Amino acids induce indicators of response to injury in glomerular mesangial cells

Abstract

High-protein diets exacerbate glomerular hyperfiltration and the progression of diabetic nephropathy. The purpose of this study was to determine whether amino acids also produce nonhemodynamic injury in the glomerulus. When rat mesangial cells were cultured with an amino acid mixture designed to replicate the composition in plasma after protein feeding, production of mRNA (Northern blot analysis) and/or protein (ELISA or Western blot analysis) for transforming growth factor-beta1 (TGF-beta1), fibronectin, thrombospondin-1 (TSP-1), and collagen IV were enhanced in a manner comparable to a culture with high glucose (30.5 mM). The bioactive portion of total TGF-beta (NRK assay) increased in response to amino acids. The TSP-1 antagonist LSKL peptide reduced bioactive TGF-beta and fibronectin, indicating the dependence of TGF-beta1 activation on TSP-1. DNA synthesis ([3H]thymidine incorporation), an index of cellular proliferation, increased in response to amino acids and was further enhanced by culture with increased levels of both amino acids and glucose. TGF-beta1 and matrix proteins increased when mesangial cells were cultured with excess l-arginine (2.08 mM) alone. Although l-arginine is the precursor of nitric oxide (NO), such responses to amino acids do not appear to be mediated through increased NO production. NO metabolites decreased in the media, and these responses to mixed amino acids or l-arginine were not prevented by NO synthase inhibition. In conclusion, amino acids induce indicators of response to injury in mesangial cells, even when hemodynamic stress is absent. In conditions associated with increased circulating amino acids, such as diabetes and/or a high-protein diet, direct cellular effects could contribute to glomerular injury.

Advanced Glycation End Products and Nephrotoxicity of High-Protein Diets

“…The strength of this in vitro approach is that the direct cellular effects of amino acids can be separated from their renal hemodynamic effects. Exposure of mesangial cells to increased levels of amino acids causes robust proliferative and fibrotic responses, both indicators of glomer-ular injury (65,66). These responses are remarkably similar to those that are induced by high glucose.…”

“…Amino acid oxidation or increased availability of free amino groups could induce sequential glycation and oxidative reactions, culminating in formation of AGE in situ. In a series of studies, we evaluated whether a key target of glomerular injury, the mesangial cell, is damaged by exposure to increased levels of amino acids that are designed to resemble a protein meal (65)(66)(67). The strength of this in vitro approach is that the direct cellular effects of amino acids can be separated from their renal hemodynamic effects.…”

Blockade of TSP1-Dependent TGF-β Activity Reduces Renal Injury and Proteinuria in a Murine Model of Diabetic Nephropathy

“…61,62 TGF-␤ is known to alter albumin permeability before up-regulation of ECM genes, 63 and TGF-␤ is increased by podoctyes exposed to excess intraglomerular albumin/proteins. 64 Treatment of mesangial cells with high levels of amino acids increases TSP1 expression and TSP1-dependent TGF-␤ activity, 65 and glycated albumin increases TSP1 expressed by cultured renal tubular cells, with increases in TSP1-dependent TGF-␤ activation. 38 These data suggest that TSP1 has a significant effect on proteinuria through regulation of TGF-␤ activity.…”

Oxidative Stress Mediates Protein Kinase C Activation and Advanced Glycation End Product Formation in a Mesangial Cell Model of Diabetes and High Protein Diet

“…AA resembles the distribution and relative concentration changes of amino acids in the circulation after a protein meal [13]. …”

“…In addition, AGE form in situ when cells are exposed to amino acid mixtures designed to resemble those typically found in the circulation or after protein feeding [11, 12]. Pro-fibrotic and proliferative responses of mesangial cells to high levels of amino acids and/or glucose are mediated by AGE [12, 13]. …”