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Inflammatory and Apoptotic Mediators are Increased in the Bladder in a Type 2 Diabetic Genetic Model
Zongwei Wang, M.D., PhD.1, Zhiyong Cheng, M.D., PhD.2, Vivian Cristofaro, PhD.3, Edward Gong, M.D.2, Maryrose Sullivan, PhD.3, Rosalyn Adam, PhD.2, Morris White, PhD.2, Aria F. Olumi, M.D.1.
1Massachusetts General Hospital, Boston, MA, USA, 2Children's Hospital, Boston, MA, USA, 3VA Boston Healthcare System, Boston, MA, USA.

BACKGROUND:
Diabetic bladder dysfunction in type 2 diabetes mellitus (DM2) affects up to 80% of individuals, however, its underlying pathophysiology is poorly understood. Here we demonstrate a genetic mouse model with DM2 with conditional knockout of Insulin receptor substrates 1 & 2 (Irs1, Irs2), which mimic the diabetic bladder dysfunction in humans. We demonstrate that inflammatory modulators are significantly upregulated during the excitatory phase of diabetic cystopathy.
METHODS:
Conditional hepatocyte knockout Irs1/Irs2 (DKO) mice develop DM2 with hyperglycemia after 5 weeks of age and remain hyperglycemic through 30 weeks of age. Bladders from 6, 10, 14 and 20 week old mice were harvested and the sensitivity of bladder muscle strips in response to various forms of stimulation (spontaneous muscle contractions, KCl and carbachol stimulation) was evaluated. Urodynamics evaluations were completed for 12-week control and DKO mice. For comparative gene analysis, Affymetrix mouse gene chip was employed to evaluate the expression of 45,000 genes in the bladder smooth muscle of 12 weeks old mice. The level of 10 cytokines in serum was determined using the Multiplex kit. The expression of proteins was assessed by Western blot, immunohistochemistry and ELISA.
RESULTS:
DKO mice exhibited abnormal spontaneous bladder contractions, with higher amplitudes of tension to different stimulation at age 6 and 12 week relative to age-matched control, while older mice (16 and 20 week of age) demonstrated lower amplitudes. Over 20 inflammatory genes were up-regulated 1.5 fold or greater, most of which belong to the TNF superfamily. In addition, we found genes that ATPase, Rho GTPase and Rho kinase, genes that are important in regulating cellular metabolism, were significantly upregulated. Since many inflammatory/apoptotic response related genes were upregulated in the bladder of the DKO animals, the ultrastructure of the mouse smooth muscle mitochondria was evaluated. We found that the mitochondria in the DKO bladder smooth muscle cells were significantly shrunken compared to the age matched controls, particularly in older mice (20 weeks of age), suggesting abnormal energy metabolism in the mice suffering from late stage diabetic cystopathy.
CONCLUSIONS:
Our findings demonstrate that diabetic cystopathy in DKO mice is a progressive process - hyperactive bladder dysfunction in early/middle aged animals and hypoactive in older animals. Mitochondria are shrunken and inflammatory/apoptotic mediated genes are upregulated in the hyperactive stage of diabetic cystopathy. Evaluating the molecular pathways that lead to diabetic cystopathy will improve our understanding of bladder dysfunction, and help devise preventive strategies for secondary complications associated with type 2 diabetes.


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