Summary Chronic kidney disease (CKD) is one of the most prominent conditions affecting approximately 15% of US adults, and most end-stage kidney disease patients have anemia of CKD. Although the causes of anemia of CKD are multifactorial, present clinical treatments could only partially reverse anemia status, indicating other mechanisms. Accumulating evidence suggests that anemia is also the result of accelerated eryptosis that shortens red blood cell (RBC) lifespan, contributing to the development and progression of anemia of CKD. We have demonstrated that the Na/K-ATPase (NKA) α1 subunit, by its interaction with c- Src under native resting condition, serves as a “receptor” for reactive oxygen species (ROS) and acts as a feed-forward ROS amplifier. In a pole-ligation 5/6th PNx mouse model, we found that PNx-induced uremic anemia was ameliorated by blocking NKA-Src signaling with a specific peptide (pNaKtide) but not with the induction of hemeoxygenase-1. pNaKtide, which was developed from the N domain of the NKA α1 subunit, binds to the α1-associated c-Src kinase domain to prevent c-Src phosphorylation, ultimately prevents the ROS amplification. Our preliminary in vivo data indicate that pNaKtide ameliorated PNx-induced anemia by blocking the increases in oxidative stress and eryptosis. However, the PNx procedure, with or without pNaKtide treatment, does not significantly affect plasma iron homeostasis. Our central hypothesis is that Accelerated eryptosis play a central role in PNx-induced anemia through stimulating NKA-Src signaling. Specifically, we hypothesize that PNx- mediated increases in cardiotonic steroids (CTS), uremic toxins, and oxidative stress stimulate the NKA-Src signaling, which further induces oxidative stress that leads to the development and progression of anemia. We further suggest that inhibition of the NKA-Src signaling could serve as a therapy for anemia in CKD. Aim 1 tests the hypothesis that PNx-stimulated NKA-Src signaling increases oxidative stress and stimulates eryptosis in vivo. We will define the role of adipocytes and the global NKA- Src signaling in the 5/6th PNx-induced anemia in C57/BL6 mice and global c-Src knock-out mice. Caveolin-1-null mice and NKA α1 knockdown mice will serve as an alternative. Aim 2 tests the hypothesis that the NKA-Src signaling stimulates oxidative stress and RBC eryptosis in isolated RBCs. We will use isolated RBCs (from C57/BL6 and global c-Src knock-out mice) to define the NKA-Src signaling complex in RBCs under native resting condition. Again, caveolin-1-null mice and NKA α1 knockdown mice will serve as an alternative. Validation of our hypothesis would further explain the development and progression of anemia under excessive oxidative stress such as hypertension, aging, obesity, and diabetes. In addition, a clearer understanding of the molecular mechanisms would have pathophysiological and therapeutic implications. Further, it also has the potential to provide the framework ...