Pyruvate kinase deficiency (PKD), caused by loss-of-function mutations in the PKLR gene, is a rare autosomal recessive disorder but is the most common red blood cell (RBC) enzymatic defect causing chronic hemolytic anemia. PKD is associated with extreme reticulocytosis that may exceed 50% and iron overload even in non-transfused patients, likely driven by reticulocyte-derived erythroferrone, which promotes intestinal iron absorption (PMID:27540014). PKLR encodes the erythroid- and liver-specific isoform of pyruvate kinase (PK), a key glycolytic enzyme producing ATP. Low ATP in PKD is considered as the cause of hemolysis. However, since other RBC low-ATP disorders do not cause hemolysis, ATP deficiency alone does not fully explain the molecular basis of PKD hemolysis.

Our previous studies of neocytolysis, the preferential destruction of hypoxia-induced young RBCs upon return to normoxia from hypoxia, demonstrated that decreased hypoxia-inducible factor (HIF) levels induce mitochondrial retention and decrease antioxidant catalase, leading to increased reactive oxygen species (ROS) in young RBCs (PMID:26017143). Based on this finding and a previous report of increased oxidative stress in PKD (PMID:34744776), we hypothesized that increased ROS due to mitochondrial retention in RBCs might contribute to PKD hemolysis.

As mitochondria are a major source of ROS generation, we measured mitochondrial mass levels in RBCs of 7 PKD patients, labeled by surface markers of RBC (CD71 and CD235a) and Mitotracker. Mitochondrial mass in both immature RBCs (CD71+/CD235a+) and late-stage RBCs (CD71-/CD235a+) was significantly (p=< 0.05)elevated in all PKD patients, suggesting the impairment of mitophagy in terminal erythropoiesis. Mitophagy-related genes (BNIP3L, ULK1, ATG5, and ATG7) were significantly decreased in PKD reticulocytes.

ROS levels in RBCs were significantly higher in CD71-/CD235a+ cells in PKD compared to controls. To evaluate possible impairment of the antioxidant scavenging system in PKD RBCs, we measured expression levels of antioxidant genes (CAT, SOD1, SOD2, HMOX1, PRDX2, TXNRD, CYBB, and GSR) in PKD reticulocytes. CYBB (NADPH oxidase 2) and GSR (glutathione reductase)transcript levels were significantly reduced in PKD reticulocytes, demonstrating a selective impairment in redox defense in PKD that further contributes to increased oxidative stress.

We used end-tidal carbon monoxide (ETCOc), a validated marker of RBC turnover (PMID:25624169), to quantitate the rate of hemolysis in five PKD patients by quantifying the amount of exhaled carbon monoxide produced by catabolized heme. ETCOc levels ranged from 5.8 to 11.6 ppm (normal range: 0.4–0.8 ppm), indicating severe hemolysis, and which positively correlated with bilirubin and lactate dehydrogenase (LDH), classic markers of hemolysis (r=0.8829, p=0.0151; r=0.9429, p=0.0167, respectively).

We investigated whether HIF might be the upstream regulator of these alterations, given our previous finding that mitophagy- and antioxidant-related genes are regulated by HIF (PMID:26017143). Expression levels of HIF-regulated genes (VEGFA, LDHA, EDN1), were significantly lower in PKD compared to controls and showed significant positive correlations with mitophagy-related genes as well as antioxidant CYBB and GSR transcripts, indicating that reduced HIF transcriptional activity decreases their expression, leading to mitochondrial retention, impaired antioxidant defense, ROS accumulation, and ultimately chronic hemolysis.

Our findings suggest that chronic hemolysis in PKD is accompanied by impaired mitophagy and redox imbalance due to suppressed HIF signaling. We observed mitochondrial retention and increased ROS in PKD erythroid cells, associated with downregulation of mitophagy-related genes and antioxidant genes. These molecular changes were associated with decreased expression of HIF target genes, suggesting reduced HIF transcriptional activity. Since HIF is negatively regulated by iron (PMID: 17557118), we propose that iron overload in PKD may contribute to HIF suppression, which in turn promotes mitochondrial retention and oxidative stress, ultimately leading to hemolysis. Our study implicates a novel HIF–mitophagy–redox axis in PKD-related extreme reticulocytosis and hemolysis and highlights the potential of targeting this pathway for therapeutic intervention, such as with vitamin C or other antioxidant administration (Valentine et al, 2025, Blood, Red Cells & Iron).

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2025
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