Tandem antifibrotic actions of L-arginine supplementation and low protein diet during the repair phase of experimental glomerulonephritis

Background. Based upon the central role transforming growth factor-beta (TG F-beta) overexpression appears to play in renal fibrotic diseases, we have recently advocated reduction of TGF-beta as a therapeutic target. As part o f efforts to determine the strength of this approach, we have undertaken st udies to quantitate the effects of currently used and promising therapies i n terms of their potential to reduce markers of disease in anti-thymocyte-s erum (ATS)-glomerulonephritis in the rat. Hers we assess the therapeutic ef fect of L-arginine supplementation. which has been shown to reduce fibrosis in a number of hypertensive models, given alone or in combination with low protein diet and started 24 hours after disease induction. Methods. Glomerulonephritis was induced by intravenous injection of OX-7 mo noclonal antibody into 200 g Sprague-Dawley rats. Twenty-four hours later a nimals were placed in groups that were either untreated, treated with 1% L- arginine in drinking water or 6% protein diets or both. On the fifth day of disease 24-hour urine specimens were collected and systemic blood pressure was measured. On the sixth day rats were anesthetized. Kidneys were perfus ed, tissue was taken for PAS staining and glomeruli were isolated. Aliquots of glomeruli were used fur RNA preparation and for culture to determine 72 -hour production of TGF-beta. fibronectin and plasminogen activator-type 1 (PAI-1), which were assayed by ELISA on culture supernatants. Measures of n itrate and nitrite (NOx) production included plasma NOx. urinary NOx and gl omerular production of NOx in culture. Results. All disease measures except proteinuria and including matrix accum ulation. TGF-beta, fibronectin and PAI-1 production and mRNA expression for TGF-beta, fibronectin and PAI-1 were significantly and similarly reduced b y about 50% in groups treated with L-arginine or with low protein diet. Pro teinuria was reduced in low protein treated but not in L-arginine supplemen ted rats. Neither systemic blood pressure nor measures of NO synthesis show ed differences between groups that could be attributed to L-arginine supple mentation. Ln contrast. disease-related increases in glomerular production of NOx were markedly reduced by low protein. Combined therapy resulted in s mall, but statistically significant decreases in most measures of disease. Conclusions. L-arginine supplementation reduces fibrotic disease in ATS-ind uced glomerulonephritis if started after disease induction. The absence of evidence for increased NO production related to L-arginine supplementation suggests that L-arginine is acting here through different pathways from tho se demonstrated in hypertensive models of disease. The data support the ide as that TGF-beta reduction is a valid therapeutic target and that quantitat ion of TGF-beta reduction is a useful approach for comparing antifibrotic d rug candidates.