Effect of glucose on stress-activated protein kinase activity in mesangial cells and diabetic glomeruli

Background. We have reported that hyperglycemia increases c-jun mRNA levels in isolated glomeruli of diabetic rats. The transcriptional activity of c;jun can be modified by phosphorylation of serine residues in the regulatory domain of the protein by stress-activated protein kinases (SAPKs), but the effect of high glucose concentrations on SAPK expression and activity is unknown. Accordingly, we studied p42/44 MAPK, p38 MAPK, and SAPK expression and activity in primary mesangial cells exposed to high glucose concentrations, as well as SAPK expression and activity in glomeruli of normal and streptozotocin-induced diabetic rats. Methods. Mesangial cells were incubated in 40 mM glucose for 30 and 60 minutes and 6, 12, 24, and 48 hours, whereas glomeruli of streptozotocin-induced diabetic rats were isolated one day and one and two weeks after the onset of hyperglycemia (blood glucose levels more than 15 mmol/liter), and were compared with age-matched normal rats. Cell lysates were subjected to Western blot analysis of SAPK and phosphorylated SAPK and an in vitro SAPK assay using recombinant c-jun. Results. Western blot analysis revealed that SAPK was expressed, but unphosphorylated, in unstimulated mesangial cells and whole glomerular lysates from normal rats. In accord with these observations, no SAPK activity was detected in lysates from mesangial cells or whole glomeruli from normal rats, although mesangial cell SAPK activity was readily induced in vitro by sorbitol. High glucose concentrations did not increase SAPK activity or lead to detectable phosphorylated SAPK either in vitro or in vivo. In contrast, short-term exposure to 40 mM of glucose activated both p42/44 MAPK and p38 MAPK. Conclusions. We conclude that high glucose concentrations do not activate SAPK in primary cultured mesangial cells or in diabetic glomeruli during the early phase of diabetic renal hypertrophy.