Role of the thioredoxin interacting protein in diabetic nephropathy and the mechanism of regulating NOD‑like receptor protein 3 inflammatory corpuscle

Abstract

Inflammatory response serves an important role in diabetic nephropathy (DN); however, the mechanism of inflammatory response results in renal damage is not yet clear. The aim of the present study was to investigate the role of the thioredoxin interacting protein (TXNIP)/NOD‑like receptor protein 3 (NLRP3) axis‑mediated activation of NLRP3 inflammasome in DN. A diabetic rat model was induced by streptozotocin injection. Hematoxylin and eosin (H&E) staining and streptavidin‑peroxidase staining were then used to examine the kidney tissue morphology, and TXNIP and NLRP3 protein expression levels, respectively. Furthermore, RNA interference technology was applied to silence the TXNIP gene. TXNIP and NLRP3 inflammasome activation‑associated proteins and mRNAs were detected by western blot analysis and RT‑qPCR, respectively. Enzyme‑linked immunosorbent assay was also used to examine interleukin (IL)‑1 and IL‑18 expression, while flow cytometry was performed to detect reactive oxygen species production. The data revealed that TXNIP and NLRP3 were overexpressed in kidney tissue of DN rats, and the level of antioxidant genes was downregulated. It was also observed that glucose promoted TXNIP expression and activation of NLRP3 inflammasome in a time‑dependent and dose‑dependent manner, therefore promoting inflammatory responses. Silencing of TXNIP gene resulted in the downregulation of NLRP3 inflammasome activation, and inhibited the expression levels of IL‑1 and IL‑18 in a high‑glucose environment. Furthermore, low expression of TXNIP promoted the levels of antioxidant genes and reduced the ROS levels. Taken together, the high‑glucose environment was able to upregulated the level of inflammatory factors by promoting the expression of TXNIP and the activation of NLRP3 inflammasome, consequently participating in DN.

Figure 1

Changes in blood glucose and renal tissue morphology in rats following streptozotocin injection. (A) Blood glucose level changes at 1, 2 and 3 weeks after the injection with streptozotocin. (B) Hematoxylin and eosin staining of the kidney tissues of the rats was observed under a microscope (magnification, ×100). (C) Glomerular volume was assessed at 1, 2 and 3 weeks after streptozotocin injection. ^**P<0.01, vs. control group. DN, diabetic nephropathy.

Figure 2

Expression levels of TXNIP and NLRP3 inflammasome-associated proteins in renal tissue of DN rats. (A) Western blot images and (B) quantified protein levels of TXNIP, NLRP3, cleaved-caspase-1, caspase-1, IL-1 and IL-18 in the control and DN groups. (C) Streptavidin-peroxidase staining was used to detect the TXNIP and NLRP3 proteins expression in kidneys tissues (magnification, ×100), and (D) the quantified staining intensity is shown. ^*P<0.05 and ^**P<0.01, vs. control group. DN, diabetic nephropathy; TXNIP, thioredoxin interacting protein; NLRP3, NOD-like receptor protein 3; IL, interleukin.

Figure 3

Antioxidant gene expression levels in DN rats. (A) Western blots and (B) quantified protein expression levels of catalase and MnSOD proteins in renal tissues in the control and DN groups were detected by performing western blot analysis. (C) mRNA expression levels of catalase and MnSOD in renal tissues in the two groups were detected by reverse transcription-quantitative polymerase chain reaction. (D) SOD and (E) MDA concentrations in the two groups, detected using ELISA. ^*P<0.05 and ^**P<0.01 vs. control group. DN, diabetic nephropathy; MnSOD, manganese superoxide dismutase; MDA, malondialdehyde.

Figure 4

Effects of different concentrations of glucose on TXNIP expression and the activation of NLRP3 inflammasome in HK-2 cells. (A) Western blots and (B) quantified expression levels of TXNIP, NLRP3, cleaved-caspase-1 and caspase-1 proteins in the control, HG1, HG2 and HG3 groups, which were treated with 5.5, 15, 30 and 50 mmol/l glucose, respectively. (C) IL-1 and (D) IL-18 expression levels in the four groups were tested using ELISA. ^*P<0.05 and ^**P<0.01, vs. control group; ^#P<0.05. TXNIP, thioredoxin interacting protein; NLRP3, NOD-like receptor protein 3; IL, interleukin.

Figure 5

Effects of glucose on TXNIP expression and activation of NLRP3 inflammasome in HK-2 cells at different times. Cells were incubated with 50 mmol/l glucose for 0, 24, 48, 72 and 96 h. (A) Western blots and (B) quantified expression levels of TXNIP, NLRP3, cleaved-caspase-1 and caspase-1 proteins at different time points. (C) IL-1 and (D) IL-18 expression levels were detected by ELISA. ^*P<0.05 and ^**P<0.01, vs. control (0 h) group; ^#P<0.05 and ##P<0.01. TXNIP, thioredoxin interacting protein; NLRP3, NOD-like receptor protein 3; IL, interleukin.

Figure 6

Effects of TXNIP gene silencing on NLRP3 inflammasome activation-associated proteins and inflammation-associated factors. (A) Western blots and (B) quantified expression levels of TXNIP, NLRP3, cleaved-caspase-1 and caspase-1 proteins in the control, HG, NC + HG, siTXNIP and siTXNIP + HG groups. (C) IL-1 and (D) IL-18 expression levels in each group were detected by ELISA. ^*P<0.05 and ^**P<0.01, vs. control group; ^#P<0.05 and ^##P<0.01. TXNIP, thioredoxin interacting protein; NLRP3, NOD-like receptor protein 3; IL, interleukin; HG, high glucose; NC, negative control siRNA; si-, small interfering RNA.

Figure 7

Effects of TXNIP gene silencing on antioxidant gene expression and ROS levels in HK-2 cells. (A) Western blots and (B) quantified protein expression levels of catalase and MnSOD in the control, HG, NC + HG, siTXNIP + HG and siTXNIP groups, detected using western blot analysis. (C) mRNA expression levels of catalase and MnSOD in the different groups, detected using reverse transcription-quantitative polymerase chain reaction. (D) ROS levels in each group and (E) flow cytometry applied to detect these levels. ^*P<0.05 and ^**P<0.01, vs. control group; ^#P<0.05 and ^##P<0.01. TXNIP, thioredoxin interacting protein; ROS, reactive oxygen species; HG, high glucose; NC, negative control siRNA; si-, small interfering RNA.