Volume 141, Issue 4, Pages 1509-1519.e3 (October 2011) Protoporphyrin Retention in Hepatocytes and Kupffer Cells Prevents Sclerosing Cholangitis in Erythropoietic Protoporphyria Mouse Model  Saïd Lyoumi, Marie Abitbol, Dominique Rainteau, Zoubida Karim, Florence Bernex, Vincent Oustric, Sarah Millot, Philippe Lettéron, Nicholas Heming, Laurent Guillmot, Xavier Montagutelli, Gilles Berdeaux, Laurent Gouya, Raoul Poupon, Jean–Charles Deybach, Carole Beaumont, Hervé Puy  Gastroenterology  Volume 141, Issue 4, Pages 1509-1519.e3 (October 2011) DOI: 10.1053/j.gastro.2011.06.078 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Liver function parameters, detection of lipoprotein-X (Lp-X) and quantification of PPIX in liver and stool in fch/fch mice. (A) Liver function parameters. AkP, alkaline phosphatase. a,b,cSignificant differences between wt and fch/fch mice (aP < .001; bP < .005; cP < .01). *Significant difference between BALB/c and C57BL/6 fch/fch mice (P < .0001). (B) Plasma Lp-X detection. Ori, Origin of migration. (C) Naked eye examination of liver homogenates. (D) Liver PPIX content. (E) Liver-to-stool PPIX ratio. Data are given as mean ± standard deviation (n = 10 in all groups). Gastroenterology 2011 141, 1509-1519.e3DOI: (10.1053/j.gastro.2011.06.078) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 PPIX deposits in BALB/c and C57BL/6 fch/fch mouse livers. Porphyrin deposits were found mainly in enlarged bile canaliculi in the livers of BALB/c fch/fch mice (A and C, black arrows), whereas porphyrin deposits were seen in hepatocytes and Kupffer cells in the livers of C57BL/6 fch/fch mice (B and D, red arrows). H&E staining; original magnification, A–C ×40; D, ×63. Electron microscopy (E and F) confirmed the presence of porphyrin deposits in the cytosol of Kupffer cells in C57BL/6 fch/fch mice (F, asterisks); high magnification, ×27,000. h, hepatocytes, k, Kupffer cells; n, nucleus. Gastroenterology 2011 141, 1509-1519.e3DOI: (10.1053/j.gastro.2011.06.078) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Periductal fibrosis, sclerosing cholangitis and cholesterol crystallization in fch/fch mice. Red picro-sirius staining shows periductal fibrosis in both fch/fch strains (A and B, asterisk). Fibrosis with portoportal bridging was observed only in BALB/c fch/fch mice (A, arrow). Interlobular bile ducts (IBD) of the BALB/c fch/fch mice (C) display greater pseudopapillary proliferation of bile duct epithelial cells (arrow) and ulceration and necrosis (arrowhead) compared with C57BL/c fch/fch mice (D). Tubular crystals (asterisk) can be seen in BALB/c fch/fch mice surrounding the dark PPIX pigment deposits (E) but not in C57BL/6 fch/fch mice (F). Note the presence of destructive cholangitis in BALB/c fch/fch mice (arrow). H&E staining was used for C and D and picro-sirius for panels A, B, E, F. Original magnification, A and B, ×10; C and D, ×20; and E and F, ×40. Gastroenterology 2011 141, 1509-1519.e3DOI: (10.1053/j.gastro.2011.06.078) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 In vitro binding of protoporphyrin (PPIX) to phosphatidylcholine (PC), bile acid (tauro-conjugated cholic acid [TCA]), or cholesterol (Chol). (A) Maximal fluorescence emissions of PPIX (20 nmol/L) in 1 mol/L Imidazol, 0.9 % NaCl buffered solution incubated alone or in the presence of PC, Chol, TCA, or a mixed solution (PC+Chol+TCA) were determined. The ratio of the fluorescence intensity of PPIX bound to either PC, TCA, or Chol (I) to PPIX alone (I0) was calculated. Significant difference between PPIX alone and PPIX bound to PC or TCA (*P < .01, **P < .001). Data are given as mean ± standard deviation. (B) In vitro binding affinity of PPIX to PC. PC and albumin at variable concentrations were added to PPIX. After excitation (λmax 400–408 nm), the fluorescence emission (λmax 620–632 nm) was measured. All experiments were performed in triplicate. Gastroenterology 2011 141, 1509-1519.e3DOI: (10.1053/j.gastro.2011.06.078) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Proposed mechanism of increased susceptibility to liver injury in an EPP mouse model. The effect of excess protoporphyrin-IX (PPIX) in hepatocytes results in different phenotypes depending on the genetic background. (A) A lower efflux of PPIX, combined with lower bile acid (BA) synthesis and reduced canalicular BA efflux, offers relative protection against bile duct epithelial cells (BEC) (cholangiocyte) destruction. (B) The presence of large amounts of PPIX in the canaliculi, associated with the absence of adaptive mechanisms affecting BA metabolism and exportation, is responsible for sclerosing cholangitis and BEC destruction. Note: Biliary secretion of bile acids (BA), phospholipids (PL), and cholesterol (Chol) is controlled at the hepatocyte canalicular membrane by Bsep, Mdr2, and Abcg5/8, respectively. BA uptake and efflux at the basolateral membrane is controlled by Oatps and Mrp4, respectively. BA synthesis in the hepatocyte is controlled by Cyp27a1, Cyp7a1, and Cyp8b1. Gastroenterology 2011 141, 1509-1519.e3DOI: (10.1053/j.gastro.2011.06.078) Copyright © 2011 AGA Institute Terms and Conditions