Melatonin modulates endoplasmic reticulum stress and Akt/GSK3-beta signaling pathway in a rat model of renal warm ischemia reperfusion

Abstract

Melatonin (Mel) is widely used to attenuate ischemia/reperfusion (I/R) injury in several organs. Nevertheless, the underlying mechanisms remain unclear. This study was conducted to explore the effect of Mel on endoplasmic reticulum (ER) stress, Akt and MAPK cascades after renal warm I/R. Eighteen Wistar rats were randomized into three groups: Sham, I/R, and Mel + I/R. The ischemia period was 60 min followed by 120 min of reperfusion. Mel (10 mg/kg) was administrated 30 min prior to ischemia. The creatinine clearance, MDA, LDH levels, and histopathological changes were evaluated. In addition, Western blot was performed to study ER stress and its downstream apoptosis as well as phosphorylation of Akt, GSK-3β, VDAC, ERK, and P38. Mel decreased cytolysis and lipid peroxidation and improved renal function and morphology compared to I/R group. Parallely, it significantly reduced the ER stress parameters including GRP 78, p-PERK, XBP 1, ATF 6, CHOP, and JNK. Simultaneously, p-Akt level was significantly enhanced and its target molecules GSK-3β and VDAC were inhibited. Furthermore, the ERK and P38 phosphorylation were evidently augmented after Mel administration in comparison to I/R group. In conclusion, Mel improves the recovery of renal function by decreasing ER stress and stimulating Akt pathway after renal I/R injury.

Figure 1

Evaluation of lactate dehydrogenase activity in plasma (a), malondialdehyde concentration in tissue (b), and creatinine clearance (c). Results are expressed as mean ± SEM (n = 6 for each group).  ^∗ p < 0.05 versus Sham. ^# p < 0.05 versus I/R.

Figure 2

Representative histological photographs of kidney tissues from Sham (a), I/R (b), and Mel + I/R (c) groups (H&E) ×400. Semiquantitative assessment of renal necrosis among the different experimental groups using the Jablonski score (d). The arrows denote brush border loss, “H” denotes hemorrhage, and “L” denotes nuclei loss. Results are expressed as mean ± SEM (n = 6 for each group).   ^∗ p < 0.05 versus Sham.  ^# p < 0.05 versus I/R.

Figure 3

Western blot of GRP 78 (a), total and phosphorylated PERK (b), XBP-1 (c), and ATF6α (d). β-actin was used as a loading control. One representative blot of six independent experiments is shown at the top whereas densitometric analysis is shown at the bottom. Results are expressed as mean ± SEM.   ^∗ p < 0.05 versus Sham.   ^# p < 0.05 versus I/R.

Figure 4

Western blot of CHOP (a) and total and phosphorylated JNK (b). The β-actin was used as a loading control. One representative blot of six independent experiments is shown at the top whereas densitometric analysis is shown at the bottom. Results are expressed as mean ± SEM.   ^∗ p < 0.05 versus Sham.   ^# p < 0.05 versus I/R.

Figure 5

Western blot of total and phosphorylated Akt (a), total and phosphorylated GSK-3β (b), and total and phosphorylated VDAC (c). One representative blot of six independent experiments is shown at the top whereas densitometric analysis is shown at the bottom. Results are expressed as mean ± SEM.  ^∗ p < 0.05 versus Sham.  ^# p < 0.05 versus I/R.

Figure 6

Western blot of total and phosphorylated ERK (a) and total and phosphorylated P38 (b). One representative blot of six independent experiments is shown at the top whereas densitometric analysis is shown at the bottom. Results are expressed as mean ± SEM.  ^∗ p < 0.05 versus Sham.  ^# p < 0.05 versus I/R.