Renal leukotriene C 4 synthase: characterization, partial purification and alterations in experimental glomerulonephritis

Abstract

Cysteinyl leukotrienes (LT) play an important role in the development of experimental glomerulonephritis (GN). We have partially purified and characterized LTC 4 synthase, the enzyme responsible for cysteinyl LT formation, from rat renal microsomes and have investigated this enzyme activity in nephritic rats. LTC 4 formation, measured in vitro, was linear for > 10 min at 25°C in the presence of 50 mM serine borate (an inhibitor of γ-glutamyl transpeptidase), with K m values for LTA 4 and GSH of 56 μM and 8.5 mM, respectively. Detergent solubilization and anion-exchange chromatography of microsomal proteins resulted in a 7-fold increase in enzyme specific activity. Enzymatic and immunoblot analysis demonstrated that cytosolic and microsomal glutathione S-transferase (GST) activities were distinct from LTC 4 synthase activity. Comparison of LTC 4 synthase activity in nephritic rats over 21 days revealed an initial increase over the first 24 h following injection of nephrotoxic sera, followed by a subsequent decline until day 7 and a gradual recovery by day 21. Inhibition of LT biosynthesis with MK-0591 (10 mg kg −1 d −1) reduced GN-associated proteinuria by 72% ( P < 0.05). These results suggest a potential mechanism for enhanced cysteinyl LT formation in the development of experimental GN and further support their causal role in the etiology of this disease.