Effects of dietary antioxidants against oxidant induced acute kidney injury

This investigation is focused on the implementation of the dietary phenylpropanoids in attenuation of acute kidney injury (AKI) in patents admitted to intensive care unit in a critical condition. Morbidity (5-7 per cent hospitalised patients, UK) and mortality rates (20-30 per cent, UK) remain high. The diagnosis of AKI has gone under review several times to try combat the difficulties in patient practice, focusing on the urine output, blood urea nitrogen levels and serum creatinine during the initiation of AKI. Treatments for AKI remain poor, focusing on restoring urine output by administration of intravenous fluids and cessation of insulting medications and often results in the need for renal replacement therapies.

This highly complex, multifaceted disease has been attributed to the induction of pre-renal or post-renal blockages by ischemia and intrinsic nephrotoxic events. These events cause damage to the renal system through induction of oxidative stress, culminating in oxidative damage to cellular systems and loss of renal function. Phenylpropaniod secondary plant metabolites, in particular cinnamic acid derivatives, hydroxycinnamates (para-, meta-, ortho- coumaric acid, caffeic, ferulic, sinapic and chlorogenic acids) and stilbenoids (resveratrol) are commonly ingested through diet. In fruits, vegetables, brewed beverages and wine. They have attracted recent attention for their well-established antioxidant properties and more recently anti-proliferative properties in cancer cell lines.

This project was intended to investigate the possible attenuation of oxidative stress mediated AKI. Hydroxycinnamates and resveratrol showed good equivalence to Trolox, TEAC values, an industrial food standard for antioxidant properties in the United States.

The test antioxidant phenylpropanoids have been tested in in vitro experiments for their potential nephrotoxicity and protection from chemical mediators of oxidative stress, free radical generators, by indirect or direct interactions within the cells. Hydroxycinnamates showed little nephrotoxicity across increasing concentrations of the antioxidants over a 24 hour period, the thermal stability in aqueous environment may determine the potential for adverse cellular toxicity in the NRK-52E cells. Paraquat, hydrogen peroxide and peroxynitrite mediated nephrotoxicity presented a concentration dependent toxicity profile using both MTT and LDH bioassays, total cell death was achieved when dosed with c.a. 2 mM over a 24 hour period, with most oxidants used.

The antioxidant defences in endogenous antioxidant molecules and enzymes within the NRK-52E cells were subdued by radical generation induced by these oxidants approaching 8-12 hours of exposure to oxidants. The paraquat dose-response suggests that superoxide generation overwhelms the endogenous antioxidant enzyme and molecule systems and induces uncontrollable oncosis or apoptosis in the cell line after 10 hours of paraquat dosing.

Pre-incubation with 10μM of each separate hydroxycinnamate or resveratrol demonstrates protection from paraquat mediated oxidative stress in the NRK-52E cells to varying degrees. Ferulic acid and meta-coumaric acid has demonstrated a high capacity of antioxidant action against reactive oxygen species within the mitochondria and cytosol of the NRK-52E cells closely followed by the protection seen with incubation with para-coumaric acid and caffeic acid and chlorogenic acid followed by resveratrol and sinapic acid.

However in most cases the protection was limited to within the first 24 hours, due to the terminal nature of the experiments. At 19 hours para-coumaric, meta-coumaric acid, ferulic, have above 50% and caffeic and chlorogenic acids had above 30% cell viability at 19 hours, but this was not reflected in the membrane integrity results, omitting ferulic acid which showed limited but significant reduction in LDH release at 24 hours.

The initial experiments with set time at 24 hours didn’t demonstrate antioxidant protection. This is thought to be due the measurement of a progressive event at one time point misses the initial protection that the antioxidants grant. The antioxidant action can be attributed to scavenging of free radicals (reactive oxygen species), however the termination products remain unknown, and they have been predicted theoretical termination products which may also have beneficial pharmacological effects.

The differences between the selected compounds in structure around the hydroxyl group, which is the active component of antioxidant action of the phenolic structure, may cause the different effects. Other pharmacological effects were noted at higher concentrations (500μM). Synergistic toxicity (higher reduction in MMT and LDH) using resveratrol demonstrated its emerging properties in anti-proliferation and use in anticancer treatments, and indications of interaction with mediators of apoptosis.

Oxidative stress is a multifaceted mechanism, driven by generation of free radical species in microsites of the NRK-52E cells. The propagation of free radicals in the paraquat model after superoxide is generated is unknown but driven by endogenous antioxidant defence mechanisms. These either terminate free radical species, or fortuitously generate stronger oxidizing agents and free radical species downstream, peroxynitrite, and hydroxyl radicals. Protection of the renal system has been demonstrated with use of hydroxycinnamates and resveratrol. However, these results need to be tested in more complex biological systems using in vivo models of AKI that can emulate the progression of AKI in humans more efficiently.