Reactive oxygen species mediate high glucose-induced plasminogen activator inhibitor-1 up-regulation in mesangial cells and in diabetic kidney.
BACKGROUND
Plasminogen activator inhibitor-1 (PAI-1) plays an important role in remodeling of extracellular matrix (ECM) in the glomeruli. PAI-1 is up-regulated by high glucose and is overexpressed in diabetic kidney. Since reactive oxygen species (ROS) mediate ECM accumulation in diabetic glomeruli and was recently found to mediate transforming growth factor-beta1 (TGF-beta1)-induced PAI-1 up-regulation in glomerular mesangial cells, we examined the role of ROS in high glucose-induced PAI-1 expression in cultured glomerular mesangial cells and in streptozotocin-induced diabetic rat glomeruli.


METHODS
Growth arrested and synchronized primary rat mesangial cells were treated with different concentrations of glucose in the presence or absence of N-acetylcysteine (NAC) or trolox, or after cellular reduced form of glutathione (GSH) depleted with DL-buthionine-(S,R)-sulfoximine (BSO). Taurine was administered to diabetic rats from 2 days to 4 weeks after streptozotocin injection. Urinary protein excretion, glomerular volume, and fractional mesangial area were measured as markers of renal injury and lipid peroxide (LPO) as an oxidative stress marker. PAI-1 mRNA expression was measured by Northern blot analysis in mesangial cells and reverse transcription-polymerase chain reaction (RT-PCR) in glomeruli, PAI-1 protein by Western blot analysis and enzyme-linked immunosorbent assay (ELISA), and plasmin activity by fluorometry.


RESULTS
High glucose significantly increased PAI-1 mRNA and protein expression and decreased plasmin activity in mesangial cells. Equimolar concentrations of l-glucose or mannitol did not affect PAI-1 expression. BSO pretreatment significantly increased basal PAI-1 expression and amplified the response to high glucose. NAC effectively inhibited high glucose-induced, but not basal, PAI-1 expression. Reduced plasmin activity in mesangial cells by high glucose was rescued by antioxidants. Anti-TGF-beta antibody inhibited both high glucose- and H(2)O(2)-induced PAI-1 up-regulation. Taurine significantly reduced plasma LPO, glomerular PAI-1 expression, glomerular volume, fractional mesangial area, and proteinuria in streptozotocin-induced diabetic rats.


CONCLUSIONS
These results demonstrate that ROS mediate high glucose-induced up-regulation of PAI-1 expression in cultured mesangial cells and in diabetic glomeruli. Since both high glucose and TGF-beta1 induce cellular ROS and ROS mediate both high glucose- and TGF-beta1-induced PAI-1, ROS appear to amplify TGF-beta1 signaling in high glucose-induced PAI-1 up-regulation. Antioxidants can prevent accumulation of ECM protein in diabetic glomeruli partly by abrogating up-regulation of PAI-1 and suppression of plasmin activity.
Lee
Eun Ah
EA
Hyonam Kidney Laboratory, Soon Chun Hyang University, Seoul, Korea.
Seo
Ji Yeon
JY
Jiang
Zongpei
Z
Yu
Mi Ra
MR
Kwon
Min Kyung
MK
Ha
Hunjoo
H
Lee
Hi Bahl
HB
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001483:Base Sequence; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D003921:Diabetes Mellitus, Experimental / Q000235:genetics / Q000378:metabolism; D003928:Diabetic Nephropathies / Q000235:genetics* / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism; D005920:Glomerular Mesangium / Q000187:drug effects / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism* / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D008297:Male; D018384:Oxidative Stress; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013654:Taurine / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000378:metabolism; D053773:Transforming Growth Factor beta1; D015854:Up-Regulation / Q000187:drug effects
D000975:Antioxidants; D018076:DNA, Complementary; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D013654:Taurine; D019328:Buthionine Sulfoximine; D005341:Fibrinolysin; D005978:Glutathione; D005947:Glucose