1. Volume 139, Issue 1, Pages 315-322.e1 (July 2010)
Hepcidin Expression Does Not Rescue the Iron-Poor Phenotype of Kupffer Cells in Hfe- Null Mice After Liver Transplantation 
Cinzia Garuti, Yinghua Tian, Giuliana Montosi, Manuela Sabelli, Elena Corradini, Rolf Graf, Paolo Ventura, Alberto Vegetti, Pierre–Alain Clavien, Antonello Pietrangelo 
Gastroenterology 
Volume 139, Issue 1, Pages e1 (July 2010)
DOI: /j.gastro
Copyright © 2010 AGA Institute Terms and Conditions

2. Figure 1
Iron status (A and B) and hepcidin expression (C) in Hfe+/+ and Hfe−/− mice after OLT. W→W: Hfe+/+ mice transplanted with Hfe+/+ livers; K→K: Hfe−/− mice transplanted with Hfe−/− livers; K→W: Hfe+/+ mice transplanted with Hfe−/− livers; W→K: Hfe−/− mice transplanted with Hfe+/+ livers. (A) Serum TS was measured in each treatment group at death as specified in the Materials and Methods section. Values are mean ± SD of triplicate measurements in at least 6 mice per group. Statistical significance of differences between different groups is also indicated (2-way ANOVA with least significant difference [LSD] post hoc test for comparisons). TS was significantly higher in K→K than in W→W mice. After transplanting Hfe+/+ mice with Hfe−/− livers (K→W group), TS increased appreciably, whereas Hfe−/− mice transplanted with Hfe+/+ livers (W→K group) showed decreased (although not fully normalized) TS levels. (B) LIC was measured in grafts' tissue specimens at death as specified in the Materials and Methods section. Values are mean ± SD of triplicate measurements made in 3 different tissue specimens from each animal. At least 6 animals per group were evaluated. Statistical significance of differences between different groups is also indicated (2-way ANOVA with LSD post hoc test for comparisons). At 6–8 months from implantation of Hfe+/+ donor livers in Hfe−/−, LIC was not significantly higher as compared with Hfe+/+ recipients (W→K vs W→W groups), whereas, after OLT with Hfe−/− donor livers, LIC remained high in Hfe+/+ recipients, similar to that of Hfe−/− recipients (K→W vs K→K). (C) Total liver RNA was extracted and subjected to RT-PCR for hepcidin and β-actin expression as specified in the Materials and Methods section. Values are mean ± SD of triplicate measurements in at least 6 mice per group. Statistical significance of differences between different groups is also indicated (2-way ANOVA with LSD post hoc test for comparisons). At the time of death, hepcidin messenger RNA expression was significantly lower in K→K mice as compared with W→W. After OLT, Hfe+/+ mice receiving an Hfe−/− liver had lower hepatic hepcidin expression, whereas Hfe−/− mice receiving Hfe+/+ mice liver fully recovered hepcidin messenger RNA expression.
Gastroenterology  , e1DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

3. Figure 2
Representative histopathology pictures at low and high power (insets) of liver iron stain by Perls' Prussian blue. (A) Iron deposits are visible in KC throughout the lobule in Hfe+/+ mice (WT); (B) iron accumulation is detected in parenchymal cells, mainly in periportal and mid zones, II, whereas KC lack iron deposits, in Hfe−/− mice (KO); (C) W→W group: iron deposits are visible in KC scattered intralobularly as occurs in untransplanted Hfe+/+ mice (see panel A); (D) K→K group: iron accumulation is detected in parenchymal cells, mainly in zones I and II, but spares KC, as occurs in Hfe−/− mice (see panel B). (E) K→W group: parenchymal cells accumulates iron, but KC remains iron rich as in Hfe+/+ mice receiving Hfe+/+ livers (see panel C). (F) W→K group: no appreciable iron deposits are detectable in parenchymal cells, but KC do not acquire the “normal” iron reach phenotype (see panel A) and remain iron poor as in untransplanted Hfe−/− mice (see panel B).
Gastroenterology  , e1DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

4. Figure 3
Spleen and intestine iron content after OLT. Iron content was assessed by chemical (A and C) or histopathologic (B and D) methods. Analysis and presentation of chemical data as specified in the legend of Figure 1B. Hfe−/− mice transplanted with Hfe−/− livers (K→K) showed much lower spleen iron content than W→W mice (A), as also indicated by the decreased iron stain in red pulp macrophages at histopathology (B). After transplanting Hfe+/+ mice with Hfe−/− livers (K→W group), spleen iron was appreciably decreased, whereas Hfe−/− mice transplanted with Hfe+/+ livers partially recovered spleen iron content (W→K group). Hfe−/− mice transplanted with Hfe−/− livers (K→K) had lower intestine iron content than Hfe+/+ mice receiving Hfe+/+ livers (W→W group) (C), as also shown by the iron-poor phenotype of absorptive enterocytes in the K→K group (D). Hfe+/+ animals lost intestine iron after OLT with Hfe−/− liver (K→W), whereas Hfe−/− mice recovered intestinal iron content after implantation of Hfe+/+ livers (W→K group).
Gastroenterology  , e1DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

5. Figure 4
Hepatic Hfe allele status before and after OLT. DNA analysis for the Hfe+/+ or Hfe−/− allele was performed in the same liver at the time of implantation (OLT) and at sacrifice (S), as indicated. Representative animals from each of the 4 OLT groups are reported (A, B, C, D). Each liver DNA sample was subjected to PCR with both Hfe+/+ (wt) and Hfe−/− (ko) primers, as reported in the Materials and Methods section. Controls: hepatic DNA from untransplanted Hfe+/+ (WT) or Hfe−/− (KO) mice were also tested by PCR with wt or ko primers, as indicated. The Hfe−/− and Hfe+/+ allele PCR products are also indicated (arrows on the left). In W→K mice, Hfe+/+ donor livers showed only the Hfe+/+ allele at the time of implantation (C) but presented also the Hfe−/− allele at the time of death (C'). The same was true for the Hfe−/− allele when Hfe−/− donor livers were implanted in Hfe+/+ mice (D vs D').
Gastroenterology  , e1DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

6. Supplementary Figure 1
Comparison of LIC values assessed in the same liver at the time of liver transplantation (OLT) and sacrifice (S). (A) Hfe+/+ donor livers; (B) Hfe−/− donor livers. See text for comments.
Gastroenterology  , e1DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions