Changes in Energy Metabolism and Cell Damage in RPTEC‐TERT1 Cells Exposed to Hypoxia or Ischemia after Shear Stress Conditioning

Abstract

Study Objective Renal proximal tubules are exposed in vivo to physiological levels of shear stress. We aim to determine how preconditioning human proximal tubule cells with shear stress affects their sensibility to hypoxia or ischemia. Hypothesis Physiological levels of shear stress would induce further differentiation and metabolic specialization resulting in changes in their response to low oxygen or nutrient conditions. Methods Immortalized human proximal tubule cells (RPTEC-TERT1 from Evercyte) were grown to confluence in Ibidi µSlides and then exposed for three days to two flow regimes providing negligible (0.01 dyn/cm2) or physiological shear stress (0.3 dyn/cm2). Hypoxia or ischemic conditions were applied for 16h, by keeping cells under 1%O2 alone or together with glucose deprivation. Results RPTEC-TERT1 cells were first adapted to a culturing regime under 5 mM glucose culture medium, instead of 17 mM glucose present in the medium recommended by this cell line vendor. Proliferation rates and expression of phenotypic markers were unchanged. RPTEC-TERT1 cells switched from proliferative-glycolytic metabolism to non-proliferative-oxidative metabolism upon reaching confluency, approximately in 6-7 days. Seven days confluent, not-perfused RPTEC-TERT1 cells exposed to hypoxia or ischemia for 16 hours exhibited 58% and 75% mortality, respectively. In cells perfused for 3 days, preconditioning with shear stress reduced their sensitivity to hypoxia (mortality = 51% no-SS vs. 35% SS) and ischemia (mortality = 83% no-SS vs. 75% SS). Shear stress conditioning increased expression of genes related to energy metabolism and cells stress (AMPK alpha-2, HIF-1alpha, KIM-1) Conclusions Preconditioning of RPTEC-TERT1 by exposing them to physiological shear-stress modifies expression of genes related to mechanisms used to cope with cellular stress and this may explain the reduction in cell mortality when exposed to hypoxia or oxygen-glucose deprivation.