The Role And Mechanism Of NLRP3 Inflammsome Activation By TXNIP In Ischemia/reperfusion Injury Of Diabetic Kidney

BackgroundAcute kidney injury(AKI)is a disease of rapid loss of renal function associated with high morbidity and mortality.Renal ischemia/reperfusion(I/R)injury is the most common cause of AKI.There are many clinical evidences that diabetes is an important risk factor for AKI,patients with diabetic nephropathy are more susceptible to AKI and at high risk of failing to recover from AKI.Oxidative stress contributes to the pathogenesis of both diabetes and ischemic AKI.So the topic of oxidative injury and perioperative in diabetic kidney from I/R injury are certainly of interest spot and important project of disciplines.A new class of signaling molecules,the thioredoxin-interacting proteins(TXNIP),has been demonstrated to be involved in both antioxidative system and insulin resistance.TXNIP is the endogenous inhibitor of cellular thioredoxin(TRX),inhibits the ability of TRX to scavenge for reactive oxygen species(ROS).In addition,experimental evidence has indicated that hyperglycaemia and diabetes could induce TXNIP expression.Recently,TXNIP has also been demonstrated as essential for the activation of the NOD-like receptor pyrin domain containing-3(NLRP3)inflammasome through its direct interaction with NLRP3.NLRP3 is a crucial pattern recognition receptor and plays pivotal roles in host defense against pathogens.The NLRP3 inflammasome is becoming increasingly recognized as integral to the pathogenesis of many renal diseases including renal I/R injury.However,whether TXNIP is a activator of NLRP3 inflammasomes in the pathophysiology of ischemia AKI in diabetic model is unknown.Therefore,the role of TXNIP and NLRP3 in diabetes-aggravated ischemia AKI calls for intensive researches.ObjectiveThe present study was designed to investigate the role of TXNIP during ischemia AKI in diabetic models and examine a key role of TXNIP in bridging redox signals with activation of NLRP3 inflammasome in AKI injury.Methods1.Male adult Sprague-Dawley(SD)rats with streptozotocin(STZ)-induced diabetes and age-matched non-diabetic control rats were used.Renal ischemia-reperfusion was induced by bilateral renal pedicle clamping for 25 minutes to produce moderate renal injury.Some of the diabetic rats were pretreated with RES prior to I/R induction.The diabetic and normal rats were randomly divided into five groups:ND Sham group(NS);ND I/R group(NI/R);DM Sham group(DS);DM I/R group(DI/R);and DM + RES I/R group(DI/R-RES).The rats were sacrificed 48 hr after reperfusion.Kidneys were harvested for H&E,TUNEL and immunohistochemical staining.Renal tissue superoxide anion redical scavenging capacity,SOD activity,MDA content and IL-1β,IL-18 levels were measured.Serum BUN and Scr levels were detected.The Western blot was adopted to detect the expression of TXNIP,NLRP3 and pro-CASPASE-15 CASPASE-1proteins.2.The HK-2 cells were divided into eight groups randomly:(1)the normal glucose group(NG group);(2)the high glucose group(HG group);(3)the normal glucose + mannitol group(M group);(4)the normal glucose + hypoxia/reoxygenation group(NH/R group);(5)the high glucose + hypoxia/reoxygenation group(HH/R group);(6)the high glucose ＋hypoxia/reoxygenation + TXNIP siRNA group(HH/R-siRNA group);(7)the high glucose + hypoxia/reoxygenation + scrambled siRNA group(HH/R-scrambled siRNA group);(8)the high glucose + hypoxia/reoxygenation + RES group(HH/R-RES group).The HK-2 cells exposed to high glucose for 72 hours were subjected to hypoxia for 4 hours and re-oxygenation for 2 hours in sequence.Therefore,the high glucose hypoxia/reoxygenation model was established of kidney proximal tubular cells of human.The CCK-8 and LDH cytotoxicity test was adopted to detect the viability of cells.Then,the level of intracellural ROS was detected,the content of MDA and the alteration of SOD and caspase-1 activity were measured.Apoptotic cells were determined by flow cytometry.The level of IL-1βwere detected by ELISA.The immunofluorescence staining and Western blot was adopted to detect the expression of TXNIP and NLRP3 proteins.Results1.Blood glucose level was signifcantly higher in STZ-induced diabetic rats than in control rats at 4 weeks.The body weight was significantly decreased in the diabetic group when compared with the non-diabetic controls.Giving RES treatment significantly ameliorated hyperglycemia in STZ-induced diabetic rats,but the plasma glucose levels in RES-treated diabetic rats still remained significantly higher in comparison with non-diabetic control.Rats that underwent renal I/R exhibited a significant increase both in renal injury which characterized by BUN,creatinine,histological severity score,apoptosis%and the level of oxidative stress which indicated by superoxide anion redical scavenging capacity,MDA,SOD compared with sham-operated rats.The levels of these markers were significantly higher in diabetic animals exposed to I/R than in non-diabetic controls.Administration of RES to diabetic rats subjected to I/R prevented the increase in the serum concentrations of BUN,creatinine,and histological severity score,apoptosis%and resulted in a significant decrease in oxidative stress level.Moreover,after 4 weeks of diabetes,kidney TXNIP expression was agumented in diabetic rats relative to non-diabetic controls.After I/R,kidney TXNIP expression was dramatically inceased in diabetic rats compared to non-diabetic control.RES treatment decreased post-ischemic kidney TXNIP protein expression in diabetic rats to a level significantly lower than that in the untreated diabetic kidney.Consistent with the changes of kidney TXNIP protein expression,kidney NLRP3,cleaved caspase-1 protein expression and the levels of IL-1β,IL-18 were significantly increased in 4 weeks diabetic rats.After I/R,kidney NLRP3/cleaved caspase-1/IL-1β,IL-18 expression were significantly higher in diabetic rats compared to non-diabetic control and RES restored post-ischemic NLRP3/cleaved caspase-1/IL-1β,IL-18 expression in diabetic rats.2.Compared with the NG group,the cell viability of those in the 72-hour HG group declined sharply,the activity of LDH and cell apoptosis%grew sharply,and the difference was statistically significant.Following 4 hrs hypoxia and 2 hrs re-oxygenation,compared with the HG group,the cell viability of those in the HH/R group dropped more sharply,while the activity of LDH and cell apoptosis%grew more rapidly,and the difference was also statistically significant.Moreover,HH/R showed a further increase of LDH,cell apoptosis%and decline in cell viability as compared to NH/R group.As oxidative stress level,in comparation with NG group,HG and NH/R significantly increased ROS production and MDA levels.Moreover,HH/R showed a further rise of ROS and MDA as compared to NH/R group.In contrast,the SOD activity was significantly reduced in NH/R or HH/R group as compared to NG or HG group respectively.Moerover,HH/R further decreased the level of SOD as compared to NH/R group.After the 72-hour stimulation by high glucose,the expressions of the TXNIP protein and NLRP3 protein as well as the activity of caspase-1 and IL-1βlevel significantly increased.Following 4 hrs hypoxia and 2 hrs re-oxygenation.The TXNIP and NLRP3 expression as well as the activity of caspase-1 and IL-1βlevel were observably increased in HG stimulating as compared with corresponding NG control group.TXNIP was knocked down prior to high glucose culture and the pretreatment of RES could inverse the cell viability detected by CCK-8 and LDH obviously.Compared with the HH/R group,the viability of HK-2 cells in the HH/R-siRNA group and HH/R-RES group ascended sharply,while the activity of LDH and cell apoptosis%dropped sharply,and the difference was statistically significant.Besides,Compared with the HH/R group,both of the knocked down of the TXNIP protein and the pretreatment of RES could strongly inverse the ROS generation,content of MDA and the activity of SOD.After the TXNIP siRNA transfection in HK-2 cells,TXNIP and NLRP3 protein expressions were significantly decreased and the activity of caspase-1 and the level of IL-1β were also significantly reduced in HK-2 cells after exposure to high glucose following H/R.Meanwhile,RES treatment significantly decreased TXNIP protein expression and correspondingly decreased NLRP3 protein expression and the activity of caspase-1,and level of IL-1β as compared to HH/R group.ConclusionTXNIP inhibition protects the diabetic kidney from I/R injury and diminishs the AKI sensitivity of diabetic kidney tissues by inhibiting oxidative stress and NLRP3 inflammasome activation.So,as an endogenous redox regulator,TXNIP may represent an important future target to develop newer therapeutics in diabetic patients that can reduce AKI sensitivity of kidney tissues.