Journal: Pharmaceuticals

Article Title: Urothelial Calcium-Sensing Receptor Modulates Micturition Function via Mediating Detrusor Activity and Ameliorates Bladder Hyperactivity in Rats

doi: 10.3390/ph14100960

Figure Lengend Snippet: The expression and location of calcium-sensing receptor (CaSR) in bladder. ( a ) Western blot analysis of CaSR (MW: 130 kDa) in the whole kidney and whole urinary bladder. ( b ) Immunohistochemistry staining of CaSR (brown signals) in the urothelium (U) and detrusor (D) of bladder, and in the proximal tubule (PT) of kidney, and the staining without applying primary antibody (Ab). ( c ) Immunofluorescence staining of CaSR (red), uroplakin III A (UPK3A) (green), and DAPI (blue) in urothelium of urinary bladder.

Article Snippet: Monoclonal CaSR antibody (1:100; NB100-1830; Novus Biologicals, Littleton, CO, USA) was used as the primary antibody.

Techniques: Expressing, Western Blot, Immunohistochemistry, Staining, Immunofluorescence

Journal: Pharmaceuticals

Article Title: Urothelial Calcium-Sensing Receptor Modulates Micturition Function via Mediating Detrusor Activity and Ameliorates Bladder Hyperactivity in Rats

doi: 10.3390/ph14100960

Figure Lengend Snippet: The effects of CaSR agonist (AC-265347, AC) and CaSR antagonist (NPS-2143 hydrochloride, NPS) on myogenic spontaneous activity in vitro . The recording of intact bladder strips myogenic spontaneous activity added with buffer ( a ), AC (from 1 × 10 −7 to 1 × 10 −4 M) ( b ), and NPS (from 1 × 10 −7 to 1 × 10 −4 M) ( c ). The recording of urothelium removal (UR) bladder strips added with buffer ( d ), AC (from 1 × 10 −7 to 1 × 10 −4 M) ( e ), and NPS (from 1 × 10 −7 to 1 × 10 −4 M) ( f ). The statistical analysis of the basal tension ( g ), amplitude of spontaneous contractions (SCs) ( h ), and spikes calculated from SCs ( i ) in intact bladder strips, and the basal tension ( j ), amplitude of spontaneous contractions (SCs) ( k ), and spikes calculated from SCs ( l ) in urothelium removal bladder strips that were affected by AC and NPS (Two-way ANOVA with Sidak’s post hoc tests, n = 6 for each group; ** p < 0.01, *** p < 0.001, as compared with the normal status of each strip) (N, normal status).

Article Snippet: Monoclonal CaSR antibody (1:100; NB100-1830; Novus Biologicals, Littleton, CO, USA) was used as the primary antibody.

Techniques: Activity Assay, In Vitro, Stripping Membranes

Journal: Pharmaceuticals

Article Title: Urothelial Calcium-Sensing Receptor Modulates Micturition Function via Mediating Detrusor Activity and Ameliorates Bladder Hyperactivity in Rats

doi: 10.3390/ph14100960

Figure Lengend Snippet: The effects of CaSR agonist (AC) and CaSR antagonist (NPS) on bladder strips contraction. The recording of contraction in response to the first and the second time of acetylcholine stimulations (sti) (from 1 × 10 −7 to 1 × 10 −3 M) in each group, which were added with different treatments before the second time of stimulations. ( a ) Contraction responses before and after added with buffer (control) (strip 1, intact strips) or 0.1 mM AC (strip 2, intact strips) or 0.1 mM AC (strip 3, urothelium removal strips). ( d ) Contraction responses before and after added with buffer (control) (strip 1, intact strips) or 0.1 mM NPS (strip 2, intact strips) or 0.1 mM NPS (strip 3, urothelium removal strips). The statistical analysis of contraction curves affected by AC ( b , c ) and NPS ( e , f ) in intact and urothelium removal strips (Two-way ANOVA with Sidak’s post hoc tests, n = 6 for each group; * p < 0.05, ** p < 0.01, *** p < 0.001, as compared with the control group).

Article Snippet: Monoclonal CaSR antibody (1:100; NB100-1830; Novus Biologicals, Littleton, CO, USA) was used as the primary antibody.

Techniques: Control, Stripping Membranes

Journal: Pharmaceuticals

Article Title: Urothelial Calcium-Sensing Receptor Modulates Micturition Function via Mediating Detrusor Activity and Ameliorates Bladder Hyperactivity in Rats

doi: 10.3390/ph14100960

Figure Lengend Snippet: The effects of different dosages of CaSR agonist (AC) on micturition. ( a ) Illustration of micturition function parameters that were measured in this study. Effects of intravesical administrations of 10 µM AC ( b ) ( n = 3), 100 µM AC ( c ) ( n = 3), and 1 mM AC ( d ) ( n = 6) on micturition with the recording of intravesical pressure (IVP) and blood pressure (BP). The statistical analysis of micturition function parameters before (saline) and after different treatments, including inter-contraction-intervals (ICI) ( e ), phase 1 IVP ( f ), amplitude of phase 2 oscillation ( g ), baseline pressure ( h ), voiding duration ( i ), amplitude of non-micturition contractions (NMCs) ( j ), and bladder compliance ( k ) (Two-way ANOVA with Sidak’s post hoc tests; * p < 0.05, ** p < 0.01, *** p < 0.001, as compared with saline treatment in each group).

Article Snippet: Monoclonal CaSR antibody (1:100; NB100-1830; Novus Biologicals, Littleton, CO, USA) was used as the primary antibody.

Techniques: Saline

Journal: Pharmaceuticals

Article Title: Urothelial Calcium-Sensing Receptor Modulates Micturition Function via Mediating Detrusor Activity and Ameliorates Bladder Hyperactivity in Rats

doi: 10.3390/ph14100960

Figure Lengend Snippet: The effects of CaSR agonist (AC), CaSR antagonist (NPS), CaCl 2 , and CaCl 2 containing NPS on micturition. Effects of intravesical administrations of 1 mM AC ( a ), 1 mM NPS ( b ), 20 mM CaCl 2 ( c ), and 20 mM CaCl 2 containing 1 mM NPS (CaCl 2 + NPS) ( d ) on micturition with the recording of IVP and BP. The statistical analysis of micturition function parameters before (saline) and after different treatments, including ICI ( e ), phase 1 IVP ( f ), amplitude of phase 2 oscillation ( g ), baseline pressure ( h ), voiding duration ( i ), amplitude of NMCs ( j ), and bladder compliance ( k ). The statistical analysis of pH values of solutions for intravesical infusion and urine from rats ( l ) (Two-way ANOVA with Sidak’s post hoc tests, n = 6 for each group; * p < 0.05, ** p < 0.01, *** p < 0.001, as compared with saline treatment in each group or among different treatments).

Article Snippet: Monoclonal CaSR antibody (1:100; NB100-1830; Novus Biologicals, Littleton, CO, USA) was used as the primary antibody.

Techniques: Saline

Journal: Pharmaceuticals

Article Title: Urothelial Calcium-Sensing Receptor Modulates Micturition Function via Mediating Detrusor Activity and Ameliorates Bladder Hyperactivity in Rats

doi: 10.3390/ph14100960

Figure Lengend Snippet: The effects of CaSR agonist (AC), CaSR antagonist (NPS), CaCl 2 , and CaCl 2 containing NPS on bladder afferent nerve activity (BANA). The effects of intravesical administrations of 1 mM AC ( a ), 1 mM NPS ( b ), 20 mM CaCl 2 ( c ), and 20 mM CaCl 2 containing 1 mM NPS (CaCl 2 and NPS) ( d ) on BANA during late storage phase. The statistical analysis of the change of BANA during late storage phase ( e ), and the mean arterial pressure (MAP) during late storage phase ( f ) before (saline) and after different treatments in each group (Two-tailed paired Student’s t -test, n = 6 for each group; ** p < 0.01, *** p < 0.001, as compared with saline treatment in each group).

Article Snippet: Monoclonal CaSR antibody (1:100; NB100-1830; Novus Biologicals, Littleton, CO, USA) was used as the primary antibody.

Techniques: Activity Assay, Saline, Two Tailed Test

Journal: Pharmaceuticals

Article Title: Urothelial Calcium-Sensing Receptor Modulates Micturition Function via Mediating Detrusor Activity and Ameliorates Bladder Hyperactivity in Rats

doi: 10.3390/ph14100960

Figure Lengend Snippet: The effects of CaSR agonist (AC), CaSR antagonist (NPS), CaCl 2 , and CaCl 2 containing NPS on bladder efferent nerve activity (BENA). The effects of intravesical administrations of 1 mM AC ( a ), 1 mM NPS ( b ), 20 mM CaCl 2 ( c ), and 20 mM CaCl 2 containing 1 mM NPS (CaCl 2 + NPS) ( d ) on BENA during voiding phase. The statistical analysis of the change of BENA ( e ) and the mean arterial pressure (MAP) ( f ) before (saline) and after different treatments in each group during voiding phase (Two-tailed paired Student’s t -test, n = 6 for each group; ** p < 0.01, as compared with saline treatment in each group).

Article Snippet: Monoclonal CaSR antibody (1:100; NB100-1830; Novus Biologicals, Littleton, CO, USA) was used as the primary antibody.

Techniques: Activity Assay, Saline, Two Tailed Test

Journal: Pharmaceuticals

Article Title: Urothelial Calcium-Sensing Receptor Modulates Micturition Function via Mediating Detrusor Activity and Ameliorates Bladder Hyperactivity in Rats

doi: 10.3390/ph14100960

Figure Lengend Snippet: The evaluation of bladder microcirculation before and after intravesical infusion of CaSR agonist (AC), CaSR antagonist (NPS), CaCl 2 , and CaCl 2 containing NPS. Images of bladder microcirculation before (saline) and after intravesical infusion of 1 mM AC ( a ), 1 mM NPS ( b ), 20 mM CaCl 2 ( c ), and 20 mM CaCl 2 containing 1 mM NPS (CaCl 2 + NPS) ( d ). The statistical analysis of bladder perfusion unit (PU) before (saline) and after intravesical infusion of AC ( e ), NPS ( f ), CaCl 2 ( g ), and CaCl 2 containing NPS (CaCl 2 + NPS) ( h ) (Two-tailed paired Student’s t -test, n = 5 for each group).

Article Snippet: Monoclonal CaSR antibody (1:100; NB100-1830; Novus Biologicals, Littleton, CO, USA) was used as the primary antibody.

Techniques: Saline, Two Tailed Test

Journal: Pharmaceuticals

Article Title: Urothelial Calcium-Sensing Receptor Modulates Micturition Function via Mediating Detrusor Activity and Ameliorates Bladder Hyperactivity in Rats

doi: 10.3390/ph14100960

Figure Lengend Snippet: The effects of CaSR agonist (AC) on cyclophosphamide (CYP)-induced bladder hyperactivity. The recording of IVP in control group ( a ), CYP group with intravesical infusion of saline ( b ), and CYP group with intravesical infusion of AC (CYP + AC) ( c ). The statistical analysis of bladder function parameters in control group, CYP group with intravesical infusion of saline and AC, including ICI ( d ), phase 1 IVP ( e ), amplitude of phase 2 oscillation ( f ), baseline pressure ( g ), voiding duration ( h ), amplitude of NMCs ( i ), and bladder compliance ( j ) (One-way ANOVA with Tukey’s post hoc tests, n = 6 for each group; * p < 0.05, ** p < 0.01, *** p < 0.001, as compared with control group; # p < 0.05, ### p < 0.001, as compared with CYP group).

Article Snippet: Monoclonal CaSR antibody (1:100; NB100-1830; Novus Biologicals, Littleton, CO, USA) was used as the primary antibody.

Techniques: Control, Saline

Journal: Frontiers in Cell and Developmental Biology

Article Title: CaSR regulates SLC26A6 expression via the PKA-FOXO4 signaling axis to promote experimental calcium oxalate kidney stone formation in rats

doi: 10.3389/fcell.2026.1746423

Figure Lengend Snippet: Calcium oxalate crystals upregulate CaSR and SLC26A6 expression in vivo and in vitro . (A) Representative images of renal tissue pathological sections from each group of rats (HE staining, Pizzolato’s staining, and polarized light microscopy). (B) Western blotting detection of CaSR and SLC26A6 protein expression levels in NRK-52E cells after COM crystal intervention. (C,D) RT-qPCR (C) and Western blotting (D) detection of CaSR and SLC26A6 mRNA and protein expression levels in renal tissues of each group of rats. (E) Immunohistochemical analysis revealed the tubular localization patterns of CaSR and SLC26A6 in rat kidney tissue. Low-power field images demonstrated their widespread distribution within both the renal cortex and medulla. Precise cellular colocalization could only be assessed through high-power confocal analysis combined with segment-specific markers, representing a limitation of this study. Data are presented as mean ± standard deviation, *p < 0.05, **p < 0.01 vs. NC group or Control group.

Article Snippet: Rabbit polyclonal antibodies against CaSR (73303S) and Phospho-PKA Substrate (9624S) were purchased from Cell Signaling Technology Company.

Techniques: Expressing, In Vivo, In Vitro, Staining, Light Microscopy, Western Blot, Quantitative RT-PCR, Immunohistochemical staining, Standard Deviation, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: CaSR regulates SLC26A6 expression via the PKA-FOXO4 signaling axis to promote experimental calcium oxalate kidney stone formation in rats

doi: 10.3389/fcell.2026.1746423

Figure Lengend Snippet: CaSR regulates SLC26A6 expression via the transcription factor FOXO4. (A) Western blotting detection of p-FOXO4 (Thr451) and SLC26A6 protein expression levels in NRK-52E cells treated with CaSR agonist (R568, 30 μM) or inhibitor (NPS-2143, 10 μM) in the presence of COM crystals. (B) Western blotting detection of SLC26A6 protein expression levels in NRK-52E cells treated with FOXO4 agonist (TNF-α, 20 ng/mL) or inhibitor (JY-2, 30 μM) in the presence of COM crystals. (C) Dual-luciferase reporter gene assay detecting the effect of FOXO4 on SLC26A6 promoter activity. Data are presented as mean ± standard deviation, *p < 0.05, **p < 0.01 vs. COM group; #p < 0.05, ##p < 0.01 vs. specified group.

Article Snippet: Rabbit polyclonal antibodies against CaSR (73303S) and Phospho-PKA Substrate (9624S) were purchased from Cell Signaling Technology Company.

Techniques: Expressing, Western Blot, Luciferase, Reporter Gene Assay, Activity Assay, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: CaSR regulates SLC26A6 expression via the PKA-FOXO4 signaling axis to promote experimental calcium oxalate kidney stone formation in rats

doi: 10.3389/fcell.2026.1746423

Figure Lengend Snippet: CaSR promotes FOXO4 phosphorylation via the PKA signaling pathway. (A) Western blotting detection of p-PKA substrate and p-FOXO4 (Thr451) protein expression levels in NRK-52E cells treated with CaSR agonist (R568, 30 μM) or inhibitor (NPS-2143, 10 μM) in the presence of COM crystals. (B) Western blotting detection of p-FOXO4 (Thr451) and SLC26A6 protein expression levels in NRK-52E cells treated with PKA agonist (8-Bromo-cAMP, 30 μM) or inhibitor (H-89 2HCl, 30 μM) in the presence of COM crystals. Data are presented as mean ± standard deviation, *p < 0.05, **p < 0.01 vs. COM group; #p < 0.05, ##p < 0.01 vs. specified group.

Article Snippet: Rabbit polyclonal antibodies against CaSR (73303S) and Phospho-PKA Substrate (9624S) were purchased from Cell Signaling Technology Company.

Techniques: Phospho-proteomics, Western Blot, Expressing, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: CaSR regulates SLC26A6 expression via the PKA-FOXO4 signaling axis to promote experimental calcium oxalate kidney stone formation in rats

doi: 10.3389/fcell.2026.1746423

Figure Lengend Snippet: Crystal deposition and tissue damage in the kidneys of rats from each group (A) Hematoxylin and eosin (HE) staining, polarized light microscopy, and Pizzolato’s calcium salt staining of kidney tissue from rats in the EG, CaSR-a, and CaSR-i groups. HE staining reveals tubular architecture and the extent of damage; polarized light microscopy reveals the birefringence of calcium oxalate crystals; Pizzolato’s staining stains calcium oxalate crystals black. (B) Hematoxylin and eosin (HE) staining, polarized light microscopy, and Pizzolato’s calcium salt staining of renal tissues from rats in the EG, PKA-i, and FOXO4-i groups. HE staining reveals the extent of tubular damage in each group; polarized light microscopy and calcium salt staining jointly confirm differences in crystal deposition among the groups.

Article Snippet: Rabbit polyclonal antibodies against CaSR (73303S) and Phospho-PKA Substrate (9624S) were purchased from Cell Signaling Technology Company.

Techniques: Staining, Light Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: CaSR regulates SLC26A6 expression via the PKA-FOXO4 signaling axis to promote experimental calcium oxalate kidney stone formation in rats

doi: 10.3389/fcell.2026.1746423

Figure Lengend Snippet: Effects of intervening in the CaSR-PKA-FOXO4 signaling axis on urinary biochemical indicators and renal protein expression in rats. (A) Western blotting detection of p-PKA substrate, p-FOXO4 (Thr451), and SLC26A6 protein expression levels in renal tissues of each group of rats. (B) Concentrations of calcium ions and oxalate in 24-h urine of each group of rats. (C) Quantitative analysis of SLC26A6 protein expression from (A) . Data are presented as mean ± standard deviation, *p < 0.05, **p < 0.01 vs. E.G., group.

Article Snippet: Rabbit polyclonal antibodies against CaSR (73303S) and Phospho-PKA Substrate (9624S) were purchased from Cell Signaling Technology Company.

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: CaSR regulates SLC26A6 expression via the PKA-FOXO4 signaling axis to promote experimental calcium oxalate kidney stone formation in rats

doi: 10.3389/fcell.2026.1746423

Figure Lengend Snippet: Schematic diagram of the mechanism by which CaSR synergistically promotes calcium oxalate stone formation through dual signaling pathways. Under stimulation by calcium oxalate crystals (COM), the calcium-sensing receptor (CaSR) on the membrane of renal tubular epithelial cells is activated. The activated CaSR activates protein kinase A (PKA) via G proteins. PKA then functions through two parallel pathways: Phosphorylates Signal Transducer and Activator of Transcription 3 (STAT3), causing its nuclear translocation and upregulation of the tight junction protein Claudin-14 expression. Claudin-14 inhibits paracellular calcium reabsorption, leading to hypercalciuria; Phosphorylates Forkhead box protein O4 (FOXO4), causing its nuclear translocation and binding to the SLC26A6 gene promoter, upregulating its expression and promoting oxalate secretion into the lumen, leading to hyperoxaluria. The increased excretion of urinary calcium and oxalate collectively exacerbates the supersaturation of calcium oxalate in urine, ultimately promoting kidney stone formation.

Article Snippet: Rabbit polyclonal antibodies against CaSR (73303S) and Phospho-PKA Substrate (9624S) were purchased from Cell Signaling Technology Company.

Techniques: Protein-Protein interactions, Membrane, Translocation Assay, Expressing, Binding Assay

Journal: Animals : an Open Access Journal from MDPI

Article Title: Synergistic Effects of 25-Hydroxyvitamin D 3 , Phytase, and Probiotics on Growth, Calcium and Phosphorus Metabolism, and Bone Development in Weaned Piglets Fed Low Ca-P Diets

doi: 10.3390/ani16020278

Figure Lengend Snippet: Effects of 25-OH-VD 3 combined with phytase and probiotics on the jejunal mRNA expression for calcium and phosphorus metabolism in weaned piglets. a, b, c Values within a row with different superscripts differ significantly ( p < 0.05; n = 6). Abbreviations: TRPV5/TRPV6, transient receptor potential cation channel subfamily V member 5/member 6; SLC34A1/SLC34A2/SLC34A3, solute carrier family 34 (type II sodium/phosphate transporter), member 1/member 2/member 3; CaBP-D9k, calcium-binding protein D9k; CaBP-D28k, calcium-binding protein D28k; CYP27B1, cytochrome P450 27B1; VDR, vitamin D receptor; CaSR, calcium sensing receptor. Same as the figures below.

Article Snippet: After transfer, the membranes were blocked with 5% ( w / v ) bovine serum albumin for 1 h at room temperature and then incubated overnight at 4 °C with the primary antibodies, including Anti-β-actin antibody (8115-1-RR, Proteintech, Rosemont, IL, USA, diluted 1:5000), Anti-CYP27B1 antibody (PA5-79128, Thermo Fisher Scientific, Waltham, MA, USA, diluted 1:2000), and Anti-CASR antibody (19125-1-AP, Proteintech, diluted 1:1000).

Techniques: Probiotics, Expressing, Binding Assay