Glucose promotes the production of interleukine-1beta and cyclooxygenase-2 in mesangial cells via enhanced (Pro)renin receptor expression

Abstract

(Pro)renin receptor (PRR) is present in renal glomeruli, and its expression is up-regulated in diabetes. Similarly, renal inflammation is increased in the presence of hyperglycemia. The linkage between PRR and renal inflammation is not well established. We hypothesized that glucose-induced up-regulation of PRR leads to increased production of the proinflammatory factors IL-1beta and cyclooxygenase-2 (COX-2). Studies were conducted in rat mesangial cells (RMCs) exposed to 30 mm D-glucose for 2 wk followed by PRR small interfering RNA knockdown, IL-1 receptor blockade with IL-1 receptor antagonist or angiotensin II type 1 receptor blockade with valsartan. The results showed that D-glucose treatment up-regulates prorenin, renin, angiotensin II, PRR, IL-1beta, and COX-2 mRNA and protein expression and increases phosphorylation of ERK1/2, c-Jun N-terminal kinase, c-Jun, and nuclear factor-kappaB (NF-kappaB) p65 (serine 276,468 and 536), respectively. PRR small interfering RNA attenuated PRR, IL-1beta, and COX-2 mRNA and protein expressions and significantly decreased angiotensin II production and phosphorylation of ERK1/2 and NF-kappaB p65 associated with high glucose exposure. Similarly, IL-1 receptor antagonist significantly reduced COX-2 mRNA and protein expression induced by high glucose. COX-2 inhibition reduced high-glucose-induced PRR expression. We conclude that glucose induces the up-regulation of PRR and its ligands prorenin and renin, leading to increased IL-1beta and COX-2 production via the angiotensin II-dependent pathway. It is also possible that PRR could enhance the production of these inflammatory cytokines through direct stimulation of ERK1/2-NF-kappaB signaling cascade.

Figure 1

PRR mRNA and protein expression and ERK1/2 phosphorylation (p-ERK) induced by high d-glucose and in response to COX-2 inhibitor in RMCs. A, PRR expression in response to different l- or d-glucose concentrations for 14 d. B, Time course for PRR expression in response to 30 mm l- or d-glucose over 14 d. C, ERK1/2 protein phosphorylation over 14 d of 30 mm l- or d-glucose exposure. D, Effect of 12 h treatment with COX-2 inhibitor NS-398 at 10 μm on PRR expression in control and glucose-treated RMCs. Control, 30 mm l-glucose; glucose, 30 mm d-glucose. *, P < 0.01; †, P < 0.05. All the results represent the average of three independent experiments.

Figure 2

Effect of high d-glucose on prorenin and renin expression in RMCs. High-glucose exposure increased prorenin mRNA (A) and prorenin and renin protein (B). Renin protein (C) increased in RMCs culture supernatants in response to high-glucose exposure as demonstrated by immunoprecipitation (IP). Immunofluorescence staining of prorenin and renin (D). NEG, negative control (no antibody control); control, 30 mm l-glucose; glucose, 30 mm d-glucose; KD, knockdown; WB, Western blot; Ab, antibody.

Figure 3

Effects of high d-glucose and PRR siRNA on PRR, IL-1β, and COX-2 mRNA and protein expression in RMCs. A and B, PRR mRNA and protein. C and D, IL-1β mRNA and IL-1β protein in culture supernatants. E and F, COX-2 mRNA and protein. Control, 30 mm l-glucose; glucose, 30 mm d-glucose.

Figure 4

Effects of high d-glucose and IL-1Ra on COX-2 expression in RMCs. A, COX-2 mRNA. B, COX-2 protein. Control, 30 mm l-glucose; glucose, 30 mm d-glucose.

Figure 5

Western blot analysis showing effects of glucose and PRR siRNA on protein phosphorylation (p-) of ERK1/2, JNK, c-Jun, and NF-κB p65. Control, 30 mm l-glucose; glucose, 30 mm d-glucose.

Figure 6

Effect of valsartan and PRR siRNA on Ang II level and IL-1β production in RMCs. A, Ang II level in response to 30 mm d-glucose and valsartan treatment. B, Ang II level and effect of PRR siRNA interference in the absence or presence of 30 mm d-glucose. Effects of valsartan on 30 mm d-glucose-induced IL-1β mRNA (C) and IL-1β protein in culture supernatants (D). Effect of PRR siRNA alone or combined with valsartan on IL-1β mRNA expression (E) and IL-1β protein production in culture supernatants (F). One micromole of valsartan (Val) was used alone and combined with PRR siRNA in SFM containing 30 mm l-glucose (control) or 30 mm d-glucose (glucose) for 12 h of serum-free culture. One hundred nanomoles of PRR siRNA were used to transfect RMCs. In PRR siRNA transfection experiments, serum-free culture and valsartan treatments were conducted simultaneously for 12 h (12 h before end of 48 h for RNA expression or 72 h for protein expression of PRR siRNA transfection). Control, 30 mm l-glucose; glucose, 30 mm d-glucose.