Fig 2: ARAP1 regulates dimeric and tetrameric PKM2 expression by reducing EGFR ubiquitination and maintaining persistent EGFR activation (A) Human glomerular mesangial cells were pre-treated with ARAP1 shRNA for 24 h and then divided into the NG group and HG group. The protein expression levels of total EGFR and EGFR phosphorylated at two phosphorylation sites (Y1068 and Y1173) were detected at 24 and 48 h after stimulation by high glucose. HRMCs were pre-treated with ARAP1 shRNA for 24 h and then divided into NG and HG groups. 0 h means that the HRMCs were only transfected with ARAP1 shRNA for 24 h under normal glucose and were not stimulated with high glucose. (B) Effect of AAV2/9-shARAP1 on the protein expression of total EGFR, and EGFR phosphorylated at two phosphorylation sites (Y1068 and Y1173) in the isolated glomeruli lysates of mouse kidney tissues was examined by Western blot analysis. (C) The mRNA expression of total EGFR in the isolated glomeruli lysates of kidney tissues from the db/m, db/db, db/db + AAV-shNC and db/db + AAV-shARAP1 mice was measured by qRT-PCR. (D) Forty-eight hours after transfection of ARAP1 shRNA in the NG group and HG group, the mRNA expression of total EGFR was measured by qRT-PCR. (E) ARAP1 knockdown with ARAP1 shRNA increased EGFR ubiquitination in human glomerular mesangial cells cultured with high glucose. (F) ARAP1 knockdown with AAV2/9-shARAP1 increased EGFR ubiquitination in the isolated glomeruli lysates of diabetic db/db mouse kidney tissues. (G) After transfection of ARAP1 overexpression plasmid accompanied by AG1478 treatment in the NG group, PKM2 monomer, dimer and tetramer in human glomerular mesangial cells were detected by cross-linking assay. In all panels, the data are representative of at least three independent experiments. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, NS, not significant.

Fig 3: ARAP1-AS2 promotes the expression of fibrosis and cell proliferation via the EGFR/TGF-ß/Smad3 pathway. A, Forty-eight hours after transfection of the ARAP1-AS2 overexpression plasmid or ARAP1-AS2 siRNA in the normal-glucose (NG) group and high-glucose (HG) group, the protein expression levels of total EGFR, EGFR phosphorylated at two phosphorylation sites (Y1068 and Y1173), TGF-ß1 and p-Smad3 were detected by Western blot analysis. B, Forty-eight hours after transfection of the ARAP1-AS2 overexpression plasmid or ARAP1-AS2 siRNA in the NG group and HG group, the mRNA expression level of total EGFR was detected by qRT-PCR. C, Forty-eight hours after transfection of the ARAP1-AS2 overexpression plasmid or ARAP1-AS2 siRNA in the NG group and HG group, the mRNA expression level of TGF-ß1 was detected by qRT-PCR. D, Forty-eight hours after transfection of the ARAP1-AS2 overexpression plasmid or ARAP1-AS2 siRNA in the NG group and HG group, the mRNA expression level of Smad3 was detected by qRT-PCR. E, Forty-eight hours after cotransfection of the ARAP1-AS2 overexpression plasmid and ARAP1 shRNA or transfection of the ARAP1-AS2 overexpression plasmid accompanied by treatment with the EGFR tyrosine kinase inhibitor AG1478 in the NG group, the protein expression levels of ARAP1, total EGFR, EGFR phosphorylated at two phosphorylation sites (Y1068 and Y1173), TGF-ß1, p-Smad3, collagen I, collagen IV and fibronectin (FN) were detected by Western blot analysis. F, After cotransfection of the ARAP1-AS2 overexpression plasmid and ARAP1 shRNA or transfection of ARAP1-AS2 overexpression plasmid accompanied by AG1478 treatment in the NG group, cell proliferation was detected by CCK-8 assay at 24, 48 and 72 h. In all panels, the data are representative of three independent experiments. Data are presented as the mean ± SD. *P < .05, **P < .01, NS, not significant

Fig 4: YY1 regulate EGFR ubiquitination and activation, HIF-1a accumulation and aberrant glycolysis and ECM accumulation in human glomerular mesangial cells through the ARAP1-AS2/ARAP1 axis. (A) Forty-eight hours after co-transfection of the YY1 overexpression plasmid and ARAP1-AS2 siRNA in the NG group, the expression level of ARAP1-AS2 was examined by qRT-PCR. (B) Forty-eight hours after co-transfection of the YY1 overexpression plasmid and ARAP1-AS2 siRNA in the NG group, the protein expression levels of ARAP1 were examined by Western blot analysis. (C) Forty-eight hours after co-transfection of the YY1 overexpression plasmid and ARAP1-AS2 siRNA in the NG group, EGFR ubiquitination were examined. (D) Forty-eight hours after co-transfection of the YY1 overexpression plasmid and ARAP1-AS2 siRNA in the NG group, the protein expression levels of total EGFR, and EGFR phosphorylated at two phosphorylation sites (Y1068 and Y1173) were examined by Western blot analysis. (E) Forty-eight hours after co-transfection of the YY1 overexpression plasmid and ARAP1-AS2 siRNA in the NG group, the protein expression levels of HIF-1a, PKM2, LDHA, HK2, COL I, COL IV and FN were examined by Western blot analysis. (F) After co-transfection of the YY1 overexpression plasmid and ARAP1-AS2 siRNA, cell proliferation was evaluated by CCK-8 assay at 0, 24, 48 and 72 h. In all panels, the data are representative of at least three independent experiments. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, NS, not significant.

Fig 5: ARAP1 knockdown inhibits high glucose-induced dimeric PKM2 expression, HIF-1a nuclear accumulation, pro-fibrotic responses and aberrant glycolysis, while partly restore tetrameric PKM2 formation in human glomerular mesangial cells. (A,B) qRT-PCR and Western blot analysis showed that the mRNA and protein levels of ARAP1 were upregulated in HG group compared with NG group and MA group. (C,D) Forty-eight hours after transfection of ARAP1 shRNA (3,000 ng) in the HG group in 6-well plates, the ARAP1 knockdown efficiency was examined by qRT-PCR and Western blot analysis. (E) HIF-1a was assessed by immunofluorescence after treatment with high glucose or ARAP1 shRNA (×400). Bar = 50 µm. Arrows indicate positive staining. (F) Forty-eight hours after transfection of ARAP1 shRNA in the NG group and HG group, the protein expression of HIF-1a, PKM2, LDHA, HK2, COL I, COL IV and FN was examined by Western blot analysis. (G) Cross-linking detection of PKM2 in human glomerular mesangial cells. In all panels, the data are representative of at least three independent experiments. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, NS, not significant.