SGLT2i and GLP1-RA exert additive cardiorenal protection with a RAS blocker in uninephrectomized db/db mice

Abstract

Introduction: Diabetic Kidney Disease (DKD) is the main cause of end-stage renal disease in the developed world. The current treatment of the DKD with renin-angiotensin system (RAS) blockade does not totally halt the progression to end stage kidney disease. Currently, several drugs have shown to delay DKD progression such as sodium-glucose co-transporter-2 inhibitors (SGLT2i) and glucagon-like-1 receptor agonists (GLP-1RA). We hypothesized that by combining several drugs that prevent DKD progression on top of RAS blockade a synergistic effect would be achieved in terms of cardiorenal protection. In the present study, we analysed if the combination of a RAS blocker (ramipril) with a SGLT2i (empagliflozin) and/or GLP-1RA (semaglutide) in a type 2 diabetic mouse model could have add-on effects in kidney and heart protection.

Methods: Male and female uninephrectomized type 2 diabetic db/db mice were treated with empagliflozin and/or semaglutide on top of ramipril during 8 weeks. During the study body weight, water and food intake were weekly monitored, glycaemia biweekly and albuminuria and glomerular filtration rate (GFR) before and after the treatment. At the end of the experiment, kidney and heart were isolated for histological and gene expression studies as well as for intrarenal RAS state assessment.

Results: Semaglutide combined with ramipril and/or empagliflozin significantly decreased albuminuria but only when combined with both compounds, semaglutide further decreased blood glucose, glomerular hyperfiltration in male mice and glomerular mesangial matrix expansion. In kidney, only the triple treatment with empagliflozin, semaglutide and ramipril reduced the expression of the proinflammatory and profibrotic genes ccl2 and TGFß1. In addition, the combination of empagliflozin and semaglutide on top of RAS blockade was superior in decreasing cardiomyocyte hypertrophy and heart fibrosis in db/db mice.

Discussion: Our results suggest that the combination of SGLT2i with GLP-1RA is superior in cardiorenal protection in DKD than the drugs administered alone on top of RAS blockade.

Keywords: chronic kidney disease (CKD); diabetes; diabetic kidney disease (DKD); glucagon-like-1 receptor agonists (GLP-1RA); renin-angiotensin system (RAS); sodium-glucose cotransporter 2 inhibitors (SGLT2i).

FIGURE 1

Metabolic parameters, blood pressure, and food intake after treatment in vehicle-treated db/m mice, vehicle-treated db/db mice, and db/db mice treated with empagliflozin, semaglutide, or their combination with ramipril. (A) 6-h fasting blood glucose measured in mg/dL. (B) Urine-to-blood glucose ratios were obtained simultaneously. (C) Systolic and (D) diastolic blood pressures measured after 8 weeks of treatment. (E) Water intake measured in mL/mouse per day. (F) Food intake measured in g/mouse per day. db/m: non-diabetic mice treated with vehicle. db/db: diabetic mice treated with vehicle. db/db EMP + RAM: diabetic mice treated with empagliflozin and ramipril. db/db SEM + RAM: diabetic mice treated with semaglutide and ramipril. db/db EMP + SEM + RAM: diabetic mice treated with empagliflozin, semaglutide, and ramipril. ^$ p < 0.05 vehicle-treated db/db mice compared to vehicle treated non-diabetic mice. * p < 0.05 db/db mice treated with empagliflozin, semaglutide, or their combination compared to vehicle-treated db/db mice.

FIGURE 2

Kidney function parameters: urine albumin-to-creatinine ratio and glomerular filtration rate (GFR). (A) Urine albumin-to-creatinine ratio measured in μg/g. (B) GFR measured in μL per 100 g of body weight per minute at the end of the treatment in all mice included in the experiment. After 8 weeks of treatment, GFRs were different in (C) female and (D) male mice. db/m: non-diabetic mice treated with vehicle. db/db: diabetic mice treated with vehicle. db/db EMP + RAM: diabetic mice treated with empagliflozin and ramipril. db/db SEM + RAM: diabetic mice treated with semaglutide and ramipril. db/db EMP + SEM + RAM: diabetic mice treated with empagliflozin, semaglutide, and ramipril. ^$ p < 0.05 vehicle-treated db/db mice compared to vehicle-treated non-diabetic mice. * p < 0.05 db/db mice treated with empagliflozin, semaglutide, or their combination compared to vehicle-treated db/db mice.

FIGURE 3

Kidney morphological studies and inflammatory and fibrotic kidney gene expression in vehicle-treated db/m mice, vehicle-treated db/db mice, and db/db mice treated with empagliflozin, semaglutide, or their combination with ramipril. (A) Kidney weight measured in mg. (B) Glomerular area and (C) representative microphotographs of PAS-stained glomeruli at ×400 magnification. (D) Glomerular mesangial matrix was also measured in PAS-stained kidney sections. (E) Representative microphotographs of WT-1 immunochemistry in glomeruli (×400 magnification) and (F) podocyte density measured through WT-1 staining. Kidney expression of (G) Ccl2 proinflammatory gene and (H) Tgfb1 profibrotic gene. db/m: non-diabetic mice treated with vehicle. db/db: diabetic mice treated with vehicle. db/db EMP + RAM: diabetic mice treated with empagliflozin and ramipril. db/db SEM + RAM: diabetic mice treated with semaglutide and ramipril. db/db EMP + SEM + RAM: diabetic mice treated with empagliflozin, semaglutide, and ramipril. ^$ p < 0.05 vehicle-treated db/db mice compared to vehicle-treated non-diabetic mice. * p < 0.05 db/db mice treated with empagliflozin, semaglutide, or their combination compared to vehicle-treated db/db mice.

FIGURE 4

Heart morphological studies in vehicle-treated db/mmice, vehicle-treated db/db mice, and db/db mice treated with empagliflozin, semaglutide, or their combination with ramipril. (A) Representative microphotographs of left ventricle (LV) cardiomyocytes (×400 magnification) for measuring and (B) cardiomyocyte size as area (μm2). (C) Representative picrosirius red staining images on a fast green background (×400) for measuring (D) collagen deposition. db/m: non-diabetic mice treated with vehicle. db/db: diabetic mice treated with vehicle. Heart expression of (E) Myh7, (F) Igfbp4, and (G) Fabp4 genes. db/db EMP + RAM: diabetic mice treated with empagliflozin and ramipril. db/db SEM + RAM: diabetic mice treated with semaglutide and ramipril. db/db EMP + SEM + RAM: diabetic mice treated with empagliflozin, semaglutide, and ramipril. ^$ p < 0.05 vehicle-treated db/db mice compared to vehicle-treated non-diabetic mice. * p < 0.05 db/db mice treated with empagliflozin, semaglutide, or their combination compared to vehicle-treated db/db mice.

FIGURE 5

Angiotensinogen and renin expression in kidney cortex of vehicle-treated db/m mice, vehicle-treated db/db mice, and db/db mice treated with empagliflozin, semaglutide, or their combination with ramipril. Kidney cortex mRNA expression of (A) angiotensinogen and (B) renin. (C) Renin immunochemistry representative glomerular images obtained at ×400 magnification and (D) renin staining area (μm²) measured in the same images. db/m: non-diabetic mice treated with vehicle. db/db: diabetic mice treated with vehicle. db/db EMP + RAM: diabetic mice treated with empagliflozin and ramipril. db/db SEM + RAM: diabetic mice treated with semaglutide and ramipril. db/db EMP + SEM + RAM: diabetic mice treated with empagliflozin, semaglutide, and ramipril. ^$ p < 0.05 vehicle-treated db/db mice compared to vehicle-treated non-diabetic mice. * p < 0.05 db/db mice treated with empagliflozin, semaglutide, or their combination compared to vehicle-treated db/db mice.

FIGURE 6

ACE2/ACE ratios in the kidney cortex and serum of vehicle-treated db/m mice, vehicle-treated db/db mice, and db/db mice treated with empagliflozin, semaglutide, or their combination with ramipril. (A) ACE2 and ACE mRNA expression and (B) ACE2/ACE activity ratios in the kidney cortex. (C) ACE2/ACE activity ratio in serum. Individual values of ACE and ACE2 gene expression and activity are shown in Supplementary Figure S2. db/m: non-diabetic mice treated with vehicle. db/db: diabetic mice treated with vehicle. db/db EMP + RAM: diabetic mice treated with empagliflozin and ramipril. db/db SEM + RAM: diabetic mice treated with semaglutide and ramipril. db/db EMP + SEM + RAM: diabetic mice treated with empagliflozin, semaglutide, and ramipril. ^$ p < 0.05 vehicle-treated db/db mice compared to vehicle-treated non-diabetic mice. * p < 0.05 db/db mice treated with empagliflozin, semaglutide, or their combination compared to vehicle-treated db/db mice.