postnatal day 10, which is 3 days after the wild-type epidermis. Our results suggest that activation of the extracellular signal-regulated kinase 1/2 (Erk1/2) pathway and Cldn1 phosphorylation are associated with the repair and maturation of the skin barrier processes. These studies provide additional support for the crucial role of Cldns in epidermal differentiation, maturation and the formation of the EPB, and describe a novel animal model for evaluating postnatal epidermal maturation and therapies that may accelerate the process.
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Involucrin–claudin-6 tail deletion mutant (CΔ206) transgenic mice: a model of delayed epidermal permeability barrier formation and repair
Disease models & mechanisms, 2010Co-Authors: Adebola Enikanolaiye, Tammy-claire Troy, Azadeh Arabzadeh, Nathalie Larivière, Elif Atasoy, Kursad TurksenAbstract:Preterm birth is a major global health problem that results in a large number of infant deaths, many of which are attributable to the complications of an immature epidermal permeability barrier (EPB), for which there is currently no effective therapeutic option. The mammalian EPB is formed during development and is essential for survival as it maintains thermoregulation and hydration, and provides a defense against infection. Using transgenic mouse technology, we have demonstrated the importance of claudin (Cldn)-containing tight junctions (TJs) in epidermal differentiation and, in particular, that epidermal suprabasal overexpression of Cldn6 results in an EPB-deficient phenotype that phenocopies the dysfunctional EPB of premature human infants. In this study, we used the same approach to target a Cldn6 tail deletion mutant to the epidermis of mice [involucrin (Inv)-Cldn6-CDelta206 transgenic mice]. The Inv-Cldn6-CDelta206 transgenic mice displayed a developmental delay in EPB formation, as shown by the expression of keratins and Cldns, and by X-Gal penetration assays. Trans-epidermal water loss measurements and immunolocalization studies indicated that the epidermal differentiation program was also perturbed in postnatal Inv-Cldn6-CDelta206 transgenic mice resulting in a delayed maturation. Notably, however, expression/localization of epidermal differentiation and maturation markers, including Cldns, indicated that the transgenic epidermis matured and normalized by postnatal day 10, which is 3 days after the wild-type epidermis. Our results suggest that activation of the extracellular signal-regulated kinase 1/2 (Erk1/2) pathway and Cldn1 phosphorylation are associated with the repair and maturation of the skin barrier processes. These studies provide additional support for the crucial role of Cldns in epidermal differentiation, maturation and the formation of the EPB, and describe a novel animal model for evaluating postnatal epidermal maturation and therapies that may accelerate the process.
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Dermatitis and aging-related barrier dysfunction in transgenic mice overexpressing an epidermal-targeted claudin 6 tail deletion mutant.
PloS one, 2009Co-Authors: Tammy-claire Troy, Azadeh Arabzadeh, Nathalie Larivière, Adebola Enikanolaiye, Kursad TurksenAbstract:The barrier function of the skin protects the mammalian body against infection, dehydration, UV irradiation and temperature fluctuation. Barrier function is reduced with the skin's intrinsic aging process, however the molecular mechanisms involved are unknown. We previously demonstrated that Claudin (Cldn)-containing tight junctions (TJs) are essential in the development of the epidermis and that transgenic mice overexpressing Cldn6 in the suprabasal layers of the epidermis undergo a perturbed terminal differentiation program characterized in part by reduced barrier function. To dissect further the mechanisms by which Cldn6 acts during epithelial differentiation, we overexpressed a Cldn6 cytoplasmic tail deletion mutant in the suprabasal compartment of the transgenic mouse epidermis. Although there were no gross phenotypic abnormalities at birth, subtle epidermal anomalies were present that disappeared by one month of age, indicative of a robust injury response. However, with aging, epidermal changes with eventual chronic dermatitis appeared with a concomitant barrier dysfunction manifested in increased trans-epidermal water loss. Immunohistochemical analysis revealed aberrant suprabasal Cldn localization with marked down-regulation of Cldn1. Both the proliferative and terminal differentiation compartments were perturbed as evidenced by mislocalization of multiple epidermal markers. These results suggest that the normally robust injury response mechanism of the epidermis is lost in the aging Involucrin-Cldn6-CΔ196 transgenic epidermis, and provide a model for evaluation of aging-related skin changes.