Ischemic Preconditioning Promotes Autophagy and Alleviates Renal Ischemia/Reperfusion Injury

Abstract

Autophagy is important for cellular survival during renal ischemia/reperfusion (I/R) injury. Ischemic preconditioning (IPC) has a strong renoprotective effect during renal I/R. Our study here aimed to explore the effect of IPC on autophagy during renal I/R injury. Rats were subjected to unilateral renal ischemia with or without prior IPC. Hypoxia/reoxygenation (H/R) injury was induced in HK-2 cells with or without prior hypoxic preconditioning (HPC). Autophagy and apoptosis were detected after reperfusion or reoxygenation for different time. The results showed that the levels of LC3II, Beclin-1, SQSTM1/p62, and cleaved caspase-3 were altered in a time-dependent manner during renal I/R. IPC further induced autophagy as indicated by increased levels of LC3II and Beclin-1, decreased level of SQSTM1/p62, and accumulation of autophagosomes compared to I/R groups at corresponding reperfusion time. In addition, IPC reduced the expression of cleaved caspase-3 and alleviated renal cell injury, as evaluated by the levels of serum creatinine (Scr), neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM-1) in renal tissues. In conclusion, autophagy and apoptosis are dynamically altered during renal I/R. IPC protects against renal I/R injury and upregulates autophagic flux, thus increasing the possibility for a novel therapy to alleviate I/R-induced acute kidney injury (AKI).

Figure 1

Hypoxia/reoxygenation (H/R) injury protocol. Control: cells were cultured constantly under normal conditions. H/R: cells received 15 h of oxygen and glucose deprivation (OGD) and 30 min, 1 h, 2 h, 6 h, or 12 h of reoxygenation in normal complete medium. Hypoxic preconditioning (HPC): transient OGD for 6 h and subsequent reoxygenation for 2 h in normal complete medium before prolonged H/R injury.

Figure 2

Autophagy induction during ischemia/reoxygenation (I/R) injury in rats with ischemic preconditioning (IPC). Male Sprague-Dawley rats were subjected to sham operation or 40 min of ischemia followed by 2 h, 6 h, 12 h, or 24 h of reperfusion with or without prior IPC, which consisted of four cycles of 8 min of clamping the left renal artery separated by 5 min of reperfusion. (a) Representative western blot gel documents and summarized data showing the levels of cleaved caspase-3, LC3II, and Beclin-1 protein expression. (b) Representative ultrastructure and semiquantification of autophagic vacuole formation in each group. N: nucleus; black arrowhead: autophagosome. Magnification ×20,000, scale bar = 1 μm. (c) Representative western blot gel documents and summarized data showing the levels of cleaved caspase-3, LC3II, Beclin-1, and SQSTM/p62 protein expression. The data are expressed as the mean ± SD. n = 3 per group. ^∗P < 0.05 versus the sham group. ^#P < 0.05 versus the I/R 6 h group. ^△P < 0.05 versus the I/R 24 h group. ^##P < 0.05 versus the I/R group at the corresponding time point. ⁺P < 0.05.

Figure 3

Ischemic preconditioning (IPC) alleviated renal ischemia/reoxygenation (I/R) injury. Male Sprague-Dawley rats were subjected to sham operation, IPC only, or 40 min of ischemia followed by 2 h, 6 h, 12 h, or 24 h of reperfusion with or without prior IPC. (a) Serum creatinine (Scr) concentrations. (b) Representative images of renal histology and quantitative analysis of tubular injury. Hematoxylin-eosin (H&E) staining. Representative slides of each group; ×200 magnification. (c) Representative images and quantitative data of terminal deoxynucleotidyl transferase-mediated nick-end labeling- (TUNEL-) positive nuclei in the kidney sections of each group. TUNEL staining; ×200 magnification. (d) Representative immunohistochemical images of tubular lesion in each group. ×400 magnification. KIM-1, kidney injury molecule-1; NGAL, neutrophil gelatinase-associated lipocalin. The data are expressed as the mean ± SD. n = 5 per group. ^∗P < 0.05 versus the sham group. ^#P < 0.05 versus the I/R group at the corresponding time point. ^△P < 0.05 versus the I/R 24 h group.

Figure 4

Hypoxic preconditioning (HPC) induced autophagy and reduced apoptosis in renal tubular cells. HK-2 cells were cultured under normal conditions or oxygen and glucose deprivation (OGD) for 15 h followed by reoxygenation in normal complete medium for 30 min, 1 h, 2 h, 6 h, and 12 h with or without prior HPC, which consisted of OGD for 6 h and subsequent reoxygenation for 2 h. ((a) and (b)) Representative western blot gel documents and summarized data showing the levels of cleaved caspase-3, LC3II, and Beclin-1 protein expression. (c) Determination and quantitative analysis of apoptotic cells by Annexin V-propidium iodide FACS analysis. (d) Renal tubular cell viability was evaluated by CCK-8 assay. The data are expressed as the mean ± SD. n = 3 per group. ^∗P < 0.05 versus the control group. ^#P < 0.05 versus the H/R 2 h group. ⁺P < 0.05.

Figure 5

Hypoxic preconditioning (HPC) promoted autophagic flux in renal hypoxia/reoxygenation (H/R) injury. Representative images showing LC3 staining in renal tubular cells of different groups infected with mRFP-GFP-LC3 adenovirus for 12 h and then exposed to H/R injury with or without HPC prior to it. The autophagosomes (APs) were represented by yellow puncta and autolysosomes (ALs) were represented by red puncta in merged images. The results were obtained from three independent experiments with at least 100 cells analyzed. The data are expressed as the mean ± SD. ^∗P < 0.05 versus the number of APs in the H/R 2 h group. ^#P < 0.05 versus the number of ALs in the H/R 2 h group.