Renal endothelium and the development of chronic kidney disease in sickle cell disorders

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PROJECT SUMMARY/ABSTRACT Chronic kidney disease (CKD), widespread among individuals with sickle cell disease (SCD), is a significant contributor to morbidity and early mortality. The progressive deterioration of renal health in SCD is associated with chronic intravascular hemolysis. However, the mechanistic approach to interpreting the hemolysis-driven pathogenesis of CKD in SCD is limited and therefore targeted therapies based on mechanistic models are lacking. Progressive loss of renal function, represented by increased albuminuria and reduced glomerular filtration rate (GFR), is associated with microvascular congestion in humans and mice with SCD. We postulated that circulating cell-free heme, a product of hemolysis, may instigate persistent endothelial damage that ensues CKD development. Endothelial protein C receptor (EPCR) maintains endothelial barrier integrity. We discovered an age-dependent as well as heme-induced loss of EPCR from renal microvascular endothelium in SCD (SS) mice. Concomitantly, the cleaved soluble form of EPCR (sEPCR) was elevated in the plasma associated with albuminuria. Using super-resolution ultrasound imaging, we found that younger SS mice challenged with the repetitive infusion of minimal doses of heme and the older SS mice without heme challenge incur substantial renal microvascular rarefaction. Heme induces endothelial P-selectin that promotes vascular congestion. Our pilot data show that reduced EPCR expression is associated with increased P-selectin on renal microvascular endothelium in the SS mice. Proteinase 3 (PR3), expressed on neutrophils, can degrade EPCR. In our repeated heme-challenged younger SS mice compared to vehicle-injected mice, we noticed increased expression of PR3 in plasma as well as in accumulated neutrophils from the kidneys. Moreover, we found that heme induces acetylation of histone 4 Lys 16 residues (H4K16ac) which are known to promote the biosynthesis of PR3. Based on these preliminary data, we hypothesize that heme-induced P-selectin and PR3 concurrently result in the loss of EPCR leading to endothelial disintegration, microvascular congestion, and CKD development in SCD. We will test this hypothesis with three specific aims that integrate experiments with human renal endothelial cells and neutrophils in vitro, murine models, and clinical samples from multiple cohorts of SCD patients. Aim 1 will determine that repeated low-dose heme insults promote renal endothelial P-selectin that suppresses EPCR leading to vascular damage and development of CKD in mice with SCD. Aim 2 will test whether heme- activated PR3 facilitates EPCR cleavage and expedite CKD in SCD. Aim 3 will determine whether sEPCR and specific genetic variants of EPCR can indicate the risk of CKD in SCD patients.