Role of Urothelial Purinergic Signaling in Diabetic Bladder Dysfunction

University Of Nevada Reno, Reno NV

Investigators

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Abstract

PROJECT SUMMARY/ABSTRACT Despite the high prevalence of Western diet-associated diseases and their clear impact on numerous systems in the body, we know very little about the origin and mechanisms of bladder dysfunction in type 2 diabetes. Current treatment strategies, although partially effective, target mainly the contractility of detrusor muscle, and have numerous unfavorable off target side effects. We propose to study cellular and molecular mechanisms of extracellular purinergic signaling in the bladder urothelium and suburothelium/lamina propria (LP) in bladder dysfunctions of two mouse models of type 2 diabetes (T2D) with obesity. We will build on four important advances made during the initial award, such as discovery of (i) mechanosensitive degradation of ATP at the luminal and abluminal sides of the urothelium, (ii) urothelial release of soluble ectonucleotidases (s-ENTDs), (iii) differential distribution of s-ENTDs in LP and lumen, and (iv) regulation of s-ENTDs release by various membrane channels, sensory neuropeptides, and receptors. In pilot studies, db/db diabetic mice and mice with T2D and obesity induced by high-fat high-sucrose diet (HFHSD-T2D) developed enlarged and underactive bladders, accompanied by extreme ATP and ADP decrease and prominent adenosine increase in the LP but not in the lumen. Such observations lay the foundation for the present proposal, aimed at identifying probable causes for severe purine disbalance in the LP of the diabetic bladder that are likely linked to considerable voiding dysfunctions. In Aim 1, we will perform comprehensive assessments of (i) voiding functions and (ii) purine availability in LP and lumen during bladder filling in control and T2D mice. Aim 2 will determine (i) the role of s- ENTDs in accelerated hydrolysis of ATP causing increased formation of adenosine in the LP of the diabetic bladders, (ii) contribution of the AC-cAMP-AMPK signaling in s-ENTDs release, and (iii) distribution and cellular sources of s-ENTDs in the bladder wall. Aim 3 will define the roles of equilibrative nucleoside transporters ENT1 and ENT2 in disproportionate accumulation of adenosine in the LP of the diabetic bladder by investigating alterations of transmembrane and transurothelial nucleoside transport and cellular localization of ENTs. We will use an interdisciplinary approach, including analytical chemistry, protein biochemistry, and molecular biology techniques and genetic tools; in vivo and ex vivo evaluation of bladder function; and sonographic imaging in nondiabetic and diabetic mice and mice with specific gene deletions. Key mechanisms of ATP hydrolysis by s- ENTDs and regulation of their mechanosensitive release will be validated in bladders of Cynomolgus monkeys (Macaca fascicularis) to translate findings in mice to primate biology. The proposed preclinical, discovery- oriented research will advance our understanding of molecular mechanisms regulating availability of the biologically active adenyl purines ATP, ADP, and adenosine at their cellular and receptor targets in the bladder wall. This work has the potential to serve as foundational research for the development of novel therapeutics that could influence or prevent the development of bladder dysfunctions associated with T2D and obesity.