Effect And Molecular Mechanism Of TXNIP Knockout On Renal Injuries In Diabetic Mice

Objectives: Diabetic kidney disease(DKD)is the most common microvascular complications and is a leading worldwide cause of chronic kidney disease(CKD).The crucial pathology underlying progressive CKD in diabetes is tubular injury.Thus,there remains an urgent request to prevent or slow the progression of diabetic kidney disease,to protect kidney function and to develop effective treatment of diabetic kidney disease.A number of studies have indicated that there was an excessive production of ROS in kidney in diabetes,which induced the happen of kidney injury.Oxidative stress occurs when production of oxidants exceeds local antioxidant capacity,then induces the happen of kidney injury.There are many antioxidant systems to clear ROS,such as glutathione,superoxide dismutase and vitamin C,and repair oxidative damage.Reducing the generation of ROS may be a potential therapy for DKD.Thioredoxin(TRX)system plays an important role in ROS.TRX is a thiol-oxidoreductase that reduces oxidized proteins,resulting in oxidation of the cysteine residues of TRX.Then,it has to be reduced back by the NADPH dependent thioredoxin reductase.That regulates cellular reduction/oxidation status.Thioredoxin interacting protein(TXNIP)was first named as thioredoxin binding protein-2(TBP-2)or vitamin D3 upregulated protein 1(VDUP-1).It is the endogenous inhibitor of cellular thioredoxin(TRX),Binding to the redox-active cysteine residues to inactivate its anti-oxidative function.Therefore,TXNIP regulates cellular reduction/oxidation status indirectly and induces the generation of reactive oxygen species.Many studies have demonstrated that TXNIP was upregulated in several DKD models.A mount of studies also suggested that knockdown or knockout of TXNIP may restrain the process of diabetic complications.TXNIP wasinvolved in the β-cell apoptosis induced by diabetes.TXNIP deficiency protects against high glucose-induced β-cell apoptosis.Our previous studies showed that the mRNA and protein expression levels of TXNIP were increased in mice mesangial cells and human kidney cells under high glucose conditions,and p38 MAPK was involved in high glucose-induced TXNIP expression.Knockdown of TXNIP reversed high glucose-induced reduction of TRX activity and inhibited high glucose-induced ROS production and activation of the p38 MAPK and ERK1/2 signal pathway.All of these studies showed that TXNIP was involved in process of DKD.TKO mice(C57BL/6 background)and WT mice(C57BL/6)were used in this study.Streptozotocin-induced diabetic animals were used to investigate the role and molecular mechanism of TXNIP on diabetic renal injuries,and to make it clear whether TXNIP maybe a new target for the treatment of DKD.Methods:1 Animal modelMale C57BL/6 mice and TKO mice(C57BL/6 background)were divided randomly into 4 groups: WT group,WT+DM group,TKO+DM group and TKO group.WT+DM group and TKO+DM group were injected intraperitoneally with STZ(50 mg/kg,dissolved in citrate buffer,pH 4.5)to induce diabetes.Diabetes was confirmed by the concentration of blood-glucose higher than 16.7mmol/L and urine glucose(++ ~ +++)or above.Mice were sacrificed in 24 week.Blood and 24 h urine samples were collected for biochemical indicator and ELISA for 8-OHdG.Partial renal tissues were fixed in 4% neutral paraformaldehyde for histology observation,immunohistochemical staining,immunofluorescence staining and TUNEL.Partial renal tissues were fixed in 2.5% glutaraldehyde for electron microscope analysis.Total RNA and total protein were extracted from partial renal cortices for Real-time PCR and Western blot to evaluate the related mRNA and protein expression levels.Results:1 Effect of knockout of TXNIP on the expression of TXNIP in the kidney of diabetic mice.The expression of TXNIP was markedly increased in WT+DM group compared with WT group.Knockout of TXNIP can disappear the expression of TXNIP in TKO+DM group and TKO group.This suggested that TXNIP knockout model is successful and knockout of TXNIP can reduce the level of TXNIP in the kidney of diabetic mice.2 Knockout of TXNIP improves general conditions in diabetic mice.In response to STZ,The mice in WT+DM group showed significantly increased blood glucose levels,reduced body weight gain,and increased urine protein.Importantly,knockout of TXNIP significantly improves general conditions.3 Knockout of TXNIP reduces the renal pathological change in diabetic mice.In the PAS-stained and MASSON-stained kidney of the four groups,the mesangial matrix was more extensive in the glomerular of WT+DM group than WT group,and expressed mesangial area broadening,glomerular capillary lumens expansion.Knockout of TXNIP reduced such pathological change.Transmission electron microscopy shows that glomerular foot process disappeared and the basement membrane become thickening in WT+DM group.Immunofluorescence for ColI showed a markedly higher expression in renal glomerular in WT+DM group than in WT group.Knockout of TXNIP reduced the expression of ColI.4 Effect of TXNIP knockout on renal cells apoptosis in diabetic mice.TUNEL staining suggested that renal cells apoptosis were markedly increased in WT+DM group compared with WT group.The expression of ratio of Bax/Bcl-2 and the ratio of cleaved caspase-3/caspase-3 increased in WT+DM group which can be inhibited by knockout of TXNIP.This suggested that knockout TXNIP can reduce renal cells apoptosis.5 Effect of knockout of TXNIP on the expression of α-SMA in kidney of diabetic mice.Results:The expression of α-SMA was markedly increased in WT+DM group compared with WT group.Knockout of TXNIP can reduce the expression ofα-SMA in diabetic mice.This suggested that knockout of TXNIP can reduce the level of α-SMA.6 Effect of knockout TXNIP on the expression of ROS in kidney of diabetic mice.The expression of Nox4 and 8-OHdG were markedly increased in WT+DM group compared with WT group.Knockout of TXNIP can reduce the expression of Nox4 and 8-OHdG in diabetic mice.This suggested that knockout of TXNIP can reduce the level of ROS.7 Effect of knockout TXNIP on the expression of p38 MAPK and ERK1/2 in kidney of diabetic mice.Compared with WT control group,the phosphorylation of p38 MAPK and ERK1/2 was markedly increased in WT+DM group.Knockout of TXNIP can reduce the phosphorylation of ERK1/2 and p38 MAPK in kidney of diabetic mice.Conclusion:1.TXNIP was markedly increased in kidney of diabetic mice.2.Knockout of TXNIP can protect kidney function,reduce glomerular capillary lumens expansion,alleviate partial sclerosis of renal tissue and inhibit diabetic mice renal fibrosis.3.Knockout of TXNIP reduce the number of apoptotic renal cells and the expression of apoptosis related indicators would decline.4.Knockout of TXNIP also can block activation of p38 MAPK and ERK1/2.5.Knockout of TXNIP can protect kidney function may be partly through inhibiting the production of ROS.