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 whet her 12-LO and fibronectin are induced in cultured rat mesangial cells (MCs) exposed to high glucose and whether they are expressed in experimental dia betic nephropathy. Methods. To determine the effect of high glucose on MC 12-LO mRNA and prote in expression, rat MCs were incubated with RPMI medium containing 100 (NG) or 450 mg/dL glucose (HG). For animal studies, rats were injected with dilu ent (control) or streptozotocin. The latter were left untreated (DM) or tre ated with insulin (DM + I). At sacrifice after four months, GAPDH, 12-LO, a nd fibronectin mRNA were measured by competitive reverse transcription-poly merase chain reaction (RT-PCR) in microdissected glomeruli (G). Renal secti ons were semiquanlitatively scored (0 to 4+) for diabetic changes and for 1 2-LO and fibronectin by immunohistochemistry. Results. 12-LO mRNA expression in MC exposed to HG (12.71 +/- 1.17 attm/mul ) and DM G (1.78 +/- 0.65 x 10(-3) attm/ glomerulus) was significantly high er than those of MCs in NG media (6.71 +/- 0.78 attm/muL) and central G (0. 34 +/- 0.12 X 10(-3) attm/glomerulus, P < 0.005), respectively. Western blo t revealed a 1.7- and a 2.8-fold increase in MC and G 12-LO protein express ion, respectively (P < 0.05). The immunohistochemistry score for G 12-LO an d diabetic nephropathy score was significantly greater in DM and DM + I tha n 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 ne phropathy. These findings suggest a role for the 12-LO pathway in the patho genesis of diabetic nephropathy.