Abstract
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 HIF1A 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 HIF1A gene up-regulation.
Highlights
IntroductionHypoxia-inducible factors (HIFs) directly control the expression of genes encoding enzymes and other proteins essential for effective glucose utilization in the latter process, including glucose transporter GLUT1, pyruvate dehydrogenase kinase
These mechanisms might be controlled at the posttranscriptional level, as under hypoxic conditions high glucose-evoked increase in Hypoxia-inducible factors (HIFs)-1α level did not result in its elevated protein content
HIF-1α content observed in epithelial cells of human renal proximal tubules cultured under normoxic conditions in the presence of 30 mM glucose
Summary
HIFs directly control the expression of genes encoding enzymes and other proteins essential for effective glucose utilization in the latter process, including glucose transporter GLUT1, pyruvate dehydrogenase kinase. On the other hand, which seems of special interest, hyperglycemia/diabetes appears to be accompanied by the state of hypoxia (cf Reference [6] for review). Hypoxia probably results mainly from elevated oxygen consumption, being a consequence of glomerular hyperfiltration and increased activity of sodium-dependent glucose transporters (SGLTs) and Na+ /K+ ATPase [1,7]. Hyperglycemia itself might cause a decline in the intracellular NAD+ /NADH ratio, the state sometimes defined as “pseudohypoxia”, leading to many deleterious metabolic consequences
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