Inhibition of the NA(+)/H(+) exchanger reduces rat hepatic stellate cell activity and liver fibrosis: an in vitro and in vivo study.
OBJECTIVE
The Na(+)/H(+) exchanger is the main intracellular pH (pH(i)) regulator in hepatic stellate cells (HSCs) and plays a key role in regulating proliferation and gene expression. We evaluated the effect of specific inhibition of this exchanger on HSC proliferation and collagen synthesis in vivo and in vitro.


METHODS
Rat HSCs were incubated in the presence of platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta1, iron ascorbate (FeAsc), and ferric nitrilotriacetate solution (FeNTA) with or without the Na(+)/H(+) exchanger inhibitor 5-N-ethyl-N-isopropyl-amiloride (EIPA). pH(i) and Na(+)/H(+) exchanger activity, cell proliferation, and type I collagen accumulation were measured by using the fluorescent dye 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein, by immunohistochemistry for bromodeoxyuridine, and by enzyme-linked immunosorbent assay, respectively. In vivo liver fibrosis was induced by dimethylnitrosamine administration and bile duct ligation (BDL) in rats treated or not treated with amiloride.


RESULTS
PDGF, FeAsc, and FeNTA increased Na(+)/H(+) exchange activity and induced HSC proliferation. TGF-beta1 had no effect on the Na(+)/H(+) exchanger and was able, as for FeAsc and FeNTA, to induce type I collagen accumulation. EIPA inhibited all the effects determined by PDGF, FeAsc, and FeNTA and had no effect on TGF-beta1-induced collagen accumulation. In vivo, amiloride reduced HSC proliferation, activation, collagen deposition, and collagen synthesis.


CONCLUSIONS
The Na(+)/H(+) exchanger can play a key role in the development of liver fibrosis and in HSC activation in vivo.
Benedetti
A
A
Department of Gastroenterology, University of Ancona, Italy. clingastro@popsci.unian.it
Di Sario
A
A
Casini
A
A
Ridolfi
F
F
Bendia
E
E
Pigini
P
P
Tonnini
C
C
D'Ambrosio
L
L
Feliciangeli
G
G
Macarri
G
G
Svegliati-Baroni
G
G
D000584:Amiloride / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D001205:Ascorbic Acid / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D003094:Collagen / Q000378:metabolism; D004232:Diuretics / Q000494:pharmacology; D005290:Ferric Compounds / Q000494:pharmacology; D005296:Ferrous Compounds / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D020287:In Situ Nick-End Labeling; D008099:Liver / Q000166:cytology* / Q000378:metabolism* / Q000473:pathology; D008103:Liver Cirrhosis / Q000378:metabolism* / Q000473:pathology*; D008297:Male; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D011347:Procollagen / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013939:Thymidine Phosphorylase / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000235:genetics
D000889:Anti-Arrhythmia Agents; D000970:Antineoplastic Agents; D002273:Carcinogens; D004232:Diuretics; D005290:Ferric Compounds; D005296:Ferrous Compounds; D011347:Procollagen; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D017923:Sodium-Hydrogen Exchangers; D016212:Transforming Growth Factor beta; C051140:iron(II)-ascorbic acid complex; D000584:Amiloride; D003094:Collagen; D013939:Thymidine Phosphorylase; D009571:Nitrilotriacetic Acid; D001205:Ascorbic Acid; C039614:ethylisopropylamiloride; C020326:ferric nitrilotriacetate