NDT Advance Access originally published online on December 4, 2008
Nephrology Dialysis Transplantation 2009 24(5):1406-1416; doi:10.1093/ndt/gfn662
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BMP-7 fails to attenuate TGF-β1-induced epithelial-to-mesenchymal transition in human proximal tubule epithelial cells
Department of Immunology Research, Centocor Research and Development, Inc., Radnor, PA 19087, USA
Correspondence and offprint requests to: Paul L. Dudas, Department of Immunology Research, Centocor Research and Development, Inc., 145 King of Prussia Road, R-4-2, Radnor, PA 19087, USA. Tel: +1-610-240-5595; Fax: +1-610-889-4418; E-mail: pdudas{at}its.jnj.com
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Abstract |
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Background. In rodent models of chronic renal disease bone morphogenetic protein-7 (BMP-7) has been shown to halt disease progression and promote recovery. Subsequent studies utilizing immortalized rodent renal cell lines showed that BMP-7 was renoprotective by antagonizing TGF-β1-stimulated epithelial-to-mesenchymal transition (EMT). The present study sought to determine if BMP-7 prevents TGF-β1-induced EMT in primary (RPTEC) and immortalized (HK-2) human proximal tubule epithelial cells.
Methods. EMT was determined by quantitative real-time PCR analysis of e-cadherin, vimentin, CTGF and TGF-β1 transcript expression and immunocytochemical analysis of ZO-1 and 
-smooth muscle actin (-SMA) protein expression following TGF-β1 treatment in RPTEC and HK-2 cells.
Results. In RPTEC and HK-2 cells, TGF-β1 significantly reduced e-cadherin expression and significantly increased vimentin, CTGF and TGF-β1 expression. TGF-β1 also diminished ZO-1 immunoreactivity and increased -SMA expression in confluent cell monolayers. Co-incubation of TGF-β1 with an anti-TGF-β1 neutralizing antibody substantially reduced the cytokine's effects, which indicated EMT in these cells was inhibitable. Co-administration of BMP-7 over a broad concentration range (0.01–100 µg/ml) with TGF-β1 failed to attenuate EMT in RPTEC or HK-2 cells, as demonstrated by no inhibition of altered e-cadherin, vimentin, CTGF and TGF-β1 expression and no restoration of ZO-1 immunoreactivity. Furthermore, when BMP-7 was applied to proximal tubule cells alone, it also decreased e-cadherin expression and increased vimentin, CTGF and TGF-β1 expression. Additionally, BMP-7 failed to induce the mesenchymal-to-epithelial transition (MET) in NRK-49F rat renal fibroblasts. BMP-7 did however prevent TGF-β1-mediated e-cadherin downregulation in TCMK-1 mouse renal tubular epithelial cells. BMP-7 activity was routinely confirmed by examining BMP-7-induced phosphorylation of SMADs 1/5/8, BMP-7 regulation of BMPR-IA, BMP-7-mediated reduction of IL-6 transcript expression and BMP-7-mediated reduction of secreted IL-6 and IL-8 proteins.
Conclusions. In the present study, despite confirming BMP-7 regulation of receptor expression and induction of downstream signalling events, we were unable to demonstrate BMP-7 inhibition of EMT in either primary or immortalized human proximal tubule cells. Moreover, we were unable to demonstrate BMP-7-stimulated MET in rat renal fibroblasts. A protective effect was however observed at an elevated BMP-7 concentration in mouse renal tubular epithelial cells.
Keywords: BMP-7; epithelial-to-mesenchymal transition; proximal tubule; TGF-β1
Received for publication: 24. 3.08
Accepted in revised form: 4.11.08