Stem cell conditioned culture media attenuated albumin-induced epithelial-mesenchymal transition in renal tubular cells

Abstract

Background: Proteinuria-induced epithelial-mesenchymal transition (EMT) plays an important role in progressive renal tubulointerstitial fibrosis in chronic renal disease. Stem cell therapy has been used for different diseases. Stem cell conditioned culture media (SCM) exhibits similar beneficial effects as stem cell therapy. The present study tested the hypothesis that SCM inhibits albumin-induced EMT in cultured renal tubular cells.

Methods: Rat renal tubular cells were treated with/without albumin (20 µmg/ml) plus SCM or control cell media (CCM). EMT markers and inflammatory factors were measured by Western blot and fluorescent images.

Results: Albumin induced EMT as shown by significant decreases in levels of epithelial marker E-cadherin, increases in mesenchymal markers fibroblast-specific protein 1 and α-smooth muscle actin, and elevations in collagen I. SCM inhibited all these changes. Meanwhile, albumin induced NF-κB translocation from cytosol into nucleus and that SCM blocked the nuclear translocation of NF-κB. Albumin also increased the levels of pro-inflammatory factor monocyte chemoattractant protein-1 (MCP)-1 by nearly 30 fold compared with control. SCM almost abolished albumin-induced increase of MCP-1.

Conclusion: These results suggest that SCM attenuated albumin-induced EMT in renal tubular cells via inhibiting activation of inflammatory factors, which may serve as a new therapeutic approach for chronic kidney diseases.

Fig. 1

Effect of SCM on albumin-induced decrease in epithelial marker E-cadherin by Western blot analysis. Upper panel: Representative gel documents; Lower Panel: summarized data. Ctrl = control cells treated with CCM, Alb = cells treated with albumin + CCM, Alb+SCM = cells treated with albumin + SCM. n=6 batches of cells, *P<0.05 vs. other groups.

Fig. 2

Effect of SCM on albumin-induced increase in mesenchymal marker FSP-1 by Western blot analysis. Upper panel: Representative gel documents; Lower panel: summarized data. Ctrl = control cells treated with CCM, Alb = cells treated with albumin + CCM, Alb+SCM = cells treated with albumin + SCM. n=6 batches of cells, *P<0.05 vs. other groups.

Fig. 3

Effect of SCM on albumin-induced increase in mesenchymal marker α-SMA by Western blot analysis. Upper panel: Representative gel documents; Lower panel: summarized data. Ctrl = control cells treated with CCM, Alb = cells treated with albumin + CCM, Alb+SCM = cells treated with albumin + SCM. n=6 batches of cells, *P<0.05 vs. other groups.

Fig. 4

Effect of SCM on albumin-induced changes of staining patterns in E-cadherin, FSP-1 and α-SMA by immunofluorescent microscopy assay. Upper panel: Representative confocal images showing the immunostaining of E-cadherin, FSP-1 and α-SMA; Lower panel: Summarized integrated optical intensity of the fluorescent staining. Ctrl = control cells treated with CCM, Alb = cells treated with albumin + CCM, Alb+SCM = cells treated with albumin + SCM. n=5 batches of cells, *P<0.05 vs. other groups.

Fig. 5

Effect of SCM on albumin-induced increase in collagen I by Western blot analysis. Upper panel: Representative gel documents; Lower panel: Summarized data. Ctrl = control cells treated with CCM, Alb = cells treated with albumin + CCM, Alb+SCM = cells treated with albumin + SCM. n=6 batches of cells, *P<0.05 vs. other groups.

Fig. 6

Effect of SCM on albumin-induced increase in NF-κB-p65 in nuclear proteins by Western blot analysis. Upper panel: Representative gel documents; Lower panel: Summarized data. Ctrl = control cells treated with CCM, Alb = cells treated with albumin + CCM, Alb+SCM = cells treated with albumin + SCM. n=6 batches of cells, *P<0.05 vs. other groups.

Fig. 7

Effect of SCM on albumin-induced changes of staining patterns in NF-κB-p65 by immunofluorescent microscopy assay. Representative confocal images showing the immunostaining of nuclear marker (green) and NF-κB-p65 (red) (from 5 batches of cells). Control = control cells treated with CCM, Albumin = cells treated with albumin + CCM, Albumin+SCM = cells treated with albumin + SCM.

Fig. 8

Effect of SCM on albumin-induced increase in MCP-1. Upper panel: mRNA levels by real-time RT-PCR analysis; Lower panel: Protein levels by Western blot analysis. Ctrl = control cells treated with CCM, Alb = cells treated with albumin + CCM, Alb+SCM = cells treated with albumin + SCM. n=4–6 batches of cells, *P<0.05 vs. other groups.