Heme Induced Progressive Loss of Endothelial Protein C Receptor Promotes Development of Chronic Kidney Disease in Sickle Cell Disorders

Abstract

Chronic kidney disease (CKD), widespread among the individuals with sickle cell disease (SCD), is a major contributor to early death in this patient population. The progressive deterioration of renal health in SCD is associated with chronic persistent intravascular hemolysis leading to anemia. We have previously reported that extracellular heme, released during acute intravascular hemolysis triggers clinically relevant acute kidney injury (AKI) in SCD mice (SS) (Blood (2020) 135 (13): 1044-1048). Although AKI is reversible, it is considered as a risk factor for CKD. The mechanistic approach elucidating the hemolysis driven pathogenesis of AKI-to-CKD transition in SCD is unknown. We found that CKD develops in the SS mice by progressive (1-10 months of age) increase in albuminuria (urinary albumin and creatinine ratio, uACR) and decrease in glomerular filtration rate (GFR) (n=5; p<0.001). Histopathology of the kidney showed age-dependent deterioration in renal peritubular vascular congestion in SS mice compared to that of the AA mice. Alongside, Evan's blue extravasation experiments showed that the SS mice are susceptible to vascular leakage that is correlated positively with age (Pearson r=0.98, p<0.001) and negatively with anemia (Pearson r= -0.46, p<0.05). We hypothesized that multiple acute hemolytic events may instigate persistent endothelial damage that ensues CKD development in SCD. To test this hypothesis, we intravenously infused vehicle or heme (14 μmoles/kg body weight; 5 times on alternate days) to 1-month old SS mice and monitored for 3 weeks following first heme injection. These mice developed severe albuminuria (n=5; p<0.01) with substantial loss of GFR (n=5; p<0.001), indicating heme induced CKD development. Next, we used ultrasound super-resolution (USR) imaging technology to determine renal microvascular changes in older SS mice (6-months) without heme challenge and in young SS mice (1-month) challenged with heme. Analysis of the USR data showed reduced renal blood volume (rBV) and substantial loss of vessel density in renal cortex as well as in corticomedullary areas of the older SS mice compared to the age-matched AA mice. Accordingly, multiple heme challenge reduced rBV and vessel density extensively in young SS mice comparable to the older SS mice without heme challenge. Since endothelial protein C receptor (EPCR) maintains vascular barrier integrity by activating protease activated receptor-1 (PAR1) signaling in the endothelium, we tested whether alterations in EPCR expression contribute to progressive endothelial damage in SS mice during CKD development. Using immunofluorescence microscopy, we determined that renal endothelium lacks expression of EPCR in older SS mice while younger SS mice retains EPCR cellular expression. In corroboration, infusion of heme in younger SS mice results in loss of renal endothelial EPCR. Shedding of EPCR from endothelium often results in a soluble form of EPCR (sEPCR). We found that SS mice had higher plasma levels of soluble EPCR (sEPCR) compared to their AA counterparts. While age dependent increase in plasma and urinary sEPCR were evident in SS mice (n=6; p<0.01), infusion of heme in younger SS mice results in significant increase in plasma sEPCR (n=6; p<0.01). In consistent with the mouse data, we discovered that the plasma sEPCR was significantly elevated in SCD patients compared to normal individuals (n=8-16; p<0.05). Moreover, the plasma sEPCR was significantly associated with the baseline albuminuria in a cohort of SCD patients (n=16; Pearson r=0.64; p<0.01). This study supports the conclusion that multiple hemolytic events may trigger CKD development in SCD by gradual loss of renal microvascular EPCR expression. Clinically, the sEPCR can be developed as risk factor for sickle CKD. Finally, our data suggest that restoration of EPCR function may protect SCD patients from CKD. Disclosures No relevant conflicts of interest to declare.

The APC-EPCR-PAR1 axis in sickle cell disease

“…Interestingly, EPCR shedding has been observed in individuals and mice with SCD (100,101), and EPCR-positive microparticles are found in the circulation of individuals with SCD (90). A recent abstract described loss of EPCR expression in the kidney vasculature and presence of soluble EPCR in the urine of aged sickle mice, a phenomenon that could also be triggered in young sickle mice by infusion of a low dose of heme to mimic an acute sickling event (102). Together, these observations describe dysfunction in regulation of the vascular endothelium.…”