Ann Surg Treat Res.  2017 Oct;93(4):209-216. 10.4174/astr.2017.93.4.209.

The optimal duration of ischemic preconditioning for renal ischemia-reperfusion injury in mice

• Choi HS
• Hwang JK
• Kim JG
• Hwang HS
• Lee SJ
• Chang YK
• Kim JI
• Moon IS

• KMID: 2392371
• DOI: http://doi.org/10.4174/astr.2017.93.4.209

Abstract

PURPOSE
The aim of the present study was to investigate the protective effects of ischemic preconditioning for different periods of time and to elucidate the optimal safe ischemic preconditioning time for renal ischemia-reperfusion (I/R) injury in mice.
METHODS
A total of 25 male C57BL/6 mice were randomly divided into 5 groups (sham, I/R, ischemic preconditioning [IP]-3, IP-5, and IP-7 groups), in which the kidney was preconditioned with IP of various durations and then subjected to I/R injury (the last 3 groups). To induce renal ischemia, the left renal pedicle was occluded with a nontraumatic microaneurysm clamp for 30 minutes followed by reperfusion for 24 hours. The effects of IP on renal I/R injury were evaluated in terms of renal function, tubular necrosis, apoptotic cell death and inflammatory cytokines.
RESULTS
Results indicated that BUN and creatinine (Cr) levels increased significantly in the I/R group, but the elevations were significantly lower in IP groups, especially in the IP-5 group. Histological analysis revealed that kidney injury was markedly decreased in the IP-5 group compared with the I/R group, as evidenced by reduced renal necrosis/apoptosis. In addition, IP significantly inhibited gene expression of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and chemokines (monocyte chemoattractant protein-1). Western blot analysis indicated that the expression levels of Toll-like receptor 4 (TLR4) and nuclear factor-kappa B (NF-κB) were upregulated in the I/R group, while expression was inhibited in the IP groups.
CONCLUSION
Five-minute IP had the greatest protective effect against I/R injury.

Keyword

Ischemic preconditioning; Renal Ischemia-reperfusion injury; TLR4/NF-κB pathway

MeSH Terms

Animals
Blotting, Western
Cell Death
Chemokines
Creatinine
Cytokines
Gene Expression
Humans
Ischemia
Ischemic Preconditioning*
Kidney
Male
Mice*
Necrosis
Reperfusion
Reperfusion Injury*
Toll-Like Receptor 4
Chemokines
Creatinine
Cytokines
Toll-Like Receptor 4

Figure

• 

  Fig. 1 Effects of ischemic preconditioning (IP) on ischemia-reperfusion (I/R)-induced acute kidney injury in mice. (A) Effects of IP on serum creatinine (Cr) and BUN levels after renal I/R injury. (B) Tubular damage was semiquantified by scoring H&E stained slides. (C) Representative images after hematoxylin and eosin (H&E) and Periodic acid-Schiff (PAS) staining of tissue taken 24 hours after I/R. Original magnification ×400. Data are presented as the mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the I/R group.

• 

  Fig. 2 Effects of ischemic preconditioning (IP) on ischemia-reperfusion (I/R)-induced apoptosis. (A) Kidney apoptosis was examined by transferase biotin-dUTP nick end labeling (TUNEL) staining (×400). (B) The level of apoptosis was expressed as the percent TUNEL-positive area. (C) The expression levels of cleaved caspase-3, Bax, and Bcl-2 were examined by Western blotting (one of three independent experiments). (D) Densitometric analysis of Western blot results. The density of each lane of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was divided by that of cleaved caspase-3, Bax, and Bcl-2. Data are presented as the mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the I/R group.

• 

  Fig. 3 IP attenuates inflammation induced by ischemia-reperfusion (I/R). Effects of ischemic preconditioning (IP) on mRNA expression of proinflammatory cytokines (A; TNF-α, B; IL-1β, and C; IL-6) and a chemokine (D; monocyte chemoattractant protein-1) were analyzed in ischemic kidneys by real-time RT-PCR analysis. Expression was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data are presented as the mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the I/R group.

• 

  Fig. 4 Effects of ischemic preconditioning (IP) on the protein levels of Toll-like receptor 4 (TLR4) and nuclear factor-kappa B (NF-κB). (A) Representative Western blotting for TLR4. (B) Quantification analysis of TLR4 protein expression compared with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference. (C) Western blotting for NF-κB p65 and (D) quantification analysis of NF-κB p65 protein expression with Histone H3.1 as the reference. Data are presented as the mean ± standard error of the mean. *P < 0.05, ***P < 0.001 compared with the I/R group.

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