Acute glucose fluctuation promotes RAGE expression via reactive oxygen species‑mediated NF‑κB activation in rat podocytes

Abstract

Diabetic nephropathy (DN) is a common chronic complication of diabetes, for which acute glucose fluctuation (AGF) is a potential risk factor. Fluctuating hyperglycemia has been confirmed to induce more serious kidney damage than hyperglycemia in diabetic rats; however, the mechanism remains unknown. The purpose of this study was to explore the potential role of AGF in the progression of DN. Viability of rat podocytes following 72‑h AGF treatment was detected using Cell Counting‑Kit‑8. The rates of apoptosis and the level of reactive oxygen species (ROS) in rat podocytes were assessed by flow cytometry. Western blotting and reverse transcription‑quantitative PCR were performed to measure relative protein and mRNA expression levels, respectively. Transfection with an mRFP‑GFP‑LC3 adenoviral vector was used to track autophagic flux under confocal microscopy. The results indicated that AGF could inhibit cell proliferation, promote TNF‑α, interleukin‑1β (IL‑1β), and reactive oxygen species (ROS) generation, and increase autophagy in rat podocytes. Moreover, AGF upregulated receptor for advanced glycation end products (RAGE) expression via activation of NF‑κB/p65 and IκBα. Pretreatment with 5 mM N‑Acetyl‑L‑cysteine or 10 µM pyrrolidine dithiocarbamate effectively reduced cellular damage and inhibited activation of the NF‑κB/RAGE signaling pathway. Thus, AGF induces rat podocyte injury by aggravating oxidative stress, promoting the inflammatory response, and regulating ROS‑mediated NF‑κB/RAGE activation.

Keywords: diabetes; acute glucose fluctuation; diabetic nephropathy; autophagy; RAGE; oxidative stress.

Figure 1.

Effect of acute blood fluctuation on rat podocyte proliferation and apoptosis. (A) Schematic diagram showing the experimental design for acute glucose fluctuation treatment in vitro. Rat podocytes were maintained under the required conditions for 72 h. (B) Cell viability was measured using a Cell Counting Kit-8 assay. (C and D) Apoptosis rate of the rat podocytes was assessed using flow cytometry using an Annexin-V/PI apoptosis kit. Data are presented as the mean ± SD. n=3. **P<0.01 vs. NG group; ^##P<0.01 vs. HG group. AGF, acute glucose fluctuation; NG, normal glucose; HG, high glucose; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 2.

AGF induces autophagy in rat podocytes. (A and B) Protein levels of LC3II/I and beclin-1 expression was examined by western blotting. (C and D) Representative fluorescence images of mRFP-LC3 (autolysosomes) and merged RFP-GFP-LC3 (autophagosomes). In green and red-merged images, autophagosomes are shown as yellow puncta (GFP⁺ mRFP⁺), autolysosomes are shown as red puncta (GFP⁻ mRFP⁺), and blue fluorescence represents DAPI-treated nuclei. (D) Image analysis of red puncta (autolysosomes) and yellow puncta (autophagosomes). n=3. *P<0.05; **P<0.01 vs. NG; ^#P<0.05; ^##P<0.01 vs. HG. AGF, acute glucose fluctuation; NG, normal glucose; HG, high glucose; GFP, green fluorescence protein; RFP, red fluorescence protein.

Figure 3.

AGF promotes pro-inflammatory cytokine generation. (A and B) Protein and (C) mRNA expression levels of TNF-α and IL-1β. Data are presented as the mean ± SD. n=3. *P<0.05 vs. NG; ^#P<0.05, ^##P<0.01 vs. HG. AGF, acute glucose fluctuation; NG, normal glucose; HG, high glucose.

Figure 4.

AGF increases the level of intracellular ROS and RAGE expression, as well as enhances the phosphorylation of p-NF-κB p65 and p-IκB-α expression in rat podocytes. (A and B) Flow cytometry analysis of intracellular ROS production. (C and D) The level of RAGE, p-NF-κB p65, NF-κB p65, p-IκB-α, and IκB-α were examined by western blotting. (E) The level of RAGE mRNA was examined by qRT-PCR. Data are presented as the mean ± SD. n=3. *P<0.05; **P<0.01 vs. NG; ^#P<0.05, ^##P<0.01 vs. HG. AGF, acute glucose fluctuation; NG, normal glucose; HG, high glucose; FITC, fluorescein isothiocyanate; RAGE, receptor for advanced glycation end products.

Figure 5.

Downregulation of intracellular ROS levels inhibits activation of the NF-κB/RAGE signaling pathway in rat podocytes. (A) Cell viability was measured using a Cell-Counting-Kit-8 assay. (B and C) ROS generation induced by AGF in rat podocytes with or without 5 mM NAC pretreatment. (D and E) Protein levels of RAGE, p-NF-κB p65, NF-κB p65, p-IκB-α, and IκB-α. (F) mRNA levels of RAGE. Data are presented as the mean ± SD. n=3. *P<0.05, **P<0.01 vs. NG; ^&P<0.05, ^&&P<0.01 vs. HG. AGF, acute glucose fluctuation; NG, normal glucose; HG, high glucose; FITC, fluorescein isothiocyanate; RAGE, receptor for advanced glycation end products; ROS, reactive oxygen species; NAC, N-Acetyl-L-cysteine; p, phosphorylated.

Figure 6.

Inhibition of NF-κB by PDTC reduced acute glucose fluctuation-induced increases in RAGE expression in rat podocytes. (A) Cell viability was measured using a CCK-8 assay. (B and C) Protein levels of RAGE, p-NF-κB p65, NF-κB p65, p-IκB-α and IκB-α. (D) mRNA levels of RAGE. Data are presented as the mean ± SD. n=3. *P<0.05, **P<0.01 vs. NG group; ^$P<0.05, ^$$P<0.01 vs. AGF. AGF, acute glucose fluctuation; NG, normal glucose; HG, high glucose; FITC, fluorescein isothiocyanate; RAGE, receptor for advanced glycation end products; PTDC, pyrrolidinedithiocarbamate; p, phosphorylated.