Hyperbaric Oxygenation Protects Against Ischemia-Reperfusion Injury in Transplanted Rat Kidneys by Triggering Autophagy and Inhibiting Inflammatory Response

Abstract

BACKGROUND Ischemia-reperfusion injury (IRI) is a clinically common pathologic process defined as the inability to improve neuronal function. This study aimed to investigate the pathological mechanism of IRI and to explore effects of hyperbaric oxygenation (HBO) on autophagy and inflammatory response in IRI. MATERIAL AND METHODS Ninety Sprague-Dawley (SD) rats were randomly divided into a Sham group, a kidney transplant group (Trans), and a kidney transplant plus HBO treatment group (Trans+HBO). The kidney was harvested from the donor and transplanted to recipient rats according to a previously reported study. Rats were anesthetized using pentobarbital-natrium, and the kidney was resected and fixed in 4% paraformaldehyde. Serum creatinine (Scr) was detected using an automatic biochemical analyzer. The interleukin-6 (IL-6) level was assessed using enzyme-linked immunosorbent assay (ELISA). LC-3 was examined using indirect immunofluorescence assay and immunochemistry assay. LC-3 mRNA levels were analyzed using real-time PCR (RT-PCR). RESULTS The kidney transplant IRI model was successfully established. Scr and IL-6 levels were significantly increased in the Trans group (P<0.05). HBO significantly enhanced Scr and IL-6 levels. Scr was positively correlated with IL-6 levels (r-0.607, P<0.05). HBO increased LC-3 protein and mRNA expression in kidney-transplanted rats compared to the Sham and Trans group (P<0.05). Moreover, immunofluorescence assay also showed that LC-3 protein mainly distributes along renal tubular epithelial cells in a linear manner. CONCLUSIONS Autophagy dysfunction and inflammatory response after renal transplantation play important roles in processes of IRI. HBO treatment protects against the renal injury of IRI in renal tissues at the early stage, which may be triggered by the IL-6 pathway.

Figure 1

The donor kidney changes pre-reperfusion and post-reperfusion in the processes of kidney transplantation. (A) Donor rat kidney (white arrow). (B) Recipient rat after resecting the kidney. (C) Transplanted kidney in the body of recipient (white arrow).

Figure 2

Observation for Scr levels and IL-6 levels and their correlation. (A) Scr levels were examined by using an automatic biochemical analyzer. (B) IL-6 levels were detected using ELISA. (C) Correlation analysis between Scr levels and IL-6 levels. * P<0.05 represents Scr or IL-6 levels compared to the Sham group. ^# P<0.05 represents Scr or IL-6 levels compared to the Trans group. ^@ P<0.05 represents the Scr or IL-6 levels compared to reperfusion for 1 h.

Figure 3

Pathological changes in kidney tissues. (A) Pathological changes at reperfusion time of 1 h. (B) Pathological changes at reperfusion time of 3 h. (C) Pathological changes at reperfusion time of 5 h.

Figure 4

LC-3 expression in kidney examined by immunohistochemistry assay. (A) Immunohistochemistry analysis for the LC-3 expression in the 3 groups at reperfusion times of 1, 3, and 5 h. Magnification, 400×. (B) Statistical analysis for LC-3 expression. * P<0.05 represents LC-3 expression compared to the Sham group. ^# P<0.05 represents the LC-3 expression compared to the Trans group. ^@ P<0.05 represents the LC-3 expression compared to reperfusion for 1 h.

Figure 5

LC-3 expression in kidney examined by immunofluorescence assay. Immunofluorescence assay for the LC-3 expression in the 3 groups at reperfusion times of 1, 3, and 5 h. Magnification, 400×.

Figure 6

LC-3 mRNA expression in kidney examined by RT-PCR in the 3 groups at reperfusion times of 1, 3, and 5 h. * P<0.05 represents LC-3 mRNA expression compared to the Sham group. ^# P<0.05 represents LC-3 mRNA expression compared to the Trans group. ^@ P<0.05 represents LC-3 mRNA expression compared to reperfusion for 1 h.