Hypothesis / aims of study
Urinary bladder dysfunction is frequent in diabetic patients and animal models thereof. An enlargement of the urinary bladder has consistently been demonstrated across >70 studies in a rat model of type 1 diabetes induced by injection of streptozotocin (STZ); this enlargement is fully reversed by treatment with insulin, whereas other anti-diabetic drugs have not been tested [1]. The sodium glucose cotransporter 2 inhibitor empagliflozin (EMPA) is an oral treatment primarily developed for the treatment of type 2 diabetes, but has also been found to improve glucose levels in patients with type 1 diabetes and animal models thereof. Therefore, we have tested effects of EMPA on bladder weight (primary outcome) and contraction and relaxation responses (secondary outcomes) in STZ-injected rats.
Study design, materials and methods
Female Sprague-Dawley rats (8-10 weeks old) were allocated to one of three groups (n = 20 each): control (CON), STZ-injected (50 mg/kg i.p.) and STZ-injected plus oral EMPA (30 mg/kg/d). EMPA was administered daily by oral gavage starting upon the first day after glucose levels of at least 300 mg/dl had been confirmed. After a total of 9-10 weeks, animals were killed and urinary bladder removed and weighed. Bladder strips were placed into organ baths and contraction in response to carbachol (10 nM – 30 µM) and to 50 mM KCl was recorded and expressed as mN/mg strip weight. Following wash-out, strips were re-exposed to KCl and when force of contraction had stabilized concentration-response curve to isoprenaline (1 nM – 30 µM), the β2-selective fenoterol (0.03 nM – 30 µM) and the β3-selective CL 316,243 (0.1 nM – 3 µM) were generated; relaxation in response to 10 µM forskolin was also assessed. Concentration-response curves were analyzed by curve fitting to derive estimates of -log EC50 and Emax from each strip. Data are shown as means ± SD of n animals and bar graphs overlaid with scatter plots. Groups are compared by effect sizes with their 95% confidence interval (CI).
Results
Model characterization: Blood glucose levels measured 9 weeks after start of treatment were increased in STZ rats, which was attenuated but not prevented by EMPA (Figure 1A; n = 12-16). End-of-study body weight was lowered by STZ and by STZ + EMPA treatment (Figure 1B; n = 16-19). Daily urine output as assessed in metabolic cage experiments was increased in STZ rats, which was attenuated in EMPA rats (Figure 1C; n = 6-9).
Primary outcome parameter: STZ rats exhibited a tripling of bladder weight (difference 193 mg [CI 126; 261]), which was attenuated but not abolished with EMPA (difference -100 mg [-24; -177]; Figure 1D; n = 16-19).
Secondary outcome parameters: Carbachol had similar potency in CON, STZ and EMPA rats (-log EC50: 5.86±0.21 vs. 6.00±0.14 vs. 6.07±0.16) but a slightly greater efficacy in STZ (Emax: 4.38±1.27 vs. 5.86±0.70 mN/mg; difference 1.48 [CI 0.20; 2.77]) but not in EMPA rats (3.91 mN/mg). Similar group differences were also observed for KCl responses (n = 6-7).
The potency (-log EC50) of the β3-adrenoceptor agonists was comparable across groups (for instance CON isoprenaline 7.03±0.29; fenoterol 5.95±0.58 and CL 316,243 7.45±0.29; n = 6-7). The efficacy (Emax) tended to be increased in the STZ and even further in the EMPA groups for all the agonists; relaxation responses to 10 µM forskolin comparable across groups (Figure 2).
Interpretation of results
Injection of STZ produced the expected changes in blood glucose, body weight, daily urine output and bladder weight; early intervention with EMPA attenuated but did not eliminate the blood glucose elevation and the bladder weight increase. Changes of contractile responses to carbachol or KCl and of relaxation responses to three β-adrenoceptor agonists and forskolin were only minor in the STZ group; however, interpretation of the organ bath findings is hampered by the chosen sample size in light of considerable variability within each group.