Upregulation of Renal Iron Metabolism in Sickle Cell Disease Mice
Authors: Vazquez-Meves Guelaguetza , Kumari Namita , Afangbedji Nowah , Khaibullina Alfia , Quezado Zena , Nekhai Sergei , Jerebtsova Marina 
Published: October 2019

Abstract
BACKGROUND: Hemolysis and frequent blood transfusions lead to the iron overload and organ iron accumulation in patients with red blood cells disorders. The pattern of iron accumulation within different organs is disease specific. Abnormalities of renal iron metabolism and cortical iron deposition is characteristic for sickle cell disease (SCD) but not for β-thalassemia. Renal iron deposition does not correlate with iron overload and blood transfusion. Iron is reabsorbed from primary urine in the renal proximal epithelial cells and released into the renal intersitium by ferroportin. Iron-regulating hormone, hepcidin controls ferroportin expression. Binding of hepcidin to the ferroportin induces ferroportin degradation and intracellular iron accumulation. Low concentrations of circulating hepcidin are common in SCD patients and do not explain paradoxical renal iron accumulation. SCD mice accumulate iron in the epithelial cells of proximal tubules and may be a suitable model to study iron metabolism in SCD.
OBJECTIVES: To characterize proteins of the renal iron metabolism in SCD mouse model.
METHODS: The SCD (Townes) mice do not express mouse α- or β-globin alleles, but carry two copies of a human α1-globin gene and two copies of a human Aγ-globin and βS-globin genes. These animals synthesize approximately 94% human sickle (HbS) and 6% human fetal hemoglobin (HbF), and no murine hemoglobin. Control animals carry two copies of the human α1-globin gene and two copies of the human hemoglobin gamma (Aγ) gene and the human wildtype hemoglobin beta (βA) gene. Kidneys were collected from 5 months old SCD and control mice. Renal cortex was used for RNA and protein isolation. Levels of renal hepcidin, ferroportin, transferrin receptor (TFR1), divalent cation receptor (DMT1), ferritin and hepheastin were determined by q-RT-PCR, WB and ELISA. Paraffin-embedded sections were used for immunostaining. Perl's Prussian blue staining was used for detection of renal iron accumulation.
RESULTS:We detected significant accumulation of iron in the epithelial cells of proximal tubules in SCD mice. Expression of renal hepcidin was increased in SCD mice compared to controls. Surprisingly mRNA levels of all other proteins involved in renal iron metabolism (ferroportin, TFR1, DMT1, ferritin and hephaestin) were decreased in SCD mice kidney. In contrast, we found increased protein levels of transferrin receptor (iron importer), ferritin (iron storage protein) and slightly increased level of ferroportin (iron exporter). We also observed significant renal macrophages infiltration in SCD mice.
CONCLUSIONS: Increased levels of renal hepcidin expression in SCD mice may be associated with renal inflammation. Higher levels of locally expressed hepcidin may lead to the partial degradation of the iron exporter (ferroportin). Increased levels of iron importers (TFR1 and DMT1) and no significant change in ferroportin expression can cumulatively saturate iron storage in ferritin and lead to the accumulation of intracellular iron.
ACKNOWLEDGMENTS: This work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005 and 5G12MD007597. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIMHD or NIH.

Disclosures
Quezado: IONIS Pharmaceuticals: Research Funding.