Renoprotective Mechanism of Remote Ischemic Preconditioning Based on Transcriptomic Analysis in a Porcine Renal Ischemia Reperfusion Injury Model

Abstract

Ischemic preconditioning (IPC) is a well-known phenomenon in which tissues are exposed to a brief period of ischemia prior to a longer ischemic event. This technique produces tissue tolerance to ischemia reperfusion injury (IRI). Currently, IPC's mechanism of action is poorly understood. Using a porcine single kidney model, we performed remote IPC with renal IRI and evaluated the IPC mechanism of action. Following left nephrectomy, 15 female Yorkshire pigs were divided into three groups: no IPC and 90 minutes of warm ischemia (control), remote IPC immediately followed by 90 minutes of warm ischemia (rIPCe), and remote IPC with 90 minutes of warm ischemia performed 24 hours later (rIPCl). Differential gene expression analysis was performed using a porcine-specific microarray. The microarray analysis of porcine renal tissues identified 1,053 differentially expressed probes in preconditioned pigs. Among these, 179 genes had altered expression in both the rIPCe and rIPCl groups. The genes were largely related to oxidation reduction, apoptosis, and inflammatory response. In the rIPCl group, an additional 848 genes had altered expression levels. These genes were primarily related to immune response and inflammation, including those coding for cytokines and cytokine receptors and those that play roles in the complement system and coagulation cascade. In the complement system, the membrane attack complex was determined to be sublytic, because it colocalized with phosphorylated extracellular signal-regulated kinase. Furthermore, alpha 2 macroglobulin, tissue plasminogen activator, uterine plasmin trypsin inhibitor, and arginase-1 mRNA levels were elevated in the rIPCl group. These findings indicate that remote IPC produces renoprotective effects through multiple mechanisms, and these effects develop over a long timeframe rather than immediately following IPC.

Fig 1. Experimental protocol.

Blue cylinders represent the time zone of clamping and clear cylinders represent the declamping. Control, no IPC and 90 minutes of warm ischemia; rIPCe, 40 minutes of remote IPC immediately followed by 90 minutes of warm ischemia; and rIPCl, 40 minutes of remote IPC with 90 minutes of warm ischemia 24 hours later.

Fig 2. Hierarchal cluster analysis of 1,053 altered genes.

The genes were divided into two clusters via K-mean hierarchal cluster analysis. Cluster 1 genes are downregulated (green) in the rIPCe and rIPCl. Cluster 2 genes are upregulated in rIPCl. rIPCe, remote ischemic preconditioning immediately prior to ischemia (early); rIPCl, remote ischemic preconditioning followed by ischemia 24 hours later (late).

Fig 3. Venn diagrams of altered gene expression.

rIPCe, remote ischemic preconditioning immediately prior to ischemia (early); rIPCl, remote ischemic preconditioning followed by ischemia 24 hours later (late).

Fig 4. Major enriched gene ontology biological processes for the 179 genes with similar expression levels in the rIPCe and rIPCl groups.

Similar gene ontology terms were excluded to avoid repetition. rIPCe, remote ischemic preconditioning immediately prior to ischemia (early); rIPCl, remote ischemic preconditioning 24 hours followed by ischemia 24 hours later (late).

Fig 5. Major enriched gene ontology biological processes for the 848 genes with altered expression levels in the rIPCl group only.

Similar gene ontology terms were excluded to avoid repetition. rIPCl, remote ischemic preconditioning followed by ischemia 24 hours later (late).

Fig 6. Heatmap signature of suggested pathways by DAVID.

rIPCe, remote ischemic preconditioning immediately prior to ischemia (early); rIPCl, remote ischemic preconditioning followed by ischemia 24 hours later (late).

Fig 7. Genes in the complement system with altered expression levels.

The yellow boxes indicate upregulated gene expression (fold change ≥2 and p < 0.05). The red lines indicate inhibition.

Fig 8. Immunohistochemical analysis of pig renal tissue.

(A) C3c (green) and MAC (red) deposition was observed in confocal images of renal tissues, scale bar = 100 μm; DAPI, 4ʹ,6-diamidino-2-phenylindole (blue). (B) Relative fluorescence intensity of C3c and MAC staining was increased in rIPCe and rIPCl, *p < 0.05 vs. Control. (C) P-ERK IHC shows that ERK is activated in rIPCe and rIPCl, scale bar = 100 μm. (D) MAC (red) and P-ERK (green) colocalization was observed in confocal images of renal tissues, scale bar = 100 μm. (E) Pearson's coefficient for MAC and P-ERK colocalization (in 12 fields) was higher in rIPCe and rIPCl, *p < 0.05 vs. Control. (F) TUNEL staining showed fewer apoptotic renal tubular cells in the rIPCl group compared with Control, scale bar = 50 μm. MAC, membrane attack complex; P-ERK, phosphorylated extracellular signal-regulated kinase; rIPCe, remote ischemic preconditioning immediately prior to ischemia (early); rIPCl, remote ischemic preconditioning followed by ischemia 24 hours later (late); TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Fig 9. Genes in the coagulation cascade with altered expression levels.

Yellow boxes indicate upregulated genes (fold change ≥2 and p < 0.05). Green boxes indicate downregulated genes (fold change ≤-2 and p < 0.05). Red lines indicate inhibition.