Volume 74, Issue 3, Pages 336-345 (September 2018) Intravesical Activation of the Cation Channel TRPV4 Improves Bladder Function in a Rat Model for Detrusor Underactivity  Yves Deruyver, Emmanuel Weyne, Karel Dewulf, Roma Rietjens, Silvia Pinto, Nele Van Ranst, Jan Franken, Matthias Vanneste, Maarten Albersen, Thomas Gevaert, Rudi Vennekens, Dirk De Ridder, Thomas Voets, Wouter Everaerts  European Urology  Volume 74, Issue 3, Pages 336-345 (September 2018) DOI: 10.1016/j.eururo.2018.05.020 Copyright © 2018 The Author(s) Terms and Conditions

Fig. 1 Bilateral pelvic nerve injury results in structural bladder wall changes 4 wk after injury in WT and Trpv4−/− rats. (A) Histological bladder sections showing thickening of the detrusor layer in WT (n=4, four sections per bladder) and Trpv4−/− (n=4, four sections per bladder) rats 4 wk after bPNI. (B) Bladder weight/body weight ratio in sham-operated and bPNI WT (n=4) and Trpv4−/− (n=4) rats. (C) Measurement of the detrusor thickness showing increase of the detrusor layer after bPNI in both genotypes. Comparisons per genotype were performed using the Mann-Whitney U test. bPNI=bilateral pelvic nerve injury; D=detrusor; KO=knockout; U=urothelium; WT=wild type. * p<0.05. European Urology 2018 74, 336-345DOI: (10.1016/j.eururo.2018.05.020) Copyright © 2018 The Author(s) Terms and Conditions

Fig. 2 Four weeks after bPNI, WT rats mimic a cystometric phenotype characteristic of DU. (A) Continuous intravesical pressure and voided volume recording in awake sham-operated and bPNI WT rats. (B) Intercontractile interval. (C) Amplitude of voiding contraction. (D) Voided volume in awake sham-operated (n=10) and bPNI (n=10) WT rats. (E) Estimated residual volume in sham-operated and bPNI WT rats. Comparisons were performed using the Mann-Whitney U test. bPNI=bilateral pelvic nerve injury; DU=detrusor underactivity; WT=wild type. ** p<0.001. European Urology 2018 74, 336-345DOI: (10.1016/j.eururo.2018.05.020) Copyright © 2018 The Author(s) Terms and Conditions

Fig. 3 mRNA expression of TRPV4 in urothelium and detrusor smooth muscle and immunohistochemical evidence for TRPV4 expression on rat urothelium. (A) mRNA expression of TRPV4 in urothelium is upregulated after bPNI. In detrusor smooth muscle, relative TRPV4 mRNA levels are very low (n=5 for each group). Comparisons were performed using the Mann-Whitney U test. (B) Urothelium from WT rats is immunoreactive for TRPV4, while urothelium from Trpv4−/− is not. Notice some nonspecific immunoreactivity in the suburothelium. bPNI=bilateral pelvic nerve injury; D=detrusor; DSM=detrusor smooth muscle; LP=lamina propria; TRP=transient receptor potential; TRPV4=transient receptor potential vanilloid 4; U=urothelium; WT=wild type. * p<0.05. European Urology 2018 74, 336-345DOI: (10.1016/j.eururo.2018.05.020) Copyright © 2018 The Author(s) Terms and Conditions

Fig. 4 GSK1016790A induces bladder hyperactivity in sham-operated rats and improves detrusor underactivity in bPNI rats. (A and B) Cystometric recordings in sham-operated (black) and bPNI (red) WT (left) and Trpv4−/− (right) rats during installation of a saline solution and 10μM GSK1016790A solution. (C–E) Effect of GSK1016790A on sham-operated (black) and bPNI (red) WT (left) and Trpv4−/−(right) rats on ICI, voided volume, and residual volume. (F and G) Boxplots summarising the effect of GSK1016790A and vehicle in WT and Trpv4−/− rats. Data presented as median ± IQR. Paired comparisons were performed using Wilcoxon signed-rank test. bPNI=bilateral pelvic nerve injury; IQR=interquartile range; WT=wild type. * p<0.05. European Urology 2018 74, 336-345DOI: (10.1016/j.eururo.2018.05.020) Copyright © 2018 The Author(s) Terms and Conditions

Fig. 5 Muscle strip contractility. (A) In response to depolarisation by a 120 mEq K+ solution, contractile force is significantly lower in bPNI bladder strips (n=7 for WT sham, n=8 for WT bPNI). Mann-Whitney U test *, p<0.05. Dose-response curves of contractile responses to cumulative doses of (B) carbachol and (C) GSK in strips from sham and bPNI rats (n=7 for each group). Two-way ANOVA, with post hoc Bonferroni test, data are presented as mean±SEM. ANOVA=analysis of variance; bPNI=bilateral pelvic nerve injury; SEM=standard error of the mean; WT=wild type. * p<0.05. ** p<0.001. European Urology 2018 74, 336-345DOI: (10.1016/j.eururo.2018.05.020) Copyright © 2018 The Author(s) Terms and Conditions