Diminished Renal Pathology in a Mouse Model of Sickle Cell Anemia in Which Fibrinogen Binding to Mac-1 Is Inhibited
Authors: Nasimuzzaman Md , Arumugam Paritha I. , Mullins Eric S. , James Jeanne M , VandenHeuvel Katherine , Narciso Marilou , Shaw Maureen A. , McGraw Sarah , Aronow Bruce J. , Flick Matthew J. , Malik Punam 
Published: October 2019

Sickle cell anemia (SCA) is caused by a point mutation in the beta-globin gene. SCA has potentially devastating consequences including chronic hemolytic anemia, episodic vascular occlusion, inflammation and oxidative stress, and cumulative multi-organ damage resulting in early mortality. In fact, with reduction of childhood mortality due to early diagnosis and infection prophylaxis, end-organ damage is the major cause of death in SCA. SCA patients show hyper coagulative state in the absence of vascular occlusion. Recently, our group has shown that reduction of circulating major clotting factor, thrombin, in SCA mice significantly improves survival, reduces inflammation, results in reduced multi-organ damage and prolongs survival (Arumugam 2015). However, the mechanism(s) by which thrombin contributes to the pathophysiology of SCA remains undefined. While fibrinogen functions primarily to occlude blood vessels after cleavage by thrombin into fibrin, and thereby prevents excessive bleeding, it also plays an important role in the pathologic inflammatory disease processes through mitogenic, chemotactic, and immune-regulatory activities by interacting with neutrophils/ macrophages via the Mac-1 receptor. To test the hypothesis that leukocyte engagement of fibrinogen via Mac-1 and secondary inflammation are drivers of SCA-associated organ pathologies, hematopoietic stem cells (HSC) from the well-characterized humanized murine model of SCA, Berkeley sickle mice (HbS or SS) were transplanted into the Fibγ390-396A mice, the later expressing a mutant fibrinogen γ-chain which does not interact with Mac-1 (named as F1M SS). Fibγ390-396 mutation has no effect on clotting functions, supports platelet adhesion, and does not cause spontaneous hemorrhagic events in mice during development or adulthood (Flick 2004). As a control, normal fibrinogen expressing mice were also transplanted with HSC from sickle mice (named as F1WT SS). One year after HSC transplant, the F1M SS and F1WT SS mice were analyzed for blood parameters, reactive oxygen species (ROS), inflammatory cytokines, and organ functions and pathologies. We found that genetically imposed inhibition of fibrinogen binding to Mac-1 resulted in mild reduction (not statistically significant) of neutrophil, monocyte, and platelet counts. There was no significant difference in RBC parameters between F1WT SS and F1M SS mice. However, we found an impressive reduction in WBC ROS and decreased circulating inflammatory cytokines, IL-6 (79.1 ± 19.5 vs. 303.5 ± 73.9, P < 0.001), KC (132.7 ± 19.4 vs. 200.7 ± 33.6, P=0.04) and TNF-α (25.9 ± 2.8 vs. 37.7 ± 3.9, P=0.02) in F1M SS mice compared to F1WT SS mice. We also found significant improvement in SCA-associated glomerular pathologies such as reduced glomerulosclerosis, inflammatory cell infiltration, ischemic lesions, mesangial thickening, mesangial hyper cellularity and glomerular enlargement; associated with reduced albuminuria (123.3 ± 19.5 vs. 322.1 ± 91.4, P=0.03) in F1M SS mice compared to F1WT SS mice. However, tubular pathologies and urine osmolality were not improved in F1M SS mice. RNA-Seq analyses of glomerular preparation revealed improved glomerular protective responses and diminished loss of podocyte/mesangial cell signatures in F1M SS mice compared to F1WT SS mice. Interestingly, unlike the pan-organ improvement with thrombin reduction (Arumugam, 2015), fibrinogen binding to Mac-1 had minimal to no effect on cardiac, lung, and liver functions and pathologies. Taken together, our data support that fibrinogen significantly contributes to the WBC-driven inflammation and ROS production, and represents a key driver of SCA-associated glomerulopathy, and may provide a novel therapeutic target against irreversible kidney damage in SCA.

Disclosures

Mullins: Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees. Malik:CSL Behring: Patents & Royalties.