Inhibition of 12/15-Lipoxygenase Reduces Renal Inflammation and Injury in Streptozotocin-Induced Diabetic Mice

Abstract

Previous studies suggest that 12/15 lipoxygenase (12/15-LO) is implicated in diabetic vascular complications. We hypothesize that 12/15-LO inhibition attenuates renal inflammation and injury in streptozotocin-induced diabetes. Diabetes was induced in wild-type C57BL/6J (WT) and 12/15-LO deficient mice using streptozotocin. Additionally, four groups of WT mice were also used; control non diabetic, diabetic, diabetic treated with the 12/15-LO inhibitor baicalein for 10 weeks and diabetic treated with baicalein only for the last 4 weeks of the experiment. After 10 weeks of induction of diabetes with streptozotocin, WT diabetic mice exhibited marked elevation in proteinuria together with elevation in the excretion levels of thiobarbituric acid reactive substance (TBARs), a marker of oxidative stress, and monocyte chemoattractant protein-1 (MCP-1), a marker of inflammation and these changes were significantly reduced in 12/15-LO deficient diabetic mice (P<0.05). Similarly, pharmacological inhibition of 12/15-LO with baicalein prevented the elevation in renal 12-HETE production, the major murine metabolic product of 12/15-LO, in diabetic mice, and this effect was associated with decreased proteinuria, TBARs excretion and renal collagen deposition compared to untreated diabetic mice. Interestingly, the protective effects of baicalein were not noticed when only administered in the last 4 weeks of diabetes compared to untreated diabetic mice. WT diabetic mice displayed elevation in renal interleukin-6 (IL-6) levels and these changes were only reduced in diabetic mice treated with baicalein for 10 weeks (P<0.05). The anti-inflammatory effects of baicalein or 12/15-LO deficiency were further confirmed in lipopolysaccharide (LPS)-induced acute renal inflammation as inhibition of 12/15-LO reduced the elevation in renal soluble epoxide hydrolase expression in LPS-injected mice. These results suggest that increased 12/15-LO activity and 12-HETE production contribute to the elevation of renal oxidative stress, inflammation and injury in streptozotocin-induced diabetic mice.

Keywords: 12/15-LO Deficient mice; Baicalein; Diabetes; Inflammation; Kidney; Oxidative stress; Proteinuria.

Figure 1

Urinary protein excretion (A), TBARs excretion (B) and renal ICAM-1 expression levels relative to β-actin (C) in WT and 12/15-LO deficient mice after 10 weeks of induction of diabetes with streptozotocin. Diabetic WT mice displayed significant elevations in proteinuria, TBARs excretion and renal ICAM-1 expression compared to control. 12/15-LO deficiency prevented the elevation in proteinuria, TBARs excretion and renal ICAM-1 during diabetes. (*P<0.05 vs. control WT mice, #P<0.05 vs. diabetic WT mice and ^λP <0.05 vs. control 12/15-LO deficient mice, n=6-8/group for excretion data and n=4 for ICAM-1 expression).

Figure 2

Renal 12-LO expression relative to β-actin (A) and renal 12-HETE levels in control and diabetic WT mice with or without baicalein treatment for 4 and 10 weeks. Renal 12-LO expression and 12-HETE levels increased significantly in diabetic versus control mice. Inhibition of 12/15-LO with baicalein treatment resulted in significant reduction in 12-LO expression and 12-HETE levels either after 4 or 10 weeks ((*P<0.05 vs. control and #P<0.05 vs. diabetic mice, n=6/group).

Figure 3

Urinary TBARs excretion (A) and MCP-1 excretion (B) in streptozotocin-induced diabetic mice. Both urinary TBARs and MCP-1 excretion levels were significantly elevated in diabetic versus control mice (*P<0.05 vs. control mice). Only baicalein treatment for 10 weeks significantly reduced TBARs excretion in diabetic mice (#P<0.05 vs. diabetic mice, n=6-8/ group).

Figure 4

Representative images (200X) of kidney sections H & E staining (A) and Masson's trichrome staining (blue staining, B), proteinuria (C) and average score for Masson's trichrome staining (D) in control and diabetic mice with or without baicalein treatment for 4 and 10 weeks. (*P<0.05 vs. control mice and #P<0.05 vs. diabetic mice; n=4/group for histology and n=6-8/group for proteinuria).

Figure 5

Effect of 12/15-LO inhibition with baicalein on renal inflammatory cytokines levels in streptozotocin-induced diabetic mice. Renal IL-6, MCP-1, VCAM-1 and ICAM-1 levels significantly increased in diabetic compared to control mice (*P<0.05 vs. control mice). Only inhibition of 12/15-LO with baicalein treatment for 10 weeks significantly reduced renal IL-6 levels in diabetic mice (#P<0.05 vs. diabetic mice, n=6/group).

Figure 6

Renal 12-LO (A) and COX2 (B) expression levels relative to β-actin in WT with or without baicalein treatment and 12/15-LO deficient mice after 24 hours of LPS injection. Renal 12-LO expression significantly increased in LPS injected mice compared with control mice and was reduced in baicalein treated WT mice or in 12/15-LO deficient mice. Renal COX2 expression was significantly reduced in baicalein treated LPS injected WT mice versus control. Renal COX2 expression was lesser in 12/15-LO deficient mice versus control and increased after LPS injection (*P<0.05 vs. control mice, #P<0.05 vs. LPS injected WT mice, and ^λP<0.05 vs. control 12/15-LO deficient mice, n=4/group).

Figure 7

A. Renal CYP4A hydroylase (A), CYP2J epoxygenase (B) and soluble epoxide hydrolase (sEH, C) expression levels relative to β-actin in WT with or without baicalein treatment and 12/15-LO deficient mice after 24 hours of injection with LPS. Renal CYP4A hydroxylase was significantly elevated in LPS injected WT mice versus control and was reduced in LPS injected 12/15-LO deficient mice. Although there was no difference in renal CYP2J epoxygenase among mice group, renal sEH increased in LPS injected WT mice. Baicalein treatment or 12/15-LO deficiency reduced the elevation in renal sEH expression in LPS injected WT mice (*P <0.05 vs. control mice, #P <0.05 vs. LPS injected WT mice, and ^λP <0.05 vs. control 12/15-LO deficient mice, n=4/group).