Fasudil inhibits epithelial-myofibroblast transdifferentiation of human renal tubular epithelial HK-2 cells induced by high glucose.

Abstract

Renal fibrosis is a crucial pathologic process underlying diabetic nephropathy (DN). Central to this process is the epithelial-mesenchymal transformation (EMT) of tubular epithelial cells. Fasudil, a Rho-associated coiled-coil forming protein serine/threonine kimase (ROCK) inhibitor, protects against renal fibrosis in a variety of renal injury models. However, fasudil's effects on renal fibrosis in DN remain unknown. The aim of the present study was to investigate the effects of fasudil on high glucose-induced EMT in human renal tubular epithelial (HK-2) cells. HK-2 cells were exposed to 5.5 or 60 mmol/L D-glucose for 72 h, or to mannitol (osmotic control). RhoA activity was assessed using a RhoA pull-down assay, and ROCK activity was determined by myosin phosphatase target subunit-1 (MYPT1) phosphorylation. Myofibroblast (vimentin and α-smooth muscle actin [α-SMA]) and epithelial (E-cadherin) markers expressions were detected by immunocytochemistry and Western blotting. Transforming growth factor (TGF)-β1 and fibronectin secretion were detected with enzyme-linked immunosorbent assay (ELISA), and connective tissue growth factor (CTGF) was analyzed by Western blotting. Results showed that high glucose levels induced morphological changes, reduced E-cadherin expression (-73%), increased expression of vimentin (+148%) and α-SMA (+226%), increased TGF-β1 (from 116.0±5.2 µg/g to 351.0±3.2 µg/g) and CTGF (from 0.26±0.01 to 0.92±0.03) secretion, and increased RhoA and ROCK activation (p<0.05 for all). All these effects of high glucose stimulation were suppressed or abolished by fasudil. In conclusion, fasudil may attenuate EMT through reduced activation of RhoA/ROCK signaling, and decreased expression of TGF-β1 and CTGF. Thus, fasudil may be a renoprotective agent for the treatment of DN.

• Authors: Lingjia Gu, Qian Gao, Liansong Ni, Meirong Wang, Feixia Shen
• Journal: Chemical & pharmaceutical bulletin (Chem Pharm Bull (Tokyo)) Vol. 61 Issue 7 Pg. 688-94 ( 2013) ISSN: 1347-5223 [Electronic] Japan
• PMID: 23812394 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Actins
• CCN2 protein, human
• Cadherins
• Transforming Growth Factor beta1
• Vimentin
• Connective Tissue Growth Factor
• 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine
• rhoA GTP-Binding Protein
• Glucose
• fasudil

Topics

• 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine (analogs & derivatives, chemistry, pharmacology)
• Actins (metabolism)
• Cadherins (metabolism)
• Cell Line
• Cell Transdifferentiation (drug effects)
• Connective Tissue Growth Factor (metabolism)
• Epithelial Cells (cytology, drug effects, metabolism)
• Glucose (toxicity)
• Humans
• Signal Transduction (drug effects)
• Transforming Growth Factor beta1 (metabolism)
• Vimentin (metabolism)
• rhoA GTP-Binding Protein (metabolism)