Reduced Flippase Activity in the Early Life of Mature Erythrocytes Is Associated with Their Shortened Lifespan in Renal Anemia

Background: The lifespan of mature erythrocytes in renal anemia patients is shortened (approximately 70 days) compared to that of healthy individuals (120 days), but its pathogenesis is unclear. Previously, we reported that in healthy senescent erythrocytes decreased intracellular K+ and ATP levels, and flippase molecule ATP11C, lead to decreased flippase activity, resulting in increased phosphatidylserine (PS) exposure, a removal sign for macrophages (Seki M, 2020). Our recent investigations have revealed that senescent erythrocytes from renal anemia patients also exhibited decreased flippase activity, leading to PS exposure, mainly due to K+ loss. This study demonstrated that senescent erythrocytes in patients were younger than the senescent erythrocytes in healthy individuals. We additionally conducted a quantitative proteome analysis of the membrane proteins obtained from young and senescent erythrocytes of patients to identify candidates involved in K+ loss. Methods: Erythrocytes from patients undergoing maintenance hemodialysis and from healthy volunteers were fractionated into young (light) and senescent (heavy) populations using a Percoll-density gradient according to our previous report. HbA1c was measured in fractionated erythrocytes as a marker of their age. We conducted a quantitative MS/MS using the Tandem Mass Tagging labeling system on membrane proteins from young and senescent erythrocytes of renal anemia patients (n=3). Results: Although the number of senescent erythrocytes in patients bore a similarity to that of healthy humans, HbA1c levels in the heavy fractions of patients were lower than levels in healthy individuals. The proteomic analysis of patients’ membranes presented oxidation-modified Transient Receptor Potential Melastatin 2 (TRPM2), an oxidation-sensitive calcium channel, and a sufficient amount of Piezo1, a mechanosensitive calcium channel. Both are potent contributors to activate calcium-dependent K+ leakage (Gardos effect). Conclusion: In the early life of mature erythrocytes in renal anemia patients, reduced flippase activity causes PS exposure due to K+ loss. We identified two responsible candidates for K+ leakage, TRPM2 and Piezo1, which could serve as novel therapeutic targets for renal anemia. Funding: Other NIH Support - Japan society for the promotion of science