Knockdown of NLRP3 alleviates high glucose or TGFB1-induced EMT in human renal tubular cells

in Journal of Molecular Endocrinology
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Tubular injury is one of the crucial determinants of progressive renal failure in diabetic nephropathy (DN), while epithelial-to-mesenchymal transition (EMT) of tubular cells contributes to the accumulation of matrix protein in the diabetic kidney. Activation of the nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome leads to the maturation of interleukin (IL)-1B and is involved in the pathogenic mechanisms of diabetes. In this study, we explored the role of NLRP3 inflammasome on high glucose (HG) or transforming growth factor-B1 (TGFB1)-induced EMT in HK-2 cells. We evaluated EMT through the expression of α-smooth muscle actin (α-SMA) and E-cadherin as well as the induction of a myofibroblastic phenotype. Reactive oxygen species (ROS) was observed using the confocal microscopy. HG was shown to induce EMT at 48 h, which was blocked by NLRP3 silencing or antioxidant N-acetyl-L-cysteine (NAC). We found that NLRP3 interference could inhibit HG-induced ROS. Knockdown of NLRP3 could prevent HG-induced EMT by inhibiting the phosphorylation of SMAD3, P38 MAPK and ERK1/2. In addition, P38 MAPK and ERK1/2 might be involved in HG-induced NLRP3 inflammasome activation. Besides, TGFB1 induced the activation of NLRP3 inflammasome and the generation of ROS, which were blocked by NLRP3 interference or NAC. Tubular cells exposed to TGFB1 also underwent EMT, and this could be inhibited by NLRP3 shRNA or NAC. These results indicated that knockdown of NLRP3 antagonized HG-induced EMT by inhibiting ROS production, phosphorylation of SMAD3, P38MAPK and ERK1/2, highlighting NLRP3 as a potential therapy target for diabetic nephropathy.

 

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    Activation of NLRP3 inflammasome in HG-treated HK-2 cells. (A) The activation of NLRP3 inflammasome in cultured HK-2 cells treated with HG (30 mM) at various time points were analyzed by Western blot (n = 6). (B) The quantitative analysis of NLRP3 inflammasome protein level in cultured HK-2 cells treated with HG (30 mM) at various time points (n = 6). (C) The mRNA expression of NLRP3 inflammasome was analyzed by RT-qPCR (n = 6). (D and E) The quantitative analysis of protein level of IL1B and IL18 in HG-treated HK-2 cells at various time points (n = 6). (F and G) The mRNA expression of IL1B and IL18 was analyzed by RT-qPCR (n = 6). Values are expressed as mean ± s.d. *P < 0.05, **P < 0.01 vs control (0 h).

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    Effects of NLRP3 shRNA plasmid transfection on HG-induced activation of NLRP3 inflammasome and ROS production in HK-2 cells. (A) The protein expression of NLRP3 inflammasome, IL1B and IL18 was analyzed by Western blot (n = 6). (B) The quantitative analysis of NLRP3 inflammasome protein level (n = 6). (C) The mRNA expression of NLRP3 inflammasome was analyzed by RT-qPCR (n = 6). (D and E) The quantitative analysis of protein level of IL1B and IL18 (n = 6). (F and G) The mRNA expression of IL1B and IL18 was analyzed by RT-qPCR (n = 6). (H) Mitochondrial ROS were detected by the confocal microscope (n = 6). (I) The quantification of mitochondrial ROS intensity. NG: 5.6 mM d-glucose; HG: 30 mM d-glucose; HG + C: HG + control shRNA plasmid; HG + shRNA: HG + NLRP3 shRNA plasmid; HG + NAC: HG + N-acetylcysteine (5 mM). Values are expressed as means ± s.d. *P < 0.05, **P < 0.01 vs NG, #P < 0.05, ##P < 0.01 vs HG + C.

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    Effects of NLRP3 silencing on HG-induced EMT in HK-2 cells. (A, B and C) The expression levels of α-SMA and E-cadherin protein were detected by Western blot (n = 6). (D and E) The mRNA levels of α-SMA and E-cadherin were analyzed by RT-qPCR (n = 6). (F) Morphological changes of HK-2 cells cultured under different conditions were analyzed by the inverted microscope. (G) The effect of NLRP3 shRNA plasmid on the expression of α-SMA and E-cadherin in HK-2 cells was detected by immunofluorescence. NG: 5.6 mM d-glucose; HG: 30 mM d-glucose; HG + C: HG + control shRNA plasmid; HG + shRNA: HG + NLRP3 shRNA plasmid; HG + NAC: HG + N-acetylcysteine (5 mM). Values are expressed as means ± s.d. **P < 0.01 vs NG, #P < 0.05, ##P < 0.01 vs HG + C.

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    Effect of NLRP3 interference on HG-induced activation of SMAD3, P38 MAPK and ERK1/2 as well as the effect of SB203580 and PD98059 on NLRP3 inflammasome. (A, B, C and D) The expression levels of p-SMAD3, SMAD3, p-P38 MAPK, P38 MAPK, pERK1/2 and ERK1/2 were analyzed by Western blot (n = 6). NG: 5.6 mM d-glucose; HG: 30 mM d-glucose; HG + C: HG + control shRNA plasmid; HG + shRNA: HG + NLRP3 shRNA plasmid; HG + NAC: HG + N-acetylcysteine (5 mM). (E, F, G and H) The expression level of NLRP3 inflammasome and the maturation of IL1B and IL18 was analyzed by Western blot (n = 4). NG: 5.6 mM d-glucose, HG: 30 mM d-glucose, HG + SB: HG + SB203580 (10 µM) and HG + PD: HG + PD98059 (50 µM). Values are expressed as means ± s.d. *P < 0.05, **P < 0.01 vs NG, #P < 0.05, ##P < 0.01, vs HG + C.

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    The effect of TGFB1 on the activation of NLRP3 inflammasome and ROS production in HK-2 cells. HK-2 cells were exposed to 4 ng/mL TGFB1 under different conditions for 48 h. (A) The protein expression of NLRP3 inflammasome, IL1B and IL18 was detected by Western blot (n = 5). (B) The quantitative analysis of NLRP3 inflammasome protein level (n = 5). (C) The mRNA expression of NLRP3 inflammasome was analyzed by RT-qPCR (n = 5). (D and E) The quantitative analysis of protein level of IL1B and IL18 (n = 5). (F and G) The mRNA expression of IL1B and IL18 was analyzed by RT-qPCR (n = 5). (H) Mitochondrial ROS were detected by the confocal microscope (n = 5). (I) The quatification of mitochondrial ROS intensity. N: normal control; T: TGFB1 (4 ng/mL); T + C: TGFB1 (4 ng/mL) + control shRNA plasmid; T + shRNA: TGFB1 (4 ng/mL) + NLRP3 shRNA plasmid; T + NAC: TGFB1 (4 ng/mL) + N-acetylcysteine (5 mM). Values are expressed as means ± s.d. **P < 0.01 vs N, #P < 0.05, ##P < 0.01 vs T + C.

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    The effect of NLRP3 silencing on TGFB1-induced EMT in HK-2 cells. HK-2 cells were exposed to 4 ng/mL TGFB1 under different conditions for 48 h. (A, B and C) The protein levels of α-SMA and E-cadherin were detected by Western blot (n = 5). (D and E) The mRNA expression levels of α-SMA and E-cadherin were analyzed by RT-qPCR (n = 5). (F) Morphological changes of HK-2 cells were analyzed by the inverted microscope. (G) The effect of NLRP3 shRNA plasmid on the expression of α-SMA and E-cadherin in HK-2 cells was detected by immunofluorescence. N: normal control; T: TGFB1 (4 ng/mL); T + C: TGFB1 (4 ng/mL) + control shRNA plasmid; T + shRNA: TGFB1 (4 ng/mL) + NLRP3 shRNA plasmid; T + NAC: TGFB1 (4 ng/mL) + N-acetylcysteine (5 mM). Values are expressed as means ± s.d. **P < 0.01 vs N, #P < 0.05, ##P < 0.01 vs T + C.

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