**3. Pathophysiology of DM-Associated OAB**

The pathophysiology of DM-associated bladder dysfunction is multifactorial and time-dependent. From experimental and human studies, these changes can be a result of an alteration in the

physiology of the detrusor smooth muscle cells, bladder innervation, extracellular matrix, or urothelial dysfunction [14]. In studies of streptozocin (STZ)-induced acute diabetic rats, the up-regulation of M2 and M3 muscarinic biosynthesis in the urinary bladder could lead to increased reactivity to acetylcholine, which results in detrusor overactivity [15,16]. In rats with type 2 DM on a high-fat diet, compared with controls, the diabetic bladders were hypertrophied and had increased volume per void and detrusor muscle contractility to the exogenous addition of carbachol in the compensated stage [17]. Progression from the compensated to decompensated state mainly involves decreased contractility to muscarinic stimulation. In addition, the alternation of the biomechanical behavior of the bladder wall induced by diuresis or diabetes is another important indicator of diabetic bladder dysfunction. In STZ-induced acute diabetic rats, the bladder wall could undergo rapid time-dependent structural and compositional remodeling, mainly including decreased collagen, increased elastin, and a nonlinear stress–strain relationship, and mechanical anisotropy, with greater tissue compliance in the circumferential direction than in the longitudinal direction [18,19].

In a prospective study of 120 type 2 DM patients using simple questionnaires, OAB severity and diabetic peripheral neuropathy were significantly correlated [20]. This finding was similar to that in another study in which the OAB group of women with type 2 DM had a significantly greater mean 5 Hz current perception threshold test value at the big toe compared to diabetic women without OAB [21]. This finding indicated that the hyposensitivity of unmyelinated C fiber afferents at the distal extremities heralded the early stages of diabetic bladder dysfunction. These studies suggest that multiple factors contribute to the occurrence and progression of diabetic bladder dysfunction.

The alternations of the urothelial and underlying lamina propria have been reported that are associated with OAB and diabetic cystopathy. In STZ diabetic rats 9 weeks after onset, scanning electron microscopy showed defective urothelial cells present in the bladders compared with controls, indicating a significant breach of the urothelial barrier [22]. In these diabetic rats, about 20% of the epithelium showed cellular disruption and death within the mucosal lining and umbrella cell loss. In addition, DM had significantly upregulated urothelial gene expression and receptors mainly for glucose metabolism (aldose reductase and sorbitol dehydrogenase), cell survival, cell-signaling receptors (acetylcholine receptors AChR-M2 and AChR-M3, purinergic receptors P2X2 and P2X3), and cell death. The compromised barrier function and alterations in urothelial mechanosensitivity and cell signaling contributed to bladder overactivity.

The findings of the animal studies have been further corroborated by a human study. Bladder mucosa was biopsied from 19 DM-associated OAB patients, 14 OAB patients without DM, and 10 healthy controls [23]. Decreased expression of urothelial junction protein (E-cadherin and ZO-1) and increased urothelial inflammation (mast cells) were noted in the non-diabetic OAB and diabetic OAB patients. The P2X3 protein expression in DM-associated OAB patients was significantly greater than that in OAB patients without DM and controls. However, E-cadherin, mast cells, ZO-1, apoptotic cells, and M2 and M3 muscarinic proteins were comparable between the OAB patients with and without DM. These findings suggest that urothelial dysfunction and chronic urothelial inflammation contribute to the pathogeneses of OAB. However, DM does not aggravate the severity of urothelial inflammation in OAB patients.

#### **4. Diabetes and Bladder Inflammation**

Chronic inflammation plays a potential role in the pathogenesis of type 2 DM [24]. The possible mechanisms to explain insulin resistance in type 2 DM include oxidative stress, endoplasmic reticulum stress, lipotoxicity, and glucotoxicity. These cellular stresses may induce an inflammatory response or they are exacerbated by inflammation. The vicious cycle of chronic inflammation and related stresses is associated with several diabetic complications, including atherosclerosis, neuropathy, retinopathy, nephropathy, and cystopathy.

Accumulating evidence suggests the roles of several inflammation biomarkers in obesity-induced insulin resistance. Acute-phase proteins such as C-reactive protein (CRP) and pro-inflammatory cytokines (interleukin (IL)-1β and IL-6, and tumor necrosis factor-α), and chemokines are increased in obese and type 2 DM patients, and these markers are reduced when patients are engaged in an intensive lifestyle intervention causing body weight loss [25]. In an 11-year cohort study, the inflammatory biomarkers C-reactive protein and pro-adrenomedullin were independently associated with cardiovascular events and all-cause mortality in type 2 DM patients [26]. Additionally, compared with controls, elevated serum levels of tumor necrotic factor-α and decreased neuregulin-4 (a novel adipokine) were found in diabetic patients and correlated with the severity of diabetic peripheral neuropathy [27,28]. Yeniel et al. assessed atherosclerosis indicators and blood perfusion in the bladder necks in women with OAB. They found that the OAB severity correlated with systemic atherosclerosis and impaired vascular perfusion of the urinary bladder [29]. In diabetic mice, Inouye et al. found that Evans blue extravasation in bladder vessels, an index of peripheral and neurogenic inflammation, correlated with bladder dysfunction [30]. Furthermore, Xiao et al. showed that compared with controls and diuretic groups, diabetic mice have bladders with higher levels of nitrotyrosine (a biomarker of NO-dependent, reactive nitrogen species-induced nitrative stress) and Mn superoxide dismutase (representing the activity of free radical scavengers) [31].

Recently, Hughes Jr. et al. showed that the NLRP3 inflammasome, an intracellular sensor that detects endogenous danger signals and environmental irritants, can sense diabetic metabolites and induce inflammation implicated in diabetic complications and neurodegeneration [32]. Compared to NLRP3 genes of knocked out non-diabetic mice, NLRP3 genes of knocked out diabetic mice had a higher serum glucose level but similar voiding volume, voiding frequency, voiding efficiency, severity of bladder inflammation, bladder Aδ-fibers, and C-fibers density. Interestingly, bladder inflammation and bladder decompression in BOO rats can be inhibited by NLRP3 inhibitor glyburide which might be effective to treat diabetic bladder dysfunction via the similar pathway [33]. In addition, Szasz et al. proposed another possible mechanism of diabetic bladder dysfunction via Toll-like receptor 4 (TLR4) activation. Innate immune system activation via TLR4 leads to inflammation and oxidative stress which causes bladder hypertrophy and hypercontractility [34]. Unlike wild type streptozotocin mice, TLR4 knock out diabetic mice were protected from diabetes-induced bladder dysfunction despite similar levels of hyperglycemia. These evidences, taken together, suggest that inflammatory pathways could be a component of a strategy to prevent or control diabetes and its associated complications.
