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Calcium oxalate crystals and oxalate induce an epithelial-to-mesenchymal transition in the proximal tubular epithelial cells: Contribution to oxalate kidney injury

View Article: PubMed Central - PubMed

ABSTRACT

TGF-β1 is the main mediator of epithelial-to-mesenchymal transition (EMT). Hyperoxaluria induces crystalluria, interstitial fibrosis, and progressive renal failure. This study analyzed whether hyperoxaluria is associated with TGF-β1 production and kidney fibrosis in mice and if oxalate or calcium oxalate (CaOx) could induce EMT in proximal tubule cells (HK2) and therefore contribute to the fibrotic process. Hyperoxaluria was induced by adding hydroxyproline and ethylene glycol to the mice’s drinking water for up to 60 days. Renal function and oxalate and urinary crystals were evaluated. Kidney collagen production and TGF-β1 expression were assessed. EMT was analyzed in vitro according to TGF-β1 production, phenotypic characterization, invasion, cell migration, gene and protein expression of epithelial and mesenchymal markers. Hyperoxaluric mice showed a decrease in renal function and an increase in CaOx crystals and Ox urinary excretion. The deposition of collagen in the renal interstitium was observed. HK2 cells stimulated with Ox and CaOx exhibited a decreased expression of epithelial as well as increased expression mesenchymal markers; these cells presented mesenchymal phenotypic changes, migration, invasiveness capability and TGF-β1 production, characterizing EMT. Treatment with BMP-7 or its overexpression in HK2 cells was effective at preventing it. This mechanism may contribute to the fibrosis observed in hyperoxaluria.

No MeSH data available.


Demonstration of epithelial-to-mesenchymal transition event through epithelial (E-cadherin and Cytokeratin) and mesenchymal markers (smooth muscle α-actin).Quantitative PCR analysis (A) and immunofluorescence images (FITC: green fluorescence, TRITC: red fluorescence and blue: nuclei) with their respective densitometric quantification using ImageJ software (B,C,D). We observed an increase in mesenchymal (smooth muscle α-actin) and a decrease in epithelial (cytokeratin and e-cadherin) marker expressions and fluorescence intensity in comparison to the control (CTL) situation in HK2-WT and HK2T + siRNA cells stimulated with potassium oxalate (Ox), calcium oxalate (CaOx) and TGF-β1. On the contrary, in HK2 receiving BMP-7 (HK2 + BMP-7) and HK2 cells overexpressing BMP-7 (HK2T) we did not observe a increase in mesenchymal (smooth muscle α-actin) and a decrease in epithelial (Cytokeratin and E-cadherin) markers. Data are presented as means ± standard errors. N = 15 for each group. (*) Significant different when compared to the control group at p < 0.05.
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f7: Demonstration of epithelial-to-mesenchymal transition event through epithelial (E-cadherin and Cytokeratin) and mesenchymal markers (smooth muscle α-actin).Quantitative PCR analysis (A) and immunofluorescence images (FITC: green fluorescence, TRITC: red fluorescence and blue: nuclei) with their respective densitometric quantification using ImageJ software (B,C,D). We observed an increase in mesenchymal (smooth muscle α-actin) and a decrease in epithelial (cytokeratin and e-cadherin) marker expressions and fluorescence intensity in comparison to the control (CTL) situation in HK2-WT and HK2T + siRNA cells stimulated with potassium oxalate (Ox), calcium oxalate (CaOx) and TGF-β1. On the contrary, in HK2 receiving BMP-7 (HK2 + BMP-7) and HK2 cells overexpressing BMP-7 (HK2T) we did not observe a increase in mesenchymal (smooth muscle α-actin) and a decrease in epithelial (Cytokeratin and E-cadherin) markers. Data are presented as means ± standard errors. N = 15 for each group. (*) Significant different when compared to the control group at p < 0.05.

Mentions: Figure 7A shows the quantitative analysis of mesenchymal e epithelial cell marker expression. There was a significant increase in mesenchymal marker smooth muscle actin (α-actin) expression and a decrease in epithelial markers expression, including E-cadherin and Cytokeratin, in HK2-WT cells treated with Ox, CaOx, and TGF-β1 compared to the control situation (Fig. 7A). In response to the overexpression of BMP-7 (HK2T) or previous treatment with BMP-7 (HK2 + BMP-7), the epithelial markers did not decrease, on the contrary, increased. The mesenchymal marker smooth muscle α-actin did not increase, on the contrary, decreased when HK2 cells were exposed to Ox, CaOx, or TGF-β1. The immunofluorescence figures and quantifications (Fig. 7B,C and D) corroborated the gene expression results of epithelial and mesenchymal markers. TGF-β1 is the main mediator of epithelial-to-mesenchymal transition. BMP-7 overexpression or treatment, significantly inhibited TGF-β1 expression. This result corroborates the epithelial and mesenchymal markers expression and protein synthesis observed in Fig. 7, showing that BMP-7 blunted EMT.


Calcium oxalate crystals and oxalate induce an epithelial-to-mesenchymal transition in the proximal tubular epithelial cells: Contribution to oxalate kidney injury
Demonstration of epithelial-to-mesenchymal transition event through epithelial (E-cadherin and Cytokeratin) and mesenchymal markers (smooth muscle α-actin).Quantitative PCR analysis (A) and immunofluorescence images (FITC: green fluorescence, TRITC: red fluorescence and blue: nuclei) with their respective densitometric quantification using ImageJ software (B,C,D). We observed an increase in mesenchymal (smooth muscle α-actin) and a decrease in epithelial (cytokeratin and e-cadherin) marker expressions and fluorescence intensity in comparison to the control (CTL) situation in HK2-WT and HK2T + siRNA cells stimulated with potassium oxalate (Ox), calcium oxalate (CaOx) and TGF-β1. On the contrary, in HK2 receiving BMP-7 (HK2 + BMP-7) and HK2 cells overexpressing BMP-7 (HK2T) we did not observe a increase in mesenchymal (smooth muscle α-actin) and a decrease in epithelial (Cytokeratin and E-cadherin) markers. Data are presented as means ± standard errors. N = 15 for each group. (*) Significant different when compared to the control group at p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384284&req=5

f7: Demonstration of epithelial-to-mesenchymal transition event through epithelial (E-cadherin and Cytokeratin) and mesenchymal markers (smooth muscle α-actin).Quantitative PCR analysis (A) and immunofluorescence images (FITC: green fluorescence, TRITC: red fluorescence and blue: nuclei) with their respective densitometric quantification using ImageJ software (B,C,D). We observed an increase in mesenchymal (smooth muscle α-actin) and a decrease in epithelial (cytokeratin and e-cadherin) marker expressions and fluorescence intensity in comparison to the control (CTL) situation in HK2-WT and HK2T + siRNA cells stimulated with potassium oxalate (Ox), calcium oxalate (CaOx) and TGF-β1. On the contrary, in HK2 receiving BMP-7 (HK2 + BMP-7) and HK2 cells overexpressing BMP-7 (HK2T) we did not observe a increase in mesenchymal (smooth muscle α-actin) and a decrease in epithelial (Cytokeratin and E-cadherin) markers. Data are presented as means ± standard errors. N = 15 for each group. (*) Significant different when compared to the control group at p < 0.05.
Mentions: Figure 7A shows the quantitative analysis of mesenchymal e epithelial cell marker expression. There was a significant increase in mesenchymal marker smooth muscle actin (α-actin) expression and a decrease in epithelial markers expression, including E-cadherin and Cytokeratin, in HK2-WT cells treated with Ox, CaOx, and TGF-β1 compared to the control situation (Fig. 7A). In response to the overexpression of BMP-7 (HK2T) or previous treatment with BMP-7 (HK2 + BMP-7), the epithelial markers did not decrease, on the contrary, increased. The mesenchymal marker smooth muscle α-actin did not increase, on the contrary, decreased when HK2 cells were exposed to Ox, CaOx, or TGF-β1. The immunofluorescence figures and quantifications (Fig. 7B,C and D) corroborated the gene expression results of epithelial and mesenchymal markers. TGF-β1 is the main mediator of epithelial-to-mesenchymal transition. BMP-7 overexpression or treatment, significantly inhibited TGF-β1 expression. This result corroborates the epithelial and mesenchymal markers expression and protein synthesis observed in Fig. 7, showing that BMP-7 blunted EMT.

View Article: PubMed Central - PubMed

ABSTRACT

TGF-&beta;1 is the main mediator of epithelial-to-mesenchymal transition (EMT). Hyperoxaluria induces crystalluria, interstitial fibrosis, and progressive renal failure. This study analyzed whether hyperoxaluria is associated with TGF-&beta;1 production and kidney fibrosis in mice and if oxalate or calcium oxalate (CaOx) could induce EMT in proximal tubule cells (HK2) and therefore contribute to the fibrotic process. Hyperoxaluria was induced by adding hydroxyproline and ethylene glycol to the mice&rsquo;s drinking water for up to 60 days. Renal function and oxalate and urinary crystals were evaluated. Kidney collagen production and TGF-&beta;1 expression were assessed. EMT was analyzed in vitro according to TGF-&beta;1 production, phenotypic characterization, invasion, cell migration, gene and protein expression of epithelial and mesenchymal markers. Hyperoxaluric mice showed a decrease in renal function and an increase in CaOx crystals and Ox urinary excretion. The deposition of collagen in the renal interstitium was observed. HK2 cells stimulated with Ox and CaOx exhibited a decreased expression of epithelial as well as increased expression mesenchymal markers; these cells presented mesenchymal phenotypic changes, migration, invasiveness capability and TGF-&beta;1 production, characterizing EMT. Treatment with BMP-7 or its overexpression in HK2 cells was effective at preventing it. This mechanism may contribute to the fibrosis observed in hyperoxaluria.

No MeSH data available.