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Objectives: Dimeric pyruvate kinase (PK) M2 (PKM2) plays an important role in promoting the accumulation of hypoxia-inducible factor (HIF)-1α, mediating aberrant glycolysis and inducing fibrosis in diabetic kidney disease (DKD). The aim of this work was to dissect a novel regulatory mechanism of Yin and Yang 1 (YY1) on lncRNA-ARAP1-AS2/ARAP1 to regulate EGFR/PKM2/HIF-1α pathway and glycolysis in DKD. Materials and methods: We used adeno-associated virus (AAV)-ARAP1 shRNA to knocked down ARAP1 in diabetic mice and overexpressed or knocked down YY1, ARAP1-AS2 and ARAP1 expression in human glomerular mesangial cells. Gene levels were assessed by Western blotting, RT-qPCR, immunofluorescence staining and immunohistochemistry. Molecular interactions were determined by RNA pull-down, co-immunoprecipitation, ubiquitination assay and dual-luciferase reporter analysis. Results: YY1, ARAP1-AS2, ARAP1, HIF-1α, glycolysis and fibrosis genes expressions were upregulated and ARAP1 knockdown could inhibit dimeric PKM2 expression and partly restore tetrameric PKM2 formation, while downregulate HIF-1α accumulation and aberrant glycolysis and fibrosis in in-vivo and in-vitro DKD models. ARAP1 knockdown attenuates renal injury and renal dysfunction in diabetic mice. ARAP1 maintains EGFR overactivation in-vivo and in-vitro DKD models. Mechanistically, YY1 transcriptionally upregulates ARAP1-AS2 and indirectly regulates ARAP1 and subsequently promotes EGFR activation, HIF-1α accumulation and aberrant glycolysis and fibrosis. Conclusion: Our results first highlight the role of the novel regulatory mechanism of YY1 on ARAP1-AS2 and ARAP1 in promoting aberrant glycolysis and fibrosis by EGFR/PKM2/HIF-1α pathway in DKD and provide potential therapeutic strategies for DKD treatments.

The persistent transactivation of epidermal growth factor receptor (EGFR) causes subsequent activation of the TGF‐β/Smad3 pathway, which is closely associated with fibrosis and cell proliferation in diabetic nephropathy (DN), but the exact mechanism of persistent EGFR transactivation in DN remains unclear. ARAP1, a susceptibility gene for type 2 diabetes, can regulate the endocytosis and ubiquitination of membrane receptors, but the effect of ARAP1 and its natural antisense long non‐coding RNA (lncRNA), ARAP1‐AS2, on the ubiquitination of EGFR in DN is not clear. In this study, we verified that the expression of ARAP1 and ARAP1‐AS2 was significantly up‐regulated in high glucose‐induced human proximal tubular epithelial cells (HK‐2 cells). Moreover, we found that overexpression or knockdown of ARAP1‐AS2 could regulate fibrosis and HK‐2 cell proliferation through EGFR/TGF‐β/Smad3 signalling. RNA pulldown assays revealed that ARAP1‐AS2 directly interacts with ARAP1. Coimmunoprecipitation, dual‐immunofluorescence and ubiquitination assays showed that ARAP1 may maintain persistent EGFR activation by reducing EGFR ubiquitination through competing with Cbl for CIN85 binding. Taken together, our results suggest that the lncRNA ARAP1‐AS2 may promote high glucose‐induced proximal tubular cell injury via persistent EGFR/TGF‐β/Smad3 pathway activation by interacting with ARAP1.