Targeting p300-mediated histone acetylation inhibits neutrophilic proteinase 3 and prevents the development of sickle chronic kidney disease Free

Chronic kidney disease (CKD) is a prevalent condition among sickle cell disease (SCD) patients and serves as a strong predictor of premature death in this population. Acute and chronic intravascular hemolysis releasing free hemoglobin and heme in circulation contributes to the development of CKD. Neutrophils are implicated in multiple complications, including inflammation and vasoocclusion in SCD. Heme-induced neutrophil activation contributes to the pathogenesis of multiorgan damage in SCD via complement activation and neutrophil extracellular trap formation. We have reported that repeated mild hemolytic events promote renovascular rarefaction by cleaving endothelial protein C receptor (EPCR) and trigger CKD development in SCD mice (SS) (Blood, 2024; PMID: 38820589). However, the mechanism underlying heme-induced EPCR loss remains unclear. Transcriptomic analysis of renal RNA sequencing revealed significant enrichment of leukocyte extravasation and neutrophil degranulation in the SS mice kidneys following heme-induced CKD. Activated neutrophils release proteinase-3 (PR3), a serine protease stored in neutrophil granules that is capable of cleaving EPCR. We discovered significant elevation of plasma PR3 in sickle patients compared to normal individuals (n=14; p<0.001) and in sickle mice with CKD (n=6; p<0.01). We hypothesized that heme-induced neutrophilic PR3 cleaves EPCR on renovascular endothelium and promotes CKD development. Using western blot, immunohistochemistry and flow cytometry, we identified substantial accumulation of neutrophils in SS mice kidneys during heme-induced CKD compared to vehicle-injected SS mice. Flow cytometry analysis of neutrophils and endothelial cells isolated from vehicle- and heme-treated SS mice showed a significant induction of neutrophilic PR3 (n=3; p<0.01) and a concomitant reduction in EPCR expression (n=3; p<0.001). To test direct activation of neutrophilic PR3 by heme, we challenged HL-60 cells, the human promyeloid neutrophil-like cells, with exogenous heme (20 µM). The PR3 expression was substantially elevated. Similarly, human renal endothelial cells (HRGEC) challenged with purified PR3 showed significant loss of EPCR. In a transwell coculture system with HL-60 and HRGEC, heme challenge exhibited significant induction of PR3 on HL-60 cell with concurrent loss of EPCR from HRGEC. Acetylation of histone (H4K16Ac) is known to be responsible for regulating PR3 expression in the neutrophils. Using gene-enrichment analysis of RNA sequencing data, heme-challenged HL-60 cells and heme-induced SS mice kidneys, we identified that heme-induced p300, a histone acetyltransferase, to stimulate H4K16Ac. We tested the efficacy of garcinol, a p300 inhibitor, in protecting SS mice from CKD development by blocking neutrophilic PR3. Pretreatment of SS mice with garcinol (i.p. 10 mg/kg) prior to heme-induced CKD, significantly reduced PR3 expression on the accumulated renal neutrophils, while enhancing the renovascular EPCR expression (n=3; p<0.01). Histopathological and biochemical analysis confirmed that garcinol attenuated the development of CKD in SS mice following heme challenge (n=6). In summary, this study demonstrates that heme-induced PR3-positive neutrophils accumulate in the kidneys, facilitating the cleavage of EPCR and consequently inducing CKD in SCD. The inhibition of PR3 using garcinol serves as a proof-of-concept for preventing CKD development in SCD.