The inflammatory cytokines TWEAK and TNFα reduce renal klotho expression through NFκB

Abstract

Proinflammatory cytokines contribute to renal injury, but the downstream effectors within kidney cells are not well understood. One candidate effector is Klotho, a protein expressed by renal cells that has antiaging properties; Klotho-deficient mice have an accelerated aging-like phenotype, including vascular injury and renal injury. Whether proinflammatory cytokines, such as TNF and TNF-like weak inducer of apoptosis (TWEAK), modulate Klotho is unknown. In mice, exogenous administration of TWEAK decreased expression of Klotho in the kidney. In the setting of acute kidney injury induced by folic acid, the blockade or absence of TWEAK abrogated the injury-related decrease in renal and plasma Klotho levels. TWEAK, TNFα, and siRNA-mediated knockdown of IκBα all activated NFκB and reduced Klotho expression in the MCT tubular cell line. Furthermore, inhibition of NFκB with parthenolide prevented TWEAK- or TNFα-induced downregulation of Klotho. Inhibition of histone deacetylase reversed TWEAK-induced downregulation of Klotho, and chromatin immunoprecipitation showed that TWEAK promotes RelA binding to the Klotho promoter, inducing its deacetylation. In conclusion, inflammatory cytokines, such as TWEAK and TNFα, downregulate Klotho expression through an NFκB-dependent mechanism. These results may partially explain the relationship between inflammation and diseases characterized by accelerated aging of organs, including CKD.

Copyright © 2011 by the American Society of Nephrology

Figures

Figure 1.

Cytokine and Klotho expression in murine model of renal tubulointerstitial inflammation. (A) Expression of inflammatory cytokines (TNFα and TWEAK) and TWEAK and TNF receptors (Fn14 and TNFR1/TNFR2, respectively) mRNA were measured by real time RT-PCR in kidneys of mice with AKI induced by a folic acid overdose at 72 hours. Expression of cytokines and their receptors is increased in this model. Mean ± SEM of six animals per group. *P < 0.05 versus control. (B) Representative immunohistochemistry of renal macrophage infiltration at 72 hour AKI mice Macrophages were identified by staining with F4/80 antibody. Increased macrophage-positive cells were noted in AKI mice with respect to healthy control mice. Controls for the technique are stained with nonspecific IgG. (C) Time course of kidney Klotho mRNA expression and plasma creatinine levels in folic acid-induced AKI. Kidney Klotho mRNA is decreased even when renal function has recovered. Mean ± SEM of six animals per group. *P < 0.005 versus control.

Figure 2.

Antagonism or absence of TWEAK prevented downregulation of Klotho in renal tubulointerstitial inflammation. (A and B) The decreased total kidney Klotho mRNA expression observed in folic acid-induced AKI at 72 hours (RT-PCR) is improved by (A) TWEAK antagonism, using a neutralizing anti-TWEAK antibody (mean ± SEM of six animals per group; *P < 0.002 versus healthy mice; #P < 0.02 versus AKI; †P < 0.02 versus AKI+Isotype IgG) or by (B) TWEAK absence as shown by comparing WT and TWEAK KO mice (mean ± SEM of five animals per group; *P < 0.005 versus WT healthy; #P < 0.03 versus WT AKI). (C) Total kidney Klotho protein, measured by Western blot, is also reduced in AKI at 72 h, and this reduction is prevented by TWEAK antagonism. Mean ± SEM of six animals per group. *P < 0.002 versus healthy mice; #P < 0.02 versus AKI; †P < 0.02 versus AKI+Isotype IgG. (D) Plasma levels of Klotho, measured by ELISA, are decreased in mice with AKI at 72 hours, and this is improved by anti-TWEAK antibody pretreatment. Mean ± SEM of six animals per group. *P < 0.005 versus healthy mice; #P < 0.05 versus AKI; †P < 0.05 versus AKI+Isotype IgG.

Figure 3.

TWEAK neutralization improves renal function during AKI. AKI was induced by a folic acid overdose in mice and renal function was assessed at 72 hours measuring plasma creatinine and BUN. (A) Plasma creatinine levels. (B) BUN. Mean ± SEM of six animals per group. *P < 0.004 versus healthy mice; #P < 0.04 versus AKI; †P < 0.03 versus AKI+Isotype IgG.

Figure 4.

Systemic TWEAK injection in healthy mice decreases renal Klotho mRNA and protein in vivo in healthy mice. TWEAK or vehicle were injected intraperitoneally. Total kidney Klotho mRNA and protein were decreased in a time-dependent manner in TWEAK-injected mice, compared with vehicle-treated control mice. This effect was reverted by pretreatment with Parthenolide (PTN). (A) Quantitative RT-PCR analyses of renal Klotho mRNA in mice 4 or 24 hours after TWEAK, PTN, or TWEAK/PTN. Mean ± SEM of six animals per group. *P < 0.01 versus control; #P < 0.05 versus TWEAK alone. (B) Total kidney Klotho protein levels in mice treated with TWEAK and PTN for 24 hours measured by Western blot. Mean ± SEM of six animals per group. *P < 0.02 versus control; #P < 0.02 versus TWEAK alone.

Figure 5.

Inflammatory cytokines decrease Klotho expression in cultured tubular cells in an NFκB-dependent manner. TWEAK (A) and TNFα (B) decrease Klotho mRNA expression in a dose-dependent manner at 3 hours in MCT cells. Mean ± SD of three independent experiments. *P < 0.0001 versus control; #P < 0.001 versus control. (C) Time course of TWEAK (100 ng/ml) and TNFα (30 ng/ml) decreased Klotho mRNA expression in tubular cells. Mean ± SD of three independent experiments. *P < 0.0001 versus control. Real time RT-PCR. (D) Representative Western blot and quantification of three independent experiments shows decreased Klotho protein in MCT cells treated with TWEAK (100 ng/ml) or TNFα (30 ng/ml). Mean ± SD of three independent experiments. *P < 0.008 versus control. (E) MCT cells were treated with 100 or 50 ng/ml TWEAK for 24 hours, and Klotho levels were measured in cell supernatants by ELISA. TWEAK decreased soluble Klotho in a dose-dependent manner. Mean ± SD of three experiments. *P < 0.05 versus control; #P < 0.05 versus TWEAK 50 ng/ml.

Figure 6.

Fn14 mediates TWEAK-induced Klotho downregulation in cultured tubular cells. (A) A neutralizing anti-TWEAK antibody (10 μg/ml) rescued Klotho levels in MCT cells treated with 100 ng/ml TWEAK for 3 hours. Mean ± SD of three independent experiments. *P < 0.002 versus control; #P < 0.02 versus TWEAK alone; †P < 0.006 versus TWEAK+Isotype IgG. (B) ITEM-4 (10 μg/ml), a neutralizing anti-Fn14 antibody, prevented Klotho downregulation induced by TWEAK in MCT cells, suggesting that this effect of TWEAK is mediated through Fn14 binding. Mean ± SD of three independent experiments. *P < 0.005 versus control; #P < 0.02 versus TWEAK alone.

Figure 7.

PTN prevents Klotho downregulation induced by TNFα or TWEAK in cultured tubular cells. (A) TNFα or TWEAK promoted DNA binding of NFκB complexes as assessed by EMSA. Quantification (mean ± SD) and representative EMSA of three independent experiments. *P < 0.05 versus control. (B) TNFα or TWEAK promote the translocation of the RelA/p65 protein to the nucleus at 60 minutes in tubular cells. This is prevented by PTN. Nuclear translocation of NFκB subunits is required for DNA binding. Images representative of three independent experiments. Confocal microscopy where RelA is green and propidium iodide (nuclei) is orange (magnification, ×320). (C) The NFκB inhibitor PTN prevents TWEAK- and TNFα-induced downregulation of Klotho mRNA in cultured MCT cells at 3 hours. Mean ± SD of three independent experiments. *P < 0.0001 versus control; #P < 0.0001 versus cytokine alone.

Figure 8.

TWEAK induces RelA binding to the Klotho promoter in cultured tubular cells. (A) Region 1; (B) region 2. MCT cells were stimulated with TWEAK for 60 minutes, and ChIP analyses were performed using an anti-RelA antibody. Promoter copy number was quantified by real-time PCR in duplicate using two specific primers that amplify two different NFκB binding sites of the Klotho promoter. Normal rabbit IgG was used as negative control for the specificity of the immunoprecipitation (intraperitoneally). As a positive control, aliquots of chromatin fragments obtained before intraperitoneally were also subjected to RT-PCR analysis (Input). Immunoprecipitated DNA with RelA binding was normalized to a 100-fold dilution of input chromatin. Data are expressed as fold enrichment of RelA binding compared with negative control antibody (normal rabbit IgG). n = 4; *P < 0.04 versus control.

Figure 9.

TWEAK- and TNFα-mediated downregulation of Klotho mRNA in cultured tubular cells requires HDAC activity. (A) The HDAC inhibitor TSA rescues TWEAK- or TNFα-induced repression of Klotho at 3 hours in MCT cells. Cells were prestimulated with TSA (100 ng/ml) 1 hour before addition of cytokines. Mean ± SD of three independent experiments. *P < 0.0001 versus control; #P < 0.001 versus cytokine alone. (B) TWEAK and TNFα induce RelA association with HDAC1 in MCT cells. Cells were stimulated with TWEAK (100 ng/ml) or TNFα (30 ng/ml) for 30 and 60 minutes, to coincide with peak RelA nuclear translocation. Nuclear extracts were immunoprecipitated with 3 μg anti-RelA antibody or control IgG and then Western blotted with anti-HDAC1. Nuclear extract input was 25 μg. (C) TWEAK induces deacetylation at the Klotho promoter in MCT cells at 60 minutes as assessed by ChIP. Anti-acetylated histone H3 and H4 antibodies were used. The presence of acetylated histone H3 and H4 at the Klotho promoter was detected by RT-PCR with specific primers for two different NFκB binding sites of the Klotho promoter. Normal rabbit IgG was used as negative control for the specificity of the immunoprecipitation (intraperitoneally). As a positive control, aliquots of chromatin fragments obtained before intraperitoneally were also subjected to RT-PCR analysis (Input). Immunoprecipitated DNA with histone modification was normalized to a 100-fold dilution of input chromatin. Data are expressed as fold enrichment of RelA binding compared with negative control antibody (normal rabbit IgG). n = 4; *P < 0.04 versus control.