Accelerated suicidal erythrocyte death in Klotho-deficient mice

Klotho, a membrane protein mainly expressed in parathyroid glands, kidney, and choroid plexus, counteracts aging and increases the life span. Accordingly, life span is significantly shorter in Klotho-deficient mice (klotho ^-/- ) than in their wild-type littermates (klotho ^+/+ ). The pleotropic effects of Klotho include inhibition of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) formation. Vitamin D-deficient diet reverses the shortening of life span in klotho ^-/- mice. In a variety of cells, 1,25(OH)₂D₃ stimulates Ca²⁺ entry. In erythrocytes, increased Ca²⁺ entry stimulates suicidal erythrocyte death, which is characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. The present study explored the putative impact of Klotho on eryptosis. According to Fluo3 fluorescence, cytosolic Ca²⁺ concentration was significantly larger in klotho ^-/- erythrocytes as compared to klotho ^+/+ erythrocytes. According to annexin V-binding, phosphatidylserine exposure was significantly enhanced, and according to forward scatter, cell volume significantly decreased in klotho ^-/- erythrocytes as compared to klotho ^+/+ erythrocytes. Energy depletion (13 h glucose depletion) and oxidative stress (35 min 1 mM tert-butyl-hydroxyl-peroxide [tert-BOOH]) increased phosphatidylserine exposure to values again significantly larger in klotho ^-/- erythrocytes as compared to klotho ^+/+ erythrocytes. Reticulocyte number was significantly increased in klotho ^-/- mice, pointing to enhanced erythrocyte turnover. Vitamin D-deficient diet reversed the enhanced Ca²⁺ entry and annexin V-binding of klotho ^-/- erythrocytes. The present observations reveal a novel function of Klotho, i.e., the at least partially vitamin D-dependent regulation of cytosolic Ca²⁺ activity in and suicidal death of erythrocytes.