The Expression And Significance Of RIP1 And RIP3 In Kidney Tissues Of Rats With Diabetic Nephropathy

Objective:To explore the expression and significance of RIP1 and RIP3 of the necroptosis signaling pathways markers in the kidney tissues of rats with diabetic nephropathy.Methods: Adult Sprague-Dalwley(SD)male rats were divided into the diabetic nephropathy(DN)and normal control groups(NC).The DN group was treated by streptozotocin.50 SD rats weretreated by streptozotocin.In the second week,40 rats were induced to the model of diabetic nephropathy,7 rats were not induced to the model of diabetic nephropathy,and there were 3 rats that those died.The 6 rats of the two groups were sacrificed at week 2,4,8,12 and 16 respectively,meanwhile blood glucose,body weight,kidney weight and 24-hours urinary proteinweremonitored at the same time points respectively.Paraffin sections of renal tissue of rats at the same time points were stained by hematoxylin-eosin(HE)and Periodic Acid-Schiff(PAS).The expression of receptor interaction protein 1(RIP1),receptor interaction protein 3(RIP3)and nuclear factor-kappa B(NF-κB)protein and m RNAwestern blot,and Real-Time PCR.Theirrelationshipwere analyzed.Results: 1.Blood and urine:1)Blood glucose:(1)The blood glucose in different time points in Group DNwere obviously higher than those of Group NC(P<0.01).(2)The blood glucose in Group DN were gradually increased,and had siginificanted(P<0.05 or P<0.01).2)24-hour urinary protein:(1)The 24-hour urinary protein in different time points in Group DN were obviously higher than those of Group NC(P<0.01).(2)The 24-hour urinary protein were gradually increased,and had siginificanted(P<0.01).3)Serum creatinine and serum urea:The serum creatinine and serum urea in different time points in Group DN were obviously higher than those of Group NC(P<0.01).(2)The serum creatinine and serum urea both had no significant difference in different time points in Group DN(P>0.05).2.Renal pathology:(1)In different points in DN group the kidneys of rats were white and sweeling.Glomerulus,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 different time points in Group DN.(2)The kidney weight index in different time points in Group DN were obviously higher than those of Group NC(P<0.01).(3)The kidney weight index were gradually increased,and have siginificanted(P<0.01 or P < 0.05).3.Immunohistochemistry and immunofluorescence:(1)RIP1,RIP3 and NF-κB were mainly expressed in glomerular.(2)The expression of RIP1,RIP3 and NF-κB in different time points in Group DN showed obviously increased than those of Group NC at the same time points(P<0.01).(3)The expression of RIP1,RIP3 and NF-κB in DN group had no significant difference between 2 weeks and 4 weeks.However,their expression at8,12 and 4 weeks were obviously higher than those at 2 and 4weeks(P<0.01).There were no significant different between 8 and 12 weeks,their expression at 16 weeks were obviously higher than those at 8 and 12 weeks(P<0.01).(4)In the DN group,There were a strong positive correlation between the levels of NF-κB,RIP1 and RIP3;and the expression levels of RIP1 an RIP3 were positively correlation with urinary protein,the kidney weight index,the serum creatinine and serum urea each other.4.Western Blot:(1)The expression of RIP1,RIP3 and NF-κB poteins in different time points in Group DN were obviously higher expression levels than that of those Group NC(P<0.01).(2)The expression of RIP1,RIP3 and NF-κB poteins have respectively compared each other in DN group,no significant difference were found between 2 weeks and 4 weeks,the expression of RIP1,RIP3 and NF-κB poteins at 8,12 and 16 weeks were obviously higher 2 and 4 at weeks(P<0.01).Also there were no significant different at 8 and 12 weeks,the expression of RIP1,RIP3 and NF-κB poteins at16 weeks were obviously higher at 8 and 12 weeks(P<0.01).(3)In the DN group,There were a strong positive correlation between the levels of NF-κB、RIP1 and RIP3;and the levels of RIP1 an RIP3 were positively correlation with urinary protein,the kidney weight index,the serum creatinine and serum urea.5.Real-Time PCR: The expression of RIP,RIP3 and NF-κB m RNA in differenttime points in Group DN showed obviously higher expression levels than that of those Group NC(P<0.01).The expression of RIP1,RIP3 and NF-κB m RNA have respectively compared each other in DN group,no significant difference were found between 2 and 4 weeks,the expression of RIP1,RIP3 and NF-κB poteins at8,12 and 16 weeks were obviously higher at 2 and 4 weeks(P<0.01).Also there were no significant different at 8 and 12 weeks,the expression of RIP1,RIP3 and NF-κB poteins at 16 weeks were obviously higher at 8 and 12 weeks(P<0.01).(3)In the DN group,There were a strong positive correlation between the levels of NF-κB、RIP1 and RIP3;and the levels of RIP1 an RIP3 were positively correlation with urinary protein and the kidney weight index,but were not correction with the serum creatinine and serum urea.Conclusion: 1.In the kidney of diabetic nephropathy Rats,the necroptosis pathway may beactivated,Which may contribute to the tissue inflammation and injury of DN rats.2.The interaction of NF-κB and the necroptosis pathway may promote inflammation and resulting in kidney tissue injury of DN rats.