Evaluation of the reno-protective effects of empagliflozin in diabetic nephropathy using in vivo imaging

Background: Diabetic nephropathy (DN) is one of the most common vascular complications associated with diabetes mellitus. In the EMPA-REG outcome trial Empagliflozin (Empa), a sodium-glucose cotransporter 2 inhibitor, not only reduced the risk of cardiovascular events but also slowed the progression of DN in patients with type 2 diabetes. Improvement of glomerular hyperfiltraion via the tubulo-glomerular feedback (TGF) mechanism is considered one of the possible reasons for renal protection by Empa in DN. However, the theory remains a matter of speculation. In this study, the in vivo multiphoton microscope (MPM) imaging technique was used to investigate the glomerular protective effects of Empa and whether Empa regulated the vascular tonus of the glomerular afferent artery via the TGF mechanism Methods: We used C57BL/6 mice and spontaneously diabetic Ins2Akita mice (Akita). In the first experiment, mice were treated with Empa (20mg/kg/day; gavage) and insulin (0.1U/body; i.s.) for four weeks. Glomerular reactive oxygen species (ROS) and nitric oxide (NO) production were evaluated in an ex vivo study. Next, we measured a single nephron GFR (SNGFR) using MPM in each group. Furthermore, the change of the SNGFRs in the same glomeruli before and two hours after medication were also studied to evaluate the acute effect of Empa. Finally, an nNOS inhibitor and a COX2 inhibitor were administered to these groups to inhibit vasodilator factors delivered from the macula densa. The SNGFR was then evaluated Results: Increased ROS and decreased NO productions in the glomeruli were observed in the Akita, but they were improved with the Empa treatment. The SNGFR in the Akita was significantly higher than in the control, however, Empa improved that alteration. In the acute study using Akita, the SNGFR in the same glomeruli were significantly reduced by Empa with blood glucose level lowering. Moreover, the diameter of the afferent artery was significantly decreased after the Empa treatment. An nNOS inhibitor and a COX2 inhibitor reduced the SNGFR in the Akita, and no additional effect regarding Empa was observed. These data indicate that Empa can reduce glomerular hyperfiltration in DN via TGF Conclusions: Empagliflozin improved glomerular hyperfiltration in DN via the TGF mechanism and contributed to glomerular protection.