Angiotensin IV stimulates plasminogen activator inhibitor-1 expression in proximal tubular epithelial cells.
BACKGROUND
Angiotensin II (Ang II) has been shown to be implicated in the development of renal fibrosis in several forms of chronic glomerulonephritides, but the precise mechanisms of its effects remain unclear. It has recently been reported that Ang II stimulates the expression of plasminogen activator inhibitor-1 (PAI-1) in several cell lines. PAI-1 is a major physiological inhibitor of the plasminogen activator/plasmin system, a key regulator of fibrinolysis and extracellular matrix (ECM) turnover. PAI-1 induction by Ang II in endothelial cells seems to be mediated by Ang IV via a receptor that is different from Ang II type 1 and 2 receptors (AT1 and AT2).


METHODS
In this study, we sought to evaluate the effects of Ang IV on PAI-1 gene and protein expression in a well-characterized and immortalized human proximal tubular cell line (HK2) by Northern blot and enzyme-linked immunosorbent assay.


RESULTS
Ang IV stimulated PAI-1 mRNA expression, whereas it did not induce a significant increase in tritiated thymidine uptake after 24 hours of incubation. This effect was dose and time dependent. Ang IV (10 nM) induced a 7.8 +/- 3.3-fold increase in PAI-1 mRNA expression. The PAI-1 antigen level was significantly higher in conditioned media and the ECM of cells treated with Ang II and Ang IV than in control cells (both P < 0.02). Although Ang II induced a 4.2 +/- 2. 1-fold increase in PAI-1 mRNA expression, its effect underwent a dose-dependent reduction when amastatin, a potent inhibitor of the endopeptidases that catalyzes the conversion of Ang II to Ang IV, was added. In contrast, amastatin was not able to prevent the expression of PAI-1 mRNA induced by Ang IV. Finally, pretreatment of HK2 cells with losartan and N-Nicotinoyl-Tyr-N3-(Nalpha-CBZ-Arg)-Lys-His-Pro-Ile, the specific antagonists of AT1 and AT2 receptors, failed to modify PAI-1 mRNA expression as induced by Ang II.


CONCLUSIONS
Our results demonstrate that Ang II stimulates PAI-1 mRNA expression and the production of its protein in human proximal tubular cells. This is mainly-if not exclusively-due to Ang IV, which acts on a receptor that is different than AT1 or AT2. Therefore, it can be hypothesized that the induction of PAI-1 by Ang IV may be implicated in the pathogenesis of renal interstitial fibrosis in several forms of chronic glomerulonephritides.
Gesualdo
L
L
Dipartimento dell'Emergenza e dei Trapianti di Organi, Sezione di Nefrologia, Policlinico, Bari, Italy. l.gesualdo@nephro.uniba.it
Ranieri
E
E
Monno
R
R
Rossiello
M R
MR
Colucci
M
M
Semeraro
N
N
Grandaliano
G
G
Schena
F P
FP
Ursi
M
M
Cerullo
G
G
D000804:Angiotensin II / Q000031:analogs & derivatives* / Q000494:pharmacology; D057911:Angiotensin Receptor Antagonists; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000959:Antihypertensive Agents / Q000494:pharmacology; D015152:Blotting, Northern; D002455:Cell Division / Q000187:drug effects; D002461:Cell Line, Transformed; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D005355:Fibrosis; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007674:Kidney Diseases / Q000201:enzymology / Q000209:etiology / Q000473:pathology; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D019808:Losartan / Q000494:pharmacology; D010455:Peptides; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D011480:Protease Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D010959:Tissue Plasminogen Activator / Q000235:genetics; D016212:Transforming Growth Factor beta / Q000235:genetics; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics
D057911:Angiotensin Receptor Antagonists; D000900:Anti-Bacterial Agents; D000959:Antihypertensive Agents; D010455:Peptides; D017395:Plasminogen Activator Inhibitor 1; D011480:Protease Inhibitors; D012333:RNA, Messenger; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D016212:Transforming Growth Factor beta; D000804:Angiotensin II; C012877:angiotensin II, des-Asp(1)-des-Arg(2)-Ile(5)-; C016977:amastatin; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator; D019808:Losartan