Amino acids injure mesangial cells by advanced glycation end products, oxidative stress, and protein kinase C.

BACKGROUND: In diabetes, high intake of dietary protein exacerbates responses associated with kidney damage. Increased levels of amino acids could injure cells by providing free amino groups for glycation reactions leading to advanced glycation end products (AGEs). METHODS: Rat mesangial cells were cultured with increased amino acids designed to resemble protein feeding, high glucose (30.5 mmol/L), and, the combination, amino acids/high glucose. AGEs, reactive oxygen species (ROS), protein kinase C (PKC) activity and production, and mitogen-activated protein (MAP) kinase-extracellular signal regulated kinase (ERK) 1,2 activity were measured. Inhibitors were used to determine roles of these processes in fibrosis and/or AGE formation. RESULTS: AGE immunostaining increased when cells were cultured in amino acids and was comparable to that observed with high glucose. In amino acids/high glucose, AGE immunostaining appeared even greater. Amino acids, high glucose, and amino acids/high glucose induced ROS production. Aminoguanidine and vitamin E prevented AGE accumulation and induction of protein and mRNA for fibrosis markers [transforming growth factor-beta1 (TGF-beta1), fibronectin, and collagen IV]. PKC and ERK 1,2 activity increased with amino acids, high glucose, and amino acids/high glucose. PKC-beta inhibition prevented ERK 1,2 activation and fibrosis induction. ERK 1,2 inhibition also blocked the fibrosis response. CONCLUSION: A profibrotic injury response occurred in mesangial cells exposed to amino acids, with or without high glucose, by formation of AGE, oxidative stress, and activation of the PKC-beta and MAP kinase-ERK 1,2 signal pathway. These observations provide new insight into cellular mechanisms of kidney damage produced by excess dietary protein, particularly in diabetes.

Cultivation and characterization of coronary microvascular endothelial cells: a novel porcine model using micropigs.

Coronary microvascular endothelial cells (CMECs) play an important role in many physiological processes. Porcine CMECs from large breed pigs have been isolated and successfully cultured. However, because micropigs offer research advantages over large breed pigs, micropig CMEC (MPCMEC) cultures may be useful as an alternative in vitro porcine model for cardiovascular studies. We isolated MPCMECs from six Panepinto micropigs using a simplified technique and developed a system for their successful culture. MPCMECs were isolated by collagenase digestion of left ventricular samples obtained using sterile techniques. Primary isolates of MPCMECs grew steadily in complete DMEM supplemented with 20% FBS, 4 mM MgSO(4), and 500 microM dibutyryl cAMP and reached confluence in 7-10 days. Endothelial origin was demonstrated by rapid (4-h) uptake of acetylated low-density lipoprotein, immunostaining for the presence of platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31), von Willebrand factor (vWf)-related antigen, vascular endothelial cadherin (VE-cadherin), endothelial nitric oxide synthase (eNOS), and by positive staining using two fluorescein isothiocyanate-labeled endothelial-specific lectins, Dolichos biflorus agglutinin and Ulex europaeus agglutinin-1. MPCMECs also exhibited immunostaining for alpha-smooth muscle actin. MPCMECs were successfully subcultured in the absence of dibutyryl cAMP and continued to express PECAM-1 and vWf, but not eNOS, to passage six. The typical morphology of subconfluent MPCMECs consisted of elongated cells that grew in a swirling, herringbone pattern.