SGLT2 Inhibitor Empagliflozin and DPP4 Inhibitor Linagliptin Reactivate Glomerular Autophagy in db/db Mice, a Model of Type 2 Diabetes

Abstract

Recent data have indicated the emerging role of glomerular autophagy in diabetic kidney disease. We aimed to assess the effect of the SGLT2 inhibitor empagliflozin, the DPP4 inhibitor linagliptin, and their combination, on glomerular autophagy in a model of type 2 diabetes. Eight-week-old male db/db mice were randomly assigned to treatment with empagliflozin, linagliptin, empagliflozin-linagliptin or vehicle for 8 weeks. Age-matched non-diabetic db/+ mice acted as controls. To estimate glomerular autophagy, immunohistochemistry for beclin-1 and LAMP-1 was performed. Podocyte autophagy was assessed by counting the volume density (Vv) of autophagosomes, lysosomes and autolysosomes by transmission electron microscopy. LC3B and LAMP-1, autophagy markers, and caspase-3 and Bcl-2, apoptotic markers, were evaluated in renal cortex by western blot. Vehicle-treated db/db mice had weak glomerular staining for beclin-1 and LAMP-1 and reduced Vv of autophagosomes, autolysosomes and lysosomes in podocytes. Empagliflozin and linagliptin, both as monotherapy and in combination, enhanced the areas of glomerular staining for beclin-1 and LAMP-1 and increased Vv of autophagosomes and autolysosomes in podocytes. Renal LC3B and Bcl-2 were restored in actively treated animals. LAMP-1 expression was enhanced in the empagliflozin group; caspase-3 expression decreased in the empagliflozin-linagliptin group only. Mesangial expansion, podocyte foot process effacement and urinary albumin excretion were mitigated by both agents. The data provide further explanation for the mechanism of the renoprotective effect of SGLT2 inhibitors and DPP4 inhibitors in diabetes.Int. J. Mol. Sci. 2020, 21, 2987 2 of 22 dedifferentiation. In its turn, the loss or damage of podocytes causes dysfunction of the filtration barrier and increases albuminuria [2]. Some recent studies have indicated an emerging role of autophagy downregulation in diabetic podocytopathy [4][5][6]. Autophagy is a cellular recycling process involving self-degradation and reconstruction of damaged organelles and proteins [6]. The process is vital for highly differentiated post-mitotic cells, such as neurons and podocytes [7]. A growing body of evidence indicates a critical role of autophagy in maintaining podocyte integrity and renal function [6]. Mice with podocyte-specific deletion of autophagic regulators, such as class III PI3K vacuolar protein sorting 34 (Vps34) and the Atg5 gene, develop early proteinuria, progressive glomerulosclerosis, and renal failure [6]. Accordingly, autophagy is considered a potential therapeutic target for renal protection [8,9].Sodium-glucose cotransporter-2 (SGLT2) inhibitors and dipeptidylpeptidase-4 (DPP4) inhibitors are promising antidiabetic agents introduced into clinical practice in the last decade. The antihyperglycemic effect of SGLT2 inhibitors is mediated by increment of glucosuria, while DPP4 inhibitors realize their activity through an increase in the half-life of incretin hormones. Both SGLT2 and DPP4 inhibitors demonstrated renal protective...

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

“…Thus, podocytes could be considered direct targets of SGLT2 inhibitors. We have shown recently that EMPA reactivated autophagic flux in the podocytes of db/db mice[ 46 ]; the promotion of autophagy could be an explanation of the renal protective effect of SGLT2 inhibitors[ 46 , 47 ].…”

Current Challenges and Future Perspectives of Renal Tubular Dysfunction in Diabetic Kidney Disease

“…decreased sodium uptake by Na + /H + exchanger isoform 3 (NHE3) expression in proximal convoluted tubules (PTs) (128-133) reduced urinary excretion of angiotensin II and angiotensinogen levels in SGLT2 inhibitor-treated T2DM rats (134) did not further activate RAS in the long term, which prevented the RAS-mediated aggravation of cardiovascular and renal events (134,135) reduced urinary angiotensinogen excretion in patients with T2DM (136) increased urinary angiotensinogen excretion in patients with T1DM (137, 138) modulated the tubular expression of proteins governing the medullary concentration activity, further had an effect on fluid and electrolyte balance (139,140) (132) blocked the activation of the apoptotic-associated protein within PT cells (141) glomerular fibrosis or injury was not alleviated in SGLT2-knockout diabetic mice (142) modulated oxidative stress and intraglomerular inflammation and could thus alleviate renal fibrosis (143) alleviated the generation of vanin-1, the biomarker for oxidative stress within the kidney (144) lessened the epithelial-to-mesenchymal transition by modulating miR21 (145) alleviated renal fibrosis by lowering lipid accumulation-induced inflammation mediated by CD68 macrophages (146) activation of tubuloglomerular feedback: alleviated apoptosis by increasing autophagosomal formation within glomerular mesangial cells and podocytes (147,148) anti-inflammatory effects: decreased the levels of several cytokines such as tumor necrosis factora (TNFa), interleukin-6, high-sensitivity C-reactive protein, and leptin (149,150) restored oxygen supply, thereby alleviating the metabolic stress state in the mitochondria and restoring the hematocrit level in patients with DM (151,152) reduced ECM fibrosis by inflammation reduction and RAAS overactivation (153) the EPO-producing ability in patients with DM might be reversed after treatment with SGLT2i (154) suppressed HIF-1a-mediated metabolic switch from lipid oxidation to glycolysis in kidney tubule cells of diabetic mice.…”

Molecular Mechanisms of SGLT2 Inhibitor on Cardiorenal Protection

“…SGLT2i also alleviates apoptosis by increasing autophagosomal formation within glomerular mesangial cells and podocytes. Korbut et al revealed that, in db/db mice, SGLT2i alone or with a DPP-4 inhibitor restored the glomerular autophagosomal formation [ 106 ]. Han et al also demonstrated that SGLT2i, along with thiazolidinedione, could alleviate the glomerular tuft area and mesangial expansion by reducing angiotensinogen and type 1 cytokine [ 107 ].…”