['Accumulation of mesangial extracellular matrix is a major characteristic of diabetic nephropathy (DN). Expression of several extracellular matrix proteins is up-regulated in human mesangial cells (HMC) cultured in high glucose. One protein, fibronectin (FN), associates to form an insoluble disulfide-linked matrix and possesses inherent protein-disulfide isomerase (PDI) activity. Bacitracin is a known PDI inhibitor. We tested the hypothesis that inhibiting FN-PDI activity with bacitracin would disrupt excessive FN-matrix assembly by cultured HMCs grown under high glucose conditions.', 'The effect of bacitracin on FN-PDI activity was tested using an RNase-refolding assay. High glucose cultures of HMC were labeled with (3)H-leucine, with and without bacitracin, and (3)H-FN immunoprecipitated from the medium and sequential extracts of cell layers to distinguish insoluble FN. FN transcription was assessed by reverse transcription-polymerase chain reaction (RT-PCR). Pericellular FN-matrix was examined by immunohistology.', 'Bacitracin inhibited the PDI activity of FN, with maximal inhibition at 1.0 mmol/L. Treatment of HMC cultures grown in high glucose with bacitracin brought about changes in the distribution of newly synthesized FN. With increasing concentrations of bacitracin there was a significant reduction in the level of FN present as an insoluble matrix of HMC cultures maintained in high glucose, and a corresponding increase in FN in medium. Decreases in FN matrix laid down by HMCs treated with different concentrations of bacitracin were seen by immunohistology. FN mRNA levels were unchanged.'] Does bacitracin inhibit fibronectin matrix assembly by mesangial cells in high glucose?