High glucose up-regulates angiotensin II subtype 2 receptors via interferon regulatory factor-1 in proximal tubule epithelial cells

Abstract

Earlier studies have reported an increase in the proximal tubule AT(2) receptor (AT(2)R) expression in diabetes, with a beneficial role in kidney function and blood pressure regulation. Here, we demonstrate that the increase in AT(2)R protein expression is associated with an increased expression of transcription factor IRF-1 in hyperglycemic rat and in high glucose-treated HK2 cells. Knock-down of IRF-1 by siRNA in HK2 cells prevented high glucose-induced AT(2)R up-regulation. The data suggest that IRF-1 is a transcriptional regulator of AT(2)R expression in hyperglycemia, and warrant further studies to understand the physiological role of IRF-1 along with AT(2)R in diabetic kidney.

Fig. 1

a HK2 cells (A) AT2 receptor expression in HK2 cells treated for 24 h with normal (5 mM) and high glucose (25 mM). Upper panel: representative western blots. Lower panel: bar graph of band density of AT2 receptor normalized with β-actin. (B) AT₂ mRNA expression measured by qRT-PCR in HK2 cells treated for 24 h with normal (5 mM) and high (25 mM) glucose. (C) AT₂ receptor expression in HK2 cells treated for 24 h with normal glucose (5 mM) and sorbitol (sorbitol 20 mM + glucose 5 mM). Upper panel: representative western blot. Lower panel: bar graph of band density of AT₂ receptor normalized with β-actin. b Proximal tubules (A) AT₂ receptor expression in the proximal tubules of lean and obese Zucker rats. Upper panel: representative western blot. Lower panel: bar graph of band density of AT₂ receptor expressed as percent of lean. (B) AT₂ receptor mRNA expression measured by qRT-PCR in proximal tubules of lean and obese Zucker rats. (C) Expression of IRF-1 in proximal tubules of lean and obese Zucker rats. Upper panel: representative western blot. Lower panel: bar graph of band density of IRF-1 normalized with β-actin. Values are represented as mean ± SEM, * significantly different from lean rats (Student’s t test, P<0.05, n = 4 in each group)

Fig. 2

Concentration and time course study of siRNA transfection: IRF-1 expression in HK2 cells transfected with a different concentrations of siRNA IRF-1 (10, 100 and 500 nM) for 48 h and b 100 nM siRNA IRF-1 for 24 and 48 h

Fig. 3

Effect of glucose (25 mM) on the expression of AT₂ receptor and IRF-1 in HK2 cells transfected with 100 nM IRF-1 siRNA. Upper Panels: western blots of IRF-1, IRF-2, AT₂ receptor and β-actin. Lower panels: bar graphs of band density of IRF-1, IRF-2, AT₂ normalized with β-actin. Values are represented as mean ± SEM, * significantly different from control HK2 cells (one-way ANOVA followed by Neuman–Keuls test, P<0.05, n = 3

Fig. 4

Effect of glucose (25 mM) on the expression of AT₂ receptor and IRF-1 in HK2 cells transfected with 500 nM IRF-1 siRNA. Upper Panels: western blots of IRF-1, IRF-2, AT₂ receptor and β-actin. Lower panels: bar graphs of band density of IRF-1, IRF-2, AT₂ normalized with β-actin. Values are represented as mean ± SEM, * significantly different from control HK2 cells (one-way ANOVA followed by Neuman–Keuls test, P<0.05, n = 3). CT control, HG high glucose, SS scrambled sequence