Haptoglobin attenuates hemoglobin-induced heme oxygenase-1 in renal proximal tubule cells and kidneys of a mouse model of sickle cell disease
Introduction
SCD is a hereditary hemolytic disorder characterized by recurring episodes of painful vaso-occlusive crises and endothelial dysfunction [5]. SCD patients express a mutation in the β-subunit of hemoglobin S (HbS) that promotes polymerization of HbS and the sickling of red blood cells (RBCs) under conditions of low oxygen. The constant sickling and unsickling cycles result in RBC lysis in the microvasculature and the release of acellular HbS [15]. Hp, an endogenous Hb scavenger protein avidly binds to αβ dimers of Hb and forms a highly stable Hb–Hp complex. Binding of Hb to Hp prevents the release of free heme and filtration of Hb by the kidneys. Plasma hemopexin (Hpx) has high affinity for free heme that might be released from metHb [6], [30]. In sickle cell disease plasma Hp and Hpx levels are low due to chronic hemolysis [25]. The Hb–Hp complex binds to CD163 receptors expressed on the macrophages of the spleen, liver, bone marrow and kidneys. The Hb–Hp complex is endocytosed and processed intracellularly. Within macrophages, HO-1 mediates the degradation of heme into ferrous iron, carbon monoxide and biliverdin [18]. The iron is safely sequestered as ferric iron by ferritin while biliverdin undergoes further degradation to bilirubin.
Under normal physiological conditions, low levels of Hp–free Hb and heme/iron are metabolized by the kidney via increased expression of HO-1 and H-ferritin [18]. Excessive hemolysis in SCD patients may overwhelm endogenous plasma Hp and other scavenging mechanisms and heme degradation pathways. Acellular Hb is a highly reactive protein which undergoes oxidation to pro-inflammatory methemoglobin and ferryl hemoglobin [26], [31]. Moreover, the oxidized Hb species readily lose heme, a highly reactive molecule [3]. Acellular Hb is primarily cleared by the proximal tubules of the kidney via megalin and cubulin receptors [16]. Thus the kidneys of SCD patients are highly susceptible not only to Hb-induced toxicity but also to the deleterious effects of highly reactive heme. Excess amounts of Hb and its degradation products such as heme/iron are implicated in the pathogenesis of SCD [28], [33]. The renal manifestations of SCD patients include hematuria, tubular abnormalities, microalbuminuria and sometimes chronic kidney disease [27], [28].
Understanding the mechanisms of Hb-induced toxicity may unravel new therapeutic avenues against hemolytic diseases in general and SCD in particular. For example, our recent study revealed that Toll-like receptor (TLR4) antagonists inhibit vaso-occlusion in a model of SCD [6]. Similarly, overexpression of HO-1 reduced hypoxia-reoxygenation induced stasis [7]. Endogenous Hb/heme scavenging proteins are increasingly being investigated for their roles in ameliorating Hb/heme-induced toxicities [30]. Hp reduced acellular Hb-induced renal damage in multiple animal models predominantly by promoting Hb clearance and metabolism [2], [4], [8]. Moreover, recent in vitro and in vivo experiments indicated that Hp shields Hb from peroxidative modifications and consequent tissue damage [9]. We hypothesized that Hp may ameliorate Hb-induced toxicity by reducing heme overload in kidney by modulating HO-1 expression as part of a well-developed anti-inflammatory response.
Section snippets
Isolation of stroma free hemoglobin
Stroma-free human adult Hb (HbA) used for in vitro studies was isolated from whole blood as reported earlier [32]. The isolated Hb was further purified on Superdex 200 column to remove catalase. A spectral analysis was performed to ascertain the quality and the oxidation state of Hb solutions prior to using them in the experiments. Stroma-free human HbA used for in vivo studies was a generous gift from Sangart, Inc. (San Diego, CA).
Haptoglobin solutions
Highly purified Hp solutions were a kind gift from Bio Products
Results and discussion
Excess free Hb is primarily cleared by the proximal tubules of the kidney. Thus, they are highly prone to Hb-induced toxicity. We used a kidney proximal tubule cell line (HK-2) to investigate their responses following exposure to Hb. HK-2 cells were exposed to 50 μM Hb and 50 μM Hp for 4 or 24 h. For studying the role of Hp in Hb-induced injury, Hb and Hp were added (in equimolar ratio) separately to the cells. Hb induced HO-1 mRNA within 4 h whereas Hp completely attenuated Hb-induced HO-1 mRNA
Conclusions
The results indicate that Hp abrogates Hb-induced HO-1 expression in proximal renal tubule cells not only in vitro but also in vivo. The results also indicate the possibility of modulating Hb clearance by administration of exogenous Hp to prevent kidney damage in SCD patients.
Acknowledgments
The authors acknowledge Dr. Dominador J. Manalo and Janvi Raichiura for help with the preliminary experiments. We acknowledge Francine Wood for providing catalase-reduced and stroma free adult human hemoglobin. We also acknowledge Bio Products Laboratory (BPL; Hertfordshire, UK) for generously providing the human haptoglobin. AIA acknowledges the support from the National Institutes of Health (NIH) under grant HL110900 and the U.S. Food and Drug Administration (MODSCI Grants). GMV and JDB were
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