Inhibition of the cation channel TRPV4 improves bladder function in mice and rats with cyclophosphamide-induced cystitis

Abstract

Reduced functional bladder capacity and concomitant increased micturition frequency (pollakisuria) are common lower urinary tract symptoms associated with conditions such as cystitis, prostatic hyperplasia, neurological disease, and overactive bladder syndrome. These symptoms can profoundly affect the quality of life of afflicted individuals, but available pharmacological treatments are often unsatisfactory. Recent work has demonstrated that the cation channel TRPV4 is highly expressed in urothelial cells and plays a role in sensing the normal filling state of the bladder. In this article, we show that the development of cystitis-induced bladder dysfunction is strongly impaired in Trpv4(-/-) mice. Moreover, we describe HC-067047, a previously uncharacterized, potent, and selective TRPV4 antagonist that increases functional bladder capacity and reduces micturition frequency in WT mice and rats with cystitis. HC-067047 did not affect bladder function in Trpv4(-/-) mice, demonstrating that its in vivo effects are on target. These results indicate that TRPV4 antagonists may provide a promising means of treating bladder dysfunction.

Fig. 1.

Cyclophosphamide induces cystitis in WT and Trpv4^−/− mice. (A) Images of WT and Trpv4^−/− mice pretreated with cyclophosphamide or vehicle (saline). (B) Bladder weight/body weight ratio in vehicle- and cyclophosphamide-pretreated WT (n = 4) and Trpv4^−/− (n = 4) mice expressed in mg/g. (C) Histological bladder sections of vehicle-pretreated (naive) or cyclophosphamide-pretreated WT and Trpv4^−/− mice. (D) Histological edema score showing the severity of edema in WT (n = 4) and Trpv4^−/− (n = 4) mice pretreated with cyclophosphamide but not in vehicle-pretreated mice. Relative expression of TRPV4 and Pacsin3 mRNA in urothelium and smooth muscle from vehicle-pretreated (n = 4) or cyclophosphamide-pretreated (n = 4) mice. (E) Levels of mRNA were normalized to the TRPV4 mRNA level in urothelium from vehicle-pretreated mice. (F) Changes in c-fos mRNA after cyclophosphamide treatment in WT and Trpv4^−/− mice.

Fig. 2.

The development of cystitis-induced detrusor overactivity is impaired in Trpv4^−/− mice. (A) Cystometry patterns in WT and Trpv4^−/− mice pretreated with vehicle or cyclophosphamide (cyp). (B and C) Voided volume and voiding frequency in naive WT (n = 12), cyp-treated WT (n = 21), naive Trpv4^−/− (n = 11), and cyp-treated Trpv4^−/− (n = 6) mice.

Fig. 3.

Characterization of the TRPV4 antagonist HC-067047. (A) Representative Fluo-4 fluorescence traces showing the effect of HC-067047 (Inset; MW = 472) on TRPV4-mediated Ca²⁺ responses in T-REx-HEK-293 cells expressing hTRPV4 stimulated with 4α-PDD (1 μM). (B) Representative whole-cell patch-clamp recording showing the dose-dependent effect of HC-067047 on TRPV4-mediated currents at +80 (red) and −80 mV (black) in T-REx-HEK-293 cells expressing hTRPV4 stimulated with 4α-PDD (1 μM). (C) Current–voltage relations of TRPV4-expressing cells obtained at the time points indicated in B. (D) Dose–response curves for the inhibition of inward TRPV4 currents at −80 mV for human, mouse and rat TRPV4. (E) Inhibition by HC-067047 of Ca²⁺ responses to 4α-PDH (1 μM) in Fura-2–loaded mouse urothelial cells. (F) Dose–response curve of the inhibition of TRPV4-mediated Ca²⁺ responses in mouse urothelial cells.

Fig. 4.

HC-067047 decreases detrusor overactivity in anesthetized WT mice. (A) Cystometry recordings in naive and cyclophosphamide-pretreated WT mice before (basal) and after (HC) i.p. administration of 10 mg/kg HC-067047. (B) Effects of HC-067047 on voided volume and voiding frequency in naive and cyclophosphamide-pretreated WT mice. Each line represents the value for a single animal before and 30 min after the i.p. administration of 10 mg/kg HC-067047. (C and D) Same experiments as in A and B with Trpv4^−/− mice. (E and F) Summary of the relative changes in voided volume and voiding frequency after the administration of different doses of HC-067047 in cyclophosphamide-treated WT and Trpv4^−/− mice, and in naive WT mice. Additional cystometric values and n numbers are provided in Table S3.

Fig. 5.

HC-067047 increases micturition volume in freely moving mice suffering from cyclophosphamide-induced cystitis. (A and B) Representative micturition pattern of a single WT (A) or Trpv4^−/− (B) mouse before (Left) and after (Center) cyclophosphamide pretreatment and after subsequent i.p. injection with HC-067047 (10 mg/kg; Right). Solid red lines delineate individual urine spots. Dotted red lines delineate an area that was discarded from analysis because of spilling of drinking water. (C) Comparison of the micturition volumes in WT (n = 10) and Trpv4^−/− (n = 13) mice before and after cyclophosphamide treatment. (D) Summary of relative changes in micturition volume after the administration of HC-067047 (n = 6 for WT; n = 5 for Trpv4^−/−) or vehicle (n = 4).

Fig. 6.

HC-067047 decreases detrusor overactivity in conscious rats. (A) Cystometry recordings in naive and cyclophosphamide-pretreated rats before (basal) and after (HC) i.p. administration of 10 mg/kg HC-067047. (B and C) Effects of HC-067047 on voided volume and voiding frequency in naive and cyclophosphamide-pretreated rats. Each line represents the value for a single animal before and 30 min after the i.p. administration of HC-067047. (D and E) Summary of the relative changes in voided volume and voiding frequency after the administration of HC-067047 or vehicle. Parameter values and n numbers are provided in Table S4.