Empagliflozin alleviates podocytopathy and enhances glomerular nephrin expression in db/db diabetic mice

Abstract

Background: Modern guidelines recommend sodium-glucose cotransporter-2 (SGLT2) inhibitors as the preferred antihyperglycemic agents for patients with type 2 diabetes and chronic kidney disease. However, the mechanisms underlying the renal protective effect of SGLT2 inhibitors are not fully understood.

Aim: To estimate the effect of the SGLT2 inhibitor, empagliflozin (EMPA), on the structure of podocytes and nephrin expression in glomeruli in db/db diabetic mice.

Methods: We treated 8-wk-old male db/db mice with EMPA (10 mg/kg/d) or vehicle for 8 wk. Age-matched male db/+ mice were included as non-diabetic controls. Parameters of body composition, glycemic and lipid control, and plasma concentrations of leptin, insulin and glucagon were assessed. We evaluated renal hypertrophy as kidney weight adjusted to lean mass, renal function as plasma levels of creatinine, and albuminuria as the urinary albumin-to-creatinine ratio (UACR). Renal structures were studied by light and transmission electron microscopy with a focus on mesangial volume and podocyte structure, respectively. Glomerular nephrin and transforming growth factor beta (TGF-β) were assessed by immunohistochemistry.

Results: Severe obesity and hyperglycemia developed in db/db mice prior to the start of the experiment; increased plasma concentrations of fructosamine, glycated albumin, cholesterol, leptin, and insulin, and elevated UACR were detected. Mesangial expansion, glomerular basement membrane thickening, and increased area of TGF-β staining in glomeruli were revealed in vehicle-treated mice. Podocytopathy was manifested by effacement of foot processes; nephrin-positive areas in glomeruli were reduced. EMPA decreased the levels of glucose, fructosamine and glycated albumin, UACR, kidney hypertrophy, mesangial expansion, glomerular basement membrane thickening, and glomerular TGF-β staining, alleviated podocytopathy and restored glomerular staining of nephrin.

Conclusion: These data indicate that EMPA attenuates podocytopathy in experimental diabetic kidney disease. The anti-albuminuric effect of EMPA could be attributed to mitigation of podocyte injury and enhancement of nephrin expression.

Keywords: Albuminuria; Chronic kidney disease; Diabetes; Empagliflozin; Podocyte; Sodium-glucose transporter 2 inhibitors.

Figure 1

Design of the experiment. The db/db diabetic mice were randomized into vehicle and empagliflozin groups. Heterozygous non-diabetic db/+ mice were included as controls. The studied drug or vehicle was administered by gavage once per day from 8 to 16 wk of age. Procedure 1 at wk 8: Weight measurement, body composition assessment, blood and urine sampling. Procedure 2 at wk 12: Weight measurement, body composition assessment, blood sampling. Procedure 3 at wk 16: Weight measurement, body composition assessment, blood and urine sampling, sacrifice and kidney sampling. EMPA: Empagliflozin.

Figure 2

Urinary albumin-to-creatinine ratio at week 0 and week 8 of the experiment in non-diabetic db/+ mice and diabetic db/db mice, treated with vehicle or empagliflozin. The data are presented as medians, lower and upper quartiles. ^aP < 0.001 vs non-diabetic control (db/+ mice); ^bP < 0.001 vs placebo(Mann-Whitney U-test); ^cP < 0.01 vs week 8 (Wilcoxon test). EMPA: Empagliflozin.

Figure 3

Podocyte foot processes. A: Non-diabetic db/+ mice; B: Vehicle-treated db/db diabetic mice demonstrate effacement of podocyte foot processes(FPs); C: Empagliflozin restores the structure of FPs in db/db mice. Transmission electron microscopy: ×100000.

Figure 4

Glomerular staining for nephrin. A:Non-diabetic db/+ mice; B: Vehicle-treated db/db mice; C: Empagliflozin-treated db/db mice; A-C: Immunohistochemistry with anti-nephrin antibody; D: Negative control.

Figure 5

Glomerular staining for TGF-β. A:Non-diabetic db/+ mice; B: Vehicle-treated db/db mice; C: Empagliflozin-treated db/db mice; A-C: Immunohistochemistry with anti-TGF-β antibody; D: Negative control.

Figure 6

Volumetric density of nephrin-positive areas and TGF-β-positive areas in glomeruli of nondiabetic db+ and diabetic EMPA-treated and vehicle-treated db/db mice. A: Nephrin-positive areas; B: TGF-β-positive areas. ^aP < 0.001 vs non-diabetic db/+ mice; ^bP < 0.01; ^cP < 0.001 vs vehicle treated db/db mice (Wilcoxon test). Vv: Volumetric density; TGF-β: Transforming growth factor β; EMPA: Empagliflozin.