Insulin alleviates mitochondrial oxidative stress involving upregulation of superoxide dismutase 2 and uncoupling protein 2 in septic acute kidney injury

Abstract

The aim of the present study was to explore the effects and mechanisms of insulin on mitochondrial oxidative stress in septic acute kidney injury (AKI). Male Sprague Dawley rats were divided randomly into four groups: Control group, sham surgery group, cecal ligation and puncture (CLP) group, and CLP plus insulin group. Blood specimens and kidney tissues were obtained at 12 and 24 h after surgery as separate experiments. Analyses of histology and indicators of renal injury [blood urea nitrogen (BUN) and serum creatinine (CRE) and neutrophil gelatinase-associated lipocalin (NGAL)], mitochondrial function [adenosine triphosphate (ATP) and mitochondrial membrane potential (MMP)], oxidative stress [inducible nitric oxide synthase (iNOS), reactive oxygen species (ROS) and nitric oxide (NO)], endogenous antioxidant systems [superoxide dismutase (SOD) and glutathione (GSH)] as well as the expression of uncoupling protein (UCP), PINK1 protein (a major mediator of mitophagy), PGC1α protein (a major regulator of mitochondrial biogenesis) were performed. Compared with CLP group, the CLP plus insulin group had milder histological damage, higher levels of ATP and MMP as well as lower levels of BUN, serum CRE and NGAL, intrarenal iNOS, mitochondrial ROS and total NO. Moreover, the CLP plus insulin group demonstrated increased expression of SOD2 and UCP2. In contrast, insulin administration suppressed mitophagy meanwhile did not upregulate total GSH and induce mitochondrial biogenesis following CLP. These findings indicated that the upregulation of SOD2 and UCP2 may be involved in insulin protecting against mitochondrial oxidative stress in septic AKI.

Keywords: acute kidney injury; endogenous antioxidant system; insulin; mitochondrial biogenesis; mitochondrial dysfunction; mitophagy; oxidative stress; sepsis; uncoupling protein 2.

Figure 1.

Effect of insulin (INS) on septic acute kidney injury. (A) Effect of INS on the levels of (a) serum neutrophil gelatinase-associated lipocalin (NGAL), (b) urea nitrogen and (c) creatinine. Results were expressed as mean ± SD. ^▲P<0.05 vs. control, ^□P<0.05 vs. cecal ligation and puncture (CLP) at same time point. (B) Effect of INS on sepsis-induced renal pathological changes. Panel a-g (H&E staining, magnification, ×400) represented control, sham surgery 12 h, sham surgery 24 h, CLP 12 h, CLP 24 h, CLP plus INS 12 h, CLP plus INS 24 h.

Figure 2.

Effect of insulin (INS) on sepsis-induced mitochondrial injury. Levels of intrarenal (A) adenosine triphosphate (ATP) content and (B) mitochondrial membrane potential (MMP). Results were expressed as mean ± SD. ^▲P<0.05 vs. control, ^□P<0.05 vs. cecal ligation and puncture (CLP) at the same time point. INS, insulin.

Figure 3.

Effect of insulin (INS) on markers of mitochondrial oxidative stress. The contents of mitochondrial (A) reactive oxygen species (ROS) and (B) nitric oxide (NO) as the activity of (C) intrarenal inducible nitric oxide synthase (iNOS) were expressed as mean ± SD. ^▲P<0.05 vs. control, ^□P<0.05 vs. cecal ligation and puncture (CLP) at the same time point.

Figure 4.

Effect of insulin (INS) on the expression of superoxide dismutase (SOD). The mRNA levels of (A) SOD1 and (B) SOD2 as well as the activity of (C) mitochondrial SOD2 were expressed as mean ± SD. ^▲P<0.05 vs. control, ^□P<0.05 vs. cecal ligation and puncture (CLP) at the same time point.

Figure 5.

Effect of insulin (INS) on the expression of glutathione (GSH). The mRNA levels of (A) glutathione synthetase (GSS) as well as intrarenal content of total (B) GSH and (C) oxidative GSH (GSSG) were expressed as mean ± SD. ^▲P<0.05 vs. control, ^□P<0.05 vs. cecal ligation and puncture (CLP) at the same time point.

Figure 6.

Effect of insulin (INS) on the expression of uncoupling protein 2 (UCP2). The mRNA levels of (A) UCP2 and the content of (B) intrarenal UCP2 were expressed as mean ± SD. ^▲P<0.05 vs. control, ^□P<0.05 vs. cecal ligation and puncture (CLP) at the same time point. (C) Immunohistochemical staining by using anti-UCP2 in brown. Nuclei are in blue. Panel a-g (magnification, ×400) represented control, sham surgery 12 h, sham surgery 24 h, CLP 12 h, CLP 24 h, CLP plus INS 12 h, CLP plus INS 24 h.

Figure 7.

Effect of insulin (INS) on the expression of PINK1. The mRNA levels of (A) PINK1 were expressed as mean ± SD. ^▲P<0.05 vs. control, ^□P<0.05 vs. cecal ligation and puncture (CLP) at the same time point. (B) Immunohistochemical staining by using anti-PINK1 in brown. Nuclei are in blue. Panel a-g (magnification, ×400) represented control, sham surgery 12 h, sham surgery 24 h, CLP 12 h, CLP 24 h, CLP plus INS 12 h, CLP plus INS 24 h.

Figure 8.

Effect of insulin (INS) on the mRNA levels of PGC1α. The results were expressed as mean ± SD. CLP, cecal ligation and puncture.

Figure 9.

Blood glucose levels in the four groups. Results were expressed as mean ± SD. ^▲P<0.05 vs. control, ^□P<0.05 vs. cecal ligation and puncture (CLP) at the same time point. INS, insulin.