Bacitracin inhibits fibronectin matrix assembly by mesangial cells in high glucose.

Abstract

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. PDI inhibition of FN reduces its association into an insoluble matrix and potentially provides a new approach to reduce excessive matrix deposition in DN.