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Transient receptor potential vanilloid 5 (TRPV5) is a highly calcium selective ion channel that acts as the rate-limiting step of calcium reabsorption in the kidney. The lack of potent, specific modulators of TRPV5 has limited the ability to probe the contribution of TRPV5 in disease phenotypes such as hypercalcemia and nephrolithiasis. Here, we performed structure-based virtual screening (SBVS) at a previously identified TRPV5 inhibitor binding site coupled with electrophysiology screening and identified three novel inhibitors of TRPV5, one of which exhibits high affinity, and specificity for TRPV5 over other TRP channels, including its close homologue TRPV6. Cryo-electron microscopy of TRPV5 in the presence of the specific inhibitor and its parent compound revealed novel binding sites for this channel. Structural and functional analysis have allowed us to suggest a mechanism of action for the selective inhibition of TRPV5 and lay the groundwork for rational design of new classes of TRPV5 modulators.

The pathogenesis of osteoporosis involves multiple factors, among which alterations in the bone microenvironment play a crucial role in disrupting normal bone metabolic balance. Transient receptor potential vanilloid 5 (TRPV5), a member of the TRPV family, is an essential determinant of the bone microenvironment, acting at multiple levels to influence its properties. TRPV5 exerts a pivotal influence on bone through the regulation of calcium reabsorption and transportation while also responding to steroid hormones and agonists. Although the metabolic consequences of osteoporosis, such as loss of bone calcium, reduced mineralization capacity, and active osteoclasts, have received significant attention, this review focuses on the changes in the osteoporotic microenvironment and the specific effects of TRPV5 at various levels.

TRPV5 and TRPV6 are calcium-selective ion channels expressed at the apical membrane of epithelial cells. Important for systemic calcium (Ca2+) homeostasis, these channels are considered gatekeepers of this cation transcellular transport. Intracellular Ca2+ exerts a negative control over the activity of these channels by promoting inactivation. TRPV5 and TRPV6 inactivation has been divided into fast and slow phases based on their kinetics. While slow inactivation is common to both channels, fast inactivation is characteristic of TRPV6. It has been proposed that the fast phase depends on Ca2+ binding and that the slow phase depends on the binding of the Ca2+/Calmodulin complex to the internal gate of the channels. Here, by means of structural analyses, site-directed mutagenesis, electrophysiology, and molecular dynamic simulations, we identified a specific set of amino acids and interactions that determine the inactivation kinetics of mammalian TRPV5 and TRPV6 channels. We propose that the association between the intracellular helix-loop-helix (HLH) domain and the TRP domain helix (TDh) favors the faster inactivation kinetics observed in mammalian TRPV6 channels.

Kidney stone disease (KSD) represents an urgent medical problem because of increasing its prevalence. Several functional polymorphisms in genes involved in the renal handling of calcium were associated with KSD pathogenesis. Among those, the rs4236480 of transient receptor potential vanilloid member 5 (TRPV5) gene, the rs1801725 of calcium-sensing receptor (CASR) gene, and the rs1801197 of calcitonin receptor (CALCR) gene appear to be of great importance. Due to the scarce data on the Egyptians, this study aimed to evaluate the association of these candidate genetic variants with the risk of developing KSD in an Egyptian population. To do so, the biochemical parameters were measured along with the genotyping of the three polymorphisms using allelic discrimination assay in 134 KSD patients and 86 age and sex-matched healthy subjects. The results showed that the genotypic distributions and allelic frequencies of the studied variants were significantly different between cases and controls. The three polymorphisms increased the risk of KSD significantly under all the tested genetic models (OR ranges from 2.152 to 5.994), except for the recessive model of the CALCR rs1801197 polymorphism after Bonferroni correction. The gene–gene interaction analyzed by multifactor dimensionality reduction selected the three-locus combination as the best model associated with the susceptibility to KSD with OR 9.706. Further, synergistic interactions were identified between TRPV5 rs4236480 and CALCR rs1801197 variants and CASR rs1801725 and CALCR rs1801197 variants. In conclusion, the TRPV5 rs4236480, CASR rs1801725, and CALCR rs1801197 polymorphisms showed a significant association with the risk of KSD in the Egyptian population. Furthermore, their complex interactions might have an impact on the genetic susceptibility to develop KSD.

Background Low barometric pressure hypoxia at high altitudes triggers vascular remodeling, resulting in high-altitude pulmonary hypertension (HAPH). The key step is the transformation of pulmonary artery smooth muscle cells (PASMCs) from a contractile to synthetic phenotype. Protein kinases and phosphatases contribute to phenotype transformation by altering phosphorylated protein expression. Objectives In this study, we aimed to investigate the role of phosphohistidine phosphatase 1 (PHPT1) in PASMC transformation and its regulatory pathway in HAPH. Methods An HAPH model was constructed in wild-type, PHPT1 − / − , and PHPT1 + / + rats by placing them in a hypobaric chamber. Evaluations included hemodynamic measurements, echocardiography, histopathological analysis, and various cellular assays. RNA-seq and western blotting were used to identify intervention targets, and co-immunoprecipitation was used to determine the interaction between PHPT1 and TRPV5. Results PHPT1 protein expression was downregulated in HAPH, and its knockdown impaired cardiopulmonary functions, including elevated mean pulmonary artery pressure (mPAP), right ventricular systolic pressure (RVSP), and increased right ventricular thickness, and enhanced PASMC proliferation and migration. PHPT1 directly interacted with TRPV5 phosphorylation sites, whereas Asp30Ala/Arg157Ala functioned to prevent this interaction. PHPT1 overexpression protected against cardiopulmonary damage, reducing mPAP, RVSP, the D/W ratio, and MWT%. Additionally, PHPT1 overexpression mitigated PASMC proliferation and migration, resulting in restored TRPV5, p-Akt, p-SMAD2/3, and p-TGF-β expression under hypoxic conditions. Conclusions These findings underscore that PHPT1 inhibits PASMC proliferation and migration through TRPV5 signaling, thereby reducing mPAP and improving right ventricular function in HAPH. Therefore, PHPT1 targeting could potentially contribute to the development of novel therapeutic approaches for treating HAPH. Graphical abstract

B-cell activation triggers the release of endoplasmic reticulum calcium stores through the store-operated calcium entry (SOCE) pathway resulting in calcium influx by calcium release-activated calcium (CRAC) channels on the plasma membrane. B-cell-specific murine knockouts of SOCE do not impact humoral immunity suggesting that alternative channels may be important. We identified a member of the calcium-permeable transient receptor potential (TRP) ion channel family, TRPV5, as a candidate channel expressed in B cells by a quantitative polymerase chain reaction (qPCR) screen. To further investigate the role of TRPV5 in B-cell responses, we generated a murine TRPV5 knockout (KO) by CRISPR-Cas9. We found TRPV5 polarized to B-cell receptor (BCR) clusters upon stimulation in a PI3K-RhoA-dependent manner. TRPV5 KO mice have normal B-cell development and mature B-cell numbers. Surprisingly, calcium influx upon BCR stimulation in primary TRPV5 KO B cells was not impaired; however, differential expression of other calcium-regulating proteins, such as ORAI1, may contribute to a compensatory mechanism for calcium signaling in these cells. We demonstrate that TRPV5 KO B cells have impaired spreading and contraction in response to membrane-bound antigen. Consistent with this, TRPV5 KO B cells have reduced BCR signaling measured through phospho-tyrosine residues. Lastly, we also found that TRPV5 is important for early T-dependent antigen specific responses post-immunization. Thus, our findings identify a role for TRPV5 in BCR signaling and B-cell activation.

Wubi Shanyao Pills (WSP) is a traditional Chinese botanical formulation known for its gastrointestinal and renal benefits, yet its pharmacological effects on postmenopausal osteoporosis (PMOP) are not well elucidated. This study aimed to evaluate the therapeutic potential of WSP in a diet-induced PMOP model and to investigate its underlying mechanisms related to calcium absorption. A PMOP-like model was established in perimenopausal mice using a low-calcium, high-phosphorus diet. The mice were treated daily with WSP (0.375, 0.75, or 1.5 g/kg) or alendronate (ALN) (0.14 g/kg). After 17 weeks of treatment, bone microstructure was assessed via small animal CT, along with evaluation of systemic physiological parameters and hematological profiles. Histopathological examinations of the ileum, kidney, and femur were conducted using hematoxylin-eosin (H&E) staining, Alcian blue-periodic acid-Schiff (AB-PAS), and Masson staining. Serum calcium and phosphorus levels were measured by enzyme-linked immunosorbent assay (ELISA). The expression levels of calcium absorption-related proteins were analyzed using immunohistochemistry (IHC), immunofluorescence (IF), Western blotting, and quantitative real-time polymerase chain reaction (qRT-PCR). WSP exhibited notable pharmacological effects by improving bone mass/quality and serum calcium/phosphorus levels in diet-induced PMOP mice, mediated via upregulating key calcium transport proteins: transient receptor potential vanilloid 5 (TRPV5) and calcium-binding protein (CABP) in the kidney, transient receptor potential vanilloid 6 (TRPV6) and CABP in the ileum, and vitamin D receptor (VDR) in the femur; moreover, WSP reversed PMOP-associated anemia and facilitated tissue structural repair in the kidney, ileum, and femur. WSP modulates diet-induced PMOP pathology by promoting calcium absorption via the restoration of organ integrity and regulation of the TRPV5/TRPV6-CABP and VDR-mediated calcium metabolism pathways, thereby underlying its pharmacological effects.

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC)was previously considered to be a paracellular channelopathy caused by mutations in the claudin-16 and claudin-19 genes. Here, we provide evidence that a missense FHHNC mutation c.908C>G (p.T303R) in the claudin-16 gene interferes with the phosphorylation in the claudin-16 protein. The claudin-16 protein carrying phosphorylation at residue T303 is localized in the distal convoluted tubule (DCT) but not in the thick ascending limb (TAL) of the mouse kidney. The phosphomimetic claudin-16 protein carrying the T303E mutation but not the wildtype claudin-16 or the T303R mutant protein increases the Trpv5 channel conductance and membrane abundance in human kidney cells. Phosphorylated claudin-16 and Trpv5 are colocalized in the luminal membrane of the mouse DCT tubule; phosphomimetic claudin-16 and Trpv5 interact in the yeast and mammalian cell membranes. Knockdown of claudin-16 gene expression in transgenic mouse kidney delocalizes Trpv5 from the luminal membrane in the DCT. Unlike wildtype claudin-16, phosphomimetic claudin-16 is delocalized from the tight junction but relocated to the apical membrane in renal epithelial cells because of diminished binding affinity to ZO-1. High-Ca2+ diet reduces the phosphorylation of claudin-16 protein at T303 in the DCT of mouse kidney via the PTH signaling cascade. Knockout of the PTH receptor, PTH1R, from the mouse kidney abrogates the claudin-16 phosphorylation at T303. Together, these results suggest a pathogenic mechanism for FHHNC involving transcellular Ca2+ pathway in the DCT and identify a molecular component in renal Ca2+ homeostasis under direct regulation of PTH.

The increasing of osteoclasts formation and activity because of oestrogen (E2) deficiency is very important in the aetiology of postmenopausal osteoporosis. Our previous studies showed that E2 inhibited osteoclastic bone resorption by increasing the expression of Transient Receptor Potential Vanilloid 5 (TRPV5) channel. However, the exact mechanism by which E2 increases TRPV5 expression is not fully elucidated. In this study, Western blot, quantitative real‐time PCR, tartrate‐resistant acid phosphatase staining, F‐actin ring staining, chromatin immunoprecipitation and luciferase assay were applied to explore the mechanisms that E2‐induced TRPV5 expression contributes to the inhibition of osteoclastogenesis. The results showed that silencing or overexpressing of TRPV5 significantly affected osteoclasts differentiation and activity. Silencing of TRPV5 obviously alleviated E2‐inhibited osteoclastogenesis, resulting in increasing of bone resorption. E2 stimulated mature osteoclasts apoptosis by increasing TRPV5 expression. Further studies showed that E2 increased TRPV5 expression through the interaction of the oestrogen receptor α (ERα) with NF‐κB, which could directly bind to the fragment of −286 nt ~ −277 nt in the promoter region of trpv5. Taken together, we conclude that TRPV5 plays a dominant effect in E2‐mediated osteoclasts formation, bone resorption activity and osteoclasts apoptosis. Furthermore, NF‐κB plays an important role in the transcriptional activation of E2‐ERα stimulated TRPV5 expression.

An exceptionally low calcium (Ca2+) concentration in the inner ear endolymph ([Ca2+]endolymph) is crucial for proper auditory and vestibular function. The endolymphatic sac (ES) is believed to critically contribute to the maintenance of this low [Ca2+]endolymph. Here, we investigated the immunohistochemical localization of proteins that are presumably involved in the sensing and transport of extracellular Ca2+ in the murine ES epithelium. Light microscopic and fluorescence immunolabeling in paraffin-embedded murine ES tissue sections (male C57BL/6 mice, 6–8 weeks old) demonstrated the presence of the calcium-sensing receptor CaSR, transient receptor potential cation channel subtypes TRPV5 and TRPV6, sarco/endoplasmic reticulum Ca2+-ATPases SERCA1 and SERCA2, Na+/Ca2+ exchanger NCX2, and plasma membrane Ca2+ ATPases PMCA1 and PMCA4 in ES epithelial cells. These proteins exhibited (i) membranous (apical or basolateral) or cytoplasmic localization patterns, (ii) a proximal-to-distal labeling gradient within the ES, and (iii) different distribution patterns among ES epithelial cell types (mitochondria-rich cells (MRCs) and ribosome-rich cells (RRCs)). Notably, in the inner ear membranous labyrinth, CaSR was exclusively localized in MRCs, suggesting a unique role of the ES epithelium in CaSR-mediated sensing and control of [Ca2+]endolymph. Structural loss of the distal ES, which is consistently observed in Meniere’s disease, may therefore critically disturb [Ca2+]endolymph and contribute to the pathogenesis of Meniere’s disease.