Benefice of the stimulation of O-GlcNAcylation on sodium potassium pump during hemorrhagic shock

Abstract

Hemorrhagic shock (HS) is the consequence of an important loss in blood volume leading to an insufficient delivery in oxygen. HS causes metabolic changes, multi-organ dysfunction and inflammation. The acute kidney injury related to HS is associated with hyperkalemia mainly through an alteration of Na/K ATPase pump. In a previous O-GlcNAcylomic study, we have identified Na/K ATPase as potentially O-GlcNAcylated protein. O-GlcNAcylation is a post-translational modification with a putative beneficial effect in shock situation. This study aims at evaluating if Na/K ATPase is O-GlcNAcylated and its potential impact during the early phase of hemorrhagic shock. Wistar rats were subjected to a HS protocol induced via exsanguination and then randomly treated or not with NButGT (10 mg/kg), a O-GlcNAcase inhibitor, to increase O-GlcNAc levels. Blood pressure and heart rate were monitored throughout the experiment. Blood was collected from the animals to perform a complete blood count and a blood gas analysis. Kidney was immediately frozen for histological and biochemical studies. Immunoprecipitation and Wheat Germ Agglutinin (WGA) based lectin affinity gel electrophoresis were used to study the O-GlcNAcylation of Na/K ATPase. NButGT increased by 2 O-GlcNAc levels in heart and kidney. This increase in O-GlcNAc restored mean arterial pressure (MAP) (P < 0.01) and bicarbonates (P < 0.01). In treated group, kalemia (Sham: 4.2 ± 0.1; HS: 4.7 ± 0.1; NButGT: 4.3 ± 0.1 mmol/L; P < 0.05) and natremia (Sham: 141.5 ± 0.4; HS: 139.8 ± 0.5; NButGT: 141.9 ± 0.4 mmol/L; P < 0.01) were restored. The study of the O-GlcNAcylation of the Na/K ATPase by immunoprecipitation and WGA gel validated its O-GlcNAcylation. Histological analysis of kidney revealed that HS reduced NA/K ATPase expression during HS and treatment by NButGT restored it (Sham: 15.1 ± 1.1; HS: 10.3 ± 0.6; NButGT: 13.78 ± 1.1; % area; P < 0.05). Our results demonstrate that increase O-GlcNAcylation during hemorrhagic shock restored MAP and reduced markers of metabolic acidosis. In our study, animal treatment with NButGT to increase O-GlcNAc levels, restored natremia and kalemia. This effect is associated with an increase in Na/K ATPase expression. To our knowledge, our study validates the O-GlcNAcylation of the Na/K ATPase. With regard to our results, now it will be relevant to study the effect of O-GlcNAcylation on the activity and localisation.