Junping Hu

Stem Cell Conditioned Culture Media Attenuated Albumin-Induced Epithelial-Mesenchymal Transition in Renal Tubular Cells

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 a-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.

Hypoxia inducible factor-1α mediates the profibrotic effect of albumin in renal tubular cells

Proteinuria is closely associated with the progression of chronic kidney diseases (CKD) by producing renal tubulointerstitial fibrosis. Over-activation of hypoxia inducible factor (HIF)-1α has been implicated in the progression of CKD. The present study tested the hypothesis that HIF-1α mediates albumin-induced profibrotic effect in cultured renal proximal tubular cells. Incubation of the cells with albumin (40 μg/ml) for 72 hrs significantly increased the protein levels of HIF-1α, tissue inhibitor of metalloproteinase (TIMP)-1 and collagen-I, which were blocked by HIF-1α shRNA. Albumin also stimulated an epithelial-mesenchymal transition (EMT) as indicated by the decrease in epithelial marker E-cadherin, and the increase in mesenchymal markers α-smooth muscle actin and fibroblast-specific protein 1. HIF-1α shRNA blocked albumin-induced changes in these EMT markers as well. Furthermore, albumin reduced the level of hydroxylated HIF-1α, indicating an inhibition of the activity of prolyl-hydroxylases, enzymes promoting the degradation of HIF-1α. An anti-oxidant ascorbate reversed albumin-induced inhibition of prolyl-hydroxylase activity. Overexpression of prolyl-hydroxylase 2 (PHD2) transgene, a predominant isoform of PHDs in renal tubules, to reduce HIF-1α level significantly attenuated albumin-induced increases in TIMP-1 and collagen-I levels. These results suggest that albumin-induced oxidative stress inhibits PHD activity to accumulate HIF-1α, which mediates albumin-induced profibrotic effects in renal tubular cells.

Inhibition of microrna-429 in the renal medulla increased salt sensitivity of blood pressure in Sprague Dawley rats

Background: We have previously shown that high salt intake suppresses the expression of prolyl hydroxylase domain-containing protein 2 (PHD2), an enzyme promoting the degradation of hypoxia-inducible factor (HIF)-1&agr;, and increases HIF-1&agr; along with its target genes in the renal medulla, which promotes sodium excretion and regulates salt sensitivity of blood pressure. However, it remains unknown how high salt inhibits the expression of PHD2. Method and results: The current study first revealed that high-salt-induced PHD2 inhibition was due to the enhanced decay of mRNA. We then found that high salt significantly increased the expression of miR-429, which was subsequently proven to target the 3′-untranslated region of PHD2 and reduce PHD2 levels, in the renal medulla. To define the functional role of renal medullary miR-429 in the regulation of PHD2/HIF-1&agr;-mediated renal adaptation to high salt intake and salt sensitivity of blood pressure, we locally inhibited miR-429 in the renal medulla by locked nucleic acid anti-miR-429 in uninephrectomized rats. Our results demonstrated that inhibition of miR-429 remarkably increased the levels of PHD2, which disrupted PHD2-associated adaptive activation of HIF-1&agr;-mediated gene expression in response to high salt in the renal medulla and consequently inhibited urinary sodium excretion, enhanced sodium retention in response to chronic sodium overloading, and as a result, produced a salt-sensitive hypertension. Conclusion: It is concluded that miR-429 is an important upstream mediator in PHD2/HIF-1&agr;-associated renal adaptation to high salt intake and that deficiency in miR-429-mediated PHD2 inhibition in response to high salt in the renal medulla may represent a pathogenic mechanism for salt-sensitive hypertension.