Activation of mesangial cell signaling cascades in response to mechanical strain

Background. Mesangial cells (MCs) are constantly exposed to pulsatile stretch and relaxation in their role as architectural support for the glomerulus. There is no cell proliferation in normal glomeruli. In contrast, animal models of increased glomerular capillary pressure are characterized by resident glomerular cell proliferation and elaboration of extracellular matrix (ECM) protein, resulting in glomerulosclerosis. This process can be ameliorated by maneuvers, such as angiotensin converting enzyme inhibition, that reduce glomerular capillary pressure. MCs grown on ECM-coated plates and exposed to cyclic stretch/relaxation proliferate and produce ECM protein, suggesting that this may be a useful in vitro model for MC behavior in response to increased physical forces. Previous work has shown induction of c-fos in response to application of mechanical strain to MCs, which may induce increases in AP-1 transcription factor activity, which, in turn, may augment ECM protein and transforming growth factor beta transcription and cell proliferation. Stimuli that lead to c-fos induction pass through mitogen-activated protein kinase (MAPK) pathways. Three MAPK cascades have been characterized in mammalian cells-p44/42 (classic MAPK), the stress-activated protein kinase/Jun terminal kinase (SAPK/JNK) pathway, and p38/HOG-and mechanical strain activates p44/42 and SAPK/JNK in cardiac fibroblasts. However, in contrast to MCs, these cells do not proliferate in response to physical force. Accordingly, we studied activation of the MAPK pathways in MCs exposed to mechanical strain. Methods. MCs (passages 5 to 10) cultured on type 1 collagen-coated, flexible-bottom plates were exposed to 30, 60, or 120 minutes of cyclic strain (60 cycles/min) by computer-driven generation of vacuums of -14 and -28 kPa, inducing 20% and 29% elongations in the diameter of the surfaces, respectively. Control MCs were grown on coated rigid bottom plates. Proliferation was assessed at 24 hours by H-3-thymidine incorporation. Protein levels (by Western blot) and activity assays for all three kinase cascades were performed at 30, 60, and 120 minutes. Results. Cyclic strain/relaxation lead to an approximate doubling of H-3-thymidine incorporation at 24 hours (N = 3, P < 0.05) only in cultures stretched 29%, but not in cultures stretched 20%. At -29% elongation, the increase in H-3-thymidine incorporation was preceded by early activation of MAPK signaling pathways. p44/42 activity increased to a maximum of eightfold greater than control at 60 minutes. p38/HOG activity was not measurable at baseline but was increased markedly at 30 minutes, which was sustained through to 120 minutes. SAPK/JNK activity was present at a very low level in MCs and was not changed by stretch. However, it was markedly increased by sorbitol. In MCs stretched to 20% elongation, lesser increases in p44/42 were seen with a similar time course, whereas no increases in p38/HOG or SAPK could be detected at the time points studied. No increase in any kinase pathway activity was seen at any time in static cultures. Conclusions. High-pressure cyclic stretch leads to MC proliferation, preceded by marked activation of p44/42 and p38/HOG MAPKs. Cell proliferation is not seen with low-pressure stretch, and there is only modest p44/42 MAPK activation, suggesting that glomerular capillary hypertension may lead to cell proliferation and injury partly through differential activation of kinase cascades.