Red Blood Cell Hemolysis and Suppression of Erythropoiesis Strongly Upregulate mRNA for Iron Binding Lipocalin (NGAL/24p3) in the Liver.

A member of the lipocalin family of proteins, NGAL/24p3, was demonstrated to bind iron and was suggested to participate in a non-transferrin dependent iron transport mechanism capable to deliver iron to the cytoplasm (Kaplan 2002, Yang et al. 2003). Therefore, we have studied mRNA for NGAL/24p3 levels in the liver tissue during its loading with iron released from senescent and damaged red blood cells. Red blood cell hemolysis was induced in mice by administration of phenylhydrazine (PHZ). Suppression of iron reutilization for erythropoiesis was achieved by a total body sublethal irradiation (5 Gy). Samples of liver tissue were collected 16 hrs or 48 hrs after PHZ and 40 hrs after irradiation. Combined treatment consisted from irradiation followed by PHZ administration 40 hrs later. The irradiation suppressed 24 hrs incorporation of ⁵⁹Fe into blood from 46,5 % in controls to 1.2 % in irradiated mice, indicating a significant suppression of erythropoiesis. PHZ administration alone decreased hematocrit from 44.7 % to 38.7 %, reflecting degree of the red blood cell hemolysis. The combined treatment by irradiation and PHZ resulted in the elevation of the liver iron content from 43.8 to 106.7 micrograms/g wet tissue 16 hours after PHZ, indicating a significant loading of the liver tissue with iron. All these treatments increased mRNA for NGAL/24p3 levels as determined by real-time PCR, significantly. After the combined treatment the increase reached almost three orders of magnitude. We further compared the response of NGAL/24p3 mRNA to the response of hepcidin and transferrin-1 receptor (TfR-1) mRNAs, both known to be sensitive to the liver iron content. Hepcidin mRNA increased significantly after the treatment with irradiation, PHZ, or combination of irradiation and PHZ but the increase was less pronounced compared to that of NGAL/24p3 mRNA. TfR-1 mRNA significantly decreased 48 hours after the combined treatment only. As an indicator of the acute phase response, the mRNA for interleukin-6 was determined and it did not change after the treatments used. The results demonstrate that mRNA for the putative iron transport molecule NGAL/24p3 was strongly upregulated by experimental maneuvers that lead to accumulation of iron in the liver tissue.