Spleen Ischemia Conditioning Attenuates Renal Ischemia-reperfusion Injury In Rats

Remote ischemia preconditioning(RIPC) and Remote ischemia postconditioning (RIPO)areprotective measures to reduce ischemia-reperfusion injury of the target organs by interrupting the blood flow of a remote organ. Only few studies have investigated protective effects of spleen ischemia preconditioning and postconditioning and the mechanisms are not reported yet. This study investigated the protective effects and the mechanism of spleen ischemia conditioning on ischemia-reperfusion injury of rat kidneys.Part 1 Spleen ischemia conditioning reduces acute kidney injury of rat induced by ischemia-reperfusionObject:To investigate the protective effect of spleen ischemia conditioning on acute kidney injury of rat induced by ischemia-reperfusionMethods:Twenty-four adult male SD rats were divided into four groups randomly:(1) sham operation (sham):open the abdominal cavity and remove the right kidney; (2) ischemia-reperfusion (IR):the blood flow of the left kidney was interrupted for 45 minutes by occlusion of the left renal pedicle; (3) spleen ischemia preconditioning (sIPC):the spleen was subjected to 3 cycles of 5min reperfusion following 5min ischemia before the ischemia-reperfusion of the left kidney; (4) spleen ischemia postconditioning (sIPO):the spleen was subjected to 3 cycles of 5min reperfusion following 5min ischemia after the ischemia-reperfusion of the left kidney. Samples of blood and kidney tissue were collected 1 day postoperation. The levels of blood urea nitrogen(BUN) and creatinine(Cr) were assayed by using kits. The kidney tissues of rats were fixed and embedded for hematoxylin and eosin(HE) staining and the pathological changes of kidneys were semi-quantitative scored.Results:The levels of BUN and Cr of IR group significantly increased compared with sham group (P<0.05). sIPC and sIPO group showed significant decreases of BUN and Cr in 1 day postoperation (P<0.05). HE staining showed that the renal structure of sham group was normal. IR group showed more severe tubular necrosis, cast formation and inflammation cells infiltration than sIPCand sIPO group. The semi-quantitative scores of sIPCand sIPO group were significantly lower compared with IR group (P<0.05). No significant difference was shown between sIPC and sIPO group (P>0.05). Conclusion:Spleen ischemia conditioning can reduce acute kidney injury induced by ischemia-reperfusion.Part 2 Analysis on mechanisms of spleen ischemia conditioning attenuating renal ischemia-reperfusion injuryObject:To investigate the mechanisms of spleen ischemia conditioning attenuating renal ischemia-reperfusion injuryMethods:Twenty-four adult male SD rats were divided into four groups randomly:(1) sham operation (sham):open the abdominal cavity and remove the right kidney; (2) ischemia-reperfusion (IR):the blood flow of the left kidney was interrupted for 45 minutes by occlusion of the left renal pedicle; (3) spleen ischemia preconditioning (sIPC):the spleen was subjected to 3 cycles of 5min reperfusion following 5min ischemia before the ischemia-reperfusion of the left kidney; (4) spleen ischemia postconditioning (sIPO):the spleen was subjected to 3 cycles of 5min reperfusion following 5min ischemia after the ischemia-reperfusion of the left kidney. Blood and kidney tissues were collected at 3 days postoperation. The levels of TNF-a, IL-6 and IL-10 of blood were measured by ELISA kit. The expression of TNF-a, IL-6 and IL-10 in kidneys were assayed using Immunohistochemistry and Western blot.Results:The levels of blood TNF-a and IL-6 of IR group were significantly higher compared with sham group (P<0.05), and sIPC and sIPO group showed significant decrease in TNF-a and IL-6 (P<0.05). The result showed that there was a significant increase in blood IL-10 level of sIPC and sIPO group compared with sham group and IR group (P<0.05). IHC and Western blot showed that the expressions of TNF-a and IL-6 in rat kidney of spleen ischemia groups were decreased compared with IR group (P<0.05) and the expressions of IL-10 in spleen ischemia conditioning groups were significantly increased (P<0.05).Conclusion:Spleen ischemia conditioning attenuates renal ischemia-reperfusion injury by suppressing inflammation.Part 3 The potential mechanism of spleen ischemia conditioning inhibiting inflammationObject:To investigate the mechanism of spleen ischemia inhibiting inflammation for protection against renal ischemia-reperfusion injuryMethods:Twenty-four adult male SD rats were divided into four groups randomly:(1) sham operation (sham):open the abdominal cavity and remove the right kidney; (2) ischemia-reperfusion (IR):the blood flow of the left kidney was interrupted for 45 minutes by occlusion of the left renal pedicle; (3) spleen ischemia preconditioning (sIPC):the spleen was subjected to 3 cycles of 5min reperfusion following 5min ischemia before the ischemia-reperfusion of the left kidney; (4) spleen ischemia postconditioning (sIPO):the spleen was subjected to 3 cycles of 5min reperfusion following 5min ischemia after the ischemia-reperfusion of the left kidney. Peripheral blood and kidney tissues were collected at 3 days postoperation. IHC and Western blot were used to assay the expression levels of IKK-P and NF-Kb p50, p56. The CD3+CD4+lymphocyte rates in peripheral blood were assayed by flow cytometry.Results:Spleen ischemia groups including sIPC and sIPO group showed a significant decrease in IKK-? and NF-?b p50, p65 expression levels compared with IR group (P<0.05). No significant differences were found between sIPC and sIPO group (P>0.05). The CD3+andCD3+CD4+lymphocyte rates of sIPC and sIPO were showed a significant increase compared with IR group (P<0.05). Compared with sIPC, The CD3+and CD3+CD4+lymphocyte rates were increased significantly in sIPO group (P<0.05).Conclusion:Spleen ischemia conditioning has the capacity of inhibiting the IKK-NF-?b pathway for suppression of inflammation and influencing the phenotype of the immune cells.