Examining the Role of Integrin-Linked Kinase in High Glucose-Induced Fibronectin Matrix Assembly

Abstract

Excess production of extracellular matrix (ECM) by kidney mesangial cells is a defining feature of the progression of diabetic nephropathy. An increase in assembly of the ECM component fibronectin (FN) by mesangial cells under the high glucose conditions associated with a diabetic state is thought to have a key role in this ECM expansion, which can eventually contribute to a loss of kidney function. Analysis of genes differentially expressed under normal and high glucose has provided insight into the pathways which may be involved in high glucose-induced FN matrix assembly. Integrin-Linked Kinase (ILK) is a pseudokinase found to be upregulated in mesangial cells under high glucose conditions. ILK possesses functions in regulating actin organization that may be relevant to FN matrix assembly, however the involvement of ILK in this process remains unclear. To understand the role of ILK in FN matrix assembly, the effects of changes in ILK expression on FN matrix assembly in NIH-3T3 fibroblast cells were measured by immunofluorescence. ILK levels were reduced by siRNA knockdown and ILK was over-expressed by transduction with a mEm-ILK fusion protein, which surprisingly both reduced FN matrix. To determine if ILK contributes to high glucose-induced FN matrix assembly in mesangial cells, ILK was knocked down using siRNA prior to growth under high glucose. FN matrix was greatly reduced, suggesting that ILK does contribute to FN matrix assembly in mesangial cells under diabetic conditions. Together these findings help to clarify the relationship between FN matrix assembly and ILK, while identifying ILK as a potential target in reducing ECM expansion during the progression of diabetic nephropathy.