| Objective: To observe the changes of renal hypoxia related indicators and evans blue(EB) content, and evaluate the effects of Ang-1 on renal hypoxia and microvascular permeability by means of treatment of adenovirus expression vector of angiopoietin-1(Ad-Ang-1) and EB through intravenous injection in diabetic nephropathy of rats.Methods: Recombinant Ad-Ang-1 was constructed and identified. SD rat diabetic model was constructed by intraperitoneal injection with streptozotocin(55mg/kg). The experimental rats were divided into four groups: normal control group(Group NC), diabetic group(Group DN), Ang-1 treatment group(Group AV, model rat was treated with 3×108PFU Ad-Ang-1 via a penile injection at 8 week), and blank vector group(Group BV, model rat was treated with 3×108PFU adenovirus vector via a penile injection at 8 week). At 12 and 16 weeks, their blood, urinary sample, and renal tissues were collected. The blood glucose, serum creatinine, urea, insulin, glucagon, growth hormone, thyroid, urinary protein and kidney injury molecule-1 were observed. The renal pathological changes was observed by conventional HE and PAS staining, levels of hypoxia-inducible factor-1α(HIF-1α), heme oxygenase-1(HO-1), and prolyl hydroxylase2(PHD2) protein and m RNA were evaluated by immunohistochemistry, immunofluorescenceand, western bloting, and Real-Time-PCR. Rats were respectively killed after intravenous injection with pimonidazole or EB, hypoxic status and EB content in renal tissue were respectively detected by Hypoxyprobe?-1immunohistochemistry or spectrophotometer.Results: 1.Blood and urine: 1The blood glucose and 24-hour urinary protein in three diabetic model groups were significantly higher than that of Group NC(P<0.01), and no significant difference was found between the model groups(P>0.05). 2 The serum creatinine had no significant difference in each groups(P>0.05), but its level in Groups AV and BV was higher at 16 W than12W(P<0.05). The serum urea in Group AV was higher than that of Groups NC and DN(P<0.05).3 The level of serum insulin in Group NC was higher than three model groups, but significant difference was only found in Group DN at 12W(P<0.05). The levels of serum glucagon and growth hormone had no significant difference in each group(P>0.05). The level of serum thyroxine in Group BV was significantly higher than that of the three groups at 12W(P<0.01).4The urinary KIM-1 levels was significantly increased in three diabetic model groups, and higher in Group AV than Groups DN and BV(P<0.01), at 16 W than 12 W in Group DN(P<0.05). 2.Renal pathology: The kidney weight index, 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 AV were relatively reduced. 3. Hypoxyprobe?-1 immunohistochemistry: Immune staining in Group NC was mainly located at inner medullary and cortico-medullary zone. The positive staining of renal cortex zone in the three diabetic model groups was found in different degrees, but its area in Group AV was significantly lower than that of Groups DN and BV(P<0.01). 4.Immunohistochemistry, immunofluorescenceand,and Western blotting of hypoxia response related indicators: 1HIF-1α was mainly expressed in renal tubular, glomerular cell nucleus and partial renal tubular cytoplasm. The HIF-1α expression in renal tissue of three diabetic model groups was significantly up-regulated as compared with Group NC(P<0.01). Its level in the Group AV was significantly higher than that of Groups DN and BV(P<0.05), and lower at 16 W than 12W(P<0.05). 2HO-1 was mainly expressed in the cytoplasm of renal tubular epithelial cells. The HO-1 expression in Group NC was significantly down-regulated as compared with three diabetic groups(P<0.05).Its level in the Group AV was significantly higher than that of Groups DN and BV(P<0.05), and lower at 16 W than 12W(P<0.05). 3PHD2 was mainly expressed in the cytoplasm of renal tubular epithelial cells. The PHD2 expression in Groups DN and BV was significantly higher than that of Group NC(P<0.01), and its level in the Group AV was significantly lower than that of Groups DN and BV(P<0.01). 5. Real-Time PCR of hypoxia related indicators: 1The expression of HIF-1α m RNA in three diabetic model groups was increased, but significant difference was only found between Groups AV and NC(P<0.05),and its level at 16 W in Group AV was significantly higher than that of Groups DN and BV(P<0.05). 2The expression of HO-1 m RNA in three diabetic model groups was increased, but significant difference was only found at 16 W between Groups AV and NC. 3The expression of PHD2 m RNA in three diabetic model groups was increased, but significant difference was only found between Groups NC and DN or BV(P<0.05),while its level at 12 W in Group AV was significantly lower than that of Group DN(P<0.05). 6.Renal microvascular leakage: After EB perfusion, the visible blue staining was mainly found in the renal pelvis and calyces of Group NC, and also in renal cortical and medullary region of three model groups, but stained degree in Group AV was reduced as compared with groups DN and BV. EB content of renal tissue in three diabetic model groups was increased, and significantly higher in Groups DN and BV than NC(P<0.05), and significantly lower in Groups AV than DN and BV(P<0.05).Conclusion: 1.Ang-1 can improve the hypoxic condition of renal tissue in diabetic rats. 2.Ang-1 can upregulate the expression of HIF-1α, and HO-1, and downregulate expression of PHD2, these effect may be very useful to the improvement of renal hypoxia in diabetic rats.3.Ang-1 can improve the microvascular leakage in the kidney of diabetic rats.4. Ang-1 do not obviously affect levels of serum insulin, glucagon, growth hormone, and thyroxin in diabetic rats. |
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