Hemopexin prevents endothelial damage and liver congestion in a mouse model of heme overload

Abstract

Intravascular hemolysis results in the release of massive amounts of hemoglobin and heme into plasma, where they are rapidly bound by haptoglobin and hemopexin, respectively. Data from haptoglobin and hemopexin knockout mice have shown that both proteins protect from renal damage after phenylhydrazine-induced hemolysis, whereas double-mutant mice were especially prone to liver damage. However, the specific role of hemopexin remains elusive because of the difficulty in discriminating between hemoglobin and heme recovery. To study the specific role of hemopexin in intravascular hemolysis, we established a mouse model of heme overload. Under these conditions, both endothelial activation and vascular permeability were significantly higher in hemopexin-null mice compared with wild-type controls. Vascular permeability was particularly altered in the liver, where congestion in the centrolobular area was believed to be associated with oxidative stress and inflammation. Liver damage in hemopexin- null mice may be prevented by induction of heme oxygenase-1 before heme overload. Furthermore, heme-treated hemopexin-null mice exhibited hyperbilirubinemia, prolonged heme oxygenase-1 expression, excessive heme metabolism, and lack of H-ferritin induction in the liver compared with heme-treated wild-type controls. Moreover, these mutant mice metabolize an excess of heme in the kidney. These studies highlight the importance of hemopexin in heme detoxification, thus suggesting that drugs mimicking hemopexin activity might be useful to prevent endothelial damage in patients suffering from hemolytic disorders.

Figure 1

Endothelial activation and vascular permeability after heme overload. A: Western blotting analysis of HO-1 (left) and ICAM-1 (right) expression on extracts of aortas from wild-type and Hx-null mice untreated (−) or treated with the high dose of hemin for 6 hours (+). B: Western blotting analysis of VCAM-1 expression on liver extracts from wild-type and Hx-null mice untreated (−) or treated with the high dose of hemin for 6 hours (+). In A and B a representative experiment for each protein is shown. Band intensities were measured by densitometry and normalized to actin or vinculin expression. Densitometry data represent mean ± SEM; n = 3 for each genotype. *P < 0.05; **P < 0.01. Results shown are representative of three independent experiments; in each experiment at least three mice per genotype were analyzed. C: Evans blue dye content of liver, kidney, and lung of wild-type and Hx-null mice untreated (−) or treated with the high dose of hemin (+) for 6 hours. Data represent mean ± SEM; n = 6 for each group. *P < 0.05; **P < 0.01; ***P < 0.0001.

Figure 2

Increased lipid peroxidation in Hx-null mice after heme overload. Lipid peroxidation, estimated as MDA levels, of liver (A) and kidney (B) homogenates of wild-type and Hx-null mice untreated or treated with the high dose of hemin for 6 hours. Data are expressed as the fold increase over a control sample (an untreated wild-type mouse). Data represent mean ± SD; n = 7 for each group; *P < 0.05; **P < 0.01.

Figure 3

Liver congestion in Hx-null mice after heme overload. Liver sections of wild-type and Hx-null mice untreated (i, ii) or treated with the high dose of hemin for 6 hours (iii–viii), stained with H&E. Note congestion around the centrolobular vein (arrows), but not in the periportal area (arrowheads), only in Hx-null mouse. Scale bars: 300 μm (i–iv); 100 μm (v–viii).

Figure 4

Increased liver inflammation in Hx-null mice after heme overload. A: Liver sections of wild-type and Hx-null mice untreated (i, ii) or treated with the high dose of hemin for 6 hours (iii, iv), stained with an antibody to CD18 antigen. Note the higher number of CD18-positive cells in Hx-null liver after hemin treatment. B: Quantification of CD18-positive cells on liver sections of wild-type and Hx-null mice. Cells were counted as reported in the Materials and Methods. Data represent mean ± SD; n = 4 for each genotype; **P < 0.01. Scale bars = 100 μm.

Figure 5

HO-1 induction after heme overload. A: Kidney sections of wild-type and Hx-null mice untreated (i, ii) or treated with the high dose of hemin for 6 hours (iii–vi), stained with an antibody to HO-1. Note the induction of HO-1 in proximal tubular cells of Hx-null kidney after hemin injection. B: Liver sections of wild-type and Hx-null mice untreated (i, ii) or treated with the high dose of hemin for 2 hours (iii, iv), 4 hours (v, vi), or 6 hours (vii–x), stained with an antibody to HO-1 (i–viii) or an antibody to F4/80 (ix–x). Note the increased number of HO-1-positive cells in Hx-null liver 6 hours after hemin injection. The last four panels show consecutive sections of wild-type (vii–ix) and Hx-null (viii–x) livers: the anti-HO-1 and anti-F4/80 antibodies stain the same cell type, ie, Kupffer cell. C: Quantification of HO-1-positive cells on liver sections of wild-type and Hx-null mice. Cells were counted as described in the Materials and Methods. Data represent mean ± SD; n = 3 for each experimental point; **P < 0.01. Scale bars: 300 μm (A, i–iv); 100 μm [A, (v, vi) B].

Figure 6

L- and H-Ft expression in kidney and liver after heme overload. Western blotting analysis of L-Ft (left) and H-Ft (right) expression on kidney (A) and liver (B) extracts from wild-type and Hx-null mice untreated (−) or treated with the high dose of hemin for 6 hours (+). A representative experiment for each protein is shown. Band intensities were measured by densitometry and normalized to vinculin expression. Densitometry data represent mean ± SEM; n = 3 for each genotype. *P < 0.05; **P < 0.01. Results shown are representative of three independent experiments; in each experiment at least three mice per genotype were analyzed.

Figure 7

Prevention of liver damage by preconditioning. A: Evans blue dye content of the liver of wild-type and Hx-null mice untreated (−) or treated with the high dose of hemin (+) for 6 hours without (open and solid bars) or with preconditioning (open and solid hatched bars). Data represent mean ± SEM; n = 8 for each group. *P < 0.05; **P < 0.01; ***P < 0.0001. B: Lipid peroxidation, estimated as MDA levels, of liver homogenates of wild-type and Hx-null mice untreated (−) or treated with the high dose of hemin (+) for 6 hours without (open and solid bars) or with preconditioning (open and solid hatched bars). Data are expressed as the fold increase over a control sample (an untreated wild-type mouse). Data represent mean ± SD; n = 8 for each group; *P < 0.05; **P < 0.01. C: Liver sections of wild-type and Hx-null mice treated with the high dose of hemin for 6 hours without (i, ii) or with (iii, iv) preconditioning, stained with H&E. Note congestion around the centrolobular vein in heme-treated Hx-null mouse, but not in preconditioned animal. Scale bars: 300 μm (C); 50 μm (C, insets).

Figure 8

A model of Hx-mediated heme recovery. In wild-type mice (top) the heme-Hx complex is taken up by hepatocytes and Kupffer cells through the LRP receptor. The latter are also able to recover free heme in an Hx-LRP-independent way. Once into cells, heme is degraded by HO-2 (hepatocytes) and HO-1 (Kupffer cells) and iron stored in ferritins. Because hepatocytes represent more than 90% of liver cells, they account for the recovery of most heme, thus preventing heme toxicity. In Hx-null mice (bottom), lack of Hx prevents heme recovery in hepatocytes. Heme is taken up by Kupffer cells that remain activated longer than in wild-type counterpart. Heme that overwhelms the recovery capacity of Kupffer cells promotes vaso-occlusion and liver damage. Part of heme is recovered by proximal tubular cells of the kidney.