Abstract
BackgroundThe objective of this study was to investigate the influence of β2-microglobulin (β2-M) on the epithelial-mesenchymal transition (EMT) in renal tubular epithelial cells.MethodsA human kidney proximal tubular cell line (HK-2) was used as the proximal tubular cell model. HK-2 cells were exposed to different concentrations of β2-M (5, 10, 25, and 50 μM) for up to 24, 48 and 72 h. The effects of β2-M on cell morphology were observed by phase contrast microscopy, and the possible associated mechanisms were assessed by immunofluorescence staining, western blot, RNA interference, immunoprecipitation, and induced coupled plasma mass spectroscopy.Resultsβ2-M induced marked morphological alterations in the HK-2 cells, accompanied by the increased expression of extracellular matrix components and α-smooth muscle actin (α-SMA), vimentin and fibronectin and the reduced expression of E-cadherin. Our results also revealed that β2-M could induce the EMT in the HK-2 cells without significant affecting cell viability. Excess β2-M in the HK-2 cells led to a decrease in iron and an increase in hypoxia inducible factor-1α (HIF-1α), which induced EMT in the HK-2 cells. Additionally, disrupting the function of the β2-M/hemochromatosis (HFE) complex by HFE knockdown was sufficient to reverse β2-M-mediated EMT in the HK-2 cells.ConclusionThese findings demonstrate that the activity of β2-M is mediated by the β2-M/HFE complex, which regulates intracellular iron homeostasis and HIF-1α and ultimately induces EMT in HK2 cells.
Highlights
The objective of this study was to investigate the influence of β2-microglobulin (β2-M) on the epithelial-mesenchymal transition (EMT) in renal tubular epithelial cells
Effect of β2-M on Proximal tubular epithelial cells (PTEC) cellular viability Considering the absorbance at 490 nm in the control group as 1, the exposure of HK-2 cells to different concentrations of β2-M (5, 10, and 25 μM) produced no significant effects on cell viability at 24, 48 or 72 h; a significant decrease in cell viability (>20%) was observed at the higher doses of ≥50 μM β2-M after 48 h (Figure 1)
We observed that in HK-2 cells exposed to 25 μM of β2-M, the expression of E-cadherin decreased after 72 h of treatment, while that of α-smooth muscle actin (α-SMA), vimentin, and fibronectin increased, as indicated by western blot analysis (Figure 3A and B), These results suggest that these cells lost their epithelial characteristics and acquired mesenchymal cell properties
Summary
The objective of this study was to investigate the influence of β2-microglobulin (β2-M) on the epithelial-mesenchymal transition (EMT) in renal tubular epithelial cells. Protein overload models as well as animal models of proteinuria induced by renal injury have suggested that excessive protein reabsorption induces EMT in tubular cells [6,7]. In vitro experiments β2-microglobulin (β2-M) is an 11-kDa nonglycosylated protein with no transmembrane domain that usually associates with cells by interacting with the extracellular regions of heavy chains. It is typically filtered through the renal glomeruli, and 99% of it is absorbed and catalyzed by the renal tubular cells, while the non-absorbed portion is excreted in the urine. Many studies have demonstrated that the serum or urine β2-M concentration is increased in a variety of diseases, including inflammatory or infectious diseases [11,12], prostate cancer, lung cancer, and
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