The Role of NF-κB and Bax/Bcl-2/Caspase-3 Signaling Pathways in the Protective Effects of Sacubitril/Valsartan (Entresto) against HFD/STZ-Induced Diabetic Kidney Disease

Abstract

LCZ696 (valsartan/sacubitril) has the potential to slow the progression of diabetic kidney disease (DKD) according to previous reports. However, the renoprotective mechanism underlying LCZ696 remains unknown. This study aimed to investigate the therapeutic potential and underlying mechanism of LCZ696 in DKD in a type 2 diabetic (T2D) rat model. This model was established in this experiment by feeding a high-fat diet (HFD) for six weeks with a single dose of streptozotocin (STZ, 30 mg/kg body weight). Valsartan or LCZ696 was orally administered to T2D animals for eight weeks. HFD/STZ rats showed hyperglycemia, impaired insulin secretion, significant increases in urea, creatinine, cytokines, nuclear factor kappa B (NF-κB), oxidative stress, caspase-3 activity, glomerular and tubular damage, glomerulsclerosis, Bax and caspese-3 expressions along with a significant decline in IL-10, antioxidant markers, and Bcl-2 expression. The administration of LCZ696 to diabetic rats reduced the serum concentrations of glucose, urea, and creatinine. In addition, ELISA results demonstrated that diabetic rats treated with LCZ696 exhibited a reduction in inflammatory (IL-1β, TNF-α, IL-6) and an increase in anti-inflammatory (IL-10) cytokine levels. In addition, a notable decrease in NF-κB and caspase-3 activity was observed. At the level of renal tissue homogenate, diabetic animals treated with LCZ696 demonstrated clear restorations in GSH content and other antioxidant enzyme levels, in addition to a significant decrease in TBARS levels. In addition, LCZ696 inhibited the expression of the Bax and cleaved caspase-3 proteins and enhanced the expression of the Bcl-2 protein. Improvements in histopathological changes in kidney tissues confirmed and significantly supported these biochemical findings. In summary, LCZ696 alleviated DKD with possible mechanisms including inhibition of inflammation and apoptosis.

Keywords: LCZ696; apoptotic markers; diabetic kidney disease; inflammation; type 2 diabetes.

Figure 1

Diagram illustrating the experimental design.

Figure 2

The effects of LCZ696 and valsartan treatment on caspase-3 activity (A) and NF-κB (B) in HFD/STZ-induced diabetic rats. The data are expressed as the mean ± SEM, n = 6, *** p < 0.001 versus control; ^### p < 0.01 versus HFD/STZ. Abbreviations: NF-κB, nuclear factor kappa-B.

Figure 3

The effects of LCZ696 and valsartan administration on renal oxidative stress markers in HFD/STZ-induced diabetic rats, including thiobarbituric acid reaction substances (TBARs) (A) and glutathione (GSH) (B). The data are expressed as the mean ± SEM, n = 6, *** p < 0.001 versus control; ^## p < 0.01 versus HFD/STZ; ⁺ p < 0.05 versus valsartan.

Figure 4

The impact of LCZ696 and valsartan treatment on the renal antioxidant status of HFD/STZ-induced diabetic rats, including superoxide dismutase (SOD) levels (A), catalase (CAT) activity (B), glutathione peroxidase (GPx) (C), and glutathione-S-transferase (GST) (D). The data are expressed as the mean ± SEM, n = 6, *** p < 0.001 versus control; ^# p < 0.05, ^## p < 0.01 versus HFD/STZ.

Figure 5

Renal cortex photomicrographs of H&E-stained kidney sections from control (A), HFD/STZ-induced rats (B), valsartan-treated group (C), and LCZ696-treated animals (D). The renal cortex of the control group displays normal proximal convoluted tubules (PT), distal convoluted tubules (DT), Bowman’s capsule, and glomerulus structures (G). The renal cortex of animals given HFD/STZ exhibited enlargement of the urinary space, thickening of the basal membrane of the glomerulus (arrow), and infiltration of inflammatory mononuclear cells (head arrow). Glomeruli and renal tubules were significantly improved in the renal cortex of HFD/STZ rats treated with valsartan and LCZ696. Damages to the glomeruli (E) and tubules (F) in the kidneys of rats from different groups were assessed semi-quantitatively, data are presented as mean ± SEM, n = 6, *** p < 0.001 versus control; ^### p < 0.001 and ^## p < 0. 01 versus HFD/STZ; ⁺ p < 0.05 and ⁺⁺⁺ p < 0.001 versus valsartan. H&E, scale bar = 50 µm.

Figure 5

Renal cortex photomicrographs of H&E-stained kidney sections from control (A), HFD/STZ-induced rats (B), valsartan-treated group (C), and LCZ696-treated animals (D). The renal cortex of the control group displays normal proximal convoluted tubules (PT), distal convoluted tubules (DT), Bowman’s capsule, and glomerulus structures (G). The renal cortex of animals given HFD/STZ exhibited enlargement of the urinary space, thickening of the basal membrane of the glomerulus (arrow), and infiltration of inflammatory mononuclear cells (head arrow). Glomeruli and renal tubules were significantly improved in the renal cortex of HFD/STZ rats treated with valsartan and LCZ696. Damages to the glomeruli (E) and tubules (F) in the kidneys of rats from different groups were assessed semi-quantitatively, data are presented as mean ± SEM, n = 6, *** p < 0.001 versus control; ^### p < 0.001 and ^## p < 0. 01 versus HFD/STZ; ⁺ p < 0.05 and ⁺⁺⁺ p < 0.001 versus valsartan. H&E, scale bar = 50 µm.

Figure 6

Representative photomicrographs of glomeruli stained with periodic acid–Schiff (PAS, scale 50 µm) from control (A), HFD/STZ (B), valsartan-treated group (C), and LCZ696-treated animals (D). The dark purple color in the glomerulus is the extracellular matrix. In different groups, the area of the glomerular matrix was measured (E). Data are presented as mean ± SEM, n = 6, *** p < 0.001 versus control; ^### p < 0.001 versus HFD/STZ; ⁺⁺⁺ p < 0.001 versus valsartan.

Figure 6

Representative photomicrographs of glomeruli stained with periodic acid–Schiff (PAS, scale 50 µm) from control (A), HFD/STZ (B), valsartan-treated group (C), and LCZ696-treated animals (D). The dark purple color in the glomerulus is the extracellular matrix. In different groups, the area of the glomerular matrix was measured (E). Data are presented as mean ± SEM, n = 6, *** p < 0.001 versus control; ^### p < 0.001 versus HFD/STZ; ⁺⁺⁺ p < 0.001 versus valsartan.

Figure 7

The effects of LCZ696 or valsartan on renal apoptosis marks in HFD/STZ-induced diabetic rats. A representative photomicrograph of renal cortex showing Bax immunohistochemistry protein distribution in different study groups. (A) control, (B) HFD/STZ, (C) valsartan-treated group, and (D) LCZ696- treated group. Area percent of immunoreactivity of Bax were quantified (E), (n = 3); data are presented as mean ± SEM, *** p < 0.001 versus control; ^### p < 0.001 versus HFD/STZ; ⁺⁺ p < 0.01 versus valsartan (scale bar 50 µm).

Figure 7

The effects of LCZ696 or valsartan on renal apoptosis marks in HFD/STZ-induced diabetic rats. A representative photomicrograph of renal cortex showing Bax immunohistochemistry protein distribution in different study groups. (A) control, (B) HFD/STZ, (C) valsartan-treated group, and (D) LCZ696- treated group. Area percent of immunoreactivity of Bax were quantified (E), (n = 3); data are presented as mean ± SEM, *** p < 0.001 versus control; ^### p < 0.001 versus HFD/STZ; ⁺⁺ p < 0.01 versus valsartan (scale bar 50 µm).

Figure 8

The effects of LCZ696 or valsartan on renal apoptosis marks in HFD/STZ-induced diabetic rats. A representative photomicrograph of renal cortex showing Bcl-2 immunohistochemistry expression in different study groups. (A) control, (B) HFD/STZ, (C) valsartan-treated group, and (D) LCZ696-treated group. Area percent of immunoreactivity of Bcl-2 were quantified (E), (n = 3); data are presented as mean ± SEM, *** p < 0.001 versus control; ^## p < 0.01 and ^### p < 0.001 versus HFD/STZ; ⁺ p < 0.01 versus valsartan (scale bar 50 µm).

Figure 9

The effects of LCZ696 or valsartan on renal apoptosis marks in HFD/STZ-induced diabetic rats. A representative photomicrograph of renal cortex showing caspase-3 immunohistochemistry expression in different study groups. (A) control, (B) HFD/STZ, (C) valsartan-treated group, and (D) LCZ696- treated group. Area percent of immunoreactivity of caspase-3 were quantified (E), (n = 3); data are presented as mean ± SEM, *** p < 0.001 versus control; ^### p < 0.001 versus HFD/STZ; ⁺⁺ p < 0.01 versus valsartan (scale bar 50 µm).