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Sources of Funding: Departmental

Introduction

Various renal cell injuries, including renal ischemia/reperfusion injury (RIRI), have been shown to be primarily attributed to oxidative stress. Particularly, to protect the kidneys from RIRI, the perioperative interventions have been routinely employed but the outcomes remain elusive. To improve the efficacy of renoprotection, certain antioxidant such as ethyl pyruvate (EPy) may more effectively prevent RIRI as its safety or beneficial and therapeutic effects have been well documented. Accordingly, we investigated the protective effects of EPy and mannitol (Mann), one of perioperative agents often used, on renal cells against oxidative stress in vitro.

Methods

Oxidative stress was exerted by hydrogen peroxide (H₂O₂) on the renal proximal tubular MDCK cells. Severity of oxidative stress was determined by malondialdehyde (MDA) assay and protective effects of EPy and Mann against H₂O₂ were assessed by cell viability test. To explore the cytotoxic mechanism of H₂O₂, the status of glycolytic parameters, cell cycle, and metabolic signaling pathways as well as induction of apoptosis was also examined.

Results

H₂O₂ (500 µM) increased the MDA level by ~3.5 times of controls, but PE (1 mM) nearly completely reduced it to the basal level. Although cell viability was reduced to merely 10% by H₂O₂ in 24 h, EPy yet maintained >90% cell viability. Two glycolytic parameters, hexokinase activity and ATP level, declined to ~45% and ~30% (compared to controls) by H₂O₂, respectively. This decline significantly (80-90%) went up with EPy. H₂O₂ also induced a G₁ cell cycle arrest and the modulations of metabolic signaling regulators such as AMP-activated protein kinase (AMPK), protein kinase B (Akt), and mammalian target of rapamycin (mTOR). EPy utterly reversed a cell cycle arrest and prevented those modulations. Analysis of two key apoptosis regulators, bcl-2 and Bax, further indicated induction of apoptosis by H₂O₂, which was yet fully prevented with EPy. Unlike EPy, Mann had little effects on any H₂O₂-induced adverse cellular effects.

Conclusions

Oxidative stress exerted by H₂O₂ can cause cell injury and death in MDCK cells. It involves the cell viability reduction, the glycolysis inhibition, a cell cycle arrest, the modulations of metabolic signaling pathways, and ultimately induction of apoptosis. However, all these adverse effects through oxidative stress were effectively and nearly completely prevented or reversed with EPy, not with Mann. Therefore, EPy should be considered as a more effective prophylactic and perioperative renoprotective agent (compared with Mann) against oxidative renal cell injury including RIRI.

Funding

Departmental