Diabetic nephropathy is among the most devastating long-term consequences of diabetes mellitus and is now the leading cause of end-stage renal disease (ESRD) in the United States. The effects of the disease are predominantly focused in the glomerulus and are characterized by increased levels of extracellular matrix (ECM) proteins that disrupt the function of the glomerular filtration apparatus. Mesangial cells, which support th glomerular capillary network, are the primary matrix-producing cells of the glomerulus. Fibronectin (FN), a major component of the ECM throughout the body, is found in the normal glomerulus and at significantly elevated levels in diabetic nephropathy. FN is an important player in cell-matrix interactions and FN matrix precedes and in part dictates the deposition of other ECM proteins. Consequently, perturbations in FN matrix assembly have profound effects on the overall integrity of tissue-appropriate ECM. Cells mediate FN matrix assembly via integrin receptors, notably ?5?1, binding to RGD cell-binding domains of FN dimers. This binding induces FN-FN interactions and formation of short fibrils that then mature into a stable insoluble matrix through continued addition of FN dimers. High glucose conditions have been shown to increase mesangial cell expression of various ECM proteins, including FN. The effects of high glucose on mesangial cell-mediated assembly of a mature matrix are as yet undetermined. Non-enzymatic glycation of matrix proteins represents another mode of glucose-mediated ECM disruption. Glycated FN, which exhibits altered binding properties, is found at elevated levels in diabetes. We propose that defects in mesangial cell matrix assembly represent a final common pathway in glomerulosclerosis, which marks diabetic nephropathy and other fibrotic diseases. The influence of high glucose on the kinetics of matrix assembly will first be determined by a time course of FN accumulation under normal and high glucose conditions by measuring deoxycholate (DOC) detergent-insoluble FN, an established marker for FN matrix maturity. Fibril density will be quantified to determine any glucose- mediated changes in ECM organization. Similar experiments will be performed in the presence of glycated FN. Since the 3D nature of a cell's environment stimulates matrix production, the effects of 3D matrix on the dynamics of mesangial cell-mediated FN fibrillogenesis will be examined to determine if a positive feedback loop exists, in which accumulation of insoluble matrix stimulates further matrix deposition. Since integrin receptors initiate the final common pathway, the roles of ?5?1 and ?v?3 integrins in high glucose-induced matrix deposition will be examined using neutralizing antibodies and knockout cells. Further insight into the mechanisms of increased matrix assembly in diabetic nephropathy may pave the way for the development of a new generation of therapeutics, which target this process in hopes of reversing the progression towards ESRD.
Chronically high blood sugar levels in diabetes mellitus can have devastating effects on the kidney's filtration apparatus. This apparatus depends on proper organization of a network of proteins called the extracellular matrix, which accumulate in excess and block filtration when exposed to high sugar levels. Using a kidney cell culture model, we plan to clarify the effects of high sugar levels on the filtration matrix in hopes of revealing new targets for future therapies aimed at reversing the progression of this increasingly common and potentially deadly disease. DESCRIPTION (provided by applicant): Diabetic nephropathy is among the most devastating long-term consequences of diabetes mellitus and is now the leading cause of end-stage renal disease (ESRD) in the United States. The effects of the disease are predominantly focused in the glomerulus and are characterized by increased levels of extracellular matrix (ECM) proteins that disrupt the function of the glomerular filtration apparatus. Mesangial cells, which support th glomerular capillary network, are the primary matrix-producing cells of the glomerulus. Fibronectin (FN), a major component of the ECM throughout the body, is found in the normal glomerulus and at significantly elevated levels in diabetic nephropathy. FN is an important player in cell-matrix interactions and FN matrix precedes and in part dictates the deposition of other ECM proteins. Consequently, perturbations in FN matrix assembly have profound effects on the overall integrity of tissue-appropriate ECM. Cells mediate FN matrix assembly via integrin receptors, notably ?5?1, binding to RGD cell-binding domains of FN dimers. This binding induces FN-FN interactions and formation of short fibrils that then mature into a stable insoluble matrix through continued addition of FN dimers. High glucose conditions have been shown to increase mesangial cell expression of various ECM proteins, including FN. The effects of high glucose on mesangial cell-mediated assembly of a mature matrix are as yet undetermined. Non-enzymatic glycation of matrix proteins represents another mode of glucose-mediated ECM disruption. Glycated FN, which exhibits altered binding properties, is found at elevated levels in diabetes. We propose that defects in mesangial cell matrix assembly represent a final common pathway in glomerulosclerosis, which marks diabetic nephropathy and other fibrotic diseases. The influence of high glucose on the kinetics of matrix assembly will first be determined by a time course of FN accumulation under normal and high glucose conditions by measuring deoxycholate (DOC) detergent-insoluble FN, an established marker for FN matrix maturity. Fibril density will be quantified to determine any glucose- mediated changes in ECM organization. Similar experiments will be performed in the presence of glycated FN. Since the 3D nature of a cell's environment stimulates matrix production, the effects of 3D matrix on the dynamics of mesangial cell-mediated FN fibrillogenesis will be examined to determine if a positive feedback loop exists, in which accumulation of insoluble matrix stimulates further matrix deposition. Since integrin receptors initiate the final common pathway, the roles of ?5?1 and ?v?3 integrins in high glucose-induced matrix deposition will be examined using neutralizing antibodies and knockout cells. Further insight into the mechanisms of increased matrix assembly in diabetic nephropathy may pave the way for the development of a new generation of therapeutics, which target this process in hopes of reversing the progression towards ESRD.
| Miller, Charles G; Pozzi, Ambra; Zent, Roy et al. (2014) Effects of high glucose on integrin activity and fibronectin matrix assembly by mesangial cells. Mol Biol Cell 25:2342-50 |
