Role And Molecular Mechanisms Of TXNIP In Renal Tubular Injury In Diabetic Nephropathy

Objectives：Diabetic nephropathy (DN) is a leading worldwide cause ofchronic kidney disease (CKD) and end-stage renal disease. The crucialpathology underlying progressive CKD in diabetes is tubulointerstitial fibrosisand atrophy. Tubulointerstitial fibrosis and tubular atrophy progression ofdiabetic nephropathy is an important process of pathological changes.Epithelial-to-mesenchymal transition (EMT) and apoptosis of tubular cellscontributes to the renal accumulation of matrix protein and tubular atrophythat is associated with interstitial fibrosis. Pathogenesis of DN is very complex,a number of studies have demonstrated that oxidative stress plays an importantrole in diabetic renal injury. Oxidative stress (or oxidant-derived tissue injury)occurs when production of oxidants or reactive oxygen species (ROS) exceedslocal antioxidant capacity. There are many antioxidant system to clear ROS,repair oxidative damage and other molecular oxidation in human andanimals.Thioredoxin (TRX) system is a ubiquitous thiol oxidoreductasesystem that regulates cellular reduction/oxidation status. TRX system, whichis composed of NADPH, thioredoxinreductase(TrxR), and thioredoxin, is akey antioxidant system in defense against oxidative stress through its disulfidereductase activity regulating proteindithiol/disulfide balance. Thioredoxininteracting protein (TXNIP) was first identified as a1,25-dihydroxyvitaminD-3inducible gene in HL-60cells, therefore, named vitamin D3upregulatedprotein1(VDUP-1) or thioredoxin binding protein-2(TBP-2), is theendogenous inhibitor of cellular thioredoxin (TRX), inactivating itsanti-oxidative function by binding to the redox-active cysteine residues.Therefore, it is not surprising that TXNIP is upregulated in most tissues indiabetes including the retina and plays a critical role in oxidative stress leadingto disease progression. Oxidant/antioxidant-control system (TRX-TXNIP) plays a vital role in the oxidation-reduction reaction, therefore, by regulatingthe expression of TXNIP, TRX can indirectly regulate the activity, so as toachieve a state of oxidative stress in the regulation of cell purposes. Previousstudies have demonstrated that TXNIP was upregulated in renal tubularepithelial cells under high glucose conditions and in the kidneys of diabeticanimals. Lack of TXNIP may slow the natural process of mouse cadiocytedeath. Similarly, ceramide exhibits a mechanism of transcriptional regulationinvolving up-regulation of TXNIP expression, which results in ASK1activation, p38and JNK phosphorylation, all leading to leukemic cellapoptosis. TXNIP deficiency protects against glucose toxicity-induced β-cellapoptosis and the gradual β-cell loss and to preserve a sufficient amount ofinsulin-producing β-cell mass. Our previous study showed that the expressionof TXNIP mRNA and protein was increased in mouse mesangial cells underhigh glucose conditions, and, meanwhile, knockdown of TXNIP reversed highglucose-induced reduction of TRX activity and inhibited high glucose-inducedROS production and increased synthesis of TGF-β1and fibronectin. Ourrecent study showed that TXNIP was involeved in high glucose-induced ROSproduction and apoptosis in mouse mesangial cells.In this study we intend to further observation, based on the original workof the role of TXNIP in diabetic renal tubular injury, and to investigate theincidence of diabetic nephropathy, the development of mechanisms of actionand to find a new target for the treatment of diabetic nephropathy anddevelopment of potential therapeutic effects to provide a scientific basis fortargeting drugs.Methods：1Determination of TXNIP, α-SMA, E-cadherin, Bcl-2and in reneltissues from patients with diabetic nephropathyTwenty five patients diagnosed as diabetic nephropathy by renal biopsyand clinical data from October2011to October2012at the third Hospital ofHebei Medical University were included in this study. Other nephropathy wasexcluded. The renal tissues (n=10) obtained from distant portions of kidneys surgically excised because of the presence of a localized neoplasm were usedas control. Blood and urinary samples were collected to detect Glu, HbA1,UPE,8-OHdG, Scr and eGFR. Protein expression of TXNIP, α-SMA,E-cadherin, Bcl-2and Bax was assessed by immunhistochemical staining. Therenal tissues apoptosis was detected by TUNEL.2Role of grape seed proanthocyanidin extract in db/db miceMale db/db diabetic mice were randomly divided into two groups:diabetic group(db/db group) and diabetic+grape seed proanthocyanidin extractgroup (db/db+GSPE). The same week-old male db/m mice as normal controls(db/m) and grape seed proanthocyanidin extract gavage treatment group(db/m+grape seed proanthocyanidin extract group, db/m+GSPE). The mice ofdb/db+GSPE group and db/m+GSPE group were administered daily withgrape seed proanthocyanidin extract (5mg/kg) by gavage. The mice of db/mgroup and db/db group were only administered daily with the same volume ofnormal sodium by gavage. Six mice from every group were respectivelysacrificed at weeks8,12,16and20. Blood and24h urine samples werecollected for biochemical indicator and enzyme linked immunosorbent assay(ELISA) for8-OHdG. Partial renal tissures were fixed in4%neutral formalinfor histochemical, TUNEL and immunohistochemical staining. Protein andRNA were extracted from partial renal cortices for Western blot and Real-timePCR. The expression of TXNIP, α-SMA, E-cadherin, Bcl-2, p38, p-p38,ERK1/2, p-ERK1/2, Caspase-3, Cleaved Caspase-3and Bax protein wasrespectively evaluated by Western blot. The mRNA levels of TXNIP, α-SMA,E-cadherin, Bcl-2, and Bax were evaluated by Real-time PCR.3Knock down TXNIP impact on high glucose-induced apoptosis andtransdifferentiation in HK-2cellsThe HK-2cells were cultured in DMEM-F12medium supplemented with5%fetal bovine serum,2mM L-glutamine,100U/ml penicillin and100μg/mlstreptomycin in a95%air,5%CO2atmosphere. Stable transfections of HK-2cells with VDUP-1shRNA plasmid or control shRNA plasmid wereperformed with Lipofectamine2000according to the manufacturer’s instructions. After24hours, cells were cultured in selection mediumcontaining4ug/ml puromycin for3weeks before single clones were isolated.HK-2cells washed once with serum-free DMEM-F12medium, and thengrowth-arrested in serum-free DMEM-F12medium for24h to synchronizethe cell growth. After this time period, cells were randomly divided into group:normal glucose group (5.5mmol/L glucose, NG), normal glucose+mannitolgroup (5.5mmol/L glucose+24.5mmol/L mannitol, NM), high glucose group(30mmol/L glucose, HG), high glucose+negative control vector (30mmol/Lglucose+control shRNA plasmid, HG+C), high glucose+VDUP-1shRNA(30mmol/L glucose+VDUP-1shRNA, HG+shRNA), high glucose+N-Acety-L-Gysteine (30mmol/L glucose+N-Acety-L-Gysteine, HG+NAC), highglucose+TGF-β1(30mmol/L glucose+4ng/mL TGF-β1, HG+T), SB203580(30mmol/L glucose+10μmol/L SB203580, HG+SB), and PD98059(30mmol/L glucose+50μmol/L PD98059, HG+PD). The groups were culturedfor0,12,24,48and72hours respectively, and then HK-2cells wereharvested. The protein expression of TXNIP, α-SMA, E-cadherin, Bcl-2, andBax was detected by immunocytochemistry. The expression of TXNIP,α-SMA, E-cadherin, Bcl-2, p38, p-p38, ERK1/2, p-ERK1/2, Caspase-3,Cleaved Caspase-3and Bax protein was respectively evaluated by Westernblot. The mRNA levels of TXNIP, α-SMA, E-cadherin, Bcl-2and Bax wereevaluated by Real-time PCR. The TGF-β1and TRX activity were examinedby ELISA. HK-2cells apoptosis was detected by TUNEL and AnnexinⅤ/PIstaining. The ROS induced by high glucose was detected by flow cytometry.Results：1Pathological manifestation and TXNIP, α-SMA, E-cadherin, Bcl-2, andBax expression of patients with diabetic nephropathy①There were no abnormal changes in glomerulus, renal tubule andinterstitium of control group by light microscopy. Pathological changesincluding glomerular enlargement, increase of glomerular basementmembrane in thickeness, increase of ECM, the presence of Kimmelstiel-Wilson lesions, focal tubular epithelial vacuolar degeneration as well as interstitial fibrosis were observed in the patients of diabetic nephropathy.②Compared with control group, the levels of Glu, HbA1, UPE,8-OhdG and Scrin diabetic nephropathy group were increased significantly. The eGFR level ofdiabetic nephropathy group was decreased than control group (P<0.05).③Immunohistochemical staining displayed that E-cadherin, Bcl-2weaklyexpressed in renal tubular epithelium in diabetic nephropathy group, whereasremarkably TXNIP, α-SMA, and Bax increased in diabetic nephropathy group(P<0.05).④Apoptotic cells by TUNEL were observed in renal tissues of thediabetic kidney.2The expression of TXNIP, α-SMA, E-cadherin, Bcl-2, and Bax in renaltissues of diabetic db/db mice and the effect of grape seed proanthocyanidinextract on renal tubular injury①Diabetic db/db mice showed slightly glomeruli hypertrophy, increasingmesangium matrix, thickened glomerular basement membrane, reduction inthe number of renal tubular epithelial cell, partial tubular epithalial vacuolardegeneration by light microscope.②Compared with control group, the FBG,weight levels upregulated in a time-dependent manner in db/db group.24hurine protein, BUN,8-OHdG and Scr was significantly increased in db/dbgroup than db/m group (P<0.05). Compared with db/db group,24h urineprotein, BUN,8-OHdG and Scr in urine of db/db+GSPE group wassignificantly decreased (P<0.05).③By immunohistochemical staining, theprotein expression of TXNIP, α-SMA, and Bax was increased in tubule cellsof db/db group than that in db/m group, whereas remarkably E-cadherin, Bcl-2,decreased in db/db group compared with db/m group (P<0.05). The mRNAlevels of TXNIP, α-SMA, and Bax increase and slightly E-cadherin, Bcl-2,decreased in db/db group (P<0.05). By Western blot analysis, Renal tissues ofdb/db diabetic mice showed increased expression of TXNIP, α-SMA and Bax,but GSPE decreased their protein expression in db/db diabetic mice (P<0.05).Renal tissues of db/db dabetic mice had a significant up-regulation in p-p38,p-ERK1/2and Cleaved Caspase-3expression and down-regulation in Bcl-2,E-cadherin expression compared with db/m group (P<0.05). However, the alternations of p-p38, p-ERK1/2, Cleaved Caspase-3and Bcl-2, E-cadherinprotein levels in db/db group were reversed by addition of grape seedproanthocyanidin extract (P<0.05). The expression of TXNIP, α-SMA,E-cadherin, Bcl-2and Bax mRNA was the same with the expression of protein.④Apoptotic cells by TUNEL were observed in renal tissues of the diabetickidney. Apoptotic rate of renal tissues in db/db group increased compared todb/m group by flow cytometry (P<0.05). Apoptotic rate was significantlylower in the db/db+GSPE group than that in db/db group (P<0.05).3Knockdown of thioredoxin-interacting protein ameliorates highglucose-induced apoptosis in HK-2cells①The results of TUNEL and AnnexinⅤ/PI staining showed that theapoptotic HK-2cells in high glucose stimulation were markedly higher thanthat in normal group (P<0.05). Downregulation of TXNIP and NAC clearlydecreased the cells to high glucose induced cell death (P<0.05).②Highglucose stimulation in HK-2cells decreased Bcl-2protein level than in normalglucose medium (P<0.05). The protein expression of the high glucose-inducedBax, p-p38, TXNIP and Cleaved Caspase-3was significantly increased thannormal glucose stimulation HK-2cells (P<0.05). Compared with the cellstreated with high glucose, Bax, p-p38and cleaved Caspase-3protein levelssignificantly decreased in cells transfected with TXNIP or NAC, while Bcl-2level increased and the protein ratio of Bax/Bcl-2decreased (P<0.05).③Highglucose stimulation in HK-2cells decreased of TRX activity and increasedintracellular ROS levels. The high glucose-induced suppression of TRXactivity and increased intracellular ROS levels were inhibited by transfectionof VDUP-1shRNA plasmid or NAC treatment (P<0.05). In addition, mannitolhad no effect on HK-2cells.4Knockdown of thioredoxin-interacting protein ameliorates highglucose-induced epithelial-mesenchymal transition in HK-2cells①It was found that high glucose significantly induced EMT change inHK-2cells, characterized by decreased expression of E-cadherin protein andmRNA, but increased expression of α-SMA protein and mRNA (P<0.05). ②The expression of TXNIP, α-SMA protein was efficiently inhibited bytransfection with VDUP-1shRNA plasmid in high glucose condition (P<0.05).In addition, antioxidant, NAC, effectively prevented high glucose-inducedTXNIP, α-SMA expression. The high glucose-induced suppression of TRXactivity and increased intracellular ROS levels were inhibited by transfectionof VDUP-1shRNA plasmid or NAC treatment (P<0.05). Compared withthose of the NG groups, the levels of phosphorylation of p38MAPK andERK1/2significantly increased in HG group, whereas transfection withUVDP-1shRNA plasmid or NAC treatment significantly suppressed highglucose-induced p38MAPK and ERK1/2phosphorylation (P<0.05).③In HGgroup showed a significant higher mRNA level of TGF-β1than those in NGgroup, whereas transfection of VDUP-1shRNA plasmid or NAC treatmentsignificantly decreased the HG-induced overexpression of TGF-β1mRNA inthe HK-2cells (P<0.05).④the high glucose-induced overexpression ofTGF-β1in culture medium was significantly inhibited by VDUP-1shRNAplasmid transfection or NAC treatment. Transfection of VDUP-1shRNAplasmid or NAC treatment significantly decreased the HG-inducedoverexpression of TGF-β1mRNA in the HK-2cells (P<0.05). The expressionlevels of TXNIP proiein and mRNA increased in the TGF-β1compared withnormal group (P<0.05). Addition of TGF-β1stimulated a robust increase inROS production that was significantly prevented by pretreatment with NAC orTXNIP interference. HK-2cells were transfected with control shRNA plasmidor VDUP-1shRNA plasmid and treated with TGF-β1respectively (P<0.05).⑤Exposure of HK-2cells to TGF-β1markedly increased both mRNA andprotein expression of α-SMA and blocked the expression of the epithelialmarker E-cadherin (P<0.05). TGF-β1-induced these changes in HK-2cellswere markedly attenuated by transfection with VDUP-1shRNA plasmid ortreatment with the antioxidant, NAC (P<0.05). In addition, mannitol had noeffect on HK-2cells.Conclusions：1The overexpression of TXNIP is found in renel tissues from patients with diabetic nephropathy, ROS increaded in diabetic mice and HK-2cellsinduced by high glucose, which suggests that TXNIP, ROS may mediate renaltubular injury of diabetic nephropathy.2Grape seed proanthocyanidin extract inhibits activation of TXNIP inrenel tissues of diabetic mice. Grape seed proanthocyanidin extract alsosuppresses proteinuria and8-OHdG level, inhibits expression of apoptoticassociated protein, decreases cell apoptosis, and inhibits accumulation ofextracellular matrical components. These findings suggest that grape seedproanthocyanidin extract provides a treatment for diabetic nephropathy.3HK-2transfected with VDUP-1shRNA and treated with NACrespectively inhibits ROS, p-p38in high glucose-induced HK-2cells, inhibitsexpression of apoptotic associated protein, increased TRX activity, decreasesHK-2cells apoptosis.4Knockdown of TXNIP prevents high glucose-induced ROS generation,EMT, TGF-β1. Knockdown of TXNIP prevents high glucose-induced EMTthrough inhibiting activation of p38MAPK and ERK1/2. TGF-β1-inducedTXNIP expression is ROS-dependent. Knockdown of TXNIP preventsTGF-β1-induced EMT via ROS. Taken together, these results suggest thatTXNIP interference inhibited TGF-β1-induced EMT through ROS in HK-2cells.