High glucose increases inducible NO production in cultured rat mesangial cells. Possible role in fibronectin production.
OBJECTIVE
Increased nitric oxide (NO) generation and action have been suggested to be associated with glomerular hyperfiltration and increased vascular permeability early in diabetes. However, previous studies have primarily focused on the constitutive nitric oxide synthase (cNOS) pathway present in endothelial cells, and the role of the inducible NOS (iNOS) pathway in diabetic nephropathy has remained unclear. This study examined whether high glucose modulates NO synthesis by the iNOS pathway in rat mesangial cells. In addition, the effect of inhibition of the iNOS pathway on fibronectin production was determined to examine the role of the iNOS pathway in high glucose-induced extracellular expansion by mesangial cells.


METHODS
NO synthesis by the iNOS pathway was evaluated by nitrite and iNOS mRNA and protein productions. The effects of protein kinase C (PKC) inhibitor and aldose reductase inhibitor on the iNOS mRNA expression and aminoguanidine, a relatively specific inhibitor of the iNOS on fibronectin protein production were examined.


RESULTS
High 30 mM glucose concentration led to significant increases in nitrite production of rat mesangial cells upon stimulation with lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) compared with control 5.6 mM glucose concentration. Mesangial iNOS mRNA expression and protein production also increased significantly in response to high glucose. The addition of calphostin C, a PKC inhibitor, and 6-bromo-1,3-dioxo-1H-benz[d,e]isoquinoline-2(3H)-acetic acid, an aldose reductase inhibitor, significantly suppressed the enhancement of iNOS mRNA expression in high glucose concentration. High glucose also significantly increased fibronectin protein production of mesangial cells upon stimulation with LPS plus IFN-gamma compared to control glucose. Aminoguanidine reversed this high glucose-induced fibronectin production at dose inhibiting iNOS mRNA expression.


CONCLUSIONS
These results indicate that high glucose enhances cytokine-induced NO production by rat mesangial cells, and that the activation of PKC and aldose reductase pathway may play a role in this enhancement. In addition, high glucose-induced NO production by the iNOS pathway may promote extracellular matrix accumulation by mesangial cells under certain condition.
Noh
Hyunjin
H
Division of Nephrology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea.
Ha
Hunjoo
H
Yu
Mi Ra
MR
Kang
Shin Wook
SW
Choi
Kyu Hun
KH
Han
Dae Suk
DS
Lee
Ho Yung
HY
D000085:Acetates / Q000494:pharmacology; D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors; D000818:Animals; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology*; D006146:Guanidines / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D009281:Naphthalenes / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D051381:Rats; D017207:Rats, Sprague-Dawley
C459702:6-bromo-1,3-dioxo-1H-benz(d,e)isoquinoline-2(3H)-acetic acid; D000085:Acetates; D016207:Cytokines; D005353:Fibronectins; D006146:Guanidines; D007546:Isoquinolines; D009281:Naphthalenes; D000449:Aldehyde Reductase; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D011493:Protein Kinase C; C058819:calphostin C; D005947:Glucose; C004479:pimagedine