Transcription Factor ChREBP Mediates High Glucose-Evoked Increase in HIF-1α Content in Epithelial Cells of Renal Proximal Tubules

• Published in: International journal of molecular sciences Vol. 22; no. 24; p. 13299
• Main Authors: Owczarek, Aleksandra, Gieczewska, Katarzyna B, Jarzyna, Robert, Frydzinska, Zuzanna, Winiarska, Katarzyna
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 10-12-2021; MDPI
• Subjects: Acetylation; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors - genetics; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors - metabolism; Binding; carbohydrate response element binding protein (ChREBP); Cell activation; Cell Line; Chromatin; Communication; Dehydrogenases; Diabetes; Diabetes mellitus; Endothelial cells; Enzymes; Epithelial cells; Epithelial Cells - metabolism; Epithelium; Gene expression; Glucose; Glucose - pharmacology; high glucose; Humans; Hyperglycemia; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit - biosynthesis; Hypoxia-Inducible Factor 1, alpha Subunit - genetics; Hypoxia-inducible factor 1a; hypoxia-inducible factor-1 (HIF-1); Immunoprecipitation; Kidney Tubules, Proximal - metabolism; Kinases; Metabolism; Metabolites; NAD+/NADH ratio; NADH; Proteins; Proximal tubules; renal proximal tubules; siRNA; sirtuin 1; Transcription factors; Up-Regulation - drug effects; sirtuin 1; hypoxia-inducible factor-1 (HIF-1); NAD+/NADH ratio; carbohydrate response element binding protein (ChREBP); high glucose; renal proximal tubules
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms222413299

Hyperglycemia/diabetes appears to be accompanied by the state of hypoxia, which especially affects kidneys. The aim of the study was to elucidate the mechanism of high glucose action on HIF-1α expression in renal proximal tubule epithelial cells. The research hypotheses included: (1) the participation of transcription factor ChREBP; and (2) the involvement of the effects resulting from pseudohypoxia, i.e., lowered intracellular NAD+/NADH ratio. The experiments were performed on HK-2 cells and primary cells: D-RPTEC (Diseased Human Renal Proximal Tubule Epithelial Cells-Diabetes Type II) and RPTEC (Renal Proximal Tubule Epithelial Cells). Protein and mRNA contents were determined by Western blot and RT-qPCR, respectively. ChREBP binding to DNA was detected applying chromatin immunoprecipitation, followed by RT-qPCR. Gene knockdown was performed using siRNA. Sirtuin activity and NAD
/NADH ratio were measured with commercially available kits. It was found that high glucose in HK-2 cells incubated under normoxic conditions: (1) activated transcription of HIF-1 target genes, elevated HIF-1α and ChREBP content, and increased the efficacy of ChREBP binding to promoter region of
gene; and (2), although it lowered NAD
/NADH ratio, it affected neither sirtuin activity nor HIF-1α acetylation level. The stimulatory effect of high glucose on HIF-1α expression was not observed upon the knockdown of ChREBP encoding gene. Experiments on RPTEC and D-RPTEC cells demonstrated that HIF-1α content in diabetic proximal tubular cells was lower than that in normal ones but remained high glucose-sensitive, and the latter phenomenon was mediated by ChREBP. Thus, it is concluded that the mechanism of high glucose-evoked increase in HIF-1α content in renal proximal tubule endothelial cells involves activation of ChREBP, indirectly capable of
gene up-regulation.