Evaluation of Glomerular Hemodynamic Function by Empagliflozin in Diabetic Mice Using In Vivo Imaging

Background: Sodium glucose cotransporter 2 inhibitors may reduce kidney hyperfiltration, thereby preventing diabetic kidney disease progression, which may in turn reduce cardiovascular risk, including heart failure. However, the mechanisms that regulate renal function responses to sodium glucose cotransporter 2 inhibition are not yet fully understood. We explored the renal protective effects of sodium glucose cotransporter 2 inhibition with empagliflozin, with a focus on glomerular hemodynamic effects and tubuloglomerular feedback using in vivo multiphoton microscopy imaging techniques. Methods: C57BL/6 mice and spontaneously diabetic Ins2(+/Akita) mice were studied. The mice were treated with empagliflozin (20 mg center dot kg(-1)center dot d(-1)) and insulin for 4 weeks, and the single-nephron glomerular filtration rate was measured using multiphoton microscope. A neuronal nitric oxide synthase inhibitor (7-nitroindazole, 20 mg center dot kg(-1)center dot d(-1)) or a cyclooxygenase-2 inhibitor (SC58236, 6 mg/L), or an A1 adenosine receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine, 1 mg center dot kg(-1)center dot d(-1)) was administered to elucidate the mechanisms of tubuloglomerular feedback signaling and single-nephron glomerular filtration rate regulation. Results: The urinary excretion of adenosine, nitric oxide metabolites, and the prostanoid prostaglandin E2 was also quantified. The single-nephron glomerular filtration rate in the Ins2(+/Akita) group was higher than in controls (C57BL/6; 4.9 +/- 1.3 nL/min versus Ins2(+/Akita); 15.8 +/- 6.8 nL/min) and lower in Ins2(+/Akita) /empagliflozin to 8.0 +/- 3.3 nL/min (P<0.01). In vivo imaging also revealed concomitant afferent arteriolar dilation (P<0.01) and increased glomerular permeability of albumin in the Ins2(+/Akita) group. Empagliflozin ameliorated these changes (P<0.01). Urinary adenosine excretion in the Ins2(+/Akita)/empagliflozin group was higher than in Ins2(+/Akita) (Ins2(+/Akita); 3.4 +/- 1.4 nmol/d, Ins2(+/Akita)/empagliflozin; 11.2 +/- 3.0 nmol/d, P<0.05), whereas nitric oxide metabolites and prostaglandin E2 did not differ. A1 adenosine receptor antagonism, but not neuronal nitric oxide synthase or cyclooxygenase-2 inhibition, blocked the effect of empagliflozin on renal function. Empagliflozin increased urinary adenosine excretion and reduced hyperfiltration via afferent arteriolar constriction, effects that were abolished by A1 adenosine receptor blockade. Conclusions: Adenosine/A1 adenosine receptor pathways play a pivotal role in the regulation of the single-nephron glomerular filtration rate via tubuloglomerular feedback mechanisms in response to sodium glucose cotransporter 2 inhibition, which may contribute to renal and cardiovascular protective effects reported in clinical trials.