Alterations in the microenvironment and the effects produced of TRPV5 in osteoporosis

Eerden et al. reported that TRPV5 plays a significant role in bone resorption and morphological changes [18]. Utilizing gene knockout techniques, they created TRPV5^−/− mice and discovered that these mice exhibited disordered bone resorption and decreased femur thickness compared to wild-type TRPV5^+/+ mice [18]. The absence of TRPV5 led to abnormal osteoclast activity and severe bone resorption disorder in TRPV5^−/− mice [115]. Although the number of osteoclasts increased and bone thickness decreased, the mice did not exhibit the phenotype of osteosclerosis, potentially due to partial compensation by the TRPV6 channel [115, 116]. TRPV5 deficiency also impacted bone resorption homeostasis, calcium deposition, and related bone metabolic factors. In male TRPV5^−/− mice, the number and volume of trabeculae decreased, the connectivity rate was suboptimal, and the thickness and volume of cortical bone diminished, with a significantly higher internal porosity compared to TRPV5^+/+ mice [117]. Quantitative backscattered electron (QBEI) measurements of the tibia revealed that CaMean, CaPeak, and CaWidth were positively correlated with age, while CaLow was negatively correlated, and CaMean and CaPeak were lower than in TRPV5^+/+ mice in young and middle-aged groups [117]. Urinary deoxypyridinoline (DPD) observations indicated that the DPD content of TRPV5^−/− mice was related to and proportional to age, and the levels of young and middle-aged TRPV5^−/− mice were significantly lower than those of the TRPV5^+/+ group, suggesting an abnormal bone resorption rate [117]. The lack of TRPV5 accelerated bone aging and disrupted bone resorption. Due to decreased abnormal mineralization of bone calcium function, the cortical bone lumen volume increased, potentially as a result of compensatory mineralization effects [118].

TRPV5 is expressed in various cells of bone tissue, including the bone matrix, osteoclasts, and chondrocytes [12]. Osteoclasts release growth factors through bone resorption, which act on chondrocytes to regulate their metabolism and degeneration, as illustrated in Fig. 3 [119]. Several factors can influence the activity of osteoclasts through TRPV5. For instance, 1,25(OH)₂D₃, a conventional drug for osteoporosis treatment, can increase Ca²⁺ absorption but also inhibit TRPV5 expression in osteoclasts, leading to the inhibition of early osteoclast differentiation, activity, and bone loss [120]. TRPV5 is also essential for calcium transport in the resorption cavity of osteoclasts. Inhibition of TRPV5 expression by econazole in osteoclasts affected the transport of Ca²⁺ and inhibited bone resorption [121]. In vitro culture of TRPV5^−/− mouse osteoclasts showed that the number of osteoclasts was approximately twice that of wild-type (WT) mice, and the number and size of osteoclast nuclei were significantly higher in TRPV5^−/− mice [18]. However, the absorptive capacity of osteoclasts was severely impaired. The absence of TRPV5 can increase the number and volume of osteoclasts, but this does not proportionally correspond to the osteoclast effect [18]. Fangjing Chen et al. found that estradiol (E₂) can increase TRPV5 expression in osteoclasts to inhibit osteoclast differentiation [122]. E₂ may induce continuous Ca²⁺ oscillations by increasing TRPV5 expression. E₂ exerts its biological activity mainly by binding with the estrogen receptor ER. Tianwen Ye et al. demonstrated that E₂ can promote TRPV5 expression and stimulate osteoclast apoptosis by interacting with NF-κB when binding with ER [123]. NF-κB can directly bind to the promoter region of the − 286 nt  to − 277 nt fragment of TRPV5 and promote TRPV5 transcription [123].

Autophagy in chondrocytes is a self-protective mechanism and metabolic mode of cartilage tissue. TRPV5 is expressed in chondrocytes and upregulated during the progression of osteoarthritis. Ismail M. Hdud et al. used immunohistochemistry to identify TRPV5 distribution primarily in the superficial and middle chondrocytes [124]. In a monosodium iodoacetate (MIA) rat model of osteoarthritis, Lunhao Bai et al. observed a positive correlation between the expression of Ca²⁺-dependent proteins, calmodulin, and TRPV5 in chondrocytes of osteoarthritis rats, substantiating the hypothesis that TRPV5 is a calmodulin-dependent channel [113]. The primary mechanism entails the upregulation of TRPV5 expression, which facilitates Ca²⁺ influx, resulting in calcium overload and subsequent activation of calmodulin and CaMKII proteins. CaMKII proteins phosphorylate Beclin1, inhibiting autophagosome formation and consequently suppressing chondrocyte autophagy, which is also stimulated by associated inflammatory factors [113]. Moreover, a subsequent investigation revealed that TRPV5 expression influences chondrocyte apoptosis [125]. The upregulation of TRPV5 promoted chondrocyte apoptosis by augmenting the expression of several apoptosis-related proteins, including calmodulin, DAP, Cleaved caspase-3, Cleaved caspase-6, Cleaved caspase-7, and Cleaved caspase-8 [125].

The presence of TRPV5 in osteoblasts is a matter of ongoing debate among researchers. Bram C. J. van der Eerden and Lieben L believe that TRPV5 is not expressed in osteoblasts, thus supporting the notion of absent of TRPV5 expression [15, 18]. In contrast, Li et al. argue that the complete absence of TRPV5 in osteoblasts cannot be unequivocally confirmed, as although direct detection of TRPV5 was not achieved, they observed downstream calcium-binding proteins and mRNA of calcium transporters. This finding implies that the expression of TRPV5 in osteoblasts may necessitate specific activation conditions [126].

The relationship between in vivo pH and osteoporosis has been a topic of interest among researchers for decades. In 1968, Pellegrino et al. suggested that inorganic salts in the bone system could function as a buffer matrix, and osteoporosis was thought to result from the absolute loss of bone minerals, which played a buffering role in absorbing acidic substances when the bone was “dissolved” [127]. However, pathological states such as uremia, renal acidosis, chronic obstructive pulmonary disease (COPD), and lung transplantation can exert complex effects on the skeletal system. In COPD patients, decreased respiratory function often leads to an elevation in inflammatory factors, which can alter the signaling pathway of bone tissue cells, further enhancing osteoclasts activity and disrupting the balance of bone metabolism, culminating in osteoporosis. Pulmonary dysfunction in COPD patients is frequently accompanied by chronic respiratory acidosis, which increases the amount of H₂CO₃ in the body and elevates the H⁺ concentration, causing a decline in tissue pH and resulting in an acidic microenvironment. In untreated or severe COPD patients, hypercapnia and hypoxia can significantly impact bone metabolism, making patients more susceptible to developing osteoporosis [128, 129].Previous investigations have demonstrated that the acidic microenvironment can induce an increase in osteoclast activity, trigger changes in downstream cell signalling pathways, inhibit osteoblast proliferation, and reduce osteoblast mineralization [130].Similarly, in patients with chronic renal insufficiency, there is a decrease in renal metabolic function, resulting in a decline in plasma HCO₃⁻ and tissue pH, leading to acidosis. Consequently, phosphate and carbonate in bone tissue are lost as buffer substances, and renal tubular epithelial cells exhibit reduced calcium reabsorption and increased urinary calcium excretion, which can contribute to osteoporosis [131]. A study revealed that elevated urinary levels of Ca²⁺ and Mg²⁺ in mice with metabolic acidosis led to a decrease in the gene and protein abundance of TRPV5 and Calbindin-D_28K in the kidney [132]. However, effective inhibition of active Ca²⁺ reabsorption was observed in TRPV5^−/− mice, indicating that metabolic acidosis did not affect urinary calcium excretion in mice. Additionally, Mg²⁺ exerted an inhibitory effect on TRPV5 activity [105]. Thus, it can be inferred that metabolic acidosis plays a critical role in urinary calcium excretion by mediating the expression abundance of TRPV5.

In recent years, numerous pH-responsive drugs have been developed to target local pH changes in osteoporotic tissues. Dou et al. reported the development of cerium bioactive nanoparticles with pH response, which can target the acidic extracellular microenvironment and inhibit the activity of mature osteoclasts (mOCs). During bone remodeling, the pH value of the bone resorption cavity can reach 3–4, with mOCs triggering the extracellular acidic microenvironment through ATPase H⁺ Transporting V0 Subunit D2 (ATP6v0d2) [133]. Yi Hu et al. argue that in the acidic microenvironment of OP, measures to neutralize acidity are unable to effectively inhibit osteoclasts [134]. Mature osteoclasts can secrete large amounts of H⁺ into the bone resorption area. Based on the unique acidic microenvironment surrounding osteoclasts, the team designed an osteoclast microenvironment-responsive nanoplatform, HA-MC/CaCO₃/ZOL@PBAE-SA (HMCZP). PBAE, a pH-responsive polymer, can release ZOL in the acidic microenvironment of OP, thereby inhibiting osteoclast activity. Thus, several studied have confirmed the extracellular acidic microenvironment caused by osteoporosis. This acidic microenvironment simultaneously inhibits the expression of TRPV5 ion channels. The exact mechanism by which TRPV5 is affected by the acid–base environment is not entirely clear, but studies have reported the involvement of an acid-sensitive receptor on the basal side of cells in the proximal tubule of the kidney (PT) [9]. When the pH decreases, basal acid-sensitive receptor GPCRs are triggered, leading to phosphorylation of the intracellular pH sensor Pyk2 and activation of the apical Na⁺–H⁺ exchanger type 3 in PT [9]. Yeh et al. found that two extracellular loops in the fifth and sixth transmembrane domains of TRPV5 may form an open structure that plays a major role in the binding and regulation of external H⁺ [73]. Additionally, the mutation from glutamic acid 522 to glutamine E522Q in TRPV5 can reduce the sensitivity of cells to external acidification and alter channel activity [73]. Therefore, it is hypothesized that glutamic acid 522 may act as a pH sensor, and external H⁺ influences the biological activity of TRPV5 by altering the conformation of TRPV5 [73]. Moreover, vesicular transport has been reported, which is termed “kiss and linger” [135]. When the extracellular environment is continuously alkalized, vesicles containing TRPV5 are rapidly recruited to the surface of the cell membrane without collapsing into the plasma membrane. The activity of these vesicles containing functional TRPV5 increases with increasing pH [135]. Conversely, when the extracellular pH value continuously decreases, vesicles containing TRPV5 are withdrawn from the plasma membrane and reach the extracellular space through the transient opening of vesicles, inhibiting the biological activity of TRPV5 [135]. Furthermore, other pathways for the inhibition of TRPV5 activity under acidic conditions have been reported. Edwin C. Fluck et al. [110] found that the activation of PI (4,5) P₂ was prevented under low pH conditions to suppress TRPV5 activity, revealing a synergistic effect of pH and lipid co-factor in gating the channel. The transition of TRPV5 channel from open to closed conformation under low pH environment was captured using cryo-electron microscopy. These structural and molecular insights provide new understanding of the interplay between pH and TRPV5 gating.

The multifaceted mechanisms through which H⁺ modulates TRPV5 ion channel activity in bone cells in response to alterations in extracellular pH may offer promising opportunities for the innovation of drug delivery approaches and the identification of novel therapeutic targets for osteoporosis treatments.

Calcium absorption in the body relies on the distal tubules of the small intestine and kidney. TRPV5 serves as the primary Ca²⁺ gated channel, and its functionality is contingent on several proteins, including CaR, CaM, 80 k-H, S100A10-annexin II, and calbindin 28 K [11]. CaR is an extracellular Ca²⁺ receptor that exhibits high sensitivity to plasma Ca²⁺ concentrations. Catalin N. Topala et al. discovered that CaR is predominantly located in the distal convoluted tubules (DCT) and connecting tubules (CNT) of the kidney [136]. Cell transfection experiments using HEK293 cells revealed that co-expression of CaR and TRPV5 notably increased intracellular Ca²⁺ concentrations. Patch clamp techniques demonstrated that CaR could stimulate the enhancement of TRPV5 activity (Fig. 3) [136]. 80 k-H is a substrate of PCK. The interaction between 80 k-H and TRPV5 can inhibit the Ca²⁺ influx, increase the TRPV5 sensitivity to Ca²⁺, and expedite channel feedback inhibition [11]. S100A10-annexin II is an essential protein in plasma membrane transport and insertion, exerting its influence through TRPV5 binding [11]. One study indicated that the membrane protein PIRT (phosphoinositide-interacting regulator of TRP) contains a cholesterol-recognition amino acid consensus (CRAC), which can play a significant role in the mechanical regulation of TRP ion channels by binding with cholecalciferol and oxytocin [137]. Although the direct regulations of TRPV5 and TRPV6 remains unclear, it is hypothesized that PIRT regulates TRPV5 through CaM binding [137]. Recently, new functional interactions between TRPV5 and CaM have been reported, characterizing dynamic lobe-specific CaM regulation and persistent interaction with apo-CaM. These two novel interactions play critical roles in rapid inhibition and conformational modulation of the TRPV5 channel, offering new insights into Ca²⁺ transport in the kidney [138].

Protein post-translational modification (PTM) is a crucial mechanism that enables proteins to function [139]. Covalent reactions occur during or after translation, and various chemical groups can be covalently linked with proteins or amino acids, such as methyl, acetyl, phosphate group, sugar chain, and ubiquitin groups. This process allows for the expression, activity, and function of proteins [140, 141]. In TRPV5, the most common modifications include phosphorylation, glycosylation, ubiquitination, and others [142].

In summary, the protein modification processes discussed above involve the coordinated interaction of numerous enzymes with protein molecules. Several protein factors, directly or indirectly related to osteoporosis, exert their effects on TRPV5. For a summary of these factors, please refer to Table 2.

Regarding agonists, the β-adrenergic receptor (β-AR) comprises β1-AR, β2-AR, and β3-AR, with β1-AR and β2-AR primarily distributed in the renal DCT2/CNT [153]. The β1-AR agonist, dobutamine, upregulates the expression of cAMP in HEK293 cells, stimulates TRPV5 in Ca²⁺ uptake, and enhances the activity of the TRPV5 channel by phosphorylating T709 residues via the PKA pathway [154]. Streptozotocin-induced diabetes significantly increases TRPV5 mRNA expression in rats, and renal immunofluorescence sections demonstrate a significant increase in TRPV5 expression [155]. Diabetic patients exhibit increased calcium and magnesium levels in their urine, which is accompanied by an increase in the expression of TRPV5 and calcium-binding protein. Insulin treatment can reverse these trends [155]. Claudin-16 (CLDN16) plays a crucial role in Ca²⁺ and Mg²⁺ transport in the renal paracellular epithelium [156]. CLDN16-deficient mice display hypercalciuria and hypomagnesemia, similar to humans, as the ion compensation pathway emerges. The response of CLDN16-deficient mice is similar to that of humans. Mice exhibit hypercalciuria and hypomagnesemia [156]. Related hormones, such as PTH and 1,25(OH)₂D₃, and some Ca²⁺ and Mg²⁺ transport channels, including TRPV5, TRPM6, and calbindin-D9k, are significantly upregulated [156]. Glucocorticoids (GCs), such as dexamethasone (Dex) and dexmedetomidine, are widely used clinical anti-inflammatory and anti-allergic drugs that can cause side effects like osteoporosis due to abnormal bone metabolism [157]. Dex administration in mice induces the expression of TRPV5 transcripts in the kidney and TRPV6 transcripts in the duodenum within 24 h. GCs may regulate the transcription of TRPV5 and TRPV6 in an organ-specific and time-dependent manner [157].

Econazole is a small molecule drug commonly used to treat skin antifungal infections and has been shown to inhibit TRPV5/6 in some studies [158, 159]. In osteoclasts, econazole inhibits the expression of TRPV5 in a dose-dependent manner, but has no effect on the activity of osteoclasts while inhibiting bone resorption in rats [121]. Freezing electron microscopy observations revealed that the binding conformation of TRPV5 and econazole results in the movement of S1-S4 and S4-S5 away from the hole axis, accompanied by the conformational change of the S6 helix, leading to the shrinkage of the lower gate and closure of the channel [159, 160]. Interestingly, the conformational changes induced by econazole do not significantly affect the outer pore region of the TRPV5 channel [159]. The concentration range of econazole, TH-1177, and other inhibitors is only in the micromolar range [161].

To achieve higher specificity, researchers continuously screen new inhibitors. ZINC9155420 and ZINC17988990 bind to TRPV5 in a nonconductive conformation along the ionic conduction pathway. The binding sites of these inhibitors do not overlap with activators, indicating that their effects do not result from competitive binding. Instead, these inhibitors lock the channels and prevent agonists from reaching the activated state [161]. Two new inhibitor binding sites have been identified in the TRPV5 structure. The inhibitory binding site of ZINC9155420 is located between lipid interfaces, a monomer S4-S5 linker, and the adjacent monomer S6 helix. The other inhibitory site mediated by ZINC17988990 occupies half of S1-S4 bundle cells, and its TRPV5 inhibitory specificity exceeds that of other reported compounds [161]. Oxoglaucine, a potential TRPV5 inhibitor, mainly blocks the calmodulin transport pathway of TRPV5 and inhibits Ca²⁺ influx, thereby inhibiting TRPV5 activity and activating chondrocyte autophagy [162]. In patients with nephrotic syndrome, calcareous deposition likely results from damage to the TRPV5 channel of DCT cells. Protease-activated receptor-1 (PAR-1) was purified from the urine of these patients by Kukiat Tudpor et al. [163]. PAR-1 can inhibit the uptake of Ca²⁺ by HEK293 cells. Plasmin-activated PAR-1-induced PKC-mediated phosphorylation of TRPV5 was found to affect the binding of calmodulin to TRPV5 and decrease the activity of the TRPV5 channel. It is speculated that urinary plasmin is an inhibitor of TRPV5 [163].