BACKGROUND: Experimental and clinical investigations suggest that oxidant stress is a critical determinant of radiocontrast nephropathy (RCN), and that N acetyl cysteine (NAC) can prevent this damage. This study addresses these issues directly at the tubular cell level. Potential alternative mechanisms for RCN have also been sought. METHODS: Isolated mouse proximal tubule segments (PTS), or cultured proximal tubule (HK-2) cells, were subjected to radiocontrast media (RCM) (Ioversol, Optiray 320) exposure, followed by assessments of cellular viability [% lactate dehydrogenase (LDH) release, tetrazolium dye (MTT), uptake] and lipid peroxidation. These experiments were conducted in the absence or presence of a variety of antioxidants [NAC, glutathione (GSH), superoxide dismutase, catalase] or pro-oxidant (GSH depletion, heme oxygenase inhibition) strategies. RCM effects on mitochondrial and plasma membrane integrity were also assessed. RESULTS: RCM exposure did not induce PTS lipid peroxidation. Neither antioxidant nor pro-oxidant interventions mitigated or exacerbated RCM-induced tubular cell injury, respectively. RCM impaired mitochondrial integrity, as assessed by ouabain-resistant ATP reductions, and by cytochrome c release (before cell death). RCM also induced plasma membrane damage, as indicated by loss of key resident proteins (NaK-ATPase, caveolin) and by increased susceptibility to phospholipase A2 (PLA2) attack (increase of >/=2 times in free fatty acid and NaK-ATPase release). Hyperosmolality could not account for RCM's toxic effects. CONCLUSION: RCM toxicity can be dissociated from tubular cell oxidant stress. Alternative mechanisms may include mitochondrial injury/cytochrome c release and plasma membrane damage. The latter results in critical protein loss, as well as a marked increase in plasma membrane susceptibility to exogenous/endogenous PLA2 attack.
BACKGROUND: Experimental and clinical investigations suggest that oxidant stress is a critical determinant of radiocontrast nephropathy (RCN), and that N acetyl cysteine (NAC) can prevent this damage. This study addresses these issues directly at the tubular cell level. Potential alternative mechanisms for RCN have also been sought. METHODS: Isolated mouse proximal tubule segments (PTS), or cultured proximal tubule (HK-2) cells, were subjected to radiocontrast media (RCM) (Ioversol, Optiray 320) exposure, followed by assessments of cellular viability [% lactate dehydrogenase (LDH) release, tetrazolium dye (MTT), uptake] and lipid peroxidation. These experiments were conducted in the absence or presence of a variety of antioxidants [NAC, glutathione (GSH), superoxide dismutase, catalase] or pro-oxidant (GSH depletion, heme oxygenase inhibition) strategies. RCM effects on mitochondrial and plasma membrane integrity were also assessed. RESULTS:RCM exposure did not induce PTS lipid peroxidation. Neither antioxidant nor pro-oxidant interventions mitigated or exacerbated RCM-induced tubular cell injury, respectively. RCM impaired mitochondrial integrity, as assessed by ouabain-resistant ATP reductions, and by cytochrome c release (before cell death). RCM also induced plasma membrane damage, as indicated by loss of key resident proteins (NaK-ATPase, caveolin) and by increased susceptibility to phospholipase A2 (PLA2) attack (increase of >/=2 times in free fatty acid and NaK-ATPase release). Hyperosmolality could not account for RCM's toxic effects. CONCLUSION:RCMtoxicity can be dissociated from tubular cell oxidant stress. Alternative mechanisms may include mitochondrial injury/cytochrome c release and plasma membrane damage. The latter results in critical protein loss, as well as a marked increase in plasma membrane susceptibility to exogenous/endogenous PLA2 attack.
Authors: John J Gildea; Brandon A Kemp; Nancy L Howell; Robert E Van Sciver; Robert M Carey; Robin A Felder Journal: Am J Physiol Renal Physiol Date: 2011-02-02