Protective effects of rapamycin induced autophagy on acute kidney injury induced by sepsis / 中华危重病急救医学

ABSTRACT

Objective To investigate the protective effect of autophagy inducer rapamycin on acute kidney injury (AKI) induced by sepsis. Methods Twenty-four Sprague-Dawley (SD) male rats were randomly divided into sham group, caecal ligation and puncture (CLP) model group, and rapamycin treatment group (Rap treatment group), with 8 rats in each group. The septic AKI model was reproduced by CLP in rats, and rats in sham group were given appendix isolation without ligation and puncture. The rats in Rap treatment group were given 1.6 mg rapamycin by intraperitoneal injection immediately after model reproduction, and the rats in CLP model group were injected with an equal amount of normal saline. The rats in all groups were sacrificed after collecting peripheral blood specimen at 24 hours after model reproduction, and the levels of blood urea nitrogen (BUN) and serum creatinine (SCr) were determined. The pathomorphology change in renal tissue was observed under light microscope after periodic acid Schiff (PAS) staining. Real-time polymerase chain reaction (real-time PCR, RT-PCR) was used to determine the mRNA expressions of renal tubular autophagy related molecules Atg-5 and Beclin-1. Western Blot was used to detect the expressions of renal tubular autophagy associated protein microtubule labeled protein 1 light chain 3-Ⅱ (LC3-Ⅱ) and Beclin-1 as well as apoptosis protein cytochrome C (Cyt C), Bax and Bcl-2. TdT-mediated dUTP nick-end labeling (TUNEL) assay was used to determine the renal tubular epithelial cell apoptosis. Results Rapamycin could alleviate pathomorphology changes in rats with septic AKI, and decrease the levels of BUN and SCr. Compared with sham group, the expressions of Atg-5, Beclin-1 and LC3-Ⅱ in CLP model group were significantly increased [Atg-5 mRNA (2-ΔΔCt) 2.34±0.04 vs. 1.00±0.03, Beclin-1 mRNA (2-ΔΔCt) 1.40±0.02 vs. 1.00±0.03, LC3-Ⅱ protein (gray value) 0.82±0.03 vs. 0.45±0.04, Beclin-1 protein (gray value) 0.59±0.06 vs. 0.29±0.03, all P < 0.01]. Rapamycin could further up-regulate the expressions of Atg-5, Beclin-1, and LC3 Ⅱ [Atg-5 mRNA (2-ΔΔCt) 3.28±0.19 vs. 2.34±0.04, Beclin-1 mRNA (2-ΔΔCt) 2.38±0.08 vs. 1.40±0.02, LC3-Ⅱ protein (gray value) 1.11±0.07 vs. 0.82±0.03, Beclin-1 protein (gray value) 0.85±0.05 vs. 0.59±0.06, all P < 0.01]. Compared with sham group, the apoptotic cells in CLP model group were increased significantly [(34.49±10.45)% vs. (2.78±1.40)%, P < 0.01], Cyt C and Bax protein expressions were significantly up-regulated (gray value 0.87±0.02 vs. 0.46±0.03, 1.20±0.06 vs. 0.46±0.01, both P < 0.01), and Bcl-2 expression was significantly down-regulated (gray value 0.64±0.02 vs. 1.33±0.09, P < 0.01). Rapamycin could effectively inhibit cell apoptosis [(15.44±5.50)% vs. (34.49±10.45)%, P < 0.01] and the protein expressions of Cyt C and Bax (gray value 0.72±0.03 vs. 0.87±0.02, 0.84±0.03 vs. 1.20±0.06, both P < 0.01), and up-regulate the protein expression of Bcl-2 (gray value 0.77±0.04 vs. 0.64±0.02, P < 0.01). Conclusion The protective effect of rapamycin on renal tissue of rat with AKI induced by sepsis was depended on cell apoptosis inhibition through inducing and promoting cell autophagy.