The Preliminary Study Of Hyperbaric Oxygenation Treatment On Autophagy In Rat Transplanted Kidney

Objective: To investigate the mechanism of ischemia-reperfusion injury (IRI) and therole of hyperbaric oxygenation (HBO)treatment in renal IRI through autophagy andinflammation pathway, in order to provide a new theory and strategies for the preventionand treatment of clinical renal IRI. A homologous kidney transplantation rat model wasestablished in this study. HBO treatment was used in transplanted model, the level ofmicrotubule associated protein1light chain3(LC3) and IL-6in serum and kidney graftwere detected.Material and Methods: SPF Sprague-Dawley (SD) male rats were randomly dividedinto three groups：sham group, kidney transplantation group and kidney transplantation+HBO treatment group（HBO treatment group）. Each group was also divided into threesubgroups：1h,3h and5h after surgery in our study. Rats in kidney transplantation groupand HBO treatment group were received kidney transplantation;1-hour-HBO treatmentwas given in HBO treatment group after modeling immediately and the2nd and4thhourafter ischemia-reperfusion. Without kidney transplantation, rats in sham group werereceived right nephrectomy only. Rats in each group were killed at the1st,3rd and5thhourrandomly. Left kidney tissue and blood samples were taken. In order to observepathological change, HE stain was conducted in our study. Both mRNA and expression ofLC3were examined by immunohistochemistry, confocal laser scanning fluorescencemicroscopy and real-time quantitative PCR. The level of IL-6in serum was examined withenzyme-linked immunosorbent assay (ELISA).Results:1. Histopathological changes in kidney tissue: There is no significant damage in kidneytissue of Sham group. With neutrophil infiltration, glomerular in kidney transplantationgroup became larger after modeling. Expansion in tubular lumen, protein cast, red cast andswelling in tubular epithelial cells were observed in kidney transplantation group. For HBO treatment group, glomerular in graft tissue also became larger after modeling. Thelevel of neutrophil infiltration in graft tissue was very small. The damages includingswelling of tubular epithelial cell, expansion in tubular lumen, protein cast and red castwere mitigated.2. The changes in renal function: At each time point including the1st,3rd,5thhour aftermodeling, compared with the sham group, the levels of Serum creatinine(Scr) in kidneytransplantation group and HBO treatment group increased significantly (P<0.05).Meanwhile, there were no significant difference for Scr level between kidneytransplantation group and HBO treatment group at the1stand3rdhour after modeling(P>0.05). The level of Scr in HBO treatment group decreased significantly at the end of5thhour after modeling (P<0.05).There was no significant difference among the rats in Shamgroup (P>0.05). The level of Scr in kidney transplantation group increased significantlyfrom the end of1sthour to5thhour with time (P<0.05).The levels of Scr in HBO treatmentgroup at the end of3rdand5thhour were significant higher than the one at the end of1sthour (P<0.05). Between the2time points including the end of3rdand5thhour aftermodeling, there was no significant difference (P>0.05).3. The change of IL-6level in serum: At the end of1st,3rdand5thhour, compared withthe sham group, the level of IL-6in HBO treatment group was significant higher at eachtime point(P<0.05). There was no significant difference between HBO treatment group andkidney transplantation group at the end of1sthour (P>0.05), however, the level of IL-6inHBO treatment group increased significantly from the end of3rdhour to the end of5thhour(P<0.05). Compared with kidney transplantation group, the level of IL-6in HBO treatmentwas significant lower at the end of1sthour (P<0.05). There was no significant difference(P>0.05) between kidney transplantation group and HBO treatment group from the end of3rdhour to the end of5thhour. The level of IL-6increased significantly (P<0.05) from theend of3rdhour after modeling in both Sham group and HBO treatment group depend onthe time. There was no significant difference for IL-6among each time point in kidneytransplantation group (P>0.05).It was shown by correlation analysis that IL-6could be positively correlated with Scr level (r=0.607，P<0.05）.4. The change of LC3in kidney tissue:(1) The expression of LC3shown by immunohistochemistry assay: It is showed that LC3protein expressed mainly in renal tubular epithelial cells, with a small amount expressionin the glomerulus. It is hown by relative quantitative analysis though mean optical densitythat LC3expression could be peaked at the end of5thhour after modeling between HBOtreatment group and kidney transplantation group. However, there was no significantdifference from the end of1sthour to3rdhour after modeling. Compared with the shamgroup, the expression of LC3in kidney transplantation group increased slightly withoutsignificant difference (P>0.05).(2) The expression of LC3shown by confocal laser scanning fluorescence microscopy: Itis shown that green fluorescent spot considered as LC3expression could be distributedmainly in renal tubular epithelial cell.(3) the change of LC3mRNA: At the end of1st,3rdand5thhour, compared with the shamgroup, there was no significant difference for LC3mRNA in HBO treatment group at eachtime point (P>0.05). At the end of1sthour, there was no significant difference for LC3mRNA in both HBO treatment group and Sham group (P>0.05), however, the level of LC3mRNA in HBO treatment group increased significantly from the end of3rdhour to5thhourafter modeling (P<0.05).There was no significant difference between HBO treatment groupand kidney transplantation group for LC3mRNA at the end of3rdand5thhour aftermodeling. At the end of5thhour after modeling, the level of LC3mRNA in HBO treatmentgroup was significant higher than the one in kidney transplantation group (P<0.05). Forsham group and kidney transplantation group, there was no significant difference foramong each group from the end of1sthour to5thhour after modeling (P>0.05). The levelof LC3mRNA in HBO treatment group increased significantly from the end of1sthour to5thhour after modeling with time (P<0.05). Conclusions:1. Inflammatory response is one of the mechanisms involved in kidney IRI after kidneytransplantation;2. Basal level of autophagy presents in normal kidney tissue, autophagy level in grafttissue after IRI also remain at basal level. Combined with these data, it is suggested thatIRI may have inhibitory effects on autophagy;3. HBO treatment may reduce inflammatory response significantly at early stage afterIRI in renal tissue. HBO treatment may protect renal tissue from IRI, possibly, thoughup-regulating autophagy pathway. It could be one of the important mechanisms thoughwhich HBO treatment could reduce renal ischemia-reperfusion injury.4. HBO treatment may play a protective role at early stage of IRI in renal tissue though avariety of pathways depend on time segment, thus it is very important for HBO treatmentto be used in early stage in renal IRI.