Targeted Induction of Hepatocyte Nuclear Factor 4α Protects Acute Kidney Injury By Upregulating Hemopexin Biosynthesis during Acute Hemolytic Events in Sickle Mice
Authors: Mondal Paritosh , Lenhart Shane C , Crosby Danielle , Lenhart Diane , Ghosh Samit 
Published: December 2024

Intravascular hemolysis, pertinent to sickle cell disease (SCD), raises extracellular heme in circulation. Multiorgan damage in SCD including acute kidney injuries (AKI) is attributed to the elevated circulating heme. Hemopexin (HPx), the primary heme scavenger, transport heme to the liver for its degradation. In SCD, intrinsic depletion of HPx results in excess heme accumulation to the kidneys leading to AKI. While hemopexin supplementation is emerging as a potential therapeutic strategy for SCD complications, the mechanism of HPx biosynthesis and its regulation by heme has not been reported. Hepatocyte nuclear factor 4 alpha (HNF4α) is a master transcription factor regulating the expression of several protein in the liver. We discovered reduced hepatic expression of HNF4α associated with low expression of HPx in preclinical SCD mice (SS), homozygous for human sickle hemoglobin S (HbSS) compared to the control AA mice, homozygous for human normal hemoglobin (HbAA). We hypothesized that extracellular heme downregulates HPx biosynthesis by repressing HNF4α in SCD. First, we tested the effect of purified ferric heme (hemin) on human hepatocyte cells (HepG2) in vitro. Extracellular heme significantly reduced the expression of HNF4α (n=4 p=0.0252) and suppressed HPx at both transcriptional and translational level in HepG2 cells (n=6 p=0.0053). The loss of HPx in HepG2 cells was corroborated with substantial reduction of HPx released into the cell supernatant. Using siRNA and an antagonist (BI6015) to HNF4α, we found that inhibition of HNF4α significantly downregulated HPx expression (n=4; p=0.0026). Alongside, we identified that heme instigates the phosphorylation of HNF4α, and thereby inhibits its binding to the HPx promoter. To test whether HNF4α is critical for HPx synthesis in hepatocytes, we generated a targeted HNF4αΔH mouse strain deficient in hepatocytic HNF4α by breeding HNF4afloxed mice with AlbCre mice. The HNF4αΔH mice invariably demonstrated marked reduction in hepatic and plasma HPx compared to the wild type mice. Next, we infused the SS mice with ferric heme (20 mmoles/kg) to trigger AKI (PMID: 32043112). The elevated circulating heme inhibits the expression of HNF4α (n=6; p&lt;0.05) and HPx in the liver, while significantly reduces plasma HPx level (n=9; p&lt;0.01) in the SS mice compared to their baseline. Oroxylin A (OA), a flavonoid compound, activates HNF4α. We found that OA prophylaxis (20 mg/kg) to the SS mice (n=9) results in the induction of HNF4α and HPx in the liver associated with increased plasma HPx compared to the SS mice treated with placebo. Moreover, histopathological and biochemical experimentation in the kidneys showed that OA prophylaxis protected the SS mice from developing heme induced AKI. Overall, these data suggest that heme regulates HPx biosynthesis by modulating HNF4α expression in the hepatocytes. Induction of HNF4α may be developed as a therapeutic strategy to enhance the bioavailability of the endogenous HPx protecting kidney injuries and other complications during acute hemolytic events in SCD.