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Background: Diabetes bladder dysfunction (DBD) is one of the major urologic complications associated with type 2 diabetes (DM2). We have shown specific molecular alterations in a mouse model that harbors hepatic insulin receptor substrate 1 and 2 deletions (double knockout: DKO) which develops type 2 diabetes. Previously, we have shown that DBD occurs progressively with hyperactivity in the early stage and hypoactivity in late stage of DBD. We showed that inflammation, especially TNF-α upregulation and enhanced macrophages appeared in the lamina propria play a critical role on DBD. Here we demonstrate that there is significant ATP release in the early stage of DBD, with an associated increase of the purinergic receptor, P2X1R, in the detrusor muscle.
Methods: The functional alterations of bladders from 12 week old DKO/ floxed control mice were evaluated by in vivo cystometry, ex vivo cystometry of intact bladder, and ex vivo muscle strip contraction measurement. The ATP release in the lumen of intact bladder during ex vivo cystometry was measured. Bladder histological changes were observed after H&E staining, Masson Trichrome staining and immunohistochemistry (IHC). The purinergic receptors were detected with qPCR at mRNA level and western blot at protein level.
Results: The DKO mice at 12 weeks of age exhibited impaired glucose tolerance and insulin resistance, a time when they also have bladder hyperactivity. In anesthetic cystometry test, the post void residual (PVR) urine volume was elevated, the voiding efficiency and bladder compliance were lowered in DKO mice compared with age-matched controls. Bladder capacity was equivalent in both diabetic and non-diabetic animals. Testing of in vitro bladder strip contractility showed that urothelium-denuded strips had significantly higher tension than urothelium-intact bladder smooth muscle strips in DKO diabetic animals responding to electrical field stimulation (EFS) and carbachol, whereas there was comparable tension in non-diabetic control mice. EFS and exogenous α-β-meATP stimulation in ex-vivo cystometry studies revealed that more ATP was released in the lumen of bladder in DKO animals than that in age-matched controls. Bladder compliance upon filling was negatively correlated with ATP release. Evaluation of the purinergic receptors, P2X1, P2X2, P2X3, P2X4, P2X5, P2X6, P2X7, in the detrusor muscle revealed that P2X1R mRNA was increased by 10-folds in the DKO animals compared to age matched control animals. Surprisingly, there was no expression of P2X1R in the urothelium. The P2X1R protein levels were also elevated as determined by IHC and Western blot analysis. NF279, the P2X1R inhibitor, dramatically suppressed the contractility of bladder smooth muscle strip in DKO animal. There was no histological alteration in DKO bladder as determined by H&E and Masson Trichrome staining.
Conclusions: During the hyperactive phase of DBD, reduced bladder compliance is associated with significant ATP release. The purinergic receptor, P2X1R, was selectively expressed in the detrusor muscle and not in the urothelial layer. Our findings signify the importance of the purinergic receptors, particularly P2X1R, which may be considered as a target for therapy during the hyperactive phase of diabetic bladder dysfunction associated with DM2.