Abstract
Background
Glomerular hypertension aggravates glomerular sclerosis by inducing growth factors, e.g., transforming growth factor-β (TGF-β) to mesangial matrix expansion. Smads are intracellular proteins that transmit signals from TGF-β to nucleus, and Smads are also negatively regulated by inhibitory Smads (I-Smads), Smad6 and Smad7. However, little is known about the role of I-Smads in glomerular hypertension. We studied I-Smad expression in cultured mesangial cells subjected to mechanical stretch as an in vitro model of glomerular hypertension.
Methods
Rat mesangial cells were cultured under cyclic mechanical stretch conditions using the Flexercell Strain Unit. Phosphorylated Smad1 and Smad2 were determined by Western blots. The expression of Smad6 and Smad7 mRNAs was determined by Northern blots. Stretch-mediated I-Smad mRNAs of cells pre-treated with MAPK-ERK kinase inhibitor, U0126, were also determined. Localization of phospho-Smad1, Smad6 and Smad7 proteins in the glomerulus of Dahl salt-sensitive rats was determined by immunohistochemistry.
Results
Stretch stress increased phospho-Smad1 levels, and significantly decreased Smad6 mRNA to 32 % of control, and increased Smad7 mRNA to 136 % of control. U0126 significantly attenuated stretch-mediated decreases in Smad6 mRNA, but had no effect on stretch-mediated increases in Smad7 mRNA. Phospho-Smad1, Smad6 and Smad7 proteins were localized in podocytes and mesangial cells of Dahl rats.
Conclusion
Mechanical stretch increases phospho-Smad1 levels and down-regulates Smad6 mRNA expression in mesangial cells. Stretch-mediated down-regulation of Smad6 is partially involved in ERK1/2 activation. These results indicate that glomerular hypertension might augment Smad1 signaling with concomitant attenuation of Smad6-mediated negative feedback.
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Acknowledgments
This work was supported by a research grant 11671042 from the Ministry of Education and Science of Japan. We thank Dr. K. Miyazono for generous donations of cDNA clones, and we thank Hiroko Ohashi and Atsuko Hashimoto for technical assistance.
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Hayashi, Y., Katoh, T., Asano, K. et al. Mechanical stretch down-regulates expression of the Smad6 gene in cultured rat mesangial cells. Clin Exp Nephrol 16, 690–696 (2012). https://doi.org/10.1007/s10157-012-0630-6
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DOI: https://doi.org/10.1007/s10157-012-0630-6