#451 The effects of early or late empagliflozin, liraglutide & combined therapy on the a-Klotho/autophagy pathway in renal and pancreatic-ß cells in diabetic db/db mice model

Abstract Background and Aims Type 2 diabetes mellitus (T2DM) can lead to kidney and pancreatic cells damage, causing diabetic nephropathy (DN) and pancreatic failure, receptively. The sodium-glucose transporter type 2 inhibitors, like Empagliflozin (EMPA) and glucagon-like peptide-1 receptor agonist, like Liraglutide (LIRA) are new T2DM medications which prevent diabetes progression and improve renal and pancreas function. However, their exact mechanisms of action are not fully understood. It is known that α-Klotho and autophagy proteins play a key role in tubule-glomerular injury, have anti-oxidative, and anti-inflammatory effects. We hypothesized that EMPA, LIRA and their combination will attenuate T2DM progression, and preserve renal and pancreatic structure and function, by affecting the α-Klotho and autophagy pathway. Method We used BKS.Cg-Dock7m +/+Leprdb/J (db/db) leptin-deficiency mutated T2DM mouse model. These mice exhibit T2DM within 4–5 weeks post-partum, characterized with blood glucose level >300 mg/dL, and considerable obesity (>35 gr). EMPA (30 mg/kg/day) was administered through drinking water, while LIRA (0.2 µg/gr/day) was administered subcutaneously. Blood glucose was measured weekly by stick, and 24 hrs urine collection took place three times along the experiment for urine albumin and electrolytes excretion, and urine glucose and creatinine. Mice were treated either at 7-weeks-old or 16-weeks-old mice for early phase or late phase, respectively, for four weeks at each phase, followed by sacrifice and organ harvesting. At sacrifice renal and pancreas samples were collected for histological and biochemical analyses. Results The preliminary results show that only EMPA+LIRA combination attenuated mice weight gain, while both EMPA and EMPA+LIRA combination reduced blood glucose levels. At the early phase, EMPA and the EMPA+LIRA combination, increased water intake, and reduced urine production. LIRA alone, attenuated water intake, decreased urinary glucose level, but did not affect urine production or urine protein level. At the Late phase, all treatments increased water intake, and reduced urine glucose level, but only the EMPA+LIRA attenuated urine production, and none affected protein urine level. All treatments moderated glucose tolerance test results. 2. Histological T2DM pancreas samples display characteristic pathological changes, including altered islets’ structure and possible signs of inflammation. 3. α-Klotho, LC3 and ATG5 immunofluorescence staining of renal tissue show reduced expression compared to the healthy specimens. LIRA prevented α-Klotho and LC3 expression reduction, while EMPA preserved ATG5 expression, in T2DM mice kidneys. Conclusion There are different effects for EMPA, LIRA and their combination when given at different time points (early phase vs. late phase). At early phase, EMPA and EMPA+LIRA combination significantly reduces T2DM mice blood glucose level and proteinuria (Microalbuminuria). At late phase all treatments affected urinary glucose level. EMPA, LIRA and their combination upregulate α-Klotho, LC3 and ATG5 proteins renal expression. These treatments alone or in combination, may delay the onset of hyperglycaemia and T2DM, preserve renal structure, and function, thus may potentially use to prevent T2DM complications such as DN, probably through their effect on both α-Klotho and autophagy pathway.