Haptoglobin Therapy Prevents Kidney Injury in Stored Blood Resuscitation of Murine Hemorrhagic Shock
Authors: Graw Jan A. , Rosales Ivy A. , Liu Yumin , Sabbisetti Venkata S. , Riley Frank E. , Rechester Osher , Bloch Kenneth D. , Warren H. Shaw , Bonventre Joseph V. , Zapol Warren M. 
Published: October 2019

Abstract
Introduction: Extracellular hemoglobin (Hb) and free heme are major breakdown products of hemolyzing Red Blood Cells (RBC). Mammals have evolved scavenger systems to bind circulating Hb with haptoglobin (Hp) and free heme with hemopexin (Hx).
During storage RBC undergo numerous morphological and biochemical changes. Transfusion of stored RBC (SRBC) increases plasma levels of heme and Hb. Increased plasma Hb levels are associated with kidney injury (KI). In prolonged hemorrhagic shock (HS) systemic hypotension and hypoperfusion impair kidney function. KI is an important complication of HS and is associated with a markedly increased mortality and morbidity rate.
In this study we employed a murine model of 14-day blood storage mimicking 42-day human blood storage. We first determined that HS-induced KI is more severe in mice resuscitated with SRBC than in those transfused fresh RBC (FRBC). Then we studied whether treatment with either Hp or Hx during transfusion reduced KI after SRBC-resuscitation from HS.
Methods: Leukoreduced, packed RBC obtained from WT C57BL6 mice were stored in CPDA-1 at 4°C for either <24 h (FRBC) or 2 weeks (SRBC). Anesthetized mice were bled approximately 50-60% of circulating blood volume over 10 min to maintain a constant mean arterial pressure of 35 mmHg and induce HS. After 120 min of HS, mice were resuscitated with either FRBC or SRBC. Sham operated mice that did not undergo hemorrhagic shock or resuscitation served as a control group. Furthermore, some mice receiving SRBC were given a co-infusion of 7.5 mg of either Hp, or Hx or albumin (Alb) during transfusion. The Alb treated group of mice served as a control for the treatment protein loading. After recovery from anesthesia, urine was collected for 24 h in a metabolic chamber. Urine creatinine, hemoglobin, and Kidney Injury Molecule-1 (KIM-1) were measured. At 48 h post HS the mice were sacrificed and plasma markers of liver and kidney injury were measured. Kidney injury was quantified by a kidney pathology scoring system.
Results: After HS and resuscitation hemoglobinuria was detected in 24 h urine samples collected from mice resuscitated with SRBC (see Table). Urine KIM-1 levels were increased after HS and resuscitation with either FRBC or SRBC (FRBC differs vs. Sham, p<0.01; SRBC differs vs. Sham, p<0.05). Plasma NGAL-levels and the kidney tubular injury score were greater in SRBC-transfused mice than in Sham operated mice or FRBC-resuscitated mice (KI score: SRBC differs vs. Sham and FRBC, p<0.01). Hemoglobinuria was present in SRBC-resuscitated mice treated with Alb or Hx but not SRBC transfused mice treated with Hp. Plasma NGAL levels at 48 h after shock did not differ in mice resuscitated with SRBC+Hp or FRBC but were greater in mice resuscitated with SRBC+Alb and SRBC+Hx. Plasma Hb levels at 48 h after resuscitation were less than 33.1±6.4 mg/ml and did not differ from Sham levels between all groups, but plasma Hb levels were 7-fold increased to 231.7±25.7 mg/ml in mice transfused with SRBC+Hp (SRBC+Hp differs vs. all other groups; p<0.001). Plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were greater after resuscitation with SRBC and with all three treatments than after resuscitation with FRBC.
Conclusion: In comparison to resuscitation from HS with FRBC, resuscitation with SRBC produced hemoglobinuria and greater KI. Treatment with exogenous Hp during transfusion but not treatment with Hx or Alb reduced SRBC-induced hemoglobinuria and prevented kidney injury after HS and resuscitation with SRBC. Treatment with exogenous Hp may prevent KI associated with high plasma Hb concentrations such as after massive transfusion of stored blood, prolonged cardiopulmonary bypass or in acute exacerbations of hemolytic disease.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.