Downregulation of chemoresistance by claudin-14 silencing in human colorectal cancer cells.

An exceptional expression of claudins (CLDNs), tight junction (TJ) proteins, is observed in various solid cancer tissues. However, the pathophysiological roles of CLDNs have not been clarified in detail. CLDN14 is highly expressed in human colorectal cancer (CRC) tissues and cultured cancer epithelial cells. We found CLDN14 silencing decreased cell viability without affecting spheroid size in the three-dimensional (3D) spheroid model of DLD-1 cells derived from human CRC. Mitochondria activity and oxidative stress level were reduced by CLDN14 silencing. Furthermore, CLDN14 silencing decreased the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and its target antioxidative genes. CLDN14 was colocalized with ZO-1, a scaffolding protein in the TJ. CLDN14 silencing induced the disruption of TJ barrier such as the reduction of transepithelial electrical resistance and elevation of fluxes of small molecules including glucose in two-dimensional (2D) cultured model,. The depletion of glucose induced the elevation of ROS generation, mitochondria activity, and Nrf2 expression. These results suggest that CLDN14 increases Nrf2 expression in spheroids mediated via the formation of paracellular barrier to glucose. The cytotoxicities of doxorubicin, an anthracycline anticancer drug, and oxaliplatin, a platinum-based agent, were augmented by an Nrf2 activator in 2D cultured cells. The anticancer drug-induced toxicity was enhanced by CLDN14 silencing in 3D spheroids. We suggest that CLDN14 may potentiate chemoresistance mediated by the suppression of paracellular glucose permeability and activation of the Nrf2 signaling pathway in CRC cells.

Preliminary study of the role of nanobacteria in the formation of renal stones in experimental rats and its mechanism.

Introduction: The aim of the study was to study the role of nanobacteria in the formation of renal calculi and the underlying mechanism. Material and methods: A total of 90 clean Wistar male rats were randomly divided into a negative control group, an experimental group, and an interference group. From the end of the first week of modelling, 10 consecutive times once a week, 3 rats in each group were randomly selected to measure the biochemical blood markers and urine metabolism. After sacrifice, the formation of kidney stones was assessed by observing the ultrastructure of the kidney by electron microscopy and pathohistology. Finally, the expression of calcium-sensitive receptor (CaSR) and claudin-14 protein in the kidney tissue was examined by western blotting. Results: Compared with the control group, the gross structure of the kidney was changed in the model group. At the fourth week of modelling, the rats in the nanobacteria group had significantly enlarged kidneys and increased kidney-to-body ratio, and the difference had statistical significance (p < 0.05). The colour of the kidney profile was dark, the structure of the skin pulp was less clear, and the accumulation of yellowish particles was observed at the junction of the cortical pulp. The creatinine, uric acid, urea nitrogen, and urinary calcium of the rats in the nanobacteria group began to increase at the third week, and the difference between the third and eighth week had statistical significance (p < 0.05). However, the difference between the 3 groups had no statistical significance after the eighth week. At the fourth week, we observed the formation of calculi, which were mainly distributed in the renal tubules and surrounding tissues. The kidney stone formation rate was 52.4% in the nanobacteria group and 27.8% in the interference group, and the difference had statistical significance (p < 0.05). Ultrastructure observations revealed that from the fourth week, the renal tissues in the nanobacteria group showed expanded renal tubules, swollen renal tubular epithelium, granular degeneration, shedding and lymphocyte infiltration of renal tubular epithelial cells, and a small amount of calcium salt crystals in renal tubules. At the third week, the expression of CaSR and Claudin-14 protein in the nanobacteria group increased, and the difference had statistical significance (p < 0.05). The expression of CaSR and Claudin-14 was positively correlated with urinary calcium (p < 0.05). Conclusions: The formation of renal calculi began in the fourth week after the model was established, and the crystals were mostly located in the renal tubules. During the formation of renal calculi, the renal tubular epithelial cells were damaged, showing granular degeneration and small amounts of calcium salt crystals, accompanied by a few renal tubules beginning to expand and epithelial swelling, granular degeneration, necrosis and shedding of renal tubular epithelial cells, lymphocyte infiltration in the renal interstitium, and small amounts of calcium salt crystals in the renal tubules, which aggravated with time. The serum creatinine, serum uric acid, urea nitrogen, and urinary calcium levels increased with time from the third week and returned to normal after the eighth week. The expression of CaSR and Claudin-14 protein was upregulated and positively correlated with the 24-h urinary calcium excretion value.

[Molecular genetic predictors of the development of urolithiasis].

Urolithiasis is one of the most urgent problems of clinical urology. Currently, there is no consensus on the causes of stone formation, as well as the role of various factors in the development of urolithiasis, however, increasingly, according to various studies, the leading role is given to genetic causes. The article presents a modern review of data on genetic polymorphisms associated with ICD: rs1801197 and rs6776158 of the CASR gene; TaqI of the VDR gene; rs1801197 of the CALCR gene, rs3752472, rs650439, rs2853744 of the Klotho gene.

Differential parathyroid and kidney Ca2+-sensing receptor activation in autosomal dominant hypocalcemia 1.

BACKGROUND: Parathyroid Ca2+-sensing receptor (CaSR) activation inhibits parathyroid hormone (PTH) release, while activation of renal CaSRs attenuates Ca2+ transport and increases expression of the pore-blocking claudin-14. Patients with autosomal dominant hypocalcemia 1 (ADH1), due to activating CASR mutations, exhibit hypocalcemia but not always hypercalciuria (elevated Ca2+ in urine). The latter promotes nephrocalcinosis and renal insufficiency. Although CaSRs throughout the body including the kidney harbor activating CASR mutations, it is not understood why only some ADH1 patients display hypercalciuria. METHODS: Activation of the CaSR was studied in mouse models and a ADH1 patient. In vitro CaSR activation was studied in HEK293 cells. FINDINGS: Cldn14 showed blood Ca2+ concentration-dependent regulation, which was absent in mice with kidney-specific Casr deletion, indicating Cldn14 is a suitable marker for chronic CaSR activation in the kidney. Mice with a gain-of-function mutation in the Casr (Nuf) were hypocalcemic with low plasma PTH levels. However, renal CaSRs were not activated at baseline but only after normalizing blood Ca2+ levels. Similarly, significant hypercalciuria was not observed in a ADH1 patient until blood Ca2+ was normalized. In vitro experiments indicate that increased CaSR expression in the parathyroid relative to the kidney could contribute to tissue-specific CaSR activation thresholds. INTERPRETATION: Our findings suggest that parathyroid CaSR overactivity can reduce plasma Ca2+ to levels insufficient to activate renal CaSRs, even when an activating mutation is present. These findings identify a conceptually new mechanism of CaSR-dependent Ca2+ balance regulation that aid in explaining the spectrum of hypercalciuria in ADH1 patients. FUNDING: Erasmus+ 2018/E+/4458087, the Canadian Institutes for Health research, the Novo Nordisk Foundation, the Beckett Foundation, the Carlsberg Foundation and Independent Research Fund Denmark.

Calcium sensitive receptor and claudin-14 expression in kidney tissues of two kidney stone models.

INTODUCTION: The differences in protein expression of calcium sensitive receptor (CaSR) and claudin-14 in a kidney stone model established by nanobacteria (NB) and ethylene glycol (EG) were compared. MATERIAL AND METHODS: Ninety Wistar male rats were randomly divided into the NB group, the EG group, and the blank control group (NC group), with 30 rats in each group. Three rats of each group were sacrificed every week after injection. Histopathology was used to evaluate the stone formation of each group. The expression of CaSR and claudin-14 protein was detected by immunohistochemistry every week. RESULTS: There was formation of bright crystals in the kidneys of the EG group and the NB group, but not the NC group. At the 3rd week, the expression of CaSR and claudin-14 in the kidney tissue of the EG group began to increase while that in the NB group increased at the 4th week. The expression of CaSR and claudin-14 protein in the EG group was stronger than that in the NB group. Meanwhile, CaSR was expressed in the NC group but did not change significantly. Claudin-14 was not expressed in the NC group. CONCLUSIONS: Our results indicate that the traditional EG kidney stone modeling method is more rapid than the NB kidney stone modeling method, with a high stone formation rate, and the CaSR and claudin-14 protein expression levels are higher. Meanwhile, the NB used to establish the kidney stone model was isolated from patients with kidney stones, which may imitate the process of natural formation of kidney stones of patients. Therefore, the results of our research are more conducive to related research on the etiology of stones.

Renal claudin-14 expression is not required for regulating Mg2+ balance in mice.

The kidneys play a crucial role in maintaining Ca2+ and Mg2+ homeostasis by regulating these minerals' reabsorption. In the thick ascending limb of Henle's loop (TAL), Ca2+ and Mg2+ are reabsorbed through the tight junctions by a shared paracellular pathway formed by claudin-16 and claudin-19. Hypercalcemia activates the Ca2+-sensing receptor (CaSR) in the TAL, causing upregulation of pore-blocking claudin-14 (CLDN14), which reduces Ca2+ and Mg2+ reabsorption from this segment. In addition, a high-Mg2+ diet is known to increase both urinary Mg2+ and Ca2+ excretion. Since Mg2+ may also activate CaSR, we aimed to investigate whether CaSR-dependent increases in CLDN14 expression also regulate urinary Mg2+ excretion in response to hypermagnesemia. Here, we show that a Mg2+-enriched diet increased urinary Mg2+ and Ca2+ excretion in mice; however, this occurred without detectable changes in renal CLDN14 expression. The administration of a high-Mg2+ diet to Cldn14-/- mice did not cause more pronounced hypermagnesemia or significantly alter urinary Mg2+ excretion. Finally, in vitro evaluation of CaSR-driven Cldn14 promoter activity in response to increasing Mg2+ concentrations revealed that Cldn14 expression only increases at supraphysiological extracellular Mg2+ levels. Together, these results suggest that CLDN14 is not involved in regulating extracellular Mg2+ balance following high dietary Mg2+ intake.NEW & NOTEWORTHY Using transgenic models and in vitro assays, this study examined the effect of Mg2+ on regulating urinary excretion of Ca2+ and Mg2+ via activation of the Ca2+-sensing receptor-claudin 14 (CLDN14) pathway. The study suggests that CLDN14 is unlikely to play a significant role in the compensatory response to hypermagnesemia.

Claudin 14/15 play important roles in early wallerian degeneration after rat sciatic nerve injury.

PURPOSE: Wallerian degeneration (WD) is an antegrade degenerative process distal to peripheral nerve injury. Numerous genes are differentially regulated in response to the process. However, the underlying mechanism is unclear, especially the early response. We aimed at investigating the effects of sciatic nerve injury on WD via CLDN 14/15 interactions in vivo and in vitro. METHODS: Using the methods of molecular biology and bioinformatics analysis, we investigated the molecular mechanism by which claudin 14/15 participate in WD. Our previous study showed that claudins 14 and 15 trigger the early signal flow and pathway in damaged sciatic nerves. Here, we report the effects of the interaction between claudin 14 and claudin 15 on nerve degeneration and regeneration during early WD. RESULTS: It was found that claudin 14/15 were upregulated in the sciatic nerve in WD. Claudin 14/15 promoted Schwann cell proliferation, migration and anti-apoptosis in vitro. PKCα, NT3, NF2, and bFGF were significantly upregulated in transfected Schwann cells. Moreover, the expression levels of the ß-catenin, p-AKT/AKT, p-c-jun/c-jun, and p-ERK/ERK signaling pathways were also significantly altered. CONCLUSION: Claudin 14/15 affect Schwann cell proliferation, migration, and anti-apoptosis via the ß-catenin, p-AKT/AKT, p-c-jun/c-jun, and p-ERK/ERK pathways in vitro and in vivo. The results of this study may help elucidate the molecular mechanisms of the tight junction signaling pathway underlying peripheral nerve degeneration.

Claudin 14/15 play important roles in early wallerian degeneration after rat sciatic nerve injury / 中华创伤杂志(英文版)

PURPOSE@#Wallerian degeneration (WD) is an antegrade degenerative process distal to peripheral nerve injury. Numerous genes are differentially regulated in response to the process. However, the underlying mechanism is unclear, especially the early response. We aimed at investigating the effects of sciatic nerve injury on WD via CLDN 14/15 interactions in vivo and in vitro.@*METHODS@#Using the methods of molecular biology and bioinformatics analysis, we investigated the molecular mechanism by which claudin 14/15 participate in WD. Our previous study showed that claudins 14 and 15 trigger the early signal flow and pathway in damaged sciatic nerves. Here, we report the effects of the interaction between claudin 14 and claudin 15 on nerve degeneration and regeneration during early WD.@*RESULTS@#It was found that claudin 14/15 were upregulated in the sciatic nerve in WD. Claudin 14/15 promoted Schwann cell proliferation, migration and anti-apoptosis in vitro. PKCα, NT3, NF2, and bFGF were significantly upregulated in transfected Schwann cells. Moreover, the expression levels of the β-catenin, p-AKT/AKT, p-c-jun/c-jun, and p-ERK/ERK signaling pathways were also significantly altered.@*CONCLUSION@#Claudin 14/15 affect Schwann cell proliferation, migration, and anti-apoptosis via the β-catenin, p-AKT/AKT, p-c-jun/c-jun, and p-ERK/ERK pathways in vitro and in vivo. The results of this study may help elucidate the molecular mechanisms of the tight junction signaling pathway underlying peripheral nerve degeneration.

Localization and regulation of claudin-14 in experimental models of hypercalcemia.

Variations in the claudin-14 (CLDN14) gene have been linked to increased risk of hypercalciuria and kidney stone formation. However, the exact cellular localization of CLDN14 and its regulation remain to be fully delineated. To this end, we generated a novel antibody that allowed the detection of CLDN14 in paraffin-embedded renal sections. This showed CLDN14 to be detectable in the kidney only after induction of hypercalcemia in rodent models. Protein expression in the kidney is localized exclusively to the thick ascending limbs (TALs), mainly restricted to the cortical and upper medullary portion of the kidney. However, not all cells in the TALs expressed the tight junction protein. In fact, CLDN14 was primarily expressed in cells also expressing CLDN16 but devoid of CLDN10. CLDN14 appeared in very superficial apical cell domains and near cell junctions in a belt-like formation along the apical cell periphery. In transgenic mice, Cldn14 promotor-driven LacZ activity did not show complete colocalization with CLDN14 protein nor was it increased by hypercalcemia, suggesting that LacZ activity cannot be used as a marker for CLDN14 localization and regulation in this model. In conclusion, CLDN14 showed a restricted localization pattern in the apical domain of select cells of the TAL.

Loss of inner hair cell ribbon synapses and auditory nerve fiber regression in Cldn14 knockout mice.

Proper functioning of the auditory nerve is of critical importance for auditory rehabilitation by cochlear implants. Here we used the Cldn14-/- mouse to study in detail the effects of Claudin 14 loss on auditory synapses and the auditory nerve. Mutations in the tight junction protein Claudin 14 cause autosomal recessive non-syndromic hearing loss (DFNB29) in humans and mice, due to extensive degeneration of outer and inner hair cells. Here we show that massive inner hair cell loss in Cldn14-/- mice starts after the third postnatal week. Immunohistochemical analysis, using presynaptic Ribeye and postsynaptic GluR2 or PSD 95 as markers, revealed the degeneration of full ribbon synapses in inner hair cells from apical cochlear regions already at postnatal day 12 (P12). At P20, significant reduction in number of ribbon synapses has been observed for all cochlear regions and the loss of synaptic ribbons becomes even more prominent in residual inner hair cells from middle and apical cochlear regions at P45, which by then lost more than 40% of all ribbon synapses. In contrast to excessive noise exposure, loss of Claudin 14 does not cause an increase in "orphan" ribbons with no postsynaptic counterpart due to a reduction of postsynaptic structures. Hair cell loss in Cldn14-/- mice is associated with regression of peripheral auditory nerve processes, especially of outer radial fibers, which normally innervate the outer hair cells. The number of spiral ganglion neurons per area, however, was unchanged between the genotypes. Different effects were observed in the cochlear nucleus complex (CNC), the central projection area of the auditory nerve. While the dorsal cochlear nucleus (DCN) showed a significant 19.7% volume reduction, VGLUT-1 input was reduced by 34.4% in the ventral cochlear nucleus (VCN) but not in the DCN of Cldn14-/- mice. Taken together, massive inner hair cell loss starts after the third postnatal week in Cldn14-/- mice, but is preceded by the loss of ribbon synapses, which may be a first sign of an ongoing degeneration process in otherwise morphologically inconspicuously inner hair cells. In addition to the regression of peripheral nerve processes, reduced levels of VGLUT-1 in the VCN of Cldn14-/- mice suggests that Claudin 14 loss does not only cause hair cell loss but also affects peripheral and central connectivity of the auditory nerve.

Claudins in Renal Physiology and Pathology.

Claudins are integral proteins expressed at the tight junctions of epithelial and endothelial cells. In the mammalian kidney, every tubular segment express a specific set of claudins that give to that segment unique properties regarding permeability and selectivity of the paracellular pathway. So far, 3 claudins (10b, 16 and 19) have been causally traced to rare human syndromes: variants of CLDN10b cause HELIX syndrome and variants of CLDN16 or CLDN19 cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis. The review summarizes our current knowledge on the physiology of mammalian tight junctions and paracellular ion transport, as well as on the role of the 3 above-mentioned claudins in health and disease. Claudin 14, although not having been causally linked to any rare renal disease, is also considered, because available evidence suggests that it may interact with claudin 16. Some single-nucleotide polymorphisms of CLDN14 are associated with urinary calcium excretion and/or kidney stones. For each claudin considered, the pattern of expression, the function and the human syndrome caused by pathogenic variants are described.

Vitamin D strengthens the bladder epithelial barrier by inducing tight junction proteins during E. coli urinary tract infection.

Tight junction proteins are pivotal to prevent bacterial invasion of the epithelial barrier. We here report that supplementation with vitamin D can strengthen the urinary bladder lining. Vitamin D deficient and sufficient mice were infected with Escherichia coli (E. coli) transurethrally to cause urinary tract infection. In addition, bladder biopsies were obtained from postmenopausal women before and after a 3-month period of supplementation with 25-hydroxyvitamin D3 (25D3) and ex vivo infected with E. coli. In biopsies, obtained before E. coli infection, vitamin D had no impact on tight junction proteins. However, during E. coli infection, vitamin D induced occludin and claudin-14 in mature superficial umbrella cells of the urinary bladder, as demonstrated by immunohistochemistry. Increased cell-cell adhesion consolidating the epithelial integrity is thereby promoted. We here describe a novel role of vitamin D in the urinary tract supporting vitamin D supplementation to restore the bladder epithelial integrity.

Detailed Clinical Features of Deafness Caused by a Claudin-14 Variant.

Tight junctions are cellular junctions that play a major role in the epithelial barrier function. In the inner ear, claudins, occludin, tricellulin, and angulins form the bicellular or tricellular binding of membrane proteins. In these, one type of claudin gene, CLDN14, was reported to be responsible for human hereditary hearing loss, DFNB29. Until now, nine pathogenic variants have been reported, and most phenotypic features remain unclear. In the present study, genetic screening for 68 previously reported deafness causative genes was carried out to identify CLDN14 variants in a large series of Japanese hearing loss patients, and to clarify the prevalence and clinical characteristics of DFNB29 in the Japanese population. One patient had a homozygous novel variant (c.241C>T: p.Arg81Cys) (0.04%: 1/2549). The patient showed progressive bilateral hearing loss, with post-lingual onset. Pure-tone audiograms indicated a high-frequency hearing loss type, and the deterioration gradually spread to other frequencies. The patient showed normal vestibular function. Cochlear implantation improved the patient's sound field threshold levels, but not speech discrimination scores. This report indicated that claudin-14 is essential for maintaining the inner ear environment and suggested the possible phenotypic expansion of DFNB29. This is the first report of a patient with a tight junction variant receiving a cochlear implantation.

Identification of claudin-2, -6, -11 and -14 as prognostic markers in human breast carcinoma.

The development of cancer occurs with various genomic and epigenetic modifications that act as indicators for early diagnosis and treatment. Recent data have shown that the abnormal expression of the claudin (CLDN) tight junction (TJ) proteins is involved in the tumorigenesis of numerous human cancers. Real-time quantitative PCR and western blotting were used to explore the differences in the expression of the CLDN TJ proteins in breast carcinoma tissues and non-neoplastic tissues. The results showed that CLDN5, CLDN9, CLDN12 and CLDN13 were not expressed in breast carcinoma tissues or non-neoplastic tissues. CLDN1, CLDN3, CLDN8 and CLDN10 were expressed in breast carcinoma and non-neoplastic tissues, but there was no significant difference between the expression of these CLDN proteins among them. The expression of CLDN2, -6, -11 and -14 varied between the breast carcinoma and non-neoplastic tissues. Moreover, 86 samples of breast carcinoma and non-neoplastic tissues were examined for the expression of CLDN2, -6, -11 and -14 by streptavidin-peroxidase immunohistochemical staining. The data revealed that the CLDN2, CLDN6, and CLDN14 were expressed in the cell membrane and the expression levels of these proteins were downregulated in breast carcinoma. The CLDN11 was expressed in cell cytoplasm and the expression level of CLDN11 was upregulated compared with those in non-neoplastic tissues. Consistent with these findings, the expression of CLDN2, CLDN6 and CLDN14 were downregulated, while the expression of CLDN11 was upregulated in breast carcinoma compared with those in non-neoplastic tissues. Furthermore, the associations between these CLDNs and clinicopathologic indicators were analyzed, and these CLDN expressions were revealed to be associated with distant metastasis and to predict a poor prognosis. In conclusion, our data showed that the expression levels of CLDN2, -6, -11 and -14 differed between breast carcinoma tissues and histologically non-neoplastic tissues, and the expression levels of these CLDNs may be useful as molecular markers for the diagnosis of breast carcinoma as well as for the determination of metastasis and prognosis.

Calcium-sensing receptor: evidence and hypothesis for its role in nephrolithiasis.

Calcium-sensing receptor (CaSR) is a plasma-membrane G protein-coupled receptor activated by extracellular calcium and expressed in kidney tubular cells. It inhibits calcium reabsorption in the ascending limb and distal convoluted tubule when stimulated by the increase of serum calcium levels; therefore, these tubular segments are enabled by CaSR to play a substantial role in the regulation of serum calcium levels. In addition, CaSR increases water and proton excretion in the collecting duct and promotes phosphate reabsorption and citrate excretion in the proximal tubule. These CaSR activities form a network in which they are integrated to protect the kidney against the negative effects of high calcium concentrations and calcium precipitates in urine. Therefore, the CaSR gene has been considered as a candidate to explain calcium nephrolithiasis. Epidemiological studies observed that calcium nephrolithiasis was associated with polymorphisms of the CaSR gene regulatory region, rs6776158, located within the promoter-1, rs1501899 located in the intron 1, and rs7652589 in the 5'-untranslated region. These polymorphisms were found to reduce the transcriptional activity of promoter-1. Activating rs1042636 polymorphism located in exon 7 was associated with calcium nephrolithiasis and hypercalciuria. Genetic polymorphisms decreasing CaSR expression could predispose individuals to stones because they may impair CaSR protective effects against precipitation of calcium phosphate and oxalate. Activating polymorphisms rs1042636 could predispose to calcium stones by increasing calcium excretion. These findings suggest that CaSR may play a complex role in lithogenesis through different pathways having different relevance under different clinical conditions.

Claudin-14 Gene Polymorphisms and Urine Calcium Excretion.

BACKGROUND AND OBJECTIVES: Claudin-16 and -19 are proteins forming pores for the paracellular reabsorption of divalent cations in the ascending limb of Henle loop; conversely, claudin-14 decreases ion permeability of these pores. Single-nucleotide polymorphisms in gene coding for claudin-14 were associated with kidney stones and calcium excretion. This study aimed to explore the association of claudin-14, claudin-16, and claudin-19 single-nucleotide polymorphisms with calcium excretion. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We performed a retrospective observational study of 393 patients with hypertension who were naïve to antihypertensive drugs, in whom we measured 24-hour urine calcium excretion; history of kidney stones was ascertained by interview; 370 of these patients underwent an intravenous 0.9% sodium chloride infusion (2 L in 2 hours) to evaluate the response of calcium excretion in three different 2-hour urine samples collected before, during, and after saline infusion. Genotypes of claudin-14, claudin-16, and claudin-19 were obtained from data of a previous genome-wide association study in the same patients. RESULTS: Thirty-one single-nucleotide polymorphisms of the 3' region of the claudin-14 gene were significantly associated with 24-hour calcium excretion and calcium excretion after saline infusion. The most significant associated single-nucleotide polymorphism was rs219755 (24-hour calcium excretion in GG, 225±124 mg/24 hours; 24-hour calcium excretion in GA, 194±100 mg/24 hours; 24-hour calcium excretion in AA, 124±73 mg/24 hours; P<0.001; calcium excretion during saline infusion in GG, 30±21 mg/2 hours; calcium excretion during saline infusion in GA, 29±18 mg/2 hours; calcium excretion during saline infusion in AA, 17±11 mg/2 hours; P=0.03). No significant associations were found among claudin-16 and claudin-19 single-nucleotide polymorphisms and calcium excretion and between claudin-14, claudin-16, and claudin-19 single-nucleotide polymorphisms and stones. Bioinformatic analysis showed that one single-nucleotide polymorphism at claudin-14 among those associated with calcium excretion may potentially influence splicing of transcript. CONCLUSIONS: Claudin-14 genotype at the 3' region is associated with calcium excretion in 24-hour urine and after the calciuretic stimulus of saline infusion.

Measurements of ionic concentrations along with endocochlear potential in wild-type and claudin 14 knockout mice.

OBJECTIVE: To examine whether the changes in endolymphatic ion concentrations were involved in hair cells degeneration in claudin-14 knockout (KO) mice (Cldn14-/-), we measured the endocochlear potential (EP) along with concentrations of K+, Na+, H+, or Ca2+ ([K]e, [Na]e, pHe, [Ca]e) in Cldn14-/-, in which hair cells were selectively damaged, and compared with measurements in wild type mice (Wt). METHODS: We used the Cldn14-/- from 3 weeks of age, in which the auditory brain responses (ABR) was severely diminished. Using double-barreled ion-selective microelectrodes, we measured [K]e, [Na]e, pHe, and [Ca]e in both Wt and Cldn14-/- at 8-10 weeks of age. RESULTS: (1) In Wt, the EP was +92mV. [K]e, [Na]e, pHe, and [Ca]e were 169mM, ∼1.0mM, 7.50, and 395nM, respectively. In the Cldn14-/-, the EP was +96mV. [K]e, [Na]e, pHe, and [Ca]e were 167mM, ∼1.0mM, 7.73, and 179nM, respectively. No significant differences in the above values were observed between Wt and Cldn14-/-. (2) A significant linear correlation between EP and [Ca]e (R=0.93) was observed for both Wt and Cldn14-/-, but no correlation was observed between EP and K+, Na+, or H+. CONCLUSION: These findings suggest that (1) the changes in endolymphatic ion concentrations might not be involved in hair cells degeneration in Cldn14-/-, (2) [Ca]e might be regulated by EP in both Wt and Cldn14-/-.

Expressions of CaSR and Claudin-14 and the formation of nanobacterial renal stone: A dynamic study / 医学研究生学报

Objective Nanobacteria are one of the factors for urinary calculi and its exact pathogenic mechanism is not yet clear.The aim of this study was to investigate the role of the CaSR -Claudin-14 regulatory channel in the formation of calculi . Methods Sixty Wistar male rats were equally randomized into a normal control group and nanobacterial group , the former injected via the tail vein with 1.2 mL of 0.9％ sodium chloride solution while the latter with 1.2 mL of nanobacterial suspension , both for once.Three of the rats in each group were sacrificed every week in the first 10 weeks after injection.Histopathological examination was performed every week to evaluate the stone formation in the kidneys of the rats , and the expressions of the CaSR and Claudin -14 proteins were determined by immunohistochemistry. Results From the 1st to the 10th week after injection, crystal particles were observed in the rat kidneys of the nanobacterial group, but not in the normal controls (52.4％ vs 0％, P<0.01).The expressions of CaSR and Claudin -14 showed no statistically significant differences between the nanobacterial and control groups in the first 3 weeks (P>0.05) but both gradually in-creased in the former group from the 4th to the 10th week as compared with the latter, mainly in membrane of the renal tubular epithelial cells. Conclusion The increased activity of the CaSR -Claudin-14 regulatory channel may play an important role in the formation of nanobacterial renal stone .

Activity-dependent formation of a vesicular inhibitory amino acid transporter gradient in the superior olivary complex of NMRI mice.

BACKGROUND: In the mammalian superior olivary complex (SOC), synaptic inhibition contributes to the processing of binaural sound cues important for sound localization. Previous analyses demonstrated a tonotopic gradient for postsynaptic proteins mediating inhibitory neurotransmission in the lateral superior olive (LSO), a major nucleus of the SOC. To probe, whether a presynaptic molecular gradient exists as well, we investigated immunoreactivity against the vesicular inhibitory amino acid transporter (VIAAT) in the mouse auditory brainstem. RESULTS: Immunoreactivity against VIAAT revealed a gradient in the LSO and the superior paraolivary nucleus (SPN) of NMRI mice, with high expression in the lateral, low frequency processing limb and low expression in the medial, high frequency processing limb of both nuclei. This orientation is opposite to the previously reported gradient of glycine receptors in the LSO. Other nuclei of the SOC showed a uniform distribution of VIAAT-immunoreactivity. No gradient was observed for the glycine transporter GlyT2 and the neuronal protein NeuN. Formation of the VIAAT gradient was developmentally regulated and occurred around hearing-onset between postnatal days 8 and 16. Congenital deaf Claudin14 -/- mice bred on an NMRI background showed a uniform VIAAT-immunoreactivity in the LSO, whereas cochlear ablation in NMRI mice after hearing-onset did not affect the gradient. Additional analysis of C57Bl6/J, 129/SvJ and CBA/J mice revealed a strain-specific formation of the gradient. CONCLUSIONS: Our results identify an activity-regulated gradient of VIAAT in the SOC of NRMI mice. Its absence in other mouse strains adds a novel layer of strain-specific features in the auditory system, i.e. tonotopic organization of molecular gradients. This calls for caution when comparing data from different mouse strains frequently used in studies involving transgenic animals. The presence of strain-specific differences offers the possibility of genetic mapping to identify molecular factors involved in activity-dependent developmental processes in the auditory system. This would provide an important step forward concerning improved auditory rehabilitation in cases of congenital deafness.

A variant in a cis-regulatory element enhances claudin-14 expression and is associated with pediatric-onset hypercalciuria and kidney stones.

The greatest risk factor for kidney stones is hypercalciuria, the etiology of which is largely unknown. A recent genome-wide association study (GWAS) linked hypercalciuria and kidney stones to a claudin-14 (CLDN14) risk haplotype. However, the underlying molecular mechanism was not delineated. Recently, renal CLDN14 expression was found to increase in response to increased plasma calcium, thereby inducing calciuria. We hypothesized therefore that some children with hypercalciuria and kidney stones harbor a CLDN14 variant that inappropriately increases gene expression. To test this hypothesis, we sequenced the CLDN14 risk haplotype in a cohort of children with idiopathic hypercalciuria and kidney stones. An intronic SNP was more frequent in affected children. Dual luciferase and cell-based assays demonstrated increased reporter or CLDN14 expression when this polymorphism was introduced. In silico studies predicted the SNP introduced a novel insulinoma-associated 1 (INSM1) transcription factor binding site. Consistent with this, repeating the dual luciferase assay in the presence of INSM1 further increased reporter expression. Our data suggest that children with the INSM1 binding site within the CLDN14 risk haplotype have a higher likelihood of hypercalciuria and kidney stones. Enhanced CLDN14 expression may play a role in the pathophysiology of their hypercalciuria.