Role And Molecular Mechanisms Of TXNIP On Lipid Accumulation In Diabetic Kidney

Objectives:Diabetic nephropathy(DN) is one of the major systemic“microvascular” complications of diabetes, which is characterized by mesangial cell proliferation and mesangial matrix deposition, tubular epithelial cell apoptosis and transdifferentiation, glomerulosclersis and interstitial fibrosis. Diabetic nephropathy has been the focus in researches of kidney disease for years due to its very complicated pathogenesis. The study over the past decade found that, lipid metabolism disorder in kidney intrinsic cells is an important pathophysiological basis of diabetic nephropathy, and abnormal lipid accumulation in renal cell under high glucose conditions leading to chronic dysfunction and injury of kidney. Knockout or pharmaceutically inhibition of the expression of genes involved in the process of lipid metabolism in renal tissue, such as sterol regulatory element binding protein(SREBPs) and peroxisome proliferator-activated receptor(PPAR), can repaire diaberes-induced kidney injury, indicating lipid metabolism disorder in kidney plays an important role in the development of diabetic nephropathy.Thioredoxin interaction protein(TXNIP) also named vitamin D3 upregulated protein 1(VDUP-1) or thioredoxin binding protein-2(TBP-2), is the endogenous inhibitor of cellular thioredoxin(TRX), inactivating its anti-oxidative function and inducing oxidative damage by binding to the redox-active cysteine residues. Some researches reported that TXNIP is involved in the development of diabetic nephropathy and the expression of TXNIP m RNA and protein was increased in renal cells under high glucose conditions. Rencent study showed that TXNIP plays an irreplaceable regulated role in the development of hepatic steatosis. Followed with abnormal lipid accumulation in liver, an up-regulation of hepatic TXNIP expression was found in STZ-induced type 1 diabetes-associated non-alcoholic fatty liver disease(NAFLD); and inhibition of hepatic TXNIP by quercetin and allopurinol contributes to the reduction in liver lipid accumulation. TXNIP-deficient mice on a high-fat diet(HFD) show decreased lipid metabolism in hepatocytes and improved hepatic steatosis mainly via the inhibition of TXNIP-PRMT1-PGC-1? anabolism pathway activation. However, the exact role of TXNIP on diabetes-induced renal lipid accumulation still remains to be defined.In the present study, we investigated the role of TXNIP on diabetesinduced lipid accumulation in renal tubular epithelial cells using diabetic mice model and cultured HK-2 cells to clarify lipid deposit location and the correlation between lipid accumulation and TXNIP expression. Further, we detected the effects of anthocyanins on the expression of TXNIP and lipid accumulation induced by high glucose in renal tubular epithelial cells, in order to provide theoretical basis for the prevention and treatment of diabetic nephropathy.Methods:1 Ectopic lipid accumulation and expression of TXNIP, SREBP-1 and PPAR? in renal tissues from patients with diabetic nephropathyTwenty patients diagnosed as diabetic nephropathy by case history, renal biopsy and clinical data from October 2010 to October 2014 at the Nephrology of First Central Hospital of Baoding were included in this study. Those cases complicated with other nephropathy were excluded. The renal tissues(n=10) obtained from distant portions of renal tumors without evident renal function impaired were used as control. Blood and urinary samples of all patients were collected to detect the level of blood sugar, glycosylated hemoglobin, total cholesterol, triglycerides, 24-hour urinary protein excretion, creatinine and glomerular filtration rate. Lipid droplets formation in renal tissues was detected by Oil Red O staining. Protein expression of TXNIP, SREBP-1 and PPAR? was assessed by immunhistochemical staining.2 Effects of TXNIP knockout on renal lipid accumulation in STZ –induced diabetic miceAdult(6-8weeks old) male C57BL/6 mice(Wild type, WT) and TXNIP?/?(TKO) mice(C57BL/6 background) were randomly divided into four groups: Wild type C57BL/6 mice group(WT), diabetes group(WT+STZ), TXNIP knockout mice group(TKO) and diabetic TXNIP knockout mice group(TKO+STZ). Diabetes was induced in 10 mice per group by intraperitoneal injections of STZ(50 mg/kg in fresh 0.1 M sodium citrate buffer, p H 4.5) daily for 5 days, and control(nondiabetic) groups received citrate buffer. The model of diabetes was considered to be successful when the blood glucose was ≥11.1mmol/L and the glucose in urine was +++~++++ after five days of the injection. All experimental animals were given free access to food and water. Blood glucose levels were monitored with a glucometer weekly. At the end of the experiments, the mice were sacrificed and the samples of blood and 24 h urine were collected for determination of the renal function, blood glucose and blood lipids. The kidneys were removed and harvested for further analysis. One portion of the kidney tissues was fixed immediately in 2.5% glutaraldehyde for transmission electron microscopy, another portion of the kidney tissues was fixed in 4% neutral formalin for histological and immunohistochemical staining. The remaining renal tissures were saved in liquid nitrogen for frozen sections and extraction of protein and m RNA. Western blot was used to detect the expression of TXNIP, SREBP-1, PPAR?, FASN, ACC, ACOX1, CPT1, p-Akt, Akt, p-m TOR and m TOR protein in kidney tissues. The levels of TXNIP, SREBP-1, PPAR?, FASN, ACC, ACOX1 and CPT1 m RNA were detected by Real-time PCR. Lipid droplets formation in renal tissues was detected by Oil Red O staining.3 Effects of TXNIP inhibition on lipid accumulation in high glucose cultured HK-2 cellsHK-2 cells were cultured at 37°C in a humidified atmosphere of 5% CO2 in DMEM-F12 medium supplemented with 10% fetal bovine serum. ⑴Effect of high glucose on the expression of TXNIP and lipid droplets formation: HK-2 cells were randomly divided into normal glucose group(5.5 mmol/L glucose, NG) and high glucose group(30 mmol/L glucose, HG). Then the cells were harvested at 0, 6, 12, 24, 48 and 72 hours after stimulation or not. Expression of TXNIP was detected by Western blot and Real-time PCR. Lipid droplets formation in HK-2 cells was detected by Oil Red O staining. ⑵Effect of TXNIP on lipid accumulation induced by high glucose in HK-2 cells: Stable transfections of HK-2 cells with TXNIP, SREBP-1 sh RNA plasmid or control vector were performed with Fu GENE-HD transfection reagent according to the manufacturer’s instructions. After 48 hours, cells were cultured in selection medium containing 4ug/ml Puromycin for 3 weeks before single clones were isolated. Cells were randomly divided into five groups: normal glucose group(5.5 mmol/L glucose, NG), normal glucose+mannitol group(5.5 mmol/L glucose+24.5 mmol/L mannitol, M), high glucose group(30 mmol/L glucose, HG), high glucose+negative control vector(30 mmol/L glucose+Vector, HG+V), high glucose+TXNIP sh RNA(30 mmol/L glucose+TXNIP sh RNA, HG+T). The groups were cultured for 48 hours respectively, and then HK-2 cells were collected. Western blot was used to detect the expression of TXNIP, SREBP-1, PPAR?, FASN, ACC, ACOX1, CPT1, p-Akt, Akt, p-m TOR and m TOR protein in kidney tissues. The levels of TXNIP, SREBP-1, PPAR?, FASN, ACC, ACOX1 and CPT1 m RNA were detected by Real-time PCR. Lipid droplets formation in HK-2 cells was detected by Oil Red O staining, and the ROS levels were detected by flow cytometry. Triglyceride content was measured using kit from Zhejiang Dongou Company according to instructions of the manufacturer.⑶Effects of activation of PI3K/Akt/m TOR pathway on lipid accumulation induced by high glucose in HK-2 cells: Cells were randomly divided into five groups: normal glucose group(5.5 mmol/L glucose, NG), normal glucose+LY294002 group(5.5 mmol/L glucose+ 10μmol/L LY294002, NG+LY), normal glucose+mannitol group(5.5 mmol/L glucose+24.5 mmol/L mannitol, M), high glucose group(30 mmol/L glucose, HG), high glucose+LY294002 group(30 mmol/L glucose+ 10μmol/L LY294002, HG+LY). Detection methods and indicators consistent with part⑵.4 Effects of natural antioxidant anthocyanins on the expression of TXNIP and lipid accumulation induced by high glucose in renal tubular epithelial cells1 Sixteen male db/db mice were randomly divided into two groups: diabetic nephropathy group(db/db, n=8) and grape seed proanthocyanidin extract treatment group(db/db+GSPE, n=8). Sixteen male non-diabetic littermate db/m mice were also randomly divided into two groups: control group(db/m, n=8) and grape seed proanthocyanidin extract treatment control group(db/m+GSPE, n=8). Mice in the db/db+GSPE group or db/m+GSPE group received 5 mg/kg grape seed proanthocyanidin extract dissolved in saline by gavage daily for eight weeks. Mice in the db/db group or db/m group received the same amount of normal saline. All experimental animals were given free access to food and water. Blood glucose levels and body weight were monitored every two weeks. At the age of fifteen weeks, the mice were sacrificed and the samples of blood and 24 h urine were collected for determination of the renal function, blood glucose and blood lipids. The kidneys were removed and harvested for further analysis. One portion of the kidney tissues was fixed immediately in 2.5% glutaraldehyde for transmission electron microscopy, another portion of the kidney tissues was fixed in 4% neutral formalin for histological and immunohistochemical staining. The remaining renal tissures were saved in liquid nitrogen for frozen sections and extraction of protein and m RNA. Western blot was used to detect the expression of TXNIP, SREBP-1, PPAR?, FASN, ACC, ACOX1 and CPT1 protein in kidney tissues. The levels of TXNIP, SREBP-1, PPAR?, FASN, ACC, ACOX1 and CPT1 m RNA were detected by Real-time PCR. Lipid droplets formation in renal tissues was detected by Oil Red O staining. 2HK-2 cells were cultured at 37°C in a humidified atmosphere of 5% CO2 in DMEM-F12 medium supplemented with 10% fetal bovine serum. ⑴ Effects of anthocyanins on cell viability and radical scavenging: HK-2 cells were cultured with different concertrations of C3G(cyanidin-3-O-?-glucoside chloride) and Cy(cyanidin chloride)(10, 20, 50, 100μM) in the absence or presence of 30 m M high glucose for 48 hours. The survival rate of HK-2 cells after anthocyanins stimulation was detected by MTT, and the ROS levels were detected by flow cytometry. ⑵ Effects of anthocyanins on lipid accumulation and expression of TXNIP induced by high glucose in HK-2 cells: Cells were randomly divided into five groups: normal glucose group(5.5 mmol/L glucose, NG), normal glucose+mannitol group(5.5 mmol/L glucose+24.5 mmol/L mannitol, M), high glucose group(30 mmol/L glucose, HG), high glucose+C3G group(30 mmol/L glucose+C3G, HG+C3G), high glucose+Cy group(30 mmol/L glucose+Cy, HG+Cy). The groups were cultured for 48 hours respectively, and then HK-2 cells were collected. Western blot was used to detect the expression of TXNIP, SREBP-1, PPAR?, FASN, ACC, ACOX1 and CPT1 protein in kidney tissues. The levels of SREBP-1, PPAR?, FASN, ACC, ACOX1 and CPT1 m RNA were detected by Real-time PCR. Lipid droplets formation in HK-2 cells was detected by Oil Red O staining, and the ROS levels were detected by flow cytometry.Results:1 Pathological manifestation and expression of TXNIP, SREBP-1 and PPAR? in renal tissues from patients with diabetic nephropathy1 No obvious morphological changes of glomeruli, renal tubule and interstitium were observed in control group by HE and PASM staining. Compared with control group, histological sections of kidneys from the patients of diabetic nephropathy revealed significant hypertrophy of glomerular, uneven increase in thickeness of glomerular basement membrane and expansion of the mesangial matrix, progressive glomerulosclerosis and interstitial fibrosis. 2 Compared with control group, the levels of Glu, Hb A1, UPE and Scr were significantly increased in diabetic nephropathy group, while the e GFR level of diabetic nephropathy group was decreased(P<0.05 or P<0.01). 3 Oil Red O staining showed that obvious red stained lipid droplet granules in the tubular cells of diabetic nephropathy, which was not seen in control group.4 Compared with control group, the expression of TXNIP and SREBP-1 were significantly increased in renal tubular epithelium in diabetic nephropathy group, whereas the expression of PPAR? was decreased in glomeruli and renal tubular epithelium in diabetic nephropathy group(P<0.05).2 Effects of TXNIP knockout on renal lipid accumulation in STZ –induced diabetic mice1 Histological sections of kidneys from diabetic mice revealed slightly glomeruli hypertrophy, mesangial cell proliferation and mesangium matrix expansion, uneven thickened glomerular basement membrane and partial tubular epithalial vacuolar formation. TKO diabetes exhibited a remarkable amelioration on these morphological changes. 2 Compared with WT mice, the levels of BUN, Scr, TG and UAE were significantly increased in diabetic group; while TKO diabetes exhibited a remarkable decrease on these parameters(P<0.05 or P<0.01). 3 Transmission electron microscopy and Oil Red O staining revealed that obvious lipid droplets formation in the tubular cells of diabetic mice. The contents of triglycerides and cholesterol in the renal tissues of diabetic mice were significantly higher than WT mice. However, lipid droplets formation and increase in the contents of triglycerides and cholesterol were prevented considerably by TXNIP knockout(P<0.01). 4Compared with WT mice, the expression of TXNIP、SREBP-1、FASN、ACC、p-Akt and p-m TOR were significantly up-regulated, whereas PPAR?、CPT1 and ACOX1 expression were decreased in the renal tissues of diabetic mice. Interestingly, increased of TXNIP、SREBP-1、FASN、ACC、p-Akt and p-m TOR and decreased of PPAR?、CPT1 and ACOX1 could be suppressed by TXNIP knockout(P<0.05 or P<0.01).3 Effects of TXNIP inhibition on lipid accumulation in high glucose cultured HK-2 cells1 HK-2 cells incubated with HG showed a time-dependent expression of TXNIP, which was efficiently inhibited by transfection with TXNIP sh RNA plasmid. Oil Red O staining revealed obvious red stained lipid droplet granules in HK-2 cells under high glucose condition, which is also increased in a time-dependent manner. 2 Transfection of TXNIP sh RNA plasmid significantly decreased the HG-induced lipid droplets formation and increase in the contents of triglycerides in HK-2 cells(P<0.01). 3 Compared with normal glucose group, the expression of SREBP-1、FASN and ACC were increased in high glucose group, at the same time, the expression of PPAR?、CPT1 and ACOX1 were decreased in HK-2 cells. Transfection of TXNIP sh RNA plasmid significantly inhibited the HG-induced increase of SREBP-1、FASN and ACC and decrease of PPAR?、CPT1 and ACOX1(P<0.05 or P<0.01). In addition, mannitol and vector sh RNA plasmid had no effect on HK-2 cells. 4 Compared with high glucose group, the phosphorylation levels of Akt and m TOR induced by high glucose were significantly inhibited by specific inhibitors of the PI3K/Akt pathway LY294002. Meanwhile, LY294002 treatment reversed high glucose-induced increase of SREBP-1, FASN and ACC and decrease of PPAR?、CPT1 and ACOX1 in HK-2 cells. Consistently, the contents of cellular triglycerides were remarkably decreased upon treatment with LY294002(P<0.05 or P<0.01).4 Effects of natural antioxidant anthocyanins on the expression of TXNIP and lipid accumulation induced by high glucose in renal tubular epithelial cells1 Histological sections of kidneys from db/db mice revealed slightly glomeruli hypertrophy, mesangial cell proliferation and mesangium matrix expansion, uneven thickened glomerular basement membrane and partial tubular epithalial vacuolar formation. These morphological changes were markedly ameliorated by treatment with grape seed proanthocyanidin extract(GSPE). Compared with db/m mice, the levels of FBG, BUN, Scr, TG, TC and UAE were significantly increased in db/db group; while db/db mice treatment with GSPE exhibited a remarkable decrease on these parameters(P<0.05 or P<0.01). 2 Transmission electron microscopy and Oil Red O staining revealed that obvious lipid droplets formation in the tubular cells of diabetic db/db mice. The contents of triglycerides and cholesterol in the renal tissues of db/db mice were significantly higher than db/m mice. However, lipid droplets formation and increase in the contents of triglycerides and cholesterol were prevented considerably by treatment with GSPE. 3 Compared with db/m mice, the expression of TXNIP、SREBP-1、FASN and ACC were significantly up-regulated, whereas PPAR?、CPT1 and ACOX1 expression were decreased in the renal tissues of db/db mice. Interestingly, increased of TXNIP、SREBP-1、FASN and ACC and decreased of PPAR?、CPT1 and ACOX1 could be suppressed by GSPE treatment(P<0.05 or P<0.01). 4 The expression of TXNIP and the levels of intracellular ROS induced by high glucose were decreased by anthocyanins in a dose-dependent manner in HK-2 cells(P<0.05 or P<0.01), meanwhile, anthocyanins treatment significantly decreased the HG-induced lipid droplets formation in HK-2 cell. 5 Compared with normal glucose group, the expression of SREBP-1、FASN and ACC were increased in high glucose group, at the same time, the expression of PPAR?、CPT1 and ACOX1 were decreased in HK-2 cells. anthocyanins treatment significantly inhibited the HG-induced increase of SREBP-1 、 FASN and ACC and decrease of PPAR? 、 CPT1 and ACOX1(P<0.05 or P<0.01). In addition, mannitol and vector sh RNA plasmid had no effect on HK-2 cells.Conclusions:1 Increased of TXNIP induced by high glucose was found in renel tissues from diabetic nephropathy patients and diabetic mouse model as well as cultured HK-2 cells. Meanwhile, obvious lipid accumulation was observed in renal tubular epithelial cells, indicating that TXNIP may participate in the process of lipid accumulation in diabetic nephropathy.2 Knockout of TXNIP prevents high glucose-induced lipid accumulation in renal tissues and cultured HK-2 cells by inhibiting the expression of proteins related with fatty acid synthesis and up-regulating the expression of proteins related with fatty acid ? oxidation, which maybe partly through inhibiting activation of Akt and m TOR.3 The expression of TXNIP induced by high glucose was decreased by anthocyanins in renal tubular epithelial cells. In addition, anthocyanins prevents high glucose-induced lipid accumulation in renal tissues and cultured HK-2 cells by inhibiting the expression of proteins related with fatty acid synthesis and up-regulating the expression of proteins related with fatty acid ? oxidation, which maybe through inhibiting activation of Akt and m TOR. Taken together, these results suggest that anthocyanins modulate lipid metabolism might partly through inhibition of TXNIP expression in diabetic nephropathy.