1. Volume 42, Issue 1, Pages 102-109 (January 2005)
Kupffer cell depletion with liposomal clodronate prevents suppression of Ntcp expression in endotoxin-treated rats 
Ekkehard Sturm, Rick Havinga, Julius F.W. Baller, Henk Wolters, Nico van Rooijen, Jan A.A.M. Kamps, Henkjan J. Verkade, Saul J. Karpen, Folkert Kuipers 
Journal of Hepatology 
Volume 42, Issue 1, Pages (January 2005)
DOI: /j.jhep
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

2. Fig. 1
Effective depletion of Kupffer cell population by liposomal clodronate. Liver sections (40× original magnification) stained with an monoclonal antibody directed against macrophages (ED2), panel A: control animal treated with vehicle (PBS/NaCl), note the red staining in the region of the portal fields representing KC; panel B: animal treated with clodronate 48h before excision of liver, portal fields are almost devoid of ED-2 signal, indicating loss of KC; panel C: animal received only vehicle before exposure to LPS (1mg/kg BW), strong positive staining for KC; panel D: pretreatment with clodronate 48h before exposure to LPS, effective KC depletion in presence of endotoxin.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

3. Fig. 2
Effect of clodronate exposure on expression of a rat-Kupffer cell specific product. Semiquantitative RT-PCR analysis for steady state mRNA levels of the rat-specific Kupffer cell receptor, RNA extracted from total liver tissue of clodronate-treated rats and vehicle treated controls (PBS); ethidium bromide staining. Liver KCR gene expression in clodronate-treated rats is significantly lower than in untreated controls, indicating disruption of Kupffer cell specific gene transcription in clodronate treated livers. LPS treatment did not alter the suppression of KCR gene expression in clodronate treated rats (data not shown).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

4. Fig. 3
Depletion of Kupffer cells diminishes cytokine expression following LPS exposure. Panel A: The hepatic content of indicated cytokine mRNA was determined using RNAse protection assay. L32 and GAPDH are RNA loading controls. Animals received clodronate (lanes 1+2) or PBS (lanes 3+4) 48h before LPS (lanes 2+4) or vehicle (lanes 1+3). Representative assay is shown. Panel B: Liver tissue Il-1β and TNFα mRNA levels and plasma levels of TNFα protein in Vehicle/LPS treated rats (grey bars) and CLO/LPS treated (black bars). (Cytokine mRNA: n=3, *P<0.04, protein: n=6, **P<0.003).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

5. Fig. 4
Residual population of ED-1 expressing cells following clodronate exposure in liver and spleen. Panel A: liver tissue of an animal receiving only vehicle before injection of LPS depicting ED-1 positive cells in portal fields indicating mixed monocyte/macrophage population. Panel B: liver tissue of an animal receiving clodronate before injection of LPS, residual population of ED-1 expressing monocytes. Panel C: spleen tissue of animal treated with vehicle before LPS, population of ED-1 expressing cells in red pulp, marginal zone and follicles. Panel D: residual ED-1 expressing cell population concentrated in red pulp in an animal receiving clodronate before LPS injection.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

6. Fig. 5
Preservation of Ntcp expression in Kupffer cell depleted, endotoxemic rats. Animals were treated with clodronate or vehicle (PBS) 48h before ip injection of LPS or vehicle (NaCl), liver tissue was harvested 16h after LPS exposure Panel A: The hepatic content of Ntcp mRNA was measured in total rat liver by using real-time RT-PCR as indicated in Section 2. Panel B: Ntcp protein level was determined by immunoblotting in crude membrane fractions from rat liver. Representative blot with mean densitometry signal intensity±SEM. Panel C: Total bile acid plasma concentration expressed as percent of PBS/LPS injected control. (n=4, *P<0.04 for panels A and B, in panel B comparison PBS/LPS and CLO/LPS groups).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

7. Fig. 6
Effect of LPS and CLO/LPS on RXR mRNA levels in rat liver. Following CLO or vehicle (PBS) pretreatment animals were injected with LPS or vehicle (NaCl 0.9%). The liver was removed 16h later. RXR mRNA was determined by real-time RT-PCR.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

8. Fig. 7
RXR:RAR and HNF1α binding in Kupffer cell depleted, endotoxin exposed animals is preserved. Analysis by mobility shift assay (see Section 2), duplicate experiment. Nuclear extract derived from whole liver of rats treated with clodronate or vehicle (PBS) 48h before injection of LPS or vehicle (NaCl 0.9%), liver tissue harvest 16h after LPS exposure. LPS inhibits RXR:RAR and HNF1α binding in vehicle treated animals (lanes 5+6) whereas RXR:RAR and HNF1α binding is normal in clodronate pretreated animals (lanes 7+8) (NSC, non-specific competition; SC, specific competition).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

9. Image 1
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2004 European Association for the Study of the Liver Terms and Conditions