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Jianghui Hou - One of the best experts on this subject based on the ideXlab platform.
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Paracellular Channel in Human Disease
The Paracellular Channel, 2019Co-Authors: Jianghui HouAbstract:Abstract Linkage analysis and positional cloning have led to the discovery of TJ genes and mutant alleles that cause various human diseases. More than 10 Mendelian diseases related to paracellular channel dysfunction have been solved on the molecular level. These include genetic disorders affecting the skin, the liver, the kidney, the inner ear, and the blood-brain barrier. In each case, identification of a gene sparks intense investigation of the cellular mechanism that can relate a genotype to a phenotype. Knowledge of the causal mechanism for disease not only facilitates the development of new treatment but also provides critical insight into the basic biology and physiology of paracellular channel. For example, the paracellular pathway in the kidney is particularly important for mineral metabolism. Genetic variations in three Claudin genes, Claudin-14, Claudin-16, and Claudin-19, are associated with renal diseases of Ca++ and Mg++ imbalance. Virtually every aspect of Claudin biology, e.g. gene transcription, protein translation, trafficking, interaction, and transport function, plays an important role in paracellular channelopathy.
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Claudins in barrier and transport function—the kidney
Pflügers Archiv - European Journal of Physiology, 2017Co-Authors: Yongfeng Gong, Jianghui HouAbstract:Claudins are discovered to be key players in renal epithelial physiology. They are involved in developmental, physiological, and pathophysiological differentiation. In the glomerular podocytes, Claudin-1 is an important determinant of cell junction fate. In the proximal tubule, Claudin-2 plays important roles in paracellular salt reabsorption. In the thick ascending limb, Claudin-14, -16, and -19 regulate the paracellular reabsorption of calcium and magnesium. Recessive mutations in Claudin-16 or -19 cause an inherited calcium and magnesium losing disease. Synonymous variants in Claudin-14 have been associated with hypercalciuric nephrolithiasis by genome-wide association studies (GWASs). More importantly, Claudin-14 gene expression can be regulated by extracellular calcium levels via the calcium sensing receptor. In the distal tubules, Claudin-4 and -8 form paracellular chloride pathway to facilitate electrogenic sodium reabsorption. Aldosterone, WNK4, Cap1, and KLHL3 are powerful regulators of Claudin and the paracellular chloride permeability. The lessons learned on Claudins from the kidney will have a broader impact on tight junction biology in other epithelia and endothelia.
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Claudins in barrier and transport function-the kidney.
Pflugers Archiv : European journal of physiology, 2016Co-Authors: Yongfeng Gong, Jianghui HouAbstract:Claudins are discovered to be key players in renal epithelial physiology. They are involved in developmental, physiological, and pathophysiological differentiation. In the glomerular podocytes, Claudin-1 is an important determinant of cell junction fate. In the proximal tubule, Claudin-2 plays important roles in paracellular salt reabsorption. In the thick ascending limb, Claudin-14, -16, and -19 regulate the paracellular reabsorption of calcium and magnesium. Recessive mutations in Claudin-16 or -19 cause an inherited calcium and magnesium losing disease. Synonymous variants in Claudin-14 have been associated with hypercalciuric nephrolithiasis by genome-wide association studies (GWASs). More importantly, Claudin-14 gene expression can be regulated by extracellular calcium levels via the calcium sensing receptor. In the distal tubules, Claudin-4 and -8 form paracellular chloride pathway to facilitate electrogenic sodium reabsorption. Aldosterone, WNK4, Cap1, and KLHL3 are powerful regulators of Claudin and the paracellular chloride permeability. The lessons learned on Claudins from the kidney will have a broader impact on tight junction biology in other epithelia and endothelia.
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Claudins and mineral metabolism.
Current opinion in nephrology and hypertension, 2016Co-Authors: Jianghui HouAbstract:PURPOSE OF REVIEW The tight junction conductance made of the Claudin-based paracellular channel is important in the regulation of calcium and magnesium reabsorption in the kidney. This review describes recent findings of the structure, the function, and the physiologic regulation of Claudin-14, Claudin-16, and Claudin-19 channels that through protein interactions confer calcium and magnesium permeability to the tight junction. RECENT FINDINGS Mutations in two tight junction genes - Claudin-16 and Claudin-19 - cause the inherited renal disorder familial hypomagnesemia with hypercalciuria and nephrocalcinosis. A recent genome-wide association study has identified Claudin-14 as a major risk gene of hypercalciuric nephrolithiasis. The crystal structure of Claudin-19 has recently been resolved allowing the reconstruction of a Claudin assembly model from cis-dimers made of Claudin-16 and Claudin-19 interaction. MicroRNAs have been identified as novel regulators of the Claudin-14 gene. The microRNA-Claudin-14 operon is directly regulated by the Ca sensing receptor gene in response to hypercalcemia. SUMMARY The paracellular pathway in the kidney is particularly important for mineral metabolism. Three Claudin proteins - Claudin-14, Claudin-16, and Claudin-19 - contribute to the structure and function of this paracellular pathway. Genetic mutations and gene expression changes in these Claudins may lead to alteration of the paracellular permeability to calcium and magnesium, ultimately affecting renal mineral metabolism.
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Biochemical and biophysical analyses of tight junction permeability made of Claudin-16 and Claudin-19 dimerization.
Molecular biology of the cell, 2015Co-Authors: Yongfeng Gong, Vijay Renigunta, Abby Sunq, Aparna Renigunta, Yi Zhou, Jinzhi Wang, Jing Yang, Lane A. Baker, Jianghui HouAbstract:The molecular nature of tight junction architecture and permeability is a long-standing mystery. Here, by comprehensive biochemical, biophysical, genetic, and electron microscopic analyses of Claudin-16 and -19 interactions--two Claudins that play key polygenic roles in fatal human renal disease, FHHNC--we found that 1) Claudin-16 and -19 form a stable dimer through cis association of transmembrane domains 3 and 4; 2) mutations disrupting the Claudin-16 and -19 cis interaction increase tight junction ultrastructural complexity but reduce tight junction permeability; and 3) no Claudin hemichannel or heterotypic channel made of Claudin-16 and -19 trans interaction can exist. These principles can be used to artificially alter tight junction permeabilities in various epithelia by manipulating selective Claudin interactions. Our study also emphasizes the use of a novel recording approach based on scanning ion conductance microscopy to resolve tight junction permeabilities with submicrometer precision.
Akira Ikari - One of the best experts on this subject based on the ideXlab platform.
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Rescue of tight junctional localization of a Claudin-16 mutant D97S by antimalarial medicine primaquine in Madin-Darby canine kidney cells.
Scientific reports, 2019Co-Authors: Kana Marunaka, Toru Kimura, Hajime Hasegawa, Satoshi Endo, Naoko Fujii, Takumi Furuta, Toshiyuki Matsunaga, Akira IkariAbstract:Magnesium ion (Mg2+) is paracellularly reabsorbed through Claudin-16 (CLDN16) in the thick ascending limb (TAL) of Henle’s loop in the kidney. Genetic disorders of CLDN16 cause mislocalization of CLDN16, resulting in hypomagnesemia. There is no effective treatment for hypomagnesemia except for magnesium administration. Here, we searched for a novel drug to restore tight junctional localization of a CLDN16 mutant. A D97S mutant, which has a mutation in the first extracellular loop (ECL) of CLDN16, was mainly colocalized with endosome marker, whereas wild-type (WT) CLDN16 was colocalized with ZO-1, an adaptor protein of tight junctions. The protein stability of the D97S mutant was lower than that of WT. The expression level of the D97S mutant was increased by lactacystin, a proteasomal inhibitor. Endocytosis inhibitors increased the tight junctional localization of the D97S mutant. We found that primaquine, an antimalarial agent, increased the protein stability and cell surface localization of the D97S mutant, but the localization of other mutants, which have mutations in the cytosolic domain or second ECL, was not affected. Transepithelial Mg2+ flux was increased by primaquine in D97S mutant-expressing cells. The expression of chaperon proteins, proteasome activity, and lactate dehydrogenase release were decreased by primaquine, and the proportion of viable cells increased. In contrast, these effects were not observed in WT CLDN16-expressing cells. These results suggested that primaquine increases the tight junctional localization of the D97S mutant, resulting in a reduction in ER stress and cellular injury. Primaquine may become an effective treatment drug for selected patients with mutant CLDN16.
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Down-regulation of magnesium transporting molecule, Claudin-16, as a possible cause of hypermagnesiuria with the development of tubulo-interstitial nephropathy
Magnesium research, 2018Co-Authors: Taisuke Shimizu, Akira Ikari, Kaori Takayanagi, Takatsugu Iwashita, Naohiko Anzai, Shimpei Okazaki, Hiroaki Hara, Minoru Hatano, Tatsuro Sano, Tomonari OgawaAbstract:Tubulo-interstitial nephropathy (TIN) is a critical pathological setting for the renal prognosis, and an increase in the urine magnesium excretion is a well-known characteristic feature as one of clinical parametets for the assessment of TIN. We examined the correlation between the development of TIN and the changes in the mRNA expression of renal magnesium-transporting molecules in rats with unilateral ureter obstruction (UUO). Ureter-ligated kidney was sampled at day-0 (control), day-1 (early phase) and day-7 (late phase). The development of TIN was assessed by immunohistochemistry and the real-time PCR of fibrosis-related genes (MCP-1: 105.1 ± 14.8% on day-0, 132.9 ± 25.7% on day-1, 302.7 ± 32.7% on day-7, TGF-β: 101.1 ± 7.6% on day-0, 93.6 ± 4.1% on day-1, 338.9 ± 20.7% on day-7) . The respective expressions of Claudin-10, 14, 16, 19, and transient receptor potential (TRP) M6 as magnesium-transporting molecules were also studied. The expression of calcium sensing receptor (CaSR) as an inhibitory regulator of Claudin-14 was additionally studied. The gene expression of Claudin-16 was decreased in the late phase of UUO (100.2 ± 2.9% at day-0, 90.3 ± 6.3% at day-1, 36.4 ± 1.6% at day-7) which was consistent with the increased urine magnesium excretion. Immunohistochemistry showed an apparent reduction of the immunoreactivity of Claudin-16 in the late phase. The expression of TRPM6 was reduced even in the early phase. The immunohistochemistry and gene expression of MCP-1 and TGF-s showed that TIN was not apparent in the early phase but was significant in the late phase of UUO. The density of peritubular capillaries was diminished in the late phase but not in the early phase. Expression of Claudin-14 and CaSR was up- and down-regulated, respectively. Our findings may indicate that the characteristic hypermagnesiuria in TIN is principally caused by the dysfunction of magnesium reabsorption in the thick ascending limb of Henle resulting from a significant decrease in the Claudin-16 expression. The down-regulation might be closely related to the development of TIN.
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Tight Junctional Localization of Claudin-16 is Regulated by Syntaxin 8 in Renal Tubular Epithelial Cells
The Journal of biological chemistry, 2014Co-Authors: Akira Ikari, Yasuhiro Yamazaki, Chie Tonegawa, Ayumi Sanada, Toru Kimura, Hideki Sakai, Hisayoshi Hayashi, Hajime Hasegawa, Masahiko Yamaguchi, Satoshi EndoAbstract:Claudin-16 (CLDN16) regulates the paracellular reabsorption of Mg2+ in the thick ascending limb of Henle's loop. However, the mechanism regulating the tight junctional localization of CLDN16 remains unknown. In yeast two-hybrid systems, we found that CLDN16 bound to syntaxin 8 (STX8), a target soluble N-ethylmaleimide-sensitive factor attachment protein receptor. We have examined the effect of STX8 on the localization and function of CLDN16 using Madin-Darby canine kidney cells expressing FLAG-tagged CLDN16. A pulldown assay showed that the carboxyl cytoplasmic region of human CLDN16 bound to STX8. CLDN16 was localized in the thick ascending limb, whereas STX8 was widely distributed throughout the rat kidney. An association between CLDN16 and STX8 was observed in rat renal homogenates and Madin-Darby canine kidney cells. STX8 siRNA decreased the cell surface localization of CLDN16 and transepithelial electrical resistance and permeability to Mg2+ but increased the co-localization of CLDN16 with early endosome and lysosome markers. Dephosphorylation of CLDN16 by protein kinase A inhibitors and S217A mutant, a dephosphorylated form, decreased the association with STX8 and the cell surface localization of CLDN16. Recycling assays indicated that STX8 siRNA decreased the trafficking of CLDN16 to the plasma membrane without affecting endocytosis. Dominant negative Rab11 and recycling inhibitor primaquine decreased the cell surface localization of CLDN16, which was similar to that in STX8 siRNA-transfected cells. These results suggest that STX8 mediates the recycling of CLDN16 and constitutes an important component of the CLDN16 trafficking machinery in the kidney.
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Extracellular Mg2+ regulates the tight junctional localization of Claudin-16 mediated by ERK-dependent phosphorylation
Biochimica et biophysica acta, 2009Co-Authors: Akira Ikari, Yohei Sasaki, Yasuhiro Yamazaki, Keishi Kinjo, Kosuke Atomi, Junko SugataniAbstract:Abstract Claudin-16 is involved in the paracellular reabsorption of Mg2+ in the thick ascending limb of Henle. Little is known about the mechanism regulating the tight junctional localization of Claudin-16. Here, we examined the effect of Mg2+ deprivation on the distribution and function of Claudin-16 using Madin-Darby canine kidney (MDCK) cells expressing FLAG-tagged Claudin-16. Mg2+ deprivation inhibited the localization of Claudin-16 at tight junctions, but did not affect the localization of other Claudins. Re-addition of Mg2+ induced the tight junctional localization of Claudin-16, which was inhibited by U0126, a MEK inhibitor. Transepithelial permeability to Mg2+ was also inhibited by U0126. The phosphorylation of ERK was reduced by Mg2+ deprivation, and recovered by re-addition of Mg2+. These results suggest that the MEK/ERK-dependent phosphorylation of Claudin-16 affects the tight junctional localization and function of Claudin-16. Mg2+ deprivation decreased the phosphothreonine levels of Claudin-16. The phosphothreonine levels of T225A and T233A Claudin-16 were decreased in the presence of Mg2+ and these mutants were widely distributed in the plasma membrane. Furthermore, TER and transepithelial Mg2+ permeability were decreased in the mutants. We suggest that the tight junctional localization of Claudin-16 requires a physiological Mg2+ concentration and the phosphorylation of threonine residues via a MEK/ERK-dependent pathway.
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Molecular physiological study of electrolyte transporters in renal tubular epithelial cells
Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan, 2009Co-Authors: Akira IkariAbstract:Patients with lifestyle-related diseases such as hypertension, diabetes, and hyperlipidemia are at high risk for the pathogenesis of a life-threatening atherosclerotic disease. The elucidation of the mechanism responsible for the pathogenesis can bring about the prevention and the cure of lifestyle-related diseases. We think that abnormal transport of electrolytes in renal tubule is involved in lifestyle-related diseases and renal failure. This review focuses on the regulatory mechanisms of Mg(2+) transport pathways in renal tubular cells. Mg(2+) filtrated by glomeruli is reabsorbed by transcellular and paracellular pathways in renal epithelial cells. Transient receptor potential melastatin 6 (TRPM6) channel is expressed in the apical membrane and involved in the reabsorption of Mg(2+). Cyclosporine A decreased TRPM6 expression and Mg(2+) influx, suggesting that the decrease in TRPM6 expression may cause hypomagnesemia. Claudin-16 is expressed in the tight junction (TJ) of the thick ascending limb of Henle and may be involved in the paracellular Mg(2+) transport. We found that the phosphorylation of Claudin-16 is necessary for its localization on the TJ and Claudin-16 is de-phosphorylated in Dahl salt-sensitive (DS) hypertensive rats. In epidemiologic studies, magnesium is the correlate of both systolic and diastolic blood pressure. Dysfunction of Claudin-16 may be involved in the salt-sensitive hypertension. Dysfunction of Mg(2+) reabsorption in renal tubule may be involved in renal failure and lifestyle-related diseases.
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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Impaired paracellular ion transport in the loop of Henle causes familial hypomagnesemia with hypercalciuria and nephrocalcinosis.
Annals of the New York Academy of Sciences, 2012Co-Authors: Lea Haisch, Martin KonradAbstract:Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare tubular disorder caused by mutations in genes coding for tight junction (TJ) proteins. TJs define the paracellular path between adjacent cells and thereby play a pivotal role for the regulation of the paracellular ion permeability of epithelia. The family of TJ proteins comprise a variety of transmembrane proteins, including the Claudins. Multiple distinct mutations in the genes for Claudin-16 and -19 have been described to be responsible for FHHNC. Both encoded proteins are especially important for the paracellular reabsorption of Mg(2+) and Ca(2+) in the thick ascending limb of Henle's loop. Interestingly, in addition to ion disturbances, FHHNC leads to chronic renal failure and may be associated with extrarenal symptoms.
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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.
Walter Hunziker - One of the best experts on this subject based on the ideXlab platform.
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Familial hypomagnesemia with hypercalciuria and nephrocalcinosis: variable phenotypic expression in three affected sisters from Mexican ancestry.
Renal failure, 2014Co-Authors: María E Arteaga, Walter Hunziker, Audrey S.m. Teo, Axel M. Hillmer, Osvaldo M MutchinickAbstract:Familial hypomagnesemia with hypercalciuria and nephrocalcinosis is a rare autosomal recessive renal disease caused by mutations in genes for the tight junction transmembrane proteins Claudin-16 (CLDN16) and Claudin-19 (CLDN19). We present the first case report of a Mexican family with three affected sisters carrying a p.Gly20Asp mutation in CLDN19 whose heterozygous mother showed evident hypercalciuria and normal low magnesemia without any other clinical, laboratory, and radiological symptoms of renal disease making of her an unsuitable donor. The affected sisters showed variable phenotypic expression including age of first symptoms, renal urinary tract infections, nephrolithiasis, nephrocalcinosis, and eye symptoms consisting in retinochoroiditis, strabismus, macular scars, bilateral anisocoria, and severe myopia and astigmatism. End stage renal disease due to renal failure needed kidney transplantation in the three of them. Interesting findings were a heterozygous mother with asymptomatic hypercalciuria warning on the need of carefully explore clinical, laboratory, kidney ultrasonograpy, and mutation status in first degree asymptomatic relatives to avoid inappropriate kidney donors; an evident variable phenotypic expression among patients; the identification of a mutation almost confined to Spanish cases and a 3.5 Mb block of genomic homozygosis strongly suggesting a common remote parental ancestor for the gene mutation reported.
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Methods to analyze subcellular localization and intracellular trafficking of Claudin-16.
Methods in molecular biology (Clifton N.J.), 2011Co-Authors: P. Jaya Kausalya, Walter HunzikerAbstract:The integral tight junction protein Claudin-16 (Cldn16) is predominantly expressed in renal epithelial cells of the thick ascending limb of Henle's loop where, together with Claudin-19, it forms a cation-selective pore that allows influx of Na+ from the interstitial fluid into the lumen of the kidney tubule. This leads to an electrochemical gradient that drives the reabsorbtion of Mg2+ and Ca2+ ions from the renal filtrate. Mutations in the Cldn16 gene have been identified in patients suffering from familial hypomagnesemia with hypercalciuria and nephrocalcinosis, with excessive renal wastage of Mg2+ and Ca2+ being a hallmark of this condition. Studies into the mechanism by which mutations impair Cldn16 function have shown that although several mutations affect paracellular ion transport, many interfere with intracellular trafficking of Cldn16, ultimately compromising its localization to TJs. Here, we describe the experimental approaches that can be used to monitor intracellular localization and trafficking of Cldn16. These methods can easily be adapted to study other Claudins, provided suitable antibodies are available.
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Claudin-16 affects transcellular Cl- secretion in MDCK cells.
The Journal of Physiology, 2009Co-Authors: Dorothee Günzel, Walter Hunziker, Salah Amasheh, Sandra Pfaffenbach, Jan F. Richter, P. Jaya Kausalya, Michael FrommAbstract:Claudin-16 (paracellin-1) is a tight junction protein localized mainly in the thick ascending limb of Henle's loop and also in the distal nephron. Its defect causes familial hypomagnesaemia with hypercalciuria and nephrocalcinosis. This had been taken as an indication that Claudin-16 conveys paracellular Mg2+ and Ca2+ transport; however, evidence is still conflicting. We studied paracellular ion permeabilties as well as effects of Claudin-16 on the driving forces for passive ion movement. MDCK-C7 cells were stably transfected with wild-type (wt) and mutant (R146T, T233R) Claudin-16. Results indicated that paracellular permeability to Mg2+ but not to Ca2+ is increased in cells transfected with wt compared to mutant Claudin-16 and control cells. Increased basolateral Mg2+ concentration activated a transcellular Cl− current which was greatly enhanced in cells transfected with wt and T233R Claudin-16, as compared to R146T Claudin-16-transfected or control cells. This current was triggered by the basolateral calcium-sensing receptor causing Ca2+ release from internal stores, thus activating apical Ca2+-sensitive Cl− channels and basolateral Ca2+-sensitive K+ channels. Immunohistochemical data suggest that the Cl− channel involved is bestrophin. We conclude that Claudin-16 itself possesses only moderate paracellular Mg2+ permeability but governs transcellular Cl− currents by interaction with apical Ca2+-activated Cl− channels, presumably bestrophin. As the transepithelial voltage generated by such a current alters the driving force for all ions, this may be the major mechanism to regulate Mg2+ and Ca2+ absorption in the kidney.
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Claudin Function in the Thick Ascending Limb of Henle's Loop
Annals of the New York Academy of Sciences, 2009Co-Authors: Dorothee Günzel, Walter Hunziker, Lea Haisch, Salah Amasheh, Sandra Pfaffenbach, Susanne M. Krug, Susanne Milatz, Dominik MüllerAbstract:During the past decade, Claudins have been established as major determinants of paracellular permeablilty in epithelia. In the kidney, each nephron segment expresses a distinct pattern of Claudins. Cells of the thick ascending limb of Henle's loop (TAL), which is characterized by high paracellular cation permeability, co-express an unusually large number of different Claudins: Claudin-10, -16, and -19 and, depending on the species, also Claudin-3, -4, -8, and/or -11. The function of most of these Claudins has been investigated in vitro. We present a summary of their function with special emphasis on Claudin-16 and -19. Mutations in the corresponding human genes lead to severely impaired renal Ca(2+) and Mg(2+) handling. To date, 42 different Claudin-16 mutations and three Claudin-19 mutations have been reported. These mutations prevent the Claudins from reaching the surface membrane, decrease membrane residence time, or render them functionless. In spite of the clear clinical symptoms such as hypomagnesemia, hypercalciuria, nephrocalcinosis, and renal insufficiency, mechanisms that link Claudin-16 and -19 to these symptoms are still unknown. Depending on the cell type used in overexpression studies, Claudin-16 appears to cause a mild increase in paracellular Mg(2+)-permeability or a pronounced increase in Na(+) permeability. Claudin-19 selectively decreases Cl(-) permeability, thus synergistically increasing relative cation permeability, or indiscriminately decreases paracellular permeability. In the light of these results it is hypothesized that the renal Mg(2+)/Ca(2+) waste may not be solely due to reduced resorption in the TAL but at least in part to paracellular back-leak of Mg(2+)/Ca(2+) into the tubular lumen of the distal convoluted tubule.
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Unusual clinical presentation and possible rescue of a novel Claudin-16 mutation.
The Journal of clinical endocrinology and metabolism, 2006Co-Authors: Dominik Müller, P. Jaya Kausalya, Iwan C. Meij, Julia Thumfart, Detlef Bockenhauer, Michael J. Dillon, William Van’t Hoff, Walter HunzikerAbstract:Context: Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is caused by a dysfunction of Claudin-16 (CLDN16) and characterized by renal wasting of Mg2+ and Ca2+. Objective: The objectives of this study were to study the clinical parameters in suspected FHHNC patients, identify mutations in the CLDN16 gene, and analyze molecular defects associated with the mutant protein. Design, Setting, and Participants: CLDN16 genes from two siblings diagnosed with FHHNC were sequenced. Expression and characterization of the mutant protein in renal MDCK cells were studied. Outcome Measures: Standard urine and serum parameters to diagnose FHHNC were determined. Mutations in the CLDN16 gene were identified. The subcellular distribution of the mutant protein was analyzed by immunofluorescence microscopy. Results: Urine and blood analysis showed signs typical for FHHNC. One patient, in addition, presented with hypocalcemic tetany, a phenomenon so far not described for FHHNC. Both siblings carry a nove...
Dorothee Günzel - One of the best experts on this subject based on the ideXlab platform.
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Deletion of Claudin-10 rescues Claudin-16-deficient mice from hypomagnesemia and hypercalciuria.
Kidney international, 2017Co-Authors: Tilman Breiderhoff, Nina Himmerkus, Dorothee Günzel, Dominik N. Müller, Hoora Drewell, Allein Plain, Kerim Mutig, Thomas E. Willnow, Markus BleichAbstract:The tight junction proteins Claudin-10 and -16 are crucial for the paracellular reabsorption of cations along the thick ascending limb of Henle's loop in the kidney. In patients, mutations in CLDN16 cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis, while mutations in CLDN10 impair kidney function. Mice lacking Claudin-16 display magnesium and calcium wasting, whereas absence of Claudin-10 results in hypermagnesemia and interstitial nephrocalcinosis. In order to study the functional interdependence of Claudin-10 and -16 we generated double-deficient mice. These mice had normal serum magnesium and urinary excretion of magnesium and calcium and showed polyuria and sodium retention at the expense of increased renal potassium excretion, but no nephrocalcinosis. Isolated thick ascending limb tubules of double mutants displayed a complete loss of paracellular cation selectivity and functionality. Mice lacking both Claudin-10 and -16 in the thick ascending limb recruited downstream compensatory mechanisms and showed hypertrophic distal convoluted tubules with changes in gene expression and phosphorylation of ion transporters in this segment, presumably triggered by the mild decrease in serum potassium. Thus, severe individual phenotypes in Claudin-10 and Claudin-16 knockout mice are corrected by the additional deletion of the other Claudin.
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Charge-selective Claudin channels
Annals of the New York Academy of Sciences, 2012Co-Authors: Susanne M. Krug, Dorothee Günzel, Salah Amasheh, Marcel P. Conrad, In-fah M. Lee, Michael FrommAbstract:Claudins are the main determinants of barrier properties of the tight junction. Many Claudins have been shown to act by tightening the paracellular pathway, but several function as paracellular channels. While some depend on the endogenous Claudin background of the analyzed cell line, for other Claudins, a distinct charge-selectivity has been shown. This paper portrays cation-selective (Claudin-2, Claudin-10b, Claudin-15) and anion-selective (Claudin-10a, Claudin-17) Claudins and Claudins with debatable channel properties (Claudin-4, Claudin-7, Claudin-16). It also describes molecular properties determining the observed charge-selectivity and pore properties in general. In leaky tissues, they widely determine overall transport characteristics by providing paracellular ion-selective pathways. In small intestine, Claudin-2 and Claudin-15 replace each other in the developing gut. In kidney proximal tubules, Claudin-2, Claudin-10, and Claudin-17 allow for paracellular reabsorption of sodium, chloride, and water.
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Targeted deletion of murine Cldn16 identifies extra- and intrarenal compensatory mechanisms of Ca2+ and Mg2+ wasting
American journal of physiology. Renal physiology, 2010Co-Authors: Constanze Will, Dorothee Günzel, Iwan C. Meij, Tilman Breiderhoff, Julia Thumfart, Marchel Stuiver, Kathrin Kopplin, Kerstin Sommer, Uwe Querfeld, Qixian ShanAbstract:Claudin-16 (CLDN16) is critical for renal paracellular epithelial transport of Ca2+ and Mg2+ in the thick ascending loop of Henle. To gain novel insights into the role of CLDN16 in renal Ca2+ and M...
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Claudin-16 affects transcellular Cl- secretion in MDCK cells.
The Journal of Physiology, 2009Co-Authors: Dorothee Günzel, Walter Hunziker, Salah Amasheh, Sandra Pfaffenbach, Jan F. Richter, P. Jaya Kausalya, Michael FrommAbstract:Claudin-16 (paracellin-1) is a tight junction protein localized mainly in the thick ascending limb of Henle's loop and also in the distal nephron. Its defect causes familial hypomagnesaemia with hypercalciuria and nephrocalcinosis. This had been taken as an indication that Claudin-16 conveys paracellular Mg2+ and Ca2+ transport; however, evidence is still conflicting. We studied paracellular ion permeabilties as well as effects of Claudin-16 on the driving forces for passive ion movement. MDCK-C7 cells were stably transfected with wild-type (wt) and mutant (R146T, T233R) Claudin-16. Results indicated that paracellular permeability to Mg2+ but not to Ca2+ is increased in cells transfected with wt compared to mutant Claudin-16 and control cells. Increased basolateral Mg2+ concentration activated a transcellular Cl− current which was greatly enhanced in cells transfected with wt and T233R Claudin-16, as compared to R146T Claudin-16-transfected or control cells. This current was triggered by the basolateral calcium-sensing receptor causing Ca2+ release from internal stores, thus activating apical Ca2+-sensitive Cl− channels and basolateral Ca2+-sensitive K+ channels. Immunohistochemical data suggest that the Cl− channel involved is bestrophin. We conclude that Claudin-16 itself possesses only moderate paracellular Mg2+ permeability but governs transcellular Cl− currents by interaction with apical Ca2+-activated Cl− channels, presumably bestrophin. As the transepithelial voltage generated by such a current alters the driving force for all ions, this may be the major mechanism to regulate Mg2+ and Ca2+ absorption in the kidney.
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Claudin Function in the Thick Ascending Limb of Henle's Loop
Annals of the New York Academy of Sciences, 2009Co-Authors: Dorothee Günzel, Walter Hunziker, Lea Haisch, Salah Amasheh, Sandra Pfaffenbach, Susanne M. Krug, Susanne Milatz, Dominik MüllerAbstract:During the past decade, Claudins have been established as major determinants of paracellular permeablilty in epithelia. In the kidney, each nephron segment expresses a distinct pattern of Claudins. Cells of the thick ascending limb of Henle's loop (TAL), which is characterized by high paracellular cation permeability, co-express an unusually large number of different Claudins: Claudin-10, -16, and -19 and, depending on the species, also Claudin-3, -4, -8, and/or -11. The function of most of these Claudins has been investigated in vitro. We present a summary of their function with special emphasis on Claudin-16 and -19. Mutations in the corresponding human genes lead to severely impaired renal Ca(2+) and Mg(2+) handling. To date, 42 different Claudin-16 mutations and three Claudin-19 mutations have been reported. These mutations prevent the Claudins from reaching the surface membrane, decrease membrane residence time, or render them functionless. In spite of the clear clinical symptoms such as hypomagnesemia, hypercalciuria, nephrocalcinosis, and renal insufficiency, mechanisms that link Claudin-16 and -19 to these symptoms are still unknown. Depending on the cell type used in overexpression studies, Claudin-16 appears to cause a mild increase in paracellular Mg(2+)-permeability or a pronounced increase in Na(+) permeability. Claudin-19 selectively decreases Cl(-) permeability, thus synergistically increasing relative cation permeability, or indiscriminately decreases paracellular permeability. In the light of these results it is hypothesized that the renal Mg(2+)/Ca(2+) waste may not be solely due to reduced resorption in the TAL but at least in part to paracellular back-leak of Mg(2+)/Ca(2+) into the tubular lumen of the distal convoluted tubule.