The transient receptor potential channels vanilloid-1 (TRPV1) and -ankyrin 1 (TRPA1) are cation channels expressed on afferent bladder nerves (1). They are involved in various pathological processes such as pain behaviour and detrusor overactivity during cystitis and neurogenic detrusor overactivity (1). Despite their pathological roles, the function of TRPV1 and TRPA1 in the normal bladder remains unclear, as TRPA1 and TRPV1 knockout (KO) mice have a normal bladder function. We hypothesized that a combined KO of TRPA1 and TRPV1 would result in more pronounced deficits than the individual knockouts and aimed to investigate the bladder function of TRPA1xTRPV1 double KO mice.

Videocystometry was performed in 10-14 weeks old female C57/Bl6J@Rj (wild type), TRPA1 KO, TRPV1 KO and TRPA1xTRPV1 double KO mice. In brief, under isoflurane anaesthesia, a midline laparotomy is performed to expose the urinary bladder. The flared end of a PE-50 tube is implanted in the bladder dome and the other end is externalized. After closure of the abdomen and skin, the animal is injected with urethane (1.2g/kg S.C.) to maintain stable anaesthesia during cystometry. The external end of the PE-50 tube is connected to an infusion pump filled with iodine contrast solution. While the bladder is being filled with contrast solution at a fixed infusion rate of 0.02ml/min, the bladder pressure is recorded and the animal is continuously imaged using X-ray imaging. Based on the contrast intensity in the X-ray images, bladder volume, voided volume, urinary flow and resistance and residual volume are calculated. Voiding efficiency is calculated as the voided volume divided by the total bladder capacity. All urodynamic parameters were compared using one-way analysis of variance. A p-value of < 0.05 was considered significant.
 
For organ bath experiments, mice are sacrificed using CO2 inhalation. The bladder is harvested and cut into strips. The muscle strips are suspended to a force transducer in an organ bath perfused with O2/CO2-bubbled Krebs solution at 37°C. Increasing doses of the muscarinic agonist carbachol are added to induce contraction of the bladder strips, while the force of contraction is measured continuously. Data were compared using two-way analysis of variance. A p-value of < 0.05 was considered significant.

Mice with a combined KO of TRPA1 and TRPV1 have a larger bladder capacity (153 microliter) than wild type mice (77 microliter, p < 0.01) but smaller voided volumes (21 microliter versus 47 microliter, p < 0.01), shorter intercontractile intervals (64 seconds versus 149 seconds, p < 0.01) and high residual volumes (132 microliter versus 30 microliter, p < 0.001). This results in a significantly lower voiding efficiency in TRPA1xTRPV1 double KO mice versus wild type mice (14% versus 62%, p < 0.001). [Figure 1]

In TRPA1 and TRPV1 single KO mice, the bladder capacity (110 and 126 microliter, respectively), voided volume (42 and 35 microliter, respectively) and intercontractile interval (138 and 119 seconds, respectively) are not significantly different from wild type mice. The residual volume is significantly higher in TRPV1 but not TRPA1 single KO mice (91 and 67 microliter, respectively) compared to wild type mice. [Figure 2] 
Likewise, the voiding efficiency is significantly lower in TRPV1 KO mice (29%) but not in TRPA1 KO mice (42%). 

The maximal bladder pressure, maximal flow and urethral conductance during voiding are not significantly different between the four different strains.

The mean bladder weight/body weight ratio is higher in TRPA1xTRPV1 double KO mice (0.0012 versus 0.0008 in wild type mice, p = 0.0015, unpaired two-tailed T-test). Carbachol-induced bladder contractions are stronger in bladder muscle strips from TRPA1xTRPV1 double KO versus wild type mice (p < 0.001).

These results show, to our knowledge for the first time, the effect of a combined genetic deletion of TRPA1 and TRPV1 on the murine bladder function. Mice with a combined knockout of TRPA1 and TRPV1 have severe bladder dysfunction with a low voiding efficiency and large residual volumes. These effects are not or only partially present in TRPA1 or TRPV1 single KO mice. 

Despite the low voiding efficiency, other urodynamic parameters such as the maximal bladder pressure, maximal flow and maximal urethral conductance do not indicate an obstructive or contractile dysfunction of the lower urinary tract. Moreover, TRPA1xTRPV1 double KO mice have a higher bladder weight and stronger carbachol-induced bladder contractions, ruling out a muscular cause of the observed voiding dysfunction.

The underlying mechanism of the decreased voiding efficiency remains therefore unknown, but after ruling out an obstructive or contractile cause, we hypothesize that the combined deletion of TRPA1 and TRPV1 might disrupt sensory signaling in the bladder nerves, leading to incomplete emptying of the bladder. This needs further investigation.

Mice with a combined genetic knockout of TRPA1 and TRPV1 show severe bladder dysfunction characterized by high residual volumes, small voided volumes and consequently very low voiding efficiencies. By contrast, there are no differences in bladder pressure, urethral conductance or bladder muscle contractility, indicating a possible sensory cause for this phenotype.

Vanneste, M., Segal, A., Voets, T. et al. Transient receptor potential channels in sensory mechanisms of the lower urinary tract. Nat Rev Urol 18, 139–159 (2021).