Renal renin is upregulated by the combination treatment of empagliflozin and ras blockade in experimental diabetic nephropathy

Abstract Background and Aims Sodium-glucose cotransporter 2 (SGLT2) inhibitors have proven to delay diabetic nephropathy (DN) progression on top of renin-angiotensin system (RAS) blockade. This protection is mainly attributed to improvement in renal hemodynamics, although direct effects on the kidney cannot be ruled out. The present study aimed to identify renal proteins differentially expressed between vehicle-treated diabetic mice and mice treated with empagliflozin, ramipril, or their combination, that could help explain the protective mechanisms of the drugs. Method Twelve weeks old diabetic db/db mice were given empagliflozin (10 mg/Kg/day), ramipril (8 mg/Kg/day), or the combination of both drugs during 8 weeks. Vehicle-treated db/db and db/m mice were used as controls. Serum glucose, blood pressure, GFR, and albuminuria were measured at baseline and at the end of the study. After 8 weeks, mice were euthanized, and the kidneys and serum were saved. A differential high-throughput proteomic analysis by mass spectrometry using isobaric tandem mass tags (TMT labelling) was performed in kidney cortex. Results Vehicle-treated db/db mice showed increased glycemia during the whole experiment, and empagliflozin reduced blood glucose. Ramipril treatment decreased blood pressure. Vehicle-treated diabetic mice also showed incipient DN with increased mesangial matrix and albuminuria compared to their non-diabetic littermates. All the treatments reduced mesangial matrix expansion and albuminuria. Only 13 proteins were differentially expressed (false discovery rate <5% and Log2FC ≥1 or ≤ −1) when comparing treated mice vs vehicle-treated db/db mice. The differentially expressed proteins were only identified between the mice treated with the combination of empagliflozin and ramipril and the vehicle-treated diabetic mice. Ramipril or empagliflozin alone did not produce significant changes in renal proteins. Kidney renin was evidently increased by the combination therapy with empagliflozin and ramipril, along with the tubular transporter scaffolding protein MAP17. The results were further validated through renin staining and renal renin concentration measurement. Renal renin concentration was increased by ramipril and further increased by the combination therapy with empagliflozin and ramipril when compared to vehicle-treated db/db mice (55 pg/μg (IQR:49–64), 94 pg/μg (IQR:71–102), and 112 pg/μg (IQR 104–116) in the groups treated with vehicle, ramipril and the combination of empagliflozin and ramipril respectively). However, renin serum concentration was similar between mice treated with ramipril and mice treated with the combination. Conclusion The combined therapy of empagliflozin with ramipril upregulated renin in the kidney of a diabetic mouse model. The increase in kidney renin suggests that other mechanisms different from RAS act in the regulation of glomerular hemodynamics and arteriolar tone. Moreover, the increased sodium delivery to the macula densa does not inhibit renal renin secretion.