Paul L. Dudas

BMP-7 fails to attenuate TGF-β1-induced epithelial-to-mesenchymal transition in human proximal tubule epithelial cells

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-beta1-stimulated epithelial-to-mesenchymal transition (EMT). The present study sought to determine if BMP-7 prevents TGF-beta1-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-beta1 transcript expression and immunocytochemical analysis of ZO-1 and alpha-smooth muscle actin (alpha-SMA) protein expression following TGF-beta1 treatment in RPTEC and HK-2 cells. RESULTS In RPTEC and HK-2 cells, TGF-beta1 significantly reduced e-cadherin expression and significantly increased vimentin, CTGF and TGF-beta1 expression. TGF-beta1 also diminished ZO-1 immunoreactivity and increased alpha-SMA expression in confluent cell monolayers. Co-incubation of TGF-beta1 with an anti-TGF-beta1 neutralizing antibody substantially reduced the cytokines effects, which indicated EMT in these cells was inhibitable. Co-administration of BMP-7 over a broad concentration range (0.01-100 microg/ml) with TGF-beta1 failed to attenuate EMT in RPTEC or HK-2 cells, as demonstrated by no inhibition of altered e-cadherin, vimentin, CTGF and TGF-beta1 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-beta1 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-beta1-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.

BMP-7 Does Not Protect against Bleomycin-Induced Lung or Skin Fibrosis

Bone morphogenic protein (BMP)-7 is a member of the BMP family which are structurally and functionally related, and part of the TGFβ super family of growth factors. BMP-7 has been reported to inhibit renal fibrosis and TGFβ1-induced epithelial-mesenchymal transition (EMT), in part through negative interactions with TGFβ1 induced Smad 2/3 activation. We utilized in vivo bleomycin-induced fibrosis models in the skin and lung to determine the potential therapeutic effect of BMP-7. We then determined the effect of BMP-7 on TGFβ1-induced EMT in lung epithelial cells and collagen production by human lung fibroblasts. We show that BMP-7 did not affect bleomycin-induced fibrosis in either the lung or skin in vivo; had no effect on expression of pro-fibrotic genes by human lung fibroblasts, either at rest or following exposure to TGFβ1; and did not modulate TGFβ1 -induced EMT in human lung epithelial cells. Taken together our data indicates that BMP-7 has no anti-fibrotic effect in lung or skin fibrosis either in vivo or in vitro. This suggests that the therapeutic options for BMP-7 may be confined to the renal compartment.

Proinflammatory/Profibrotic Effects of Interleukin-17A on Human Proximal Tubule Epithelium

Background/Aims: Interleukin-17A (IL-17A) is a T cell-derived inflammatory cytokine that is upregulated during renal allograft rejection. The present study sought to further describe the IL-17A-mediated proinflammatory/profibrotic activity of proximal tubule epithelium that may contribute to allograft rejection. Methods: Immortalized (HK-2) and primary (HRPTEpiC) human proximal tubule epithelial cells were utilized for this study. Profibrotic gene alterations were examined by real-time quantitative PCR. Inflammatory mediator secretion was examined by multiplex bead-based detection of secreted proteins. Immunofluorescence microscopy and immunoblotting were utilized to examine alterations in junctional protein expression and cell morphology. Results: In HK-2 cells IL-17A significantly downregulated the expression of the proepithelial gene CDH1 (E-cadherin) while the proinflammatory/profibrotic genes CTGF, CD44 and TGFBR1 were significantly increased. IL-17A also increased the secretion of fractalkine, G-CSF, GM-CSF, VEGF, IL-6 and IL-8. In HRPTEpiC 100 ng/ml IL-17A upregulated the proinflammatory/profibrotic genes ACTA2, CCL2, CHMP1A, CTGF, FN1, IL6, FSP1, SMAD1, SMAD5, TGFB1 and TGFBR2 while treatment with a reduced concentration of IL-17A (0.1 ng/ml) decreased SMAD5, TGFB1 and PDGFRB expression. Changes in ZO-1 and E-cadherin protein expression and cell morphology were examined following IL-17A treatment as indicators of epithelial-to-mesenchymal transition. IL-17A decreased ZO-1 expression in HK-2 and HRPTEpiC; however, E-cadherin was only reduced in HK-2 cells. Neither HK-2 nor HRPTEpiC assumed an elongated, fibroblast-like morphology following IL-17A treatment. Conclusions: IL-17A directly mediates proximal tubule epithelial cell proinflammatory/profibrotic activity as demonstrated by the alteration in genes associated with extracellular matrix remodeling and cell-cell interaction, and stimulation of inflammatory mediator and immune cell chemoattractant secretion. Additionally, IL-17A may have a negative impact on barrier integrity as indicated by ZO-1 downregulation.