Glucose-Induced Stiffening of Extracellular Matrix in Diabetic Nephropathy

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The goal of this project is to determine how changes in kidney extracellular matrix (ECM) stiffness caused by advanced glycation impact progression of diabetic nephropathy. Our overarching hypothesis is that advanced glycation end products (AGEs) form crosslinks that increase ECM stiffness in the kidney and alter integrin signaling to trigger increased extracellular matrix synthesis. The newly synthesized matrix is further crosslinked, thus creating a feed forward cycle of increased stiffness and ECM synthesis. We propose that this mechanism contributes to accumulation of ECM in the glomerulus and tubulointerstitium that is characteristic of diabetic kidney disease. Our preliminary studies show that incubating renal tubules with glucose or ribose significantly increases the stiffness of the tubular basement membrane. The aims of this application are to (1) Determine the mechanism by which sugar exposure alters the stiffness of kidney tubular and glomerular extracellular matrix and (2) Determine the mechanism by which increased stiffness alters integrin signaling and leads to increased ECM production. The methods employed in aim 1 will include measurement of tubular basement membrane stiffness in normal and sugar modified kidney tubules using our newly developed microcantilever-based stiffness measurement technique. Glomerular stiffness will be measured using microscale compression testing. Mechanical characterization of the tissue will be correlated with biochemical analysis via mass spectrometry to evaluate crosslink formation. For aim 2, we will use in vitro cell culture models that mimic the mechanical properties of normal and sugar modified tubular and glomerular ECM. Stiffness induced changes in cell phenotype, cell proliferation and ECM production will be evaluated in wild type and integrin α1 and α2-null tubular epithelial and glomerular mesangial cells. Successful completion of these aims will establish the role of AGE- mediated ECM stiffening as a potential contributor to progression of diabetic kidney disease and will inform subsequent studies in diabetic and integrin-null mouse models.