Astaxanthin attenuates early acute kidney injury following severe burns in rats by ameliorating oxidative stress and mitochondrial-related apoptosis

Abstract

Early acute kidney injury (AKI) is a devastating complication in critical burn patients, and it is associated with severe morbidity and mortality. The mechanism of AKI is multifactorial. Astaxanthin (ATX) is a natural compound that is widely distributed in marine organisms; it is a strong antioxidant and exhibits other biological effects that have been well studied in various traumatic injuries and diseases. Hence, we attempted to explore the potential protection of ATX against early post burn AKI and its possible mechanisms of action. The classic severe burn rat model was utilized for the histological and biochemical assessments of the therapeutic value and mechanisms of action of ATX. Upon ATX treatment, renal tubular injury and the levels of serum creatinine and neutrophil gelatinase-associated lipocalin were improved. Furthermore, relief of oxidative stress and tubular apoptosis in rat kidneys post burn was also observed. Additionally, ATX administration increased Akt and Bad phosphorylation and further down-regulated the expression of other downstream pro-apoptotic proteins (cytochrome c and caspase-3/9); these effects were reversed by the PI3K inhibitor LY294002. Moreover, the protective effect of ATX presents a dose-dependent enhancement. The data above suggested that ATX protects against early AKI following severe burns in rats, which was attributed to its ability to ameliorate oxidative stress and inhibit apoptosis by modulating the mitochondrial-apoptotic pathway, regarded as the Akt/Bad/Caspases signalling cascade.

Figure 1

Histological and serum evaluations of renal function in the early stages after severe burn and ATX application. Representative HE-stained images of rat renal tissues are shown for all groups (n = 12 per group) at a magnification of 200× (A). Furthermore, the tubular damage scores provided quantitative verification (n = 8 per group) (B). Random-tested serum Cr (C) and NGAL (D) levels showed similar substantial elevations in the burn and vehicle groups at 24 h post burn, which indicated burn-induced early renal dysfunction, and ATX showed a dose-dependent effect on decreasing burn-induced elevations in serum Cr and NGAL levels (n = 8 per group). The results are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, vs. sham; ^# p < 0.05, ^## p < 0.01, vs. burn + vehicle; ^ns p > 0.05.

Figure 2

Assessment of oxidative stress and inner antioxidant enzyme levels in rat kidneys post burn and ATX application. After the burn insults, the levels of ORP and MDA in the burn and vehicle groups increased significantly, whereas the activities of the inner antioxidant enzymes (SOD and CAT) decreased remarkably. Following various doses of ATX, a marked reduction in the ORP and MDA levels and elevated activities of inner antioxidant enzymes were observed in the 20 mg/kg groups. The sample size was n = 8 for each group. The results are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, vs. sham; ^# p < 0.05, ^## p < 0.01, vs. burn + vehicle; ^ns p > 0.05.

Figure 3

Effect of ATX on tubular apoptosis in rats post burn. Representative TUNEL stained images are shown for the various groups (A). The burn insults caused significant increases in green-fluorescent labelled nuclei in renal tubular cells of both burn and vehicle groups. The administration of ATX at different doses significantly decreased the number of green-fluorescent labelled nuclei post burn (B). LY294002 abolished the protective effect of ATX on apoptotic tubular cells (B). The indices of apoptosis in the different groups were consistent with the TUNEL staining results. The most effective ATX dose was 20 mg/kg. The sample size was n = 8 for each group. The results are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, vs. sham; ^## p < 0.01, vs. burn + vehicle; ^@@ p < 0.01, vs. burn + ATX 20 mg/kg group; ^ns p > 0.05.

Figure 4

Effect of ATX on the distribution and protein expression of p-Akt and p-Bad in the kidneys of burned rats. As shown in (A,B), representative graphs show that severe burn insults resulted in reactive elevations in p-Akt or p-Bad positive staining in renal tubular cells as a protection against early AKI, compared with the sham group (n = 12 per group). With changes in the ATX doses, the number of positive-stained tubular cells increased further (n = 12 per group). The results of western blotting exposed similar tendencies of p-Akt or p-Bad protein expression post burn or ATX application (C–E) (n = 6 per group). The results are expressed as the mean ± SD. ** p < 0.01, vs. sham; ^## p < 0.01, vs. burn + vehicle; ^ns p > 0.05.

Figure 5

Expression changes in mitochondria pro-apoptotic proteins post burn with ATX administration. Although the expression of Bcl-xL decreased significantly post burn, all doses of ATX did not increase declined Bcl-xL levels (A,B). With regard to cytochrome c and cleaved caspase-3/9, burn insults caused obvious elevations in their protein expression in both burn and vehicle groups (A,C–E). ATX administration remarkably reduced the changes of protein expression, and the 20 mg/kg dose was the most effective (A,C–E). The sample size was n = 6 for each group. The results are expressed as the mean ± SD. ** p < 0.01, vs. sham; ^## p < 0.01, vs. burn + vehicle; ^ns p > 0.05.

Figure 6

Effect of LY294002 on Akt and Bad phosphorylation in the kidneys of severe-burned rats with ATX management. Representative staining images of burn + 20 mg/kg ATX and burn + 20 mg/kg ATX + LY294002 groups are shown in (A) (n = 12 per group). With LY294002 pretreatment, the distribution of both p-Akt and p-Bad stained tubular cells decreased substantially. The results of western blotting indicated that LY294002 significantly reduced ATX-induced phosphorylation of p-Akt and p-Bad (n = 6 per group) (B–D). The results were expressed as the mean ± SD. ** p < 0.01.

Figure 7

Effect of LY294002 on the renal expression of mitochondria pro-apoptotic proteins after ATX administration. The representative results of western blotting are shown in (A) (n = 6 per group). The analytic outcomes show that LY294002 pretreatment significantly increased the ATX-decreased protein expression of Bcl-xL (B); cytochrome c (C); and cleaved caspase 3/9 (D,E), compared with those of the burn + 20 mg/kg ATX group. The results are expressed as the mean ± SD. ** p < 0.01.

Figure 8

Schematic diagram of the potential mechanisms of burn-induced AKI and the routes by which ATX exerts its effects. ATX = astaxanthin.

Figure 9

Schematic illustration of the experimental design and animal group classification. ATX = astaxanthin, Cr = creatinine, iv = intravenous injection, NGAL = neutrophil gelatinase-associated lipocalin, TUNEL = terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling.