Introduction: Bladder denervation and bladder outlet obstruction are urological conditions that promote bladder growth. Unbiased transcriptomics have identified differentially expressed genes in obstruction, but similar studies following denervation have not been done. Partial denervation has been reported in the obstructed bladder, suggesting that the conditions are intertwined. This study aimed to define gene activation following denervation and compare this with obstruction.
Methods: Female Sprague Dawley rats (200-250g) were used and bladder denervation was induced by cryoablation of pelvic ganglia. Rats with denervated (n=6) and obstructed (n=8) bladders, and sham-operated rats (n=6 and 8, respectively), were euthanized 10 days after surgery. RNA was isolated from bladders and microarrays were run. Concordantly regulated genes were identified by applying stringent cut-offs (Q=0, SAM analysis) and fold-change (<0.5 or >3). Protein level changes were confirmed by western blotting. Bladder weights were correlated to the collagen triple helix repeat containing 1 (Cthrc1) mRNA level derived from the microarrays using the Spearman method. Immunofluorescence was used to locate Cthrc1 in rat bladder sections. Expression of Cthrc1 in human bladder smooth muscle cells was manipulated using an adenovirus, and cell proliferation was measured using the MTT assay.
Results: Mean bladder weight increased 5.6-fold and 2890 mRNAs were differentially expressed following denervation. Comparison with array data from obstructed bladders demonstrated overlap between the conditions and 10% of mRNAs changed significantly and in the same direction. Discordantly regulated transcripts in the two models were rare, making up for less than 0.07% of all changes. Numerous genes were upregulated in both conditions, including Cthrc1, protein regulator of cytokinesis 1 (Prc1) and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (Plod2). Western blotting demonstrated directionally consistent changes at the protein level, with increases of Cthrc1, Prc1 and Plod2. Individual bladder weights in the array experiment correlated with the Cthrc1 mRNA level. Immunofluorescence showed that Cthrc1 was induced in the smooth muscle layer following denervation. Knockdown and overexpression of Cthrc1 reduced and increased smooth muscle cell proliferation, respectively.
Conclusions: This work defines the transcriptomic response to bladder denervation and identifies an expression signature shared with bladder outlet obstruction. Mechanistic studies suggest a role for Cthrc1 induction in bladder growth following denervation. Source of
Funding: This work was supported by grants from The Swedish Research Council [Vetenskapsrådet, https://www.vr.se/, K2015–65X-22662–01–3 (K. Swärd)], the Hillevi Fries Foundation (B. Uvelius), and the Gösta Jönsson Foundation (B. Uvelius)
