Protective Role Of Hypoxia Inducible Factor-1 And Heme Oxygenase-1 In Rat Kidney Transplants From Ischemia/Reperfusion Injury

With current immunosuppressive protocols resulting in significant reduction in the rate of acute rejection, increased attention has been directed toward Ag-independent factors influencing allograft survival. Ischemia/reperfusion injury (IRI) remains a major problem in clinical transplantation. The short-term consequences of IRI may be delayed graft function (DGF) due to acute tubular necrosis. While in the long term, this may lead to increased immunogenicity of the graft, resulting in acute rejection or chronic renal graft dysfunction. Hypoxia inducible factor-1 (HIF-1) is a key oxygen-dependent transcription factor that up-regulates many protective genes against hypoxia-induced injury. One of HIF-1 's target gene, heme oxygenase-1 (HO-1), is a rate-limiting enzyme that catalyze the degradation of heme. An increasing number of reports suggest that endogenous HO-1 expression inhibits IRI associated with the heart, liver and small bowel transplantation. In this study we will explore the protective role of the endogenous overexpression of HO-1 induced by stable expression of HIF-1 in the renal microvascular endothelial cells from cold hypoxia/reoxygenation (HR) injury, and further in the rat kidney transplantation model from IRI.Part IMethods of Renal Microvascular Endothelial Cell Culture andEstablishment of Cell Model from Hypoxia/Reoxygenation InjuryObjective: To explore the methods of culturing renal microvascular endothelial cells,and establishing cell model from cold hypoxia/reoxygenation injury to mimic theischemia/reperfusion injury in vivo.Methods: After the highly purified glomeruli have been acquired by three steps grads filtrated net, the obtained renal microvascular endothelial cells were cultured in vitro. The renal microvascular endothelial cells were identified by immuno-histochetest for CD34 antigen, indirect immunofluorescence for factor Ⅷ-related antigen and flow cytometry method for CD31 antigen. The cell model from cold hypoxia/reoxygenation injury was established with the renal microvascular endothelial cells, which were cultured in the anoxia and cold condition first and thentransferred to normal condition.Results: The primary cells were identified as endothelium by the presence of factorVlll-related antigen, CD34 and CD31 antigen and by typical endothelial morphologyat confluence. 97.2+1.7% of these cells were shown to be CD34 antigen positive,and 96.3% ± 2.7% were CD31 antigen positive. The cell model from coldhypoxia/reoxygenation injury was established to mimic the ischemia/reperfusioninjury in vivo.Conclusion: Renal micro vascular endothelial cell can be cultured successfully invitro, and the cold hypoxia/reperfusion cell system was stably established.Part II Antiapoptotic and Immunoprotective Roles of Hypoxia Inducible Factor-1 andHeme Oxygenase-1 on Renal Microvascular Endothelial Cell from ColdHypoxia/Reoxygenation InjuryObjective: To explore the influence of hypoxia inducible factor-la (HIF-la) on the transcriptional regulation of heme oxygenase-1 in the renal glomerular microvascular endothelial cells (RMVEC) from cold hypoxia/reoxygenation (HR) injury, and test the hypothesis that HIF-1-induced-overexpression of HO-1 has an antiapoptotic and immunoprotective roles on the RMVEC from cold HR injury.Methods: Prolyl 4-hydroxylase inhibitor and cyclooxygenase enzymes-2 blocker were used to intervene the expression of HIF-la and HO-1. RMVECs were divided into 4 groups (control, cold HR, 3,4-DHB and NS-398 group) according to different intervention. Apoptosis of RMVEC was evaluated by Terminal deoxynucleotidyl transferase-mediated dUTP Nick End labeling assay and by flow cytometry method, while the expresson of activated Caspase-3 was tested by Western-blotting. Proliferative rate of lymphocytes stimulated by the RMVEC was measured by MTT test, while supernatant levels of IL-2 and ICAM-1 were detected by ELISA. Results:There is no significant statistical difference in the expression levels of HIF-la mRNA in RMVEC with different intervention. When compared with the normal cultures, cold HR injury significantly upregulated HIF-la (/><0.001) and HO-1 in both mRNA (PO.001) and protein (P=0.002) levels in RMVEC. Compared with cold HR group, the expression levels of HIF-la and HO-1 were increasedsignificantly in 3,4-DHB group; on the contrary, the mRNA and protein levels of HO-1 in NS-398 group were decreased significantly (PO.05).Compared with normoxic cultures, cold HR injury significantly upregulated the activated Caspase-3 expression level (P<0.001) and the percentage of apoptotic cells (18.3 ±6.1% vs. 4.0+1.0%, P=0.003). When compared with the cold HR group, the Caspase-3 expression level (P=0.005) and the percentage of apoptotic cells (9.3 + 2.9%, P- 0.028) in the 3,4-DHB group were significantly decreased; While the Caspase-3 expression level (PO.001) and the percentage of apoptotic cells (31.0 + 4.6%, P—0.005) in the NS-398 group group were significantly increased.When compared with normoxic cultures, cold HR injury significantly upregulated the supernatant levels of IL-2 (40.0 + 5.0 vs. 18.0±3.0pg, P=0.004), sICAM-1 (14.55 + 1.13 v.s.7.90+ 1.61ng, P=0.003) and the lymphocyte proliferative rate (0.191 +0.053 vs. 0.069 + 0.032, P=0.002).Compared with RMVEC from cold HR injury, the supernatant levels of IL-2 (25.7±6.1pg, P=0.032)> sICAM-1 (8.71 ± 1.32ng, P=0.006) and the lymphocyte proliferative rate (0.079 ±0.038, P=0.003) in the RMVEC pretreated with 3,4-DHB were significantly decreased. On the contrary, the supernatant levels of IL-2 (51.0+ ll.Opg, P=0.025^ sICAM-1 (20.33 +3.05ng, P =0.006) and the lymphocyte proliferative rate (0.268 ±0.079, P=0.038) in RMVEC pretreated with NS-398 were much increased than those in cold HR injury cells. Conclusion: In the RMVEC from cold HR injury, stable existence of HIF-la can induce endogenous overexpression of HO-1. HO-l's overexpression induced by HIF-1 has antiapoptotic and immunoprotective roles on the RMVEC from cold HR injury.Part III Overexpression of Heme Oxygenase-1 Induced by Hypoxia Inducible Factor-1Protects Rat Kidney Transplants from Ischemia/Reperfusion Injury Objective: To induce overexpression of HO-1 by stable existence of HIF-la in vivo; and testify its protective roles of HIF-1 and HO-1 overexpression in rat renal transplantation model from ischemia/reperfusion injury.Methods: 42 LEW male rats ( 8 to 10 weeks, body weight of 200 to 250 g) were divided into 4 groups randomly. 6 rats served as controls in control group. The...