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The targeted activation or inhibition of specific cell populations using chemogenetics allows the precise dissection of cellular signaling and function. Designer receptors exclusively activated by designer drugs (DREADDs) is a chemogenetic platform initially developed by mutating human muscarinic receptors to be unresponsive to endogenous acetylcholine but exclusively activated by an “inert” designer drug. Compound 21 (C21) is a new and potent DREADD agonist; however, radioligand assays from a recent report indicated its ability to bind to endogenous G protein-coupled receptors (GPCRs), including muscarinic M1–3 receptors. Whether this binding causes off-target effects is unclear. Renal innervation is important for the regulation of renal function, and the advent of chemogenetic tools provides significant opportunities for the mechanistic understanding of renal innervation and function. GPCRs such as adrenergic and muscarinic receptors play a role in renal function; thus, a careful pharmacological characterization of C21 in renal function is a prerequisite for this approach. Unexpectedly, an infusion of 1.0 mg/kg C21 in anesthetized mice caused an ∼4-fold increase in urine output and correspondingly increased the glomerular filtration rate (GFR), suggesting a C21-mediated acute diuretic effect. This acute diuresis effect was further confirmed in awake mice using voiding spot assays. The exact molecular mechanism for C21-mediated diuresis is unclear; however, we demonstrated by in vitro myography that C21 can effectively inhibit bladder smooth muscle contraction by antagonizing M3 receptors at the micromolar level, causing increased voiding size in vivo. In summary, C21 functions as a GPCR antagonist and has significant dose-dependent off-target effects in the renal system.

Lower urinary tract symptoms are extremely common in people with diabetes and obesity, but the causes are unclear. Furthermore, it has proven difficult to reliably demonstrate bladder dysfunction in diabetic mouse models, thus limiting the ability to gain mechanistic insights. Therefore, the main objective of this experimental study was to characterize diabetic bladder dysfunction in three promising polygenic mouse models of type 2 diabetes. We performed periodic assessments of glucose tolerance and micturition (void spot assay) for eight to twelve months. Males and females and high-fat diets were tested. NONcNZO10/LtJ mice did not develop bladder dysfunction over twelve months. TALLYHO/JngJ males were severely hyperglycemic from two months of age (fasted blood glucose ~550 mg/dL), while females were moderately so. Although males exhibited polyuria, neither they nor the females exhibited bladder dysfunction over nine months. KK.Cg-Ay/J males and females were extremely glucose intolerant. Males exhibited polyuria, a significant increase in voiding frequency at four months (compensation), followed by a rapid drop in voiding frequency by six months (decompensation) which was accompanied by a dramatic increase in urine leakage, indicating loss of outlet control. At eight months, male bladders were dilated. Females also developed polyuria but compensated with larger voids. We conclude KK.Cg-Ay/J male mice recapitulate key symptoms noted in patients and are the best model of the three to study diabetic bladder dysfunction.