EXTENDED CYTOPROTECTIVE EFFECT OF AUTOPHAGY IN THE LATE STAGES OF SEPSIS AND FLUCTUATIONS IN SIGNAL TRANSDUCTION PATHWAYS IN A RAT EXPERIMENTAL MODEL OF KIDNEY INJURY.

Abstract

The impact of a potential autophagy (LC3a/b) deregulation in hyper and in hypo stages during sepsis-induced kidney injury and the temporal profile of phosphorylated extracellular signal-related kinase, P38 (pP38), Akt (pAKT), and 13-3-3β protein were investigated in the current study, using a rat cecal ligation and puncture (CLP) model, by means of flow cytometry and immunohistochemistry. Cell viability was assessed by protein C zymogen concentrate (PC), 7-aminoactinomycin D (7-AAD) staining and inflammation by S100 protein immunostaining. The impact of reduced kidney inflammation in autophagy was assessed by PC administration, an anti-inflammatory and cytoprotective substance. Sepsis induction increased LC3a/b expression, which presented two peaks at 6 and 36 h after CLP, both in the percentage of positive cells (P = 0.024, P = 0.025, respectively) and in fluorescence intensity. At 6 h when inflammation was already apparent, LC3a/b increase was escorted by phosphorylated extracellular signal-related kinase stimulation and high cell viability (65%), designating autophagy as a cytoprotective mechanism against microbial infection. The phosphorylation of P38 was delayed to 12 h after CLP, when autophagy was reduced. pAkt and 14-3-3β expression was stimulated between 6 and 36 h after CLP, although a slight inhibition of pAkt within each cell was detected (lower MnIX value). During the second peak, inflammation was intensified, necrosis was significantly increased with LC3a/b+/7-AAD + cells to present a 1.5-fold increase. Protein C zymogen concentrate administration declined autophagy at 6 and 36 h after CLP and reduced necrosis, whereas double positive LC3a/b and 7-AAD cells were increased by 1.68 and 2.78-fold, respectively. These data open new prospectives in sepsis treatment, since they further support that autophagy represents a cytoprotective mechanism triggered by stress conditions, rather than an alternative cell death pathway.