Diabetic nephropathy is one of the major complications of diabetes, and the leading cause of end stage renal disease in the United States. Histologically diabetic nephropathy is characterized by fibrosis of the glomeruli due to an expansion of the mesangial extracellular matrix (ECM). This excess ECM disrupts the glomerular filtration apparatus leading to a decline in renal function. Mesangial cells produce the ECM that surrounds glomerular capillaries. They express fibronectin (FN) and collagen, and both of these ECM components are up- regulated in diabetic nephropathy. FN matrix also provides a framework for the deposition of other ECM components including collagens. Therefore, deregulation of normal FN matrix assembly can have adverse effects on the overall structure of the ECM, as occurs in glomerulosclerosis. Elevated glucose is a known risk factor for diabetic nephropathy. We have shown that high glucose conditions increase FN matrix assembly by mesangial cells through activation of integrin receptors by a protein kinase C pathway. Integrin binding to FN initiates FN assembly into fibrils by promoting FN-FN interactions. As more FN dimers are incorporated, the nascent fibrils mature into a stable, insoluble FN matrix. This assembly process can be followed by measuring FN in the detergent-insoluble fraction and by analyzing fibril formation using immunofluorescence. In addition to cell signaling, other mechanisms are activated by increases in glucose such as non-enzymatic glycation and lysine acetylation and these might also contribute to excess ECM production. Advanced glycation end-products (AGEs) accumulate on ECM proteins exposed to high glucose conditions. AGEs induce a range of cellular responses usually through the receptor for AGEs (RAGE). Both AGEs and RAGE have been implicated in the development and progression of diabetic nephropathy and our preliminary results show a role for AGEs in stimulating mesangial cell matrix assembly. We will determine the effect of AGEs on FN matrix assembly by treatment of mesangial cells with AGE-modified proteins, and whether the effects result from binding to RAGE by using blocking agents and siRNA knockdown of RAGE. Another protein modification that is affected by hyperglycemic conditions is lysine acetylation, which is increased in animal models of diabetic nephropathy and has been shown to occur on the cytoplasmic tail of ?1 integrins. We will determine whether high glucose induces increased lysine acetylation using anti-acetyl lysine antibodies and whether this alters the level of FN matrix assembly by mesangial cells using deacetylase inhibitors combined with our standard matrix assembly assays. In addition, the effects of acetylation on ?1 integrin activity will be tested by point-mutation studies with established integrin activity assays. There are currently no therapies to reverse the effects of fibrosis. Understanding the mechanisms for increased FN matrix assembly in response to hyperglycemic conditions may provide novel therapeutic targets to stop or slow the accumulation of ECM.
In diabetic kidney disease, elevated blood sugar levels cause the over-production of extracellular matrix (ECM) proteins, which clog the glomeruli that filter blood into urine. This is the leading cause of end-stage renal disease in the United States but unfortunately current therapies are unable to reverse the accumulation of the glomerular ECM. Using a kidney cell culture model, we plan to clarify how high glucose can promote the accumulation of the extracellular matrix, in hopes to discover new targets for potential therapeutic for this deadly disease.
| Goyal, Ritu; Vega, Maria E; Pastino, Alexandra K et al. (2017) Development of hybrid scaffolds with natural extracellular matrix deposited within synthetic polymeric fibers. J Biomed Mater Res A 105:2162-2170 |
| Vega, Maria E; Schwarzbauer, Jean E (2016) Collaboration of fibronectin matrix with other extracellular signals in morphogenesis and differentiation. Curr Opin Cell Biol 42:1-6 |
