TRPA1 channel mediates methylglyoxal-induced mouse bladder dysfunction

Abstract

Introduction: The transient receptor potential ankyrin 1 channel (TRPA1) is expressed in urothelial cells and bladder nerve endings. Hyperglycemia in diabetic individuals induces accumulation of the highly reactive dicarbonyl compound methylglyoxal (MGO), which modulates TRPA1 activity. Long-term oral intake of MGO causes mouse bladder dysfunction. We hypothesized that TRPA1 takes part in the machinery that leads to MGO-induced bladder dysfunction. Therefore, we evaluated TRPA1 expression in the bladder and the effects of 1 h-intravesical infusion of the selective TRPA1 blocker HC-030031 (1 nmol/min) on MGO-induced cystometric alterations. Methods: Five-week-old female C57BL/6 mice received 0.5% MGO in their drinking water for 12 weeks, whereas control mice received tap water alone. Results: Compared to the control group, the protein levels and immunostaining for the MGO-derived hydroimidazolone isomer MG-H1 was increased in bladders of the MGO group, as observed in urothelium and detrusor smooth muscle. TRPA1 protein expression was significantly higher in bladder tissues of MGO compared to control group with TRPA1 immunostaining both lamina propria and urothelium, but not the detrusor smooth muscle. Void spot assays in conscious mice revealed an overactive bladder phenotype in MGO-treated mice characterized by increased number of voids and reduced volume per void. Filling cystometry in anaesthetized animals revealed an increased voiding frequency, reduced bladder capacity, and reduced voided volume in MGO compared to vehicle group, which were all reversed by HC-030031 infusion. Conclusion: TRPA1 activation is implicated in MGO-induced mouse overactive bladder. TRPA1 blockers may be useful to treat diabetic bladder dysfunction in individuals with high MGO levels.

Keywords: MG-H1; cystometry; glyoxalase; lamina propria; urothelium; void spot assay.

FIGURE 1

Void spot analysis in female mice exposed to 0.5% methylglyoxal (MGO) for 12 weeks. (A) displays representative images of the void spot assay in both the control group (receiving tap water alone) and the MGO-exposed groups. (B–D) show data on the number of voids, volume per void, and total void volume, respectively. The distribution of void spots across different volume ranges is shown in (E) (<25 µL), (F) (between 25 and 100 µL), and (G) (>100 µL). The number of voids in the corner and the center on the filter paper is illustrated in (H,I), respectively. The data are expressed as the mean ± SEM (n = 6–7 animals per group). *p < 0.05, **p < 0.01 compared to control group (unpaired t-test).

FIGURE 2

Quantification of methylglyoxal (MGO)-derived hydroimidazolone MG-H1 and glyoxalase 1 (Glo1) in the bladders of MGO-treated mice. (A,B) display densitometry analyses and representative Western blots of MG-H1, respectively. (C) depicts immunohistochemistry for MG-H1, demonstrating negative staining (absence of primary antibody binding) and positive immunostainings in control and MGO groups. In the control group, there is a weak positive staining observed in the urothelium, while in the MGO group, a strong positive staining is observed in both the urothelium and detrusor smooth muscle. In (C), black bars represent a scale of 10 μm (×10 objectives). (D,E) display the protein expression, whereas (F) shows the Glo1 activity. The data are expressed as mean ± SEM (n = 5–6 animals per group). *p < 0.05 compared to control group (unpaired t-test).

FIGURE 3

Protein expression of TRPA1 in the bladders of mice was assessed following a 12-week treatment with 0.5% methylglyoxal (MGO) or in control animals receiving tap water alone. (A) illustrates the results of protein expression analysis using Western Blotting analysis. (B) displays the immunohistochemistry for TRPA1 in bladder, showing negative immunostaining (absence of the antibody signal), and positive immunostainings in control and MGO groups. Positive immunostaining is observed in lamina propria and urothelial cells. The black bars in (B) represent a scale of 10 μm, as viewed under ×20 and ×40 objectives. In (A), data are expressed as mean ± SEM (n = 7–8 animals per group). *p < 0.05 compared to control group (unpaired t-test).

FIGURE 4

Effect of continuous infusion of the selective TRPA1 antagonist HC-030031 on cystometric changes in mice following 12-week treatment with 0.5% methylglyoxal (MGO) or control animals receiving tap water alone. In control and MGO groups, animals were infused continuously with saline (0.01 mL/min), vehicle (0.001% DMSO) or HC-030031 (1 nmol/min) for 1 h. (A) shows representative cystometric tracings from each sub-group, with arrows indicating micturition peaks. (B–H) show data on voiding frequency, bladder capacity, voided volume, compliance, basal pressure, threshold pressure and maximum pressure, respectively. All data are expressed as mean ± SEM (n = 5–7 animals per group). *p < 0.05, **p < 0.01, ***p < 0.001 compared to respective control group; ^## p < 0.01, ^### p < 0.001 compared to saline infusion in MGO group; ^&& p < 0.01, ^&&& p < 0.001 compared vehicle infusion in MGO group (one-way ANOVA followed by Dunnett’s multiple comparisons test for comparison to the control group and Bonferroni’s multiple comparisons test to compare all groups).

FIGURE 5

Effects of the selective TRPA1 antagonist HC-030031 on the contractile responses induced by electrical-field stimulation (EFS) and the muscarinic agonist carbachol in intact bladder strips obtained from mice treated with 0.5% methylglyoxal (MGO, 12 weeks) or tap water (control group). (A) illustrates the contractions induced by EFS at frequencies ranging from 1 to 32 Hz in both the control and MGO-treated groups in the presence of vehicle (0.001% DMSO) or HC-030031 (10 μM, 30 min). (B) displays the contractions induced by carbachol at concentrations ranging from 10⁻¹⁰ to 3 × 10⁻⁵ M in both the control and MGO-treated groups, in the presence of vehicle or HC-030031. (C) shows the maximal responses (E_max) to carbachol in all experimental groups. The data are expressed as mean ± SEM (n = 7 animals per group). *p < 0.05, **p < 0.01 compared to corresponding control vehicle group. ^# p < 0.05 ^## p < 0.01 ^### p < 0.001 compared to respective MGO vehicle group (One-way ANOVA followed by the Tukey).