Lipid peroxidation in isolated rat nephron segments

Elevated levels of lipid peroxides (LPO) in tissues have been considered an index of increased reactive oxygen metabolites, which are important pathological mediators also found in the kidney. By adopting the quantification of malondialdehyde-thiobarbituric acid adduct as a standard, using a fluorometer, a microassay was developed that enabled us to measure LPO in tissue having <1 μg protein. By this method, basal levels of LPO along the rat nephron showed that proximal tubules bear more LPO per millimeter of tubule than distally located segments (~0.2 pmol/mm tubule for proximal tubules and 0.02 for thick ascending limbs) and that S3 was the highest LPO per tissue protein (2.2 ± 0.1 pmol/μg protein, n = 8). In addition, the levels of LPO were stimulated by 10 μM phorbol 12-myristate 13-acetate (PMA) in both glomeruli and S3 (P < 0.001) and in S2 (P < 0.05). Furthermore, sphingosine (100 μM), a protein kinase C (PKC) inhibitor, totally blocked the LPO increment by PMA without any effect on the basal LPO in glomeruli, suggesting the involvement of PKC in LPO formation. Taken together, the results indicate the applicability of LPO assay to the nephron for evaluation of site-specific nephrotoxic insult and its mechanisms in renal pathophysiology.