Gentamicin Inhibits Ca2+ Channel TPRV5 and Induces Calciuresis Independent of the Calcium-Sensing Receptor-Claudin-14 Pathway

Background Treatment with the aminoglycoside antibiotic gentamicin can be associated with severe adverse effects, including renal calcium wasting. The underlying mechanism is unknown but it has been proposed to involve activation of the Ca2+-sensing receptor (CaSR) in the thick ascending limb, which would increase expression of claudin-14 (CLDN14) and limit Ca2+ reabsorption. However, no direct evidence for this hypothesis has been presented. Methods We studied the effect of gentamicin in vivo using mouse models with impaired Ca2+ reabsorption in the proximal tubule and the thick ascending limb. We used a Cldn14 promoter luciferase-reporter assay to study CaSR activation and investigated the effect of gentamicin on activity of the distal nephron Ca2+ channel transient potential receptor vanilloid 5 (TPRV5), as determined by patch-clamp in HEK293 cells. Results Gentamicin increased urinary Ca2+ excretion in wild-type mice following acute and chronic administration. This calciuretic effect was unaltered in mice with genetic CaSR overactivation and was present in furosemide-treated animals, whereas the calciuretic effect in Cldn14-/-mice and mice with impaired proximal tubular Ca2+ reabsorption (claudin-2 [CLDN2]-deficient Cldn2-/- mice) was equivalent to that of wild-type mice. In vitro, gentamicin failed to activate the CaSR. In contrast, patch-clamp analysis revealed that gentamicin strongly inhibited rabbit and human TRPV5 activity and that chronic gentamicin administration downregulated distal nephron Ca2+ transporters. Conclusions Gentamicin does not cause hypercalciuria via activation of the CaSR-CLDN14 pathway or by interfering with proximal tubular CLDN2-dependent Ca2+ reabsorption. Instead, gentamicin blocks distal Ca2+ reabsorption by direct inhibition of the Ca2+ channel TRPV5. These findings offer new insights into calcium wasting in patients treated with gentamicin.

Preliminary Evaluation of Nanobacteria on Crystal Retention, CaSR, and Claudin-14 Expression in HK-2 Cells

To evaluate cell morphology, crystal adhesion, cell damage, Calcium sensitive receptor (CaSR), and Claudin protein-14 (Claudin-14) expression at different time intervals and explore the role of nanobacteria in the formation of urinary calculi. In this experiment, HK-2 cells were cocultured with nanobacteria (NB) in the absence or presence of tetracycline (Tet). Cells treated with calcium oxalate monohydrate (COM) crystals were used as a positive control of urinary stone-induced cell damage. After which, cell morphology was evaluated by hematoxylin-eosin staining in comparison to untreated HK-2 cells (negative control). Use different methods to assess cell damage, crystal adhesion, and protein expression. (The degree of cell damage, crystal adhesion, and protein expression were evaluated by various methods). It was found that the degree of cell damage observed in Tet + NB-treated cells was significantly lower than that in NB-treated cells. Lactate dehydrogenase (LDH) leakage was higher in COM-exposed than in control cells ( P < 0.05 ). However, LDH release from both NB- and Tet + NB-treated cells was significantly lower than from COM-treated cells ( P < 0.05 ). The relative expression of CaSR and Claudin-14 proteins was higher in NB, COM, and TET + NB cells than in control cells ( P < 0.001 ) and was lower in Tet + NB than in NB cells ( P < 0.01 ). And P < 0.05 means that the difference was statistically significant, and P < 0.001 means that there was a significant difference between the both things. From the cell morphology, the cell damage in the COM group was greater than that in the NB group, and the cell damage markers in the COM group and the NB group were elevated. NB caused damage to HK-2 cells by inducing lipid peroxidation, and the degree of damage was increased in processing time. The adhesion of HK-2 cells to COM crystals increased after injury and was proportional to the duration of NB coculture. NB upregulated the expression of CaSR and Claudin-14 in HK-2 cells.

Renal claudin-14 expression is not required for regulating magnesium balance in mice

The kidneys play a crucial role in maintaining calcium (Ca2+) and magnesium (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 the pore-blocking claudin-14 (CLDN14) that reduces Ca2+ and Mg2+ reabsorption from this segment. Additionally, a high Mg2+ diet is known to increase both urinary Mg2+ and Ca2+ excretion. Since Mg2+ may also activate the 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 nor 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.

Claudin-12 Knockout Mice Demonstrate Reduced Proximal Tubule Calcium Permeability

The renal proximal tubule (PT) is responsible for the reabsorption of approximately 65% of filtered calcium, primarily via a paracellular pathway. However, which protein(s) contribute this paracellular calcium pore is not known. The claudin family of tight junction proteins confers permeability properties to an epithelium. Claudin-12 is expressed in the kidney and when overexpressed in cell culture contributes paracellular calcium permeability (PCa). We therefore examined claudin-12 renal localization and its contribution to tubular paracellular calcium permeability. Claudin-12 null mice (KO) were generated by replacing the single coding exon with β-galactosidase from Escherichia coli. X-gal staining revealed that claudin-12 promoter activity colocalized with aquaporin-1, consistent with the expression in the PT. PTs were microperfused ex vivo and PCa was measured. PCa in PTs from KO mice was significantly reduced compared with WT mice. However, urinary calcium excretion was not different between genotypes, including those on different calcium containing diets. To assess downstream compensation, we examined renal mRNA expression. Claudin-14 expression, a blocker of PCa in the thick ascending limb (TAL), was reduced in the kidney of KO animals. Thus, claudin-12 is expressed in the PT, where it confers paracellular calcium permeability. In the absence of claudin-12, reduced claudin-14 expression in the TAL may compensate for reduced PT calcium reabsorption.

Comparative study of a nano-bacterial rat kidney stone model and the traditional ethylene glycol rat kidney stone model

Objective To Compare a nanobacterial (NB) rat kidney stone model and the traditional ethylene glycol (EG) rat kidney stone model to assess its significance. Methods Ninety Wistar male rats were randomly divided into three groups. After the first week of modeling, three rats of every groups were randomly selected to measure the biochemical blood and urine markers. After the sacrifice, the renal tissues were observed to assess the pathological changes. The expression levels of CaSR and Claudin-14 protein were detected by kinds of technology. Results The biochemical metabolic indices of rats in NB group began to increase at the third week and decreased to normal at the 9th week. In the EG group they began to increase at the second week and did not return to normal later. At the 7th week, the creatinine levels of rats in the EG group were higher than in the NB group, and the difference was statistically significant. The formation rates of kidney stones in the NB group and EG group were 52.4 and 66.7%, respectively, but the difference of the two rates was not statistically significant. The protein expression CaSR and Claudin-14 in the EG group began to strengthen at 3rd week, and at 4th week in the NB group. CaSR was continuously expressed in the NC group, but Claudin-14 was not expressed. Conclusion The formation of stones in the NB group began slightly later. CaSR and Claudin-14 proteins play a role in the formation of kidney stones.

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.