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Background: The underlying mechanisms by which diabetes and dyslipidemia contribute to diabetic nephropathy (DN) are not fully understood. In this study, we aimed to investigate the role of high glucose (HG) on intracellular cholesterol accumulation in glomerular endothelial cells (GEnCs) and its potential mechanism. Methods: Oil red O staining, RT-qPCR, Western blotting, and immunocytofluorescence analyses were used to determine cholesterol accumulation and the expressions of LXRs and ABCA1 in GEnCs under high cholesterol (HC) and/or HG conditions, and the effect of these treatments was compared to that of low glucose without adding cholesterol. LncRNA microarrays were used to identify a long non-coding RNA (LncRNA OR13C9), of which levels increased in cells treated with the LXR agonist, GW3965. Fluorescence in situ hybridization (FISH) was conducted to confirm subcellular localization of LncOR13C9 and a bioinformatics analysis was used to identify competing endogenous RNA (ceRNA) regulatory networks between LncOR13C9 and microRNA-23a-5p (miR-23a-5p). Gain and loss of function, rescue assay approaches, and dual-luciferase reporter assay were conducted to study interactions between LncOR13C9, miR-23a-5p, and ABCA1. Results: We showed that HG could decrease the response ability of GEnCs to cholesterol load, specifically that HG could downregulate LXRs expression in GEnCs under cholesterol load and that the decrease in LXRs expression suppressed ABCA1 expression and increased cholesterol accumulation. We focused on the targets of LXRs and identified a long non-coding RNA (LncOR13C9) that was downregulated in GEnCs grown in HG and HC conditions, compared with that grown in HC conditions. We speculated that LncRNAOR13C9 was important for LXRs to increase cholesterol efflux via ABCA1 under HC. Furthermore, using gain of function, loss of function, and rescue assay approaches, we showed that LncOR13C9 could regulate ABCA1 by inhibiting the action of miR-23a-5p in the LXR pathway. Furthermore, dual-luciferase reporter assay was conducted to study the interaction of LncOR13C9 with miR-23a-5p.

Background Lipid metabolism disorder is closely related to diabetic nephropathy (DN), but the mechanism remains unclear. The aim of this study was to investigate the effect of high glucose on intracellular accumulation of lipids and to clarify the possible mechanism. Methods We found that in human glomerular endothelial cells (GEnCs), cholesterol could reduce cell activity, lead to abnormal lipid deposition in cells and high glucose can aggravate lipid deposition under high cholesterol load. Results CCK8 showed that soluble cholesterol could reduce GEnCs cells activity. Oil red O staining and cholesterol quantification experiment showed that the intracellular lipid deposition did not obvious after the intervention of high glucose (HG), but the intracellular lipid deposition increased under HG combined with high cholesterol (HC). The results of RT-qPCR,WB and immunofluorescence experiment showed the expression of ABCA1 in HC group increased significantly. However, compared with the HC group, the expression of ABCA1in the HG and HC group were decreased. And then we found HG affected ABCA1 up-regulation by LXRs. Based on Gene Microarray, we found that LXRs regulation of ABCA1 transcription requires the involvement of LncRNAOR13C9. Conclusions HG could enhance intracellular lipid deposition by interfering with cellular response to HC to up-regulate the expression of ABCA1, and HG blocked the expression of ABCA1 by down-regulating LXRs, and LncRNAOR13C9 also played an important role in this process. It further elucidate the pathogenesis of DN and provide a new target for the prevention and treatment of DN.