| Objective: To observe the changes of renal hypoxia and angiogenesis-related factors, and assess the protective effect of inhibiting HIF-1α degradation with L- mimosine on diabetic kidney in rats.Methods: Diabetic model was constructed by intraperitoneal injection with streptozotocin(STZ). The experimental rats were divided into three groups: normal control group(N group), diabetic group(D group), L- mimosine treatment group(L group). Diabetic rats were received with L- mimosine treatment every other day by intraperitoneal injection at 8 weeks. After 12 and 16 weeks, the samples of blood, urine, and renal tissue were collected. The blood glucose and 24- hour urinary protein were observed, renal pathological changes were observed by conventional HE and PAS staining, hypoxic status in renal tissue was detected by Hypoxyprobe?-1 probe, and expressional levels of m RNA and proteins including hypoxia inducible factor-1α(HIF-1α), heme oxygenase-1(HO-1), prolyl hydroxylase 2(PHD2), vascular endothelial growth factor(VEGF), angiopoietin-1(Ang-1), and angiopoietin-2(Ang-2) were detected by immunohistochemistry, immunofluorescence, western bloting, and RT- PCR.Results: 1. The blood glucose and 24-hour urinary protein in two diabetic model groups were significantly higher than that of N group(P<0.05), and 24-hour urinary protein in L group was lower than that of D group(P <0.01).2. 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 two diabetic model groups, but these changes in L group were relatively reduced.3. Hypoxyprobe?-1 immune staining of renal tissue was found in each groups. Immune staining in N group was mainly located at inner medullary and cortico- medullary zone, and in two diabetic model groups it was extended to cortex of kidney. The positive staining area in two diabetic groups was significantly higher than that of N group(P <0.01), but its area in L group was significantly lower than that of D group(P<0.01).4.Immunohistochemistry, immunofluorescenceand, and Western blotting : 1HIF-1α was mainly expressed in renal tubular, glomerular cell nucleus and partial renal tubular cytoplasm. The HIF-1α expression in renal tissue of two diabetic model groups was significantly up-regulated as compared with N group(P <0.01). HIF-1α level in L group was significantly higher than that of D group(P <0.01), and lower at 16 than 12 weeks(P<0.01). 2HO-1 was mainly expressed in the cytoplasm of renal tubular epithelial cells. The HO-1 expression in renal tissue of two diabetic model groups was significantly up-regulated as compared with N group(P <0.05). HO-1 level in L group was significantly higher than that of D group(P <0.01), and lower at 16 than 12 weeks(P<0.01).3PHD2 was mainly expressed in the cytoplasm of renal tubular epithelial cells. The PHD2 expression in renal tissue of two diabetic model groups was significantly up-regulated as compared with N group(P<0.01), and its level in L group was significantly higher than that of D group(P <0.01). 4VEGF was mainly expressed in the cytoplasm of renal tubular epithelial cells and partial glomerular. The expression of VEGF in renal tissue of two diabetic model groups was significantly enhanced as compared with N group(P <0.01), and its level in L group was higher than that of D group with no statistical significance. 5Ang-1 was mainly expressed in the glomerular, renal tubule and some tiny blood vessels.The expression of Ang-1 in renal tissue of two diabetic model groups was significantly enhanced as compared with N group(P <0.05), and its level in L group was higher than that of D group(P <0.05). 6Ang-2 was mainly expressed in cytoplasm and nucleus of renal tubular, partial glomerular cell, and some tiny blood vessels. The expression of Ang-2 in renal tissue of two diabetic renal tissue was significantly increased as compared with N group(P<0.05), but no statistical significance was found between two diabetic model groups(P> 0.05).5.RT-PCR :1The expression of HIF-1α mRNA in renal tissue of two diabetic model groups was increased, but significant difference was only found between L and N groups(P<0.05), and its level in L group was lower at 16 week than that at 12 week(P<0.05).2The expression of HO-1 m RNA in renal tissue of two diabetic model groups was increased, but significant difference was only found between L and N groups at 16 week(P<0.05). 3The expression of PHD2 m RNA in renal tissue of two diabetic model groups was increased(P<0.05), and its level in L group was higher than that of D group with no statistical significant difference(P>0.05). 4The expression of VEGF m RNA in renal tissue of two diabetic model groups was increased, and its level in L group was higher than that of D group, but significant difference was not found(P>0.05). 5The expression of Ang-1 m RNA in renal tissue of two diabetic model groups was increased, but significant difference was only found between L and N groups(P<0.05). Its level in L group was higher than that of D group(P<0.05).6The expression of Ang-2 m RNA in renal tissue of two diabetic model groups was increased, and its level in L group was higher than that of D group, but significant difference was not found(P>0.05).6. Correlation analysis: 1The area of Hypoxyprobe?-1 staining was correlated with the levels of urine protein(r= 0.808, P<0.05), HIF-1α(r=-0.726, P <0.05), and HO-1(r=-0.826, P <0.05). The level of urine protein was correlated with HIF-1α and HO-1(r=-0.882, P<0.01; r=-0.793, P<0.05).2The area of Hypoxyprobe?-1 staining was negatively correlated with the levels of Ang-1, Ang-2, and VEGF(r=-0.757, r=-0.818, r=-0.791, P<0.05). The levels of HIF-1α and HO-1 were positively correlated with Ang-1(r=0.960, P<0.01; r=0.793, P<0.05), Ang-2(r=0.739, P<0.05; r= 0.941, P<0.01), and VEGF(r=0.962, P<0.05; r=0.872,P<0.01).Conclusion: 1. L- mimosine may improve hypoxic condition of kidney in type 1 diabetic rats.2. Effect of improving renal hypoxia in type 1 diabetic rats with L- mimosine may be associated with upregulating the expression of HIF-1α and HO-1.3. L- mimosine can significantly upregulate the expression of Ang-1 in kidney of type 1 diabetic rat, and this effect can help to improve the diabetic renal microvascular disease. |
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