Plasma protein haptoglobin modulates renal iron loading

Abstract

Haptoglobin is the plasma protein with the highest binding affinity for hemoglobin. The strength of hemoglobin binding and the existence of a specific receptor for the haptoglobin-hemoglobin complex in the monocyte/macrophage system clearly suggest that haptoglobin may have a crucial role in heme-iron recovery. We used haptoglobin-null mice to evaluate the impact of haptoglobin gene inactivation on iron metabolism. Haptoglobin deficiency led to increased deposition of hemoglobin in proximal tubules of the kidney instead of the liver and the spleen as occurred in wild-type mice. This difference in organ distribution of hemoglobin in haptoglobin-deficient mice resulted in abnormal iron deposits in proximal tubules during aging. Moreover, iron also accumulated in proximal tubules after renal ischemia-reperfusion injury or after an acute plasma heme-protein overload caused by muscle injury, without affecting morphological and functional parameters of renal damage. These data demonstrate that haptoglobin crucially prevents glomerular filtration of hemoglobin and, consequently, renal iron loading during aging and following acute plasma heme-protein overload.

Figure 1

Plasma clearance and organ distribution of ¹²⁵I-hemoglobin. A: ¹²⁵I-hemoglobin was injected intravenously through the tail vein of mice as described in Materials and Methods. The radioactivity of blood samples obtained after 45 seconds was in the range of 88,300 to 122,300 cpm/ml. Three mice of each genotype were used and the average (± SD) at each data point is indicated. B: Mice were sacrificed 20 minutes after ¹²⁵I-hemoglobin injection and total radioactivity of various organs was counted as described in Materials and Methods. Three mice of each genotype were analyzed. Data represent mean ± SD.

Figure 2

Identification of cell types taking up ¹²⁵I-hemoglobin. A: ¹²⁵I-hemoglobin was injected intravenously through the tail vein, mice were sacrificed 20 minutes later, and total radioactivity of the kidney, liver, and spleen was counted as described in Materials and Methods. Three mice of each genotype were analyzed. Data represent the mean ± SD. B: Autoradiography of kidney, liver and spleen sections of a wild-type mouse. Sections were prepared as described in Materials and Methods. Note silver grain accumulation in proximal tubular cells (arrows) in the kidney, in hepatocytes (arrows) and Kupffer cells (arrowheads) in the liver and in macrophages (arrows) in the spleen. The same pattern was seen in Hp-null mice (data not shown). Scale bar = 25 μm.

Figure 3

Iron loading during aging. A: Liver iron content was measured in wild-type and Hp-null mice at increasing ages. At least six mice of each genotype were analyzed. Data represent mean ± SD. B: Kidney iron content was measured in wild-type and Hp-null mice at increasing ages. At least six mice of each genotype were analyzed. Data represent mean ± SD. C: Kidney sections of a wild-type and a Hp-null mouse at 3 months of age stained with Perl’s reaction. Note iron loading in tubular cells in Hp-deficient mouse (at high magnification in the inset). Scale bar = 100 μm.

Figure 4

Hp expression after IRI. A: Western blotting of plasma proteins of a wild-type mouse before and one day after IRI assayed with an anti-Hp antibody. B: Northern blotting of total RNA extracted from the liver of a non-treated wild-type mouse and of a mouse subjected to IRI one day after surgery, sequentially analyzed with ³²P-dCTP-labeled probes to Hp and β-actin. C: Northern blotting of poly-A⁺ RNA extracted from the kidney of a non-treated wild-type mouse and from the contralateral and ischemic kidney of a wild-type mouse subjected to IRI one day after surgery, sequentially analyzed with ³²P-dCTP-labeled probes to Hp and β-actin. D: Western blot analysis to test anti-CD163 antibody specificity. Total protein extracts from E. coli expressing GST-CD163 fragment 1069–1121 and GST alone were subjected to SDS-PAGE, blotted on nitrocellulose membrane and probed with purified anti-CD163 polyclonal antibodies. Only recombinant CD163 (35 kd), but not GST (29 kd), was recognized by the purified anti-CD163 polyclonal antibody. E: Western blot on protein extracts from the kidney and the spleen of a non-treated wild-type mouse and of a mouse subjected to IRI one day after surgery, sequentially analyzed with the purified anti-CD163 and anti-vinculin antibodies. A CD163 band (130 kd) was detected in the ischemic kidney and in the spleen.

Figure 5

Iron loading after IRI. A: Kidney iron content was measured in non-treated mice and in mice subjected to IRI 1 and 7 days after surgery both in contralateral and ischemic organ. Six mice of each genotype were analyzed. Data represent mean ± SD. B: Kidney sections from non-treated and ischemized (both ischemic and contralateral organ 7 days after injury) of wild-type and Hp-null mice stained with Perl’s reaction. Note weak staining of some tubular cells in non-treated Hp-null mouse compared to more pronounced labeling of proximal tubules in both ischemic and contralateral kidney of Hp-null mouse subjected to IRI (arrowheads). Infiltrating macrophages in renal medulla of the ischemic kidney (arrows) were labeled in both wild-type and Hp-deficient mice. Scale bar = 100 μm.

Figure 6

Iron loading after glycerol-induced acute muscle injury. A: Kidney iron content was measured in non-treated mice and in glycerol-treated mice 1 and 7 days after injection. Six mice of each genotype were analyzed. Data represent the mean ± SD. B: Kidney sections from non-treated and glycerol-treated (7 days after injection) wild-type and Hp-null mice stained with Perl’s reaction. Note weak staining of some tubular cells in non-treated Hp-null mouse compared to strong labeling of proximal tubules in Hp-null mouse 7 days after glycerol injection. In wild-type mouse only a weak stain of some proximal tubules was detected after glycerol treatment. Scale bar = 100 μm.

Figure 7

Model to explain renal iron loading in Hp-null mice. In wild-type mice free plasma hemoglobin is bound by Hp and transported to the monocyte/macrophage system. In Hp-null mice, free hemoglobin is filtered through the glomerular barrier and reabsorbed by proximal tubular cells through the endocytic receptors megalin and cubilin. In tubular cells, hemoglobin is degraded in the endosomal compartment and heme is metabolized by heme oxygenase. Iron derived from heme catabolism is stored associated to ferritin. Hb, hemoglobin; Hp, haptoglobin; HO, heme oxygenase.