| Background and Objective: Evidence showed that the abnormal angiogenesis and chronic hypoxia in kidney were the important feature of diabetic nephropathy, which was associated with the imbalance of angiogenesis regulatory factors. Our previous study has confirmed that angiopoietin-1( Ang-1) can improve the abnormal expression of angiogenesis regulatory factors,and relieve the microvascular disease in diabetic kidney.In present study, Ang-1 and L-mimosine(L-mim),a inhibitor of hypoxia-inducible factor-1α(HIF-1α) degradation, were applied, and assessed for their effects on hypoxia and its related factors in diabetic rats with nephropathy.Methods: Recombinant adenovirus vector expressing Ang-1(Ad-Ang-1) was constructed and identified in our previous study. SD diabetic rat was built by intraperitoneal injection with streptozotocin(STZ). The experimental rats were divided into four groups: normal control group(Group N), diabetic group(Group D), Ang-1 + L-mim treatment group(Group AL, model rat was treated with both 3×108PFU Ad-Ang-1 via penis vein injection and 50mg/kg L-mim via intraperitoneal injection every other day at 8 week), and empty vector group(Group K, model rat was only treated with 3×108PFU adenovirus vector via a caudal vein injection at 8 week). At 12 and 16 weeks,the serum glucose, urea, creatinine and urinary protein were collected and observed.Their renal tissues was collected,the renal pathological changes was observed by conventional HE and PAS staining, levels of Ang-1, vascular endothelial growth factor(VEGF), HIF-1α, and heme oxygenase-1(HO-1) were evaluated by immunohistochemistry, fluorescence, western bloting, and RT-PCR. Hypoxic conditions in kidney was detected with intravenous Hypoxyprobe?-1 injection by immunohistochemistry.Results: 1.The serum glucose and 24-hour urinary protein in three diabetic modelgroups were significantly higher than that of Group N(P<0.01), and the 24-hour urine protein in Group AL was less than that in Groups D and K(P<0.05). 2.The kidney weight index,glomerular size, mesangial matrix, and number of cells were increased with dilatation of partial tubular and Bowman’s capsule, tubular epithelial cell granules and vacuolar degeneration in the three diabetic model groups, but their changes in Group AL were relatively reduced. 3. The Hypoxyprobe?-1 of renal tissues by immunohistochemistry showed that the area of positive staining in three diabetic model groups increased significantly at 12 week as compared with the Group N(P<0.01), and the area of positive staining was significantly lower in Group AL than Groups D and K(P<0.01), and at 16 than 12 week in Group AL(P<0.05). 4.The results displayed by immunohistochemistry,fluorescent, and Western blotting:1Ang-1 was mainly expressed in glomerular and renal tiny vessel.The level of Ang-1 expression in three diabetic model groups was significantly higher than that of Group N(P<0.05), and increased Ang-1 in Group AL was higher than that of Groups D and K at 16 week(P<0.01).2VEGF was mainly expressed in glomerular.The expression of VEGF in three diabetic model groups was significantly enhanced as compared with Group N(P<0.01), and its level in Group AL was significantly higher than that of Groups D and K(P<0.05).3HIF-1α was mainly expressed in renal tubular epithelial cell.The expression of HIF-1α in three diabetic model groups was significantly enhanced as compared with Group N(P<0.01), but the expression in Groups D and K at 16 week was significantly lower than that of Group AL(P<0.01).4HO-1 was mainly expressed in renal tubular epithelial cell. The expression of HO-1 in three diabetic model groups was significantly increased as compared with Group N(P<0.05), and its level in Group AL was significantly higher than that of Groups D and K(P<0.05). 5. The result of RT-PCR showed:1The expression of Ang-1 m RNA in Group AL was upregulated as compared with Group N(P<0.05), with downregulated at 16 week(P<0.05),while increased Ang-1 m RNA in Groups D and K was not statistical significance(P>0.05).2The expression of VEGF m RNA in Group AL at 12 week was enhanced as compared with Group N(P<0.05).3The expression of HIF-1α m RNA in Group AL was increased as compared with GroupN(P<0.05),and higher than that of Groups D and K at 16 week(P<0.05), while increased HIF-1α m RNA in Groups D and K was not statistical significance(P>0.05).4The expression of HO-1 m RNA in Group AL at 16 week was upregulated as compared with Groups D, K and N(P<0.05), while increased HO-1 m RNA in Groups D and K was not statistical significance(P>0.05).6. VEGF level in AL Group was positively correlated with Ang-1 and HIF-1(P<0.05), and their levels were negatively correlated with staining degree of hypoxia probes(P<0.05).Conclusion: 1.Ang-1 combined with L-mim can improve the hypoxic condition of renal tissue in diabetic rats. 2. Ang-1 combined with L-mim can upregulate the expression of hypoxia-related factors including HIF-1, VEGF, and HO-1, and increased factors were related to the improvement of renal hypoxia in diabetic rats. |
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