Three Months of Human Haptoglobin Treatment Decreases Iron Deposition in the Kidneys of Townes Sickle Mice
Authors: Shi Patricia A , Choi Erika , Nguyen Julia , Guo Xinhua , Mohandas Narla , Alayash Abdu , Manci Elizabeth , Belcher John D. , Vercellotti Gregory M 
Published: October 2019

Abstract
Introduction: Haptoglobin (Hp), the scavenger for hemoglobin, and hemopexin (Hx), the scavenger for heme, are depleted in most patients with sickle cell disease due to chronic hemolysis. There is mounting evidence of the crucial role of free hemoglobin and/or free heme in mediating inflammatory and oxidative damage in sickle cell disease, including vaso-occlusion and acute chest syndrome. Purified Hp has been used in Japan for a variety of hemolytic conditions and has been proposed as a potential treatment for sickle cell disease. Although infusions of Hp or Hx have been shown to ameliorate vaso-occlusion, acute lung injury, and heme toxicity in sickle cell mouse models, no prior studies have examined the utility of chronic Hp treatment for amelioration of organ damage. We therefore studied the effect of 3 months of chronic Hp treatment in the Townes sickle mouse model.
Methods: Male and female Townes mice (Stock number 013071, The Jackson Laboratory) were used for all experiments, starting at 1 or 3 months of age. SS genotype was confirmed by PCR and HPLC. Organ damage in the spleen, liver, and kidneys as previously described was confirmed. Human Hp solution was a kind gift from Bio Product Laboratory (BPL, Hertfordshire, UK). Hp or equivalent volume PBS control was administered intraperitoneally (IP) in the first cohort of 5 mice and then subcutaneously (SC) in the next two cohorts of 7 and 12 mice on a 48-72 hr dosing schedule of Monday, Wednesday, Friday for a period of 3 months. At the end of 3 months treatment, mice were evaluated by the following studies (with concurrent blinding to treatment group for most studies): plasma Hp (ELISA), plasma heme (QuantiChrom heme assay), urine osmolality (osmometer), urine albumin (ELISA), CBC (Advia 120), WBC differential (Advia 120 and manual count), red blood cell ektacyometry (ektacyometer), organ mass (percent of body weight), and organ histology.
Results: Mouse Hp levels in SS Townes mice were confirmed to be markedly low compared to Townes AA mice (mean ± SD: SS 2 ± 1 versus AA 39 ± 4 ug/mL). Dose-finding experiments determined that a dose of 200-400 mg/kg IP or SC in SS mice resulted in a 24 hr peak concentration that was 5-14X supraphysiologic, variably physiologic at 48 hr, and absent or almost absent at 72 hr. Chronic dosing at the 400 mg/kg IP in SA mice showed no CBC or organ toxicity. Three successive cohorts of SS mice were treated with Hp (or equivalent volume of PBS): 200 mg/kg IP in 3-month old mice, 400 mg/kg SC in 3-month old mice, and 400 mg/kg SC in 1-month old mice. At the 400 mg/kg dosing levels, there was a significant decrease in iron deposition in the kidneys of both 4-month and 6-month old mice (treatment started at 1-month and 3 months, respectively) (Table 1). There was also a trend towards decreased liver infarction in 6-month old mice (Table 2).
Discussion: Functional binding of the administered human Hp to the human Hb of the Townes mice likely occurred, as evidenced by the decrease in iron deposition in the kidneys, suggesting that formation of the complex prevents filtration of Hb into the kidneys. Surprisingly, kidney function as measured by urine osmolality or albumin excretion was not improved, which may be explained by continued heme-laden red cell microparticle filtration (Camus SM, Blood 2015). Encouragingly, however, a trend towards decreased liver infarction in older mice was observed. The less-than-expected effect of Hp on mouse disease severity may also be explained by: 1) continuous physiologic Hp concentrations not being maintained with the dosing frequency while continued hemolysis releases Hb every minute of the day, and 2) CD163-mediated uptake in mice seems to only account for a part of the Hb clearance as opposed to in humans (Etzerodt A, Antioxid Redox Signal 2013). Despite the limitations of the SCD mouse model, the current study suggests haptogobin infusions could be beneficial in SCD patients.
Acknowledgment: The authors are grateful to Sandra Suzuka for performing the HPLC.
Table 1. Table 1. Kidney iron deposition (scale 1-10) Treatment group 6-month old SS 4-month old SS 400 mg/kg Hp 4.0 ± 1.4 1 ± 1.1 PBS 9.3 ± 0.6 5 ± 2.9 p-value 0.002 0.02
Table 2. Liver infarction (scale 1-10) Treatment group 6-month old SS 4-month old SS 400 mg/kg Hp 2.6 ± 2.0 3.7 ± 2.8 PBS 6.3 ± 2.4 3.8 ± 2.3 p-value 0.07 0.91

Disclosures
Belcher: Biogen Idec: Research Funding; Seattle Genetics: Research Funding; CSL Behring: Research Funding. Vercellotti:CSL Behring: Research Funding; Seattle Genetics: Research Funding; Cydan: Research Funding; Biogen Idec: Research Funding.