ApoHemoglobin-Haptoglobin protects from hemoglobin induced nephropathy

Abstract

Hemolysis, the process by which red blood cell membranes rupture, releasing hemoglobin (Hb), is typically managed in mammalian organisms by intrinsic proteins such as hemopexin (Hpx) and haptoglobin (Hp). Their presence in response to circulating heme and hemoglobin within the bloodstream aids in mitigating the potential adverse effects of minor hemolysis. However, their concentrations in the plasma are very limited, and, in instances of severe hemolysis, they are unable to protect against multi-organ toxicity due to the Hb and heme leakage and the generation of reactive oxygen species, resulting in cellular and tissue damage. In response to this challenge, our research group has developed a novel dual scavenger protein named ApoHemoglobin-Haptoglobin (ApoHb-Hp). This engineered protein is a protective mechanism by binding to free heme and Hb, thereby preventing tissue damage induced by Hb and heme. Previous studies have shown that ApoHb-Hp can reduce microvascular constriction and diminish the levels of inflammatory cytokines in critical organs, highlighting its potential as a therapeutic candidate. To further investigate the protective function of ApoHb-Hp, we focused on podocytes, specialized cells enveloping the capillaries of glomeruli and crucial in establishing the filtration barrier within the kidney. The dimerization of hemoglobin often disrupts this filtration barrier, causing detrimental effects on the glomerulus and its podocytes. In our study, we cultured podocytes in various environments, including a control group with human serum albumin, a group with hemoglobin, a group with ApoHb-Hp, and a combination group with both ApoHb-Hp and hemoglobin. The results demonstrated a significant reduction in the production of reactive oxygen species (ROS) in the ApoHb-Hp with Hb group, 264.6 AU ± 21.9, compared to the group exposed to Hb alone, 468.2 AU ± 50.2, illustrating the protective role of ApoHb-Hp. A decrease in ROS was consistent with percentage of cell viability, showing a significant decrease in cell viability for the Hb group, 55.1% ± 8.2, compared to the ApoHb-Hp group with Hb, 77.4% ± 9.8. Moreover, the percent of positive nephrin cells, the protein in charge of creating the filtration barrier in between podocyte processes, seem to be significantly decreased in the Hb group, 18.3% ± 4.5, compared to the ApoHb-Hp with Hb group, 29.6% ± 3.7. Lastly, there was an observed increase in the production of heme oxygenase 1 and nuclear factor erythroid 2 in the ApoHb-Hp with Hb group, which are crucial proteins in shielding cells against oxidative stress and toxicity. Both the control group and the ApoHb-Hp group displayed no significant differences in these results, underscoring the therapeutic's biocompatibility. This study highlights the toxic characteristics of Hb in kidney cells. It demonstrates how our ApoHb-Hp therapy may act as a protective mechanism in severe hemolysis, mitigating kidney injury by preserving cellular integrity and functionality. Title of the project: Bioengineering a Dual Function Protein Construct to Detoxify Heme and HemoglobinProject number: R01HL159862Title of the project: Bioengineering a novel therapeutic protein complex to minimize the effects of medical device induced hemolysis.Project number: R01HL162120. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.