GSK-3β Regulates Positively RANKL, NF-κB, OPN Through The Cross-talk Of GSK-Wnt And RANKL-NF-κB Pathway In Renal Tissue Of The Rats With DKD

Objective: To explore the role of GSK-3β regulating NF-κBp65, OPN, OPG, RANKL, β-catenin and SM22 of in renal tissue of rats with DKD. Methods: 10 rats were ramdonly chosen from 40 healthy SD rats in 8 weeks as Normal Control group(NC group, n=10). DKD model group( DKD group, n=10). The non-ATP competitive GSK-3β inhibitor lithium chloride( Li Cl) DKD treatment group( Li Cl group, n=10), and the ATP competitive GSK-3β inhibitor treatment group(Ar-a 014418 group, n=10). The rat models with DM was established by intraperitoneal injection of sterptozotocin(STZ) after two weeks adaptive feed. The rat models with DKD was verified to be successful after ten weeks, provided that three consecutive blood glucose results in a row were all higher than 16.7 mmol/L, meanwhile 24-hour urine protein were not lower than 30mg/L. 10 rats were ramdonly chosen from 30 rats with DKD as DKD model group( DKD group, n=10). The second 10 rats were ramdonly chosen from 30 rats with DKD as non-ATP competitive GSK-3β inhibitor lithium chloride( Li Cl) group( Li Cl group, n=10), treated by intraperitoneal injection 15 mg / Kg( every other day for 10 days on end). The last 10 rats were from 30 rats with DKD as ATP competitive GSK-3β inhibitor Ar-a 014418 group(ARA group, n=10), treated by intraperitoneal injection 1mg / Kg(every three day for 10 days on end). The weight, 24-hour proteinuria and blood glucose of the experimental animals were tested on time, in different stage. The kidney tissue was kept after all the rats were killed, and the pathological morphology of kidney tissue was observed under the light microscopy was observed under the light microscopy by Hematoxyline-eosin staining(HE). The expression of GSK-3β, NF-κB p65, OPG, RANKL, β-catenin and SM22 in the kidney tissue were tested by immunohistochemistry(IHC). The level of m RNA in OPN, OPG, RANKL and β-catenin inthe kidney tissue were determined by the quantitative Real-time Polymerase chain reaction(RT-q PCR). The protein expression of p GSK-3β(ser9), β-catenin, NF-κBp65 and OPN in the kidney tissue were tested by Western blot(WB). Results: 1. Rat models of DKD were constructed successfully and token the medical intervention. Compare with the treatment group and DKD group, rats in GSK-3β inhibitor groups were better in general state and survival rate( 95%) than those( 90% survival) in DKD group. 2. The blood glucose and urine protein in DKD group and the both GSK-3β inhibitor groups were higher than those in NC group, the blood glucose and urine protein in the both GSK-3β inhibitor groups were lower than those in DKD group(P<0.05). 3. The pathological morphology in the DKD group showed that: the glomerular hypertrophy, mesangial matrix increased, edema of renal tubuleepithelia and inflammatory cell infiltration were obvious and the lumen out of the way. The pathological changes in the DKD-related kidney were alleviated to some extend in Li Cl and ARA groups. 4. The Following were shown in IHC:(1) The expression of GSK-3β in DKD group was higher than that in NC group, but the expression of GSK-3β in Li Cl group and ARA group were weaker than that in DKD group(P<0.05).(2) the expression of NF-κBp65 and OPN increased and some NF-κBp65 entered nucleus in DKD group compared with that in NC group, but the expression of NF-κBp65 and OPN in Li Cl group and ARA group decreased compared with that in DKD group(P<0.05).(3) The expression of OPG and RANKL increased in DKD and other two treatment groups compared with that in NC group, the ration of OPG protein/ RANKL protein in DKD group decreased while the ration of OPG protein/ RANKL protein in both GSK-3β inhibitor groups increased(P<0.05).(4) The expression of SM22 and β-catenin in kidney tubules epithelial cells showed weakly positive in the DKD group. No significant different existed in the Li Cl group and the NC group. The expression of SM22 and β-catenin in ARA group were significantly stronger than other 3 groups. 5. The Following were shown in RT-q PCR:(1) The expression of OPN m RNA in DKD group was higher than that in NC group, and the expression of OPN m RNA in Li Cl group and ARA group were lower than that in DKD group(P<0.05).(2) The expression of OPG m RNA and RANKL m RNA increased in DKD and other two treatment groups compared with that in NC group, the ration of OPG m RNA / RANKL m RNA in DKD group decreased while the ration of OPG m RNA / RANKL m RNA in both GSK-3β inhibitor groups increased(P<0.05).(3) The expression of β-catenin in DKD group, Li Cl group and ARA group were higher than that in NC group, and the expression of β-catenin in ARA group was much higher than that in Li Cl group and DKD group(P<0.05). 6. The Following were shown in WB:(1) The expression of p GSK-3β(ser9) in Li Cl group and ARA group were stronger than that in NC group and DKD group(P<0.05).(2) The expression of β-catenin in DKD group, Li Cl group and ARA group were stronger than that in NC group, and the expression of β-catenin in ARA group was much stronger than that in Li Cl group and DKD group(P<0.05).(3) The expression of NF-κB and OPN in DKD group were stronger than that in NC group, and the expression of NF-κB and OPN in Li Cl group and ARA group were weaker than that in DKD group(P<0.05). Conclusion: GSK-3β regulates positively NF-κB signaling pathway activity at least in part through the Cross-Talk of GSK-Wnt signaling pathway and RANKL-NF-κB signaling pathway. 1. High blood glucose can active GSK-3β increasing in renal tissues of rats with DKD, upregulation of RANKL, NF-κB and OPN. It has shown that GSK-3β activated NF-κB signaling pathway activity at least in part through the Cross-Talk of GSK-Wnt pathway and RANKL-NF-κB pathway. 2. Li Cl and Ar-a014418 can cause GSK-3β ser9 phosphorylation to make GSK-3β inactive, upregulation of p GSK-3 beta(ser9), RANKL, NF-κB and OPN. 3. The both of two GSK-3β inhibitors could improve the DKD rats conditions, GSK-3β inhibitor weaken the progress of DKD, and greatly increase survival rates.