L-Arginine supplementation increases mesangial cell injury and subsequent tissue fibrosis in experimental glomerulonephritis

Background. Mesangial cell lysis in the antithymocyte serum (ATS)-induced m odel of glomerulonephritis is dependent on the generation of cytotoxic nitr ic oxide (NO) through transient induction of NO synthase (iNOS). We hypothe sized that increased availability of L-arginine (L-Arg) during mesangial ce ll lysis might provide iNOS with increased substrate leading to increased l ysis, and that this increased lysis would be reflected in more severe fibro tic disease at day 6. Methods. To ensure whole body equilibration with high L-Arg at the time of injury, rats were pretreated with 1% L-Arg in drinking water for one week p rior to the administration of ATS. Animals were sacrificed six hours after ATS injection when previous experiments had indicated iNOS induction had oc curred and at six days. At six hours, plasma was obtained for L-Arg levels and nitrite/nitrate (NOx) content. Renal tissues were taken for histologica l evaluation of glomerular cell counts, macrophage infiltration (ED-1), and iNOS expression. Glomeruli were isolated for detection of iNOS mRNA and pl aced in culture to study the dependence of NO production on L-Arg concentra tion. In rats sacrificed at six days, L-Arg supplementation was stopped 16 hours after ATS injection. Fibrotic disease was evaluated by urinary protei n excretion, histological assessment of glomerular cell number, matrix accu mulation, and production of transforming growth factor-pi and matrix compon ents fibronectin and plasminogen activator inhibitor type-1 (PAI-1) by isol ated glomeruli in culture. Results. At six hours, the glomerular cell number was significantly reduced by ATS injection (P < 0.01) and further significantly (P < 0.05) reduced b y L-Arg feeding [normal control (NC) = 64.2 +/- 1, ATS = 53.4 +/- 0.7, ATS + L-Arg = 50.8 +/- 0.7]. Disease increased macrophage infiltration and iNOS protein and iNOS mRNA levels markedly (P < 0.01), whereas L-Arg feeding di d not further increase these variables. Plasma L-Arg levels (nmol/ml) were reduced by disease (NC = 121 + 9, ATS = 84 +/- 13, P < 0.01) and elevated b y L-Arg feeding (ATS + L-Arg = 166 +/- 12, P < 0.01). Plasma NOx was signif icantly increased by ATS and further increased by ATS + L-Arg (P < 0.05). P roduction of NOx by cultured glomeruli showed striking L-Arg concentration dependence in six hours but not in normal glomeruli. In the group sacrifice d at day 6, day 2 proteinuria was higher in the ATS + L-Arg group compared with the ATS alone group (P < 0.05). Measures of fibrotic disease at day 6 all showed large increases over control with ATS alone (P < 0.01), and furt her small, but significant increases when L-Arg was combined with ATS (P < 0.05). Conclusions. The results indicate that if given during disease induction, L -Arg supplementation can enhance iNOS-dependent tissue injury by providing increased substrate. Although the increase in injury with L-Arg supplementa tion was small, it led to increased fibrosis at day 6. These data predict t hat in diseases with repeated iNOS-dependent tissue injury, L-Arg supplemen tation may produce cumulative increases in tissue fibrosis.