TNF-α-induced Inflammation Stimulates Apolipoprotein-A4 via Activation of TNFR2 and NF-κB Signaling in Kidney Tubular Cells

Abstract

Apo-A4 expression was increased in tissues from chronic kidney disease (CKD) patients compared to that in normal kidney tissue. We determined the association of apo-A4 and its regulatory signals following acute kidney injury and elucidated the effects of apo-A4 on cell signaling pathways related to kidney injury in vitro and in vivo. Tumor necrosis factor (TNF)-α, which causes inflammatory cell injury, induced significantly increased expression of apo-A4 protein levels, and these levels were related to pro-inflammatory acute kidney injury in human kidney cells. Apo-A4 expression was also increased in experimented rat kidney tissues after ischemic reperfusion injury. The expression of tumor necrosis factor receptor (TNFR) 2 was increased in both kidney cell lines and experimented rat kidney tissues following acute kidney injury. The expression of apo-A4 and TNFR2 was increased upon treatment with TNF-α. Immunohistochemistry revealed positive apo-A4 and TNFR2 staining in ischemic reperfusion injury rat kidneys compared with levels in the sham operation kidneys. After neutralization of TNF-α, NF-κB expression was only observed in the cytoplasm by immunofluorescence. Therefore, the apo-A4 expression is increased by stimulation of injured kidney cells with TNF-α and that these effects occur via a TNFR2-NFκB complex.

Figure 1

Kidney cell lines and human kidney tissues expressed apo-A4. (A) Endogenous apo-A4 protein expression was measured in Caki-1, Caki-2, HK-2, and HEK-293 cells by western blotting. The loading control was α-tubulin. (B) IHC of apo-A4 in Formalin-fixed, paraffin-embedded human normal and CKD kidney tissue.

Figure 2

Effect of renal injury model expressed apo-A4 in kidney cell lines. (A) Hypoxic cell injury; HIF-1α and apo-A4 expression in Caki-1, HK-2, and HEK-293 cells exposed to hypoxia for 72 h. (B) Chemical cell injury; HIF-1α and apo-A4 expression in HK-2 cells treated with different concentrations of CoCl₂ (0, 100, and 200 µM) for 8, 16, and 24 h. (C) Cytotoxic cell injury and cytokaine cell injury; Apo-A4 expression in HK-2 cells treated with cisplatin (0, 10, 20, and 30 µM), A23187 (0, 0.1, 0.5, and 1 μg/ml), TNF-α (0, 50, 100, and 200 ng/ml), and IL-1β (0, 50, 100, and 200 ng/ml) for 24 h (see Supplementary Figs S1, 2 and 3). (D) Immunofluorescence assay showing localization of apo-A4 (red) in HK-2 cells after 24 h of treatment with TNF-α.

Figure 3

Inflammatory AKI induced by TNF-α increased apo-A4 expression in vivo. (A) Graphical presentation of in vivo experiment method with male SD rat. (B) Expression of apo-A4 protein detected by western blotting of kidney tissue from an ischemic reperfusion injury rat model. (C) Apo-A4 expression in rat plasma measured by ELISA. Results represent the mean (±SEM) of ten independent experiments (n = 10). The statistical signification was marked as * for p < 0.05 compared with the control.

Figure 4

Inflammatory AKI induced by TNF-α increased apo-A4 expression via TNFR2. (A) HK-2 cells were treated with different concentrations of TNF-α (0, 50, and 200 ng/ml) for 24 h. Apo-A4 protein expression was detected by western blotting. (B) Apo-A4 and TNFR2 immunohistochemistry on formalin-fixed, paraffin-embedded tissue from ischemic reperfusion injury rat kidneys.

Figure 5

Inflammatory AKI increases apo-A4 expression through the NF-κB complex. (A) Western blotting revealed an inverse relationship between apo-A4 and IκB protein expression in HK-2 cells following TNF-α treatment. The loading control was GAPDH and the normalized ratio of each extract pair (A set to 1) determined using Image Jv1.42q software (http://rsb.info.nih.gov/ij). (B) Immunofluorescence assay for apo-A4 and NF-κB expression in HK-2 cells after 24-h TNF-α treatment. TNF-α antibody was used to neutralize the TNF-α effect. NF-κB (green) immune-localization is shown. And NF-κB regulates apo-A4 (see Supplementary Fig. S4).

Figure 6

Proposed model of apo-A4 mediated signaling pathway in kidney injury. (A) TNF-α mediates an increase in apo-A4 expression during inflammatory AKI. TNF-α activates apo-A4 expression via the TNFR2-NFκB pathway. (B) Continuous kidney damage caused by ischemic reperfusion injury promotes apo-A4 deposition in kidney cells.