BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 229, 243–248 (1996) ARTICLE NO. 1787 Chemically Modified Tetracyclines Inhibit Inducible Nitric Oxide Synthase Expression and Nitric Oxide Production in Cultured Rat Mesangial Cells Howard Trachtman, 1 Stephen Futterweit, Robert Greenwald, Susan Moak, Pravin Singhal, Nicholas Franki, and Ashok R. Amin Department of Pediatrics, Division of Nephrology, Schneider Children’s Hospital and Department of Medicine, Divisions of Nephrology and Rheumatology, Long Island Jewish Medical Center, Long Island Campus for the Albert Einstein College of Medicine, New Hyde Park, New York 11040; and Departments of Rheumatology, Surgery, and Medicine, New York University Medical Center, New York, New York 10016 Received October 4, 1996 Tetracyclines inhibit matrix metalloproteinases (MMP) and attenuate connective tissue degradation in a wide variety of human and animal disorders. Chemically modified tetracyclines (CMT) have been synthe- sized in which the antibacterial potency has been eliminated but in which the anti-MMP efficacy is retained. Nitric oxide (NO) modulates MMP synthesis and activity in mesangial cells in vitro. Therefore, we examined whether CMT inhibit iNOS gene and protein expression and NO production in cultured rat mesangial cells. Mesangial cells were maintained in media containing IFN-g and LPS for 24–72 h. Test media contained either no further additives or CMT-1, 3, 5, or 8 at concentrations of 1, 2.5, 5, and 10 mg/ml. iNOS gene and protein expression were assessed and NO production was determined by the Griess reaction. Incubation of mesangial cells with CMT-3 and CMT-8 resulted in time- and dose-dependent inhibition of NO production that was maximal at 48 h (õ20% of control) and at a drug concentration of 5 mg/ml (Põ0.05). Addition of CMT-1 had a modest (40%) inhibitory effect and CMT-5 did not alter NO production. The impact of CMT on NO production was directly related to their potency as collagenase inhibitors. Moreover, CMT- induced changes in NO synthesis were associated with parallel alterations in steady-state iNOS mRNA abundance and protein expression. These agents may be useful to ameliorate NO-dependent glomerular inflammation.  1996 Academic Press, Inc. NO is a messenger molecule that serves many functions including smooth muscle relaxation, neurotransmission, platelet aggregation, immune cell activation, and tumor cell killing (1). NO is synthesized from the guanidino nitrogen of L-arginine by NOS (2,3). This enzyme has three isoforms: two constitutive species are present in neuronal (NOS1) and endothelial cells (NOS3) and an inducible species, iNOS (NOS2), is present in a wide variety of cells including macro- phages, hepatocytes, and dermal fibroblasts (3,4). The inducible isoform of the enzyme is also expressed in renal cells including glomerular mesangial cells and tubular epithelial cells (3,5,6,7). In glomerulonephritis induced by the intravenous injection of anti-thymocyte serum, admin- istration of the NOS inhibitor, L-NMMA, reduces urinary nitrite and protein excretion and attenuates mesangial cell injury and matrix expansion (8). This suggests that NO contributes to renal damage in immune-mediated glomerulonephritis. We recently demonstrated that NO stimulates the synthesis and activity of a 72-kDa neutral MMP in cultured rat mesangial cells (9). MMP activity is an important modulator of glomerular injury (10). Amin et al (11,12) recently demonstrated that several tetracyclines, notably minocy- cline and doxycycline, inhibit the expression of an osteoarthritis NOS isoform in human chondrocytes and iNOS in LPS-stimulated macrophages. CMT have been synthesized that are 1 Correspondence address: Schneider Children’s Hospital, Division of Nephrology, 269-01 76th Ave., New Hyde Park, NY 11040-1432. Fax: 718-470-0887. E-mail: trachtma@lij.edu. 0006-291X/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved. 243