The bladder urothelium has traditionally been regarded as a highly impermeable barrier. On the other hand, emerging evidence suggests that it may also contribute to the transport and reabsorption of water and ions. For example, nocturnal urine absorption across the bladder urothelium has been proposed as a mechanism influencing nocturnal urine volume [1], and hibernating bears reportedly absorb water and ammonia across the bladder urothelium during prolonged torpor [2]. Although several molecules, including claudins, ENaCs, and aquaporins, are thought to regulate bladder urothelial permeability, evidence remains limited. In this study, we focused on hypothermia during hibernation and the cold-sensitive transient receptor potential ankyrin 1 (TRPA1) channel, and investigated changes in urothelial permeability using isolated bladders from rats, a non-hibernating animal.

Female Sprague-Dawley rats were used. Organ bath study was performed using both intact and inside-out isolated whole-bladder preparations to evaluate whether exposure to low temperatures or activation of TRPA1 increases bladder urothelial permeability. Inside-out preparations were created by inverting the bladder. KCl was applied to the bath, and the bath temperature was lowered stepwise to 37, 27, 17, and 7 °C while intravesical pressure changes were recorded. In addition, allyl isothiocyanate (AITC), a TRPA1 agonist, and HC-030031, a TRPA1 antagonist, were used to examine their effects on intravesical pressure. Ussing chamber study using urothelium–lamina propria sheets was performed to measure transepithelial electrical resistance (TEER) as an index of bladder urothelial ionic permeability.

In the organ bath study, KCl-induced contractions in the intact preparations (n = 5) showed no significant change as the temperature was lowered from 37 °C to 7 °C (Figure 1A, B). In contrast, in the inside-out preparations (n = 6), KCl applied to the urothelial side did not induce contraction at 37 °C, but elicited marked contractions at 17 °C and 7 °C (Figure 1C, D). These responses disappeared when the temperature was returned to 37 °C. The KCl-induced contractions at 17 °C were attenuated by HC-030031 in a concentration-dependent manner. At 37 °C, pretreatment of the urothelial side with AITC enhanced KCl-induced contractions. In the ussing chamber study, urothelial application of AITC (n = 6) produced a progressive decline in TEER, reaching 62.14 ± 3.17% at 90 min, compared with the control group (n = 5, 100.92 ± 9.03%). This decline was attenuated in the HC + AITC group (n = 4, 87.12 ± 8.90%) (Figure 2).

In the organ bath study, cooling did not increase intravesical pressure in intact preparations, indicating that the intrinsic contractile function of the smooth muscle was not altered by low temperature. In contrast, in the inside-out preparations, urothelial application of KCl elicited marked contractions at low temperatures. Thus, these findings suggest that cold stimulation enhances urothelial ionic permeability, and notably, such temperature-dependent changes were reversible. Moreover, HC-030031 inhibited the KCl-induced contractions at 17 °C, whereas AITC facilitated them at 37 °C, supporting the involvement of TRPA1 in this response. In the ussing chamber study, the decrease in TEER induced by AITC at 37 °C reflects impaired urothelial barrier function and increased ionic permeability across the bladder urothelium. A previous study using cultured epithelial cells have reported that TRPA1 activation induces reversible opening of tight junctions [3].Thus, the ionic permeation observed in the present study may have occurred through the paracellular pathway as a result of structural alterations in tight junctions.

The present findings suggest that low temperatures (< 17 °C) and TRPA1 activation reversibly increases ionic permeability across the bladder urothelium. Regulation of urothelial barrier function may provide new insight into bladder physiology and may also have implications for intravesical drug delivery.

Torimoto K, Matsushita C, Itami Y, Iwamoto T, Owari T, Gotoh D, Miyake M, Hori S, Nakai Y, Aoki K et al: Assessment of bladder function for stabilizing urinary volume overnight with recording of brain waves (ABSORB study). Low Urin Tract Symptoms 2022, 14(1):72-77.
Spector DA, Deng J, Coleman R, Wade JB: The urothelium of a hibernator: the American black bear. Physiol Rep 2015, 3(6).
Kanda Y, Mukaiyama M, Yamasaki Y, Usui T, Nagumo Y: Capsaicin indirectly regulates TRPA1 via the arachidonic acid cascade, resulting in TJ opening. Biosci Biotechnol Biochem 2023, 88(1):44-52.