Supplementary Table 1. Cystometric parameters in sham-operated wild type and Trpv4 -/- rats during saline infusion and

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1 WT sham Trpv4 -/- sham Saline 10µM GSK A P value Saline 10µM GSK A P value Number Intercontractile interval (sec) 143 ( ) 98.4 ( ) (92 121) 109 (95 123) 0.3 Voided volume (ml) 0.23 ( ) 0.18 ( ) ( ) 0.21 ( ) 0.06 Amplitude (cmh2o) 60 (46 70) 50 (49 60) (32 42) 38 (33 43) 0.3 Peak pressure (cmh2o) 67 (55 82) 60 (57 72) 1 43 (42 52) 49 (30 53) 0.3 Basal pressure (cmh2o) 8 (7 10) 10 (9 11) (9 12) 10 (8 13) 0.6 Post-void residual (ml) 0.05 ( ) 0.02 ( ) ( ) 0.04 ( ) 0.8 Supplementary Table 1. Cystometric parameters in sham-operated wild type and Trpv4 -/- rats during saline infusion and infusion of a 10 µm GSK A solution. Data are presented as median ± IQR. Paired comparisons per genotype were performed using the Wilcoxon signed-rank test.

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3 Supplementary Fig. 1 A. Representative ultrasound images of PVR in urethaneanesthetized sham and bpni rats. The total volume was estimated using the formula for the % volume (V) of an ellipsoid (V = $ &'()) L: length, H: height, W: width. B. n=2. Estimated bladder volumes after instillation of 100l, 300ml, 500ml, 700ml and 1000ml of saline. C-D. PVR estimated by ultrasound (C) and direct measurement from the catheter (D) in sham (black) and bpni (red) rats (n=4 in each group). Comparisons were performed using the Mann-Whitney U test. *, P<0.05. E-H Changes in PVR after instillation of 10 µm GSK A using the ultrasound technique (E,F) and direct measurement from the catheter (G,H) in sham-operated (black) and bpni (red) WT (diamond) and Trpv4 -/- (circle) rats (n= 4 for each group, 3 PVR measurements per animal). Boxplots summarizing effect of GSK A in WT and Trpv4 -/- rats. Data presented as median ± IQR. Mann-Whitney U test was used to compared differences between wild type and Trpv4 -/- rats. *, P<0.05

4 Supplementary Fig. 2 Trpm7 expression after bpni remains unchanged compared to sham.

5 Rat TRPV4 gene ENSRNOG CTCTCCGGTGTTAGATCCTCTTCAAAGATCTCTTCCGCTTTCTGCTGGTCTACCTGCTTT tm5 exon 12 TTATGATTGGCTATGCCTCAGGTGAGTGTGGGCCTGGGGTGCTGGTGGCAGTAACTAGGG CTATAGGGGAAGCTACCATGGGCTGGAGGAAATGGGTATCCCTGCCTGAGATCCTTATGT GTGGGCCAAGGCCAGCCAAGCTGCGTTCCCCCTCCCAGCAAGCCTGAAAAGTGAAAGGCT CATCATTCCACTTACAGAGGATCGGTCTCAGGTCAGAGAGGCCATGTAAAGCCACAGGTG TCCAAAGGTCAGCGGTCAGTGAGATGCCTCCATGGGTAACAGCGCTTGCTGTCAAGCATG ATGACCTGAGTTTGACCTCCATGACCCATAGATTGGAAGGAGATACCTGACTCATGAACT TTGTCCTCTTGTCCCCACATGTGTGCCATGGTGTGTAGATGCTCATGCCCATAGGCACAC CCAAGTAAACGAATGTAATGAAAACATTGTTTTAAGCCAGTCAATGCGGAAATGGGATGT GAGCTACTTCTGTGAAGCAAACTGAAGCTGAAACTTACAGGTCAGGTTGGGTACACTAGG TCACAGGTCAGGGCCAGATATAACACAGAGCACACATGCTCCTGTCACTCATTTTTGTGC CCAAAGCAGACATTACTAATGGATCACAGAATGATTTTTTTTTCTGTTGGATGTAGATCA V4LgDel-F GGTCTCAAAACACATACACTGTTTGGGCATCCACTACACAATTAGATTGCATCGgtgtgt ggattgaaaagtgttcactatgttctctagctgaaggcagtgcagcctcccagactcccc tgctgaggtctgcctccctgccctggtggctagttgctatcatctgggtttagcaggaaa agatggtgagaacagcttggaattgttgcaggcacaggggctcttagagtagggcctttg ggactagcaagacagggcaattagtcttgtgcttccactctggctagtgtggagggaagg aggaccctgcatggagtctgcaagtaacaggctgtgttaagggcttctggggacagatgg V4LgDel- RvKO ggtctggctatccttgtcctatgagctgtaggatgtagcagcatccttgactccacccac tggatgctggcacacccaccctcctgtcactcgaaggtgccacatgtgccctaagagaaa atgatgggggctgggtagctctagactccattctgaaaatccctaactcctcagagagtt ttgggagagagcagtggcttggcttcctgctggccgaccggtgcgtacccctcatcccca tccagtggggtagcacctctcagccttcccgcccctcccgcagctctggtcaccctcctg tm5 exon 13 aatccgtgcaccaacatgaaggtctgtaacgaggaccagagcaactgcacggtgccctca taccccgcgtgccgggacagcgagaccttcagcgccttcctactggacctcttcaagctc pore accatcggcatgggcgacctggagatgctgagcagcgctaagtaccccgtggtcttcatt tm6 ctcctgctggttacctacatcatcctcaccttcgtgctcctgctgaacatgctcatcgcc ctcatgggtgagaccgtgggccaggtgtccaaggagagcaagcacatctggaagctgcag GTGAGGTCGGCCGCTCGGAGTGTTCTTCCACCTTCCATGTTTACCACAGGCTGGTGAGGC CACTGTTTTAGAATGGCCTCATTTCCCCCTCCCCACCCCCCACCCCAGTTTAGTGCCCTT CATTTCAGCTGTGGTGCAATTCAGGGGCCCCAGCTGGCTCCATTCTGCGTGACTTCATGA CCTCTGTCAGCACGGAGCATGCTCAGAGAGTCAGCTGTCAGCACTGAGCATGCTCAGAGA GCCTGCTTCTGTTTTCAGTCCCCGTCACATAAATCCCTAAAACAGGGTCATTTATAAAGA ATATAGATTCTGGGTAGTGTGGTGGCCCATGCCTTTGATCCCAGCACTTGGGAGGCAGAG GCAGATGAATTTCTGTGAGTTCGAGGCCAGCCTGGTCTACAGAGTGAGTTCCAGAATAGC CAAGGCTGTTACCCAGAGAAACTCTGCCTTTAAAAAAAAATGGAGAATCACTTGGTTCAC GGCTTTGGGGGCTGGGAAGTTCAGGTATGGTGTCGTGTCTGCCAAGAGCCCTCTTGCTTC V4LgDel- RvWT ATCTGGCCGTGGTGGAGGGCTTGTGCTGCTCGCAGAGCAGGCCTCTTAGCTTGAGTCTCT TTTCTGGTGCTGCCTCATGGGCGCATACCACAGGCCTCATCTAACCTTAATCTTGGCTAC AATGAGTGCCCCACATTTCGAAGCCCATGGACACAGGCATTTGCTCAAGACACAGCAGGC AGCACGGTCATTTTAATGGGTTTTACGCTGAGCCGATGCCCCACCTTGATTCAGAAATGA GACCAGGGGCTGGTGAAGTGCTTGCCACTCAACAAGCAAAAGGACCCGAGTTCGGATCCC CCAAACCATTTTTTTTAAAAGATTTCTTTTATTCATTTCATGTTTATGAGTAGTACACTG TCGCTCTCTTCAGACACACTAGAAAAGGGCATCAGACACTGTTATATATGGTTGTGAGCC ACCATGTGGTTGCTGGGAATTAAACTCAGGACCTCTGGAAGAGCAATCAGTGCTCTTAAC Shown is an excerpt from the genomic sequence of the rat TRPV4 gene (ENSRNOG ). Exons are indicated in Bordeaux text. Underlined text indicate exon sequence that encodes transmembrane domains (exon number is indicated). Text marked in blue encodes the putative pore region. Text marked in green indicates location of primers for genotyping (names are indicated). Sequence in lowercase italic is deleted in TRPV4 KO rats.

6 Note that this portion includes exon 13 completely, plus part of the intron between exon 12 and exon 13. As such, in the mutant rat part of Transmembrane domain 5, Transmembrane domain 6, and the putative pore region of the TRPV4 protein are lacking. Example genotyping gel: A litter of 12 animals from a heterozygous TRPV4-KO couple. On the left WT and KO control samples are loaded. WT KO WT-reaction: V4LgDel-F + V4LgDel-R1: 409bp KO-reaction: V4LgDel-F + V4LgDel-R2: 592bp Supplementary Fig. 3. Detailed description of the alterations in the TRPV4 gene of TRPV4 KO rats and genotyping protocol.

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