Advanced oxidation protein products inhibit the autophagy of renal tubular epithelial cells

Abstract

It is well known that autophagy serves a crucial role in renal tubular epithelial cell (RTEC) injury in the pathogenesis of chronic kidney disease (CKD). The accumulation of advanced oxidation protein products (AOPPs) also participates in the progression of CKD. However, the effects of AOPPs on autophagy remain unknown. To clarify the underlying mechanism of RTEC injury in CKD, the effect of AOPPs on HK-2 cells, an RTEC cell line, was investigated. The results of the present study revealed that AOPP exposure downregulated the expression of B-cell lymphoma-2-interacting myosin-like coiled-coil protein 1, reduced the conversion of microtubule-associated proteins 1 light chain 3B (LC3)-I to LC3-II and the formation of autophagosomes, and lead to an accumulation of p62. These results suggest that AOPPs may inhibit the autophagic activity of HK-2 cells. Furthermore, the aforementioned changes were mediated by the AOPP-phosphorylated phosphoinositide 3-kinase3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway; this was verified by treatment with LY294002, a PI3K inhibitor, which reversed the AOPP-induced changes. The present study also demonstrated that the activation of autophagy with rapamycin led to an improvement in the AOPP-induced overexpression of kidney injury molecule 1 and neutrophil gelatinase-associated lipocalin, two biomarkers of RTEC injury, whereas inhibiting autophagy with chloroquine further increased their expression during AOPP treatment. Collectively, these results indicate that AOPPs may inhibit autophagy in RTECs via activation of the PI3K/AKT/mTOR pathway and that autophagy inhibition serves a role in AOPP-induced RTEC injury.

Keywords: advanced oxidation protein products; autophagy; chronic kidney disease; phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway; renal tubular epithelial cell injury.

Figure 1.

AOPPs inhibit autophagic activity in HK-2 cells. HK-2 cells were treated with (A) vehicle control, unmodified BSA (200 µg/ml) and BSA + AOPPs for 12 h, or (B) 200 µg/ml of AOPPs for 3, 6, 12 or 24 h and levels of LC3-I, LC3-II, Beclin1 and p62 were assessed using western blotting. (C and D) Quantified results of western blotting. Data are expressed as the mean ± standard deviation of three independent experiments; *P<0.05 vs. Ctrl. AOPPs, advanced oxidation protein products; BSA, bovine serum albumin; LC3, microtubule-associated proteins 1 light chain 3B; Beclin1, B-cell lymphoma-2-interacting myosin-like coiled-coil protein; Ctrl, control.

Figure 2.

Immunofluorescent staining and TEM observation of autophagy. HK-2 cells were treated with the vehicle control or 200 µg/ml AOPPs for 12 h in the absence or presence of LY294002 (10 µM). (A) Indirect immunofluorescent staining revealed that AOPP treatment decreased positive LC3B staining, whereas LY294002 addition reversed this effect. Magnification, ×100. Scale bar, 10 µm. (B) TEM visualization revealed that AOPP treatment inhibited autophagic vacuole formation, whereas LY294002 treatment improved it. Top row, magnification, ×10,000 and scale bar, 2 µm; bottom row, magnification, ×20,000 and scale bar, 1 µm (C) LC3B-positive cells were counted in individual HK-2 cells and the mean of ≥30 cells was calculated. (D) Images of 10 random TEM fields were captured in a grid (top left, middle left, bottom left, center, top right, middle right and bottom right) and the number of autophagosomes, autolysosomes and cells were counted. Data are expressed as the mean ± standard deviation of three independent experiments; *P<0.05 vs. control, ^#P<0.05 vs. AOPP-treatment group. TEM, transmission electron microscopy; AOPPs, advanced oxidation protein products; LC3B, microtubule-associated proteins 1 light chain 3B; Ctrl, control.

Figure 3.

AOPPs activate the PI3K/AKT/mTOR signaling pathway. HK-2 cells were treated with (A) vehicle, unmodified BSA (200 µg/ml) and 100, 200 or 400 µg/ml AOPPs for 12 h, or (B) 200 µg/ml of AOPPs for 3, 6, 12 or 24 h and the phosphorylation of PI3K, AKT and mTOR was assessed using western blotting (C-F). Western blotting results were quantified using densitometry. Data are presented as the mean ± standard deviation of three independent experiments; *P<0.05 vs. Ctrl. AOPPs, advanced oxidation protein products; PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; mTOR, mammalian target of rapamycin; BSA, bovine serum albumin; Ctrl, control; p, phosphorylated; t, total.

Figure 4.

AOPPs inhibit autophagy via the PI3K/AKT/mTOR signaling pathway. (A) HK-2 cells were treated with the vehicle control or 200 µg/ml AOPPs for 12 h in the absence or presence of 10 µM LY294002 and the expression of (B) PI3K, (C) AKT and mTOR was quantified. The PI3K/AKT/mTOR signaling pathway was significantly blocked by LY294002. (D) Western blotting revealed that the AOPP-induced decrease in LC3-I to LC3-II conversion, suppression of Beclin1 and overexpression of p62 were partly reversed by treatment with LY294002. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. Ctrl, ^#P<0.05 vs. AOPP-treatment group. AOPPs, advanced oxidation protein products; PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; mTOR, mammalian target of rapamycin; LC3, microtubule-associated proteins 1 light chain 3B; Beclin1, B-cell lymphoma-2-interacting myosin-like coiled-coil protein; Ctrl, control; p, phosphorylated; t, total.

Figure 5.

AOPPs induce HK-2 cell injury. HK-2 cells were cultured in control medium, native BSA (200 µg/ml) and 100, 200 and 400 µg/ml AOPPs for 24 h, or 200 µg/ml of AOPPs for 6, 12, 24 and 48 h. (A) Reverse transcription-quantitative polymerase chain reaction demonstrated that AOPP treatment increases the expression of KIM-1 and NGAL mRNA. (B) ELISA assays revealed that AOPP treatment ≥200 µg/ml induced a significant increase in KIM-1 and NGAL expression following 24 h. Data is expressed as the mean ± standard deviation of three independent experiments. *P<0.05 vs. Ctrl. AOPPs, advanced oxidation protein products; BSA, bovine serum albumin; KIM-1, kidney injury molecular; NGAL, neutrophil gelatinase-associated lipocalin; PI3K, phosphoinositide 3-kinase; ctrl, control.

Figure 6.

Autophagy inhibition mediates AOPP-induced HK-2 injury. HK-2 cells were treated with vehicle control or 200 µg/ml AOPPs for 24 h, in the absence or presence of rap (1 µM) or CQ (1 mM). (A) Western blotting demonstrated that rap increased autophagy in AOPP-treated HK-2 cells, whereas CQ decreased it. (B) Reverse transcription-polymerase chain reaction revealed that the AOPP-induced over-expression of KIM-1 and NGAL mRNA was reversed by rap and further aggravated by CQ. ELISA assays demonstrated that the AOPP-induced over-expression of (C) KIM-1 and (D) NGAL protein was reversed by rap or further aggravated by CQ. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. Ctrl, ^#P<0.05 vs. AOPP-treatment group. AOPP, advanced oxidation protein product; rap, rapamycin; CQ, chloroquine; KIM-1, kidney injury molecular; NGAL, neutrophil gelatinase-associated lipocalin; PI3K, phosphoinositide 3-kinase; Beclin1, B-cell lymphoma-2-interacting myosin-like coiled-coil protein; LC3, microtubule-associated proteins 1 light chain 3B;ctrl, control.