Volume 120, Issue 2, Pages 545-556 (February 2001) Inhibition of the Na+/H+ exchanger reduces rat hepatic stellate cell activity and liver fibrosis: An in vitro and in vivo study  Antonio Benedetti, Antonio Di Sario, Alessandro Casini, Francesco Ridolfi, Emanuele Bendia, Paola Pigini, Cecilia Tonnini, Letizia D'Ambrosio, Giuseppe Feliciangeli, Giampiero Macarri, Gianluca Svegliati-Baroni  Gastroenterology  Volume 120, Issue 2, Pages 545-556 (February 2001) DOI: 10.1053/gast.2001.21203 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Effect of PDGF, TGF-β1, FeNTA, and FeAsc on (A–C) Na+/H+ exchange activity and (D) the slope of the curve measuring the relationship between acid extrusion rate and nadir pHi. Cells were cultured for 24 hours in Iscove's medium containing 0.5% serum and then incubated in the same medium containing the indicated substances. pHi was measured in the absence of bicarbonate (HEPES solution) using the fluorescent indicator BCECF (12 μmol/L). Na+/H+ exchange activity was evaluated by measuring pHi recovery from an acute acid load. Transmembrane H+ fluxes (JH+), expression of the activity of the Na+/H+ exchanger, were calculated by multiplying the rate of pHi recovery in the unit of time (pH units/min) by the intracellular buffering power. (A) PDGF, (B) FeNTA, and (C) FeAsc increased the Na+/H+ exchange activity, whereas (A) TGF-β1 had no effect. The increase was inhibited by EIPA in all experimental conditions. (D) Bar values, which are a measure of the slope of the curve indicating the relationship between acid extrusion rate and nadir pHi, are significantly higher in HSCs incubated with either PDGF, FeNTA, or FeAsc. Data are expressed as mean ± SD. *P < 0.01 vs. controls, TGF-β1, PDGF + EIPA 20 μmol/L, FeNTA + EIPA 20 μmol/L, and FeAsc + EIPA 20 μmol/L; #P < 0.05 vs. PDGF + EIPA 1 and 10 μmol/L, FeNTA + EIPA 1 and 10 μmol/L, and FeAsc + EIPA 1 and 10 μmol/L. Gastroenterology 2001 120, 545-556DOI: (10.1053/gast.2001.21203) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Effect of (A) PDGF on HSC proliferation, (B) TGF-β1, FeAsc, and FeNTA on type I collagen accumulation in HSCs culture medium, and (C and D) EIPA on TGF-β1 mRNA expression. Cells were cultured for 24 hours in Iscove's medium + 0.5% serum and then incubated in the same medium containing the indicated substances for an additional 4 hours (for TGF-β1 mRNA expression) or 24 hours (for HSC proliferation type I collagen accumulation). HSC proliferation was measured by indirect immunoperoxidase staining of nuclei that had incorporated BrdU, added during the last 4 hours of incubation. Type I collagen concentration was measured in HSC culture media by enzyme-linked immunosorbent assay. TGF-β1 mRNA expression was evaluated by Northern blot analysis of 20 μg of total RNA extracted from rat HSCs. Equal loading of the sample was checked by reprobing the blots with cyclophilin cDNA after the hybridization procedure (lane 1, controls; lane 2, controls + EIPA 10 μmol/L; lane 3, TGF-β1 + EIPA 10 μmol/L; and lane 4, TGF-β1). Data are expressed as mean ± SD. *P < 0.05 vs. all other groups. Gastroenterology 2001 120, 545-556DOI: (10.1053/gast.2001.21203) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Morphometric evaluation of proliferating HSCs in the (A) DMN and (B) BDL models of liver fibrosis. To identify proliferating HSCs, a sequential double immunoenzymatic reaction was performed for the simultaneous detection of α-SMA, as a marker of activated HSCs, and of PCNA expression, as a marker of S-phase nuclei, in methanol-fixed sections. Proliferating HSCs were defined as interstitial cells with at least one α-SMA–positive cytoplasmic process and a labeled nucleus, and they were expressed as the number of cells/mm2. Data are expressed as mean ± SD. *P < 0.05 vs. all other groups. Gastroenterology 2001 120, 545-556DOI: (10.1053/gast.2001.21203) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Histologic features of the liver at the end of treatment with (A and C) DMN alone, (B and D) DMN + amiloride, (E) BDL, and (F) BDL + amiloride as seen after immunostaining for (A and B) α-SMA and (C–F) Sirius red. (A) Reactivity of the cells associated with altered terminal hepatic veins and with the septa, whereas in B, only limited areas of immunoreactivity are evident. (C) The pattern of micronodular cirrhosis in DMN-treated rats is evident, with thick fibrotic septa bridging between terminal hepatic veins, and elements of the portal space are spared in the center of the nodule. (D) Only incomplete fibrotic septa are evident that form thin parenchymal extension without a nodular pattern. (E) The lobular architecture is massively modified by proliferating ductules accompanied by fibrotic tissue, whereas in F, only a limited ductular proliferation from the portal tract is observed. (Original magnification 25X.) Gastroenterology 2001 120, 545-556DOI: (10.1053/gast.2001.21203) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 Morphologic evaluation of (A and B) HSC activation and (C and D) collagen deposition after DMN and BDL treatment. The extension of (A and B) α-SMA– and (C and D) Sirius red–positive parenchyma (measured by point-counting technique and expressed as a percentage of area) was significantly increased in DMN- and BDL-treated animals compared with the amiloride groups. Data are expressed as mean ± SD. *P < 0.05 vs. all other groups. Gastroenterology 2001 120, 545-556DOI: (10.1053/gast.2001.21203) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 Effect of treatment with amiloride on the stimulation of α1(I) procollagen mRNA expression induced by (A and C) DMN and (B and D) BDL in rat liver (n = 5 for each group). (A and B) α1(I) procollagen mRNA expression was evaluated by Northern blot analysis of 20 μg of total RNA extracted from rat livers (upper panels). Equal loading of the sample was checked by reprobing the blots with cyclophilin cDNA after the hybridization procedure (lower panels). The size of transcripts (expressed in kilobases) is indicated at right. (A) Lane 1, control rat; lane 2, DMN + amiloride (1 week); lane 3, DMN (1 week); lane 4, DMN + amiloride (3 weeks); lane 5, DMN (3 weeks). (B) Lane 1, control rat; lane 2, BDL + amiloride (1 week); lane 3, BDL (1 week); lane 4, BDL + amiloride (4 weeks); and lane 5, BDL (4 weeks). (C and D) Laser densitometric analysis of Northern blots. Values obtained by laser scanning densitometry of both mRNA hybridization bands were added and normalized to cyclophilin bands. Data (mean ± SD) are expressed as percentage of increase of controls. *P < 0.05 vs. DMN + amiloride and BDL + amiloride. Gastroenterology 2001 120, 545-556DOI: (10.1053/gast.2001.21203) Copyright © 2001 American Gastroenterological Association Terms and Conditions