Mesangial cell signaling cascades in response to mechanical strain and glucose
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BACKGROUND: Elevated glucose levels and glomerular hypertension (Pgc) are considered to contribute to the elaboration of matrix protein by mesangial cells (MCs) in diabetic glomeruli. MCs grown in 30 mM of glucose produce excessive matrix protein, as do MCs exposed to cyclic strain, and the combination of the two exacerbates this. Tight glucose control or reduction in Pgc clinically delays progression of diabetic nephropathy. MC c-fos is induced in response to either application of strain or high ambient glucose, inducing increases in activated protein-1 transactivational activity and extracellular matrix production. Stimuli that lead to c-fos induction pass through the three mitogen-activated protein (MAP) kinase pathways: p44/42, SAPK/JNK, and p38/HOG. We studied MAP kinase activation in MCs exposed to mechanical strain and a high glucose. METHODS: MCs (passage 5 through 10) cultured for 96 hours on type 1 collagen-coated flexible-bottom plates in either 5.6 or 30 mM glucose were exposed to 5, 10, or 30 minutes of cyclic strain (60 cycles per min) by computer-driven generation of vacuums of -14 kPa, inducing 20% elongation in the diameter of the surface. Control MCs were grown on both coated rigid and flexible-bottom plates. Protein levels (by Western blot) and activity assays for all three kinase cascades were performed at baseline and after 5, 10, and 30 minutes. All experiments were performed in triplicate. RESULTS: MAP kinase signaling was seen in response to stretch, and high ambient glucose affected the pattern of activation. Both p44/42 and p38HOG kinase activities showed small increases to a maximum of 2.5- to 3.5-fold greater than static MCs at 10 minutes. Activity in both kinase cascades was slightly suppressed by 30 mM glucose. In contrast, SAPK/JNK activity was present at a very low level in static MCs and increased markedly by 10 minutes of stretch. Thirty micromolars of glucose augmented this effect by a factor of six over MCs cultured in 5.6 mM glucose after 10 minutes of stretch. Neither glucose concentration nor mechanical strain had any effect on the protein expression of any of the kinases by Western blot. CONCLUSIONS: MAP kinase cascade signaling is seen when physical force is applied to MCs, and glucose affects the pattern of activity. Thirty micromolars of glucose markedly increase the level of SAPK/JNK activation. This may have implications in diabetic signal transduction and matrix protein production.
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