Kidney disorders comprising acute kidney injury (AKI), chronic kidney disease (CKD) and end-stage renal disease (ESRD) account for significant morbidity and mortality in sickle cell disease (SCD). AKI, a potent risk factor for CKD and ESRD, develops primarily in SCD patients hospitalized with vasoocclusive pain crisis (VOC) or acute chest syndrome (ACS). These characteristic SCD events are associated with rapid drop in hemoglobin implying acute intravascular hemolysis releasing free circulating heme as a potential trigger for AKI. However, the precise mechanisms of this association have not been investigated per se, and therefore targeted therapies based on mechanistic models have not emerged for kidney injuries in SCD. Excess circulating heme is primarily scavenged by hemopexin (Hx) and delivered to liver for degradation by heme oxygenase-1 (HO-1). Due to chronic hemolysis, Hx is depleted in SCD. We reasoned that during acute intravascular hemolysis in SCD, excess extracellular heme will preferentially bind to alpha-1-microglobulin (A1M), a secondary plasma heme scavenger, which carries free heme to the kidneys. Consequently, renal proximal tubular epithelial cells (RPTECs) will be exposed to high amount of toxic heme. Induction of intracellular HO-1 normally protects RPTECs from heme toxicity and averts AKI. We have recently discovered that both patients and mice with SCD have elevated plasma A1M compare to normal controls. This discovery leads to the development of a clinically relevant model of AKI in humanized sickle mice by modest elevation of circulating heme through intravenous injection of purified heme (hemin). Pilot data suggests that SCD patients with higher A1M/Hx ratio posses the risk of developing AKI following VOC. Heme suppresses hepatocyte nuclear factor 4 alpha (HNF4a) expression associated with reduced hemopexin expression in liver following acute hemolysis. Preliminary data also showed that persistent exposure to excess heme renders RPTECs refractory to HO-1 induction during acute hemolysis in SCD. Moreover, we found that heme induces kruppel-like factor 9 (KLF9) associated with amplification of mitochondrial ROS (mtROS) that triggers renal tubular epithelial cell death. Based on these data we hypothesized that enhanced clearance of circulating heme to the kidneys and impaired induction of HO-1 in the renal tubular epithelium during intravascular hemolysis in SCD trigger tubular cell death and AKI development. We will test this hypothesis with three specific aims that integrate experiments with cultured and primary human RPTECs, murine models and clinical biorepository samples including serum, plasma and urine from multiple cohorts of SCD patients.
Aim 1 will determine whether altered concentration of circulating heme scavenger proteins, can serve as risk factor for AKI in individuals with SCD.
This aim will also determine if multiple hemolytic events develop CKD.
Aim 2 will test the hypothesis that heme regulates the biosynthesis of Hx by down-regulating the expression of HNF4a.
Aim 3 will utilize human RPTECs and specific gene knockout mouse strains to determine if heme induced KLF9 amplification accelerates cell death that involves overproduction of mtROS.
This aim will use targeted HO-1 knockout or overexpression mice to determine whether amplified KLF9 blocks sufficient HO-1 induction and promotes heme induced AKI in SCD. This study will delineate the cellular and molecular pathogenesis of excess circulating heme mediated AKI in SCD during intravascular hemolysis, and identify potential therapeutic targets. This project will also elucidate a novel mechanism of heme-induced KLF9 mediated renal tubular epithelial cell death. Most importantly, rigorous analysis of clinical samples collected at baseline, during hospitalizations or following AKI incidences will establish whether A1M and Hx can serve as risk factors for AKI in SCD patients.

Public Health Relevance

Kidney disorders including acute kidney injury (AKI), chronic kidney disease (CKD) and end-stage renal diseases (ESRD) are prevalent in sickle cell disease (SCD) and account for significant morbidity and mortality in this patient population. Incidences of AKI, compelling risk factors for CKD and ESRD, are major clinical concerns among SCD patients hospitalized for acute hemolytic crisis. Enhanced clearance of excess circulating heme to the kidneys during hemolysis promotes clinically relevant AKI in murine model of SCD. The role of altered heme scavenging system, clearing free circulating heme, in the pathogenesis of AKI is unknown. This project will determine i) the association of two heme scavengers, hemopexin and alpha-1-microglobulin, with AKI in SCD patients, ii) the mechanism by which heme regulates the biosynthesis of heme scavengers, and iii) the putative pathway that impairs heme degradation by heme oxygensase-1 in SCD kidneys. This proposal will determine if ratio of plasma heme scavengers? concentration serves as a prognostic risk factor for AKI in SCD. The results of this proposal will also identify specific intermediate therapeutic targets to block AKI pathogenesis in order to improve adverse outcomes of kidney injuries in SCD.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Pathobiology of Kidney Disease Study Section (PBKD)
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Sadusky, Anna Burkart
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University of Pittsburgh
Internal Medicine/Medicine
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United States
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