12-lipoxygenase is increased in glucose-stimulated mesangial cells and in experimental diabetic nephropathy.

Background: Arachidonic acid-derived 12-lipoxygenase (12-LO) products have potent growth and chemotactic properties. The present studies examined whether 12-LO and fibronectin are induced in cultured rat mesangial cells (MCs) exposed to high glucose and whether they are expressed in experimental diabetic nephropathy.

Methods: To determine the effect of high glucose on MC 12-LO mRNA and protein expression, rat MCs were incubated with RPMI medium containing 100 (NG) or 450 mg/dL glucose (HG). For animal studies, rats were injected with diluent (control) or streptozotocin. The latter were left untreated (DM) or treated with insulin (DM + I). At sacrifice after four months, GAPDH, 12-LO, and fibronectin mRNA were measured by competitive reverse transcription-polymerase chain reaction (RT-PCR) in microdissected glomeruli (G). Renal sections were semiquantitatively scored (0 to 4+) for diabetic changes and for 12-LO and fibronectin by immunohistochemistry.

Results: 12-LO mRNA expression in MC exposed to HG (12.71 +/- 1.17 attm/microL) and DM G (1.78 +/- 0.65 x 10-3 attm/glomerulus) was significantly higher than those of MCs in NG media (6.71 +/- 0.78 attm/microL) and control G (0.34 +/- 0.12 x 10-3 attm/glomerulus, P < 0.005), respectively. Western blot revealed a 1.7- and a 2.8-fold increase in MC and G 12-LO protein expression, respectively (P < 0.05). The immunohistochemistry score for G 12-LO and diabetic nephropathy score was significantly greater in DM and DM + I than controls. MC and G GAPDH mRNA remained unchanged.

Conclusions: In MCs exposed to HG and in diabetic rat glomeruli, increments in 12-LO mRNA and protein are associated with changes modeling diabetic nephropathy. These findings suggest a role for the 12-LO pathway in the pathogenesis of diabetic nephropathy.