Indoxyl sulfate induces transdifferentiation of peritoneal mesenchymal cells through TGF-β1

doi:10.3877/cma.j.issn.2095-1221.2022.06.002

Abstract

Abstract:

Objective

To investigate the role of transforming growth factor (TGF-β1) in the induction of indoxyl sulfate in the transdifferentiation of peritoneal mesenchymal cells.

Methods

Human peritoneal mesenchymal cell line HMrSV5 was cultured in vitro using RPMI1640 medium containing 10% fetal bovine serum. 1 000 μmol/L indoxyl sulfate and 4.25% peritoneal dialysate (PDF) were added into the medium, respectively. 2 μmol/L TGF-β1 receptor inhibitor LY364947 (TRI) +PDF, TRI+1 000 μmol/ L indoxyl sulfate co-culture. The morphologic changes of cells at 0, 4, 24, 48 and 72 h were observed by immunofluorescence method. The relative expression levels of TGF-β1, Twist1 and E-cadherin at each time point were measured by qRT-PCR method. One-way analysis of variance was used for comparison between multiple groups, and SNK-q test was used for pairwise comparison between different groups.

Results

After the stimulation of indoxyl sulfate, the cells became thin and long like paving stones, but the changes were not obvious after the intervention of TRI +1 000 μmol/L indoxyl sulfate. Immunofluorescence staining showed that the expression of alpha-smooth muscle actin (α-SMA) was increased from 24 h after indoxyl sulfate intervention, and the expression was most obvious at 48 h, while the expression of α-SMA was decreased after indoxyl sulfate+TRI intervention at the same time. The results of qRT-PCR showed that compared with the control, the relative expression of TGF-β1 and Twist (1.03±0.01, 1.07±0.01 vs 0.28±0.01) gene was increased after 48 h intervention with indoxyl sulfate or PDF. E-cadherin (0.86± 0.02, 0.88± 0.02 vs 3.34±0.02) gene expression was decreased (P < 0.05) ; Compared with indoxyl sulfate culture medium, the relative gene expression of TGF-β1 and Twist1 (0.14±0.00 vs 1.03±0.01) was decreased after being added with indoxyl sulfate+TRI culture for 48 h. The relative expression level of E-cadherin (1.69±0.03 vs 0.86±0.02) was increased, and the difference was statistically significant (P < 0.05) . Compared with PDF culture, TGF-β1 and Twist1 48 h (0.14±0.00 vs 1.07±0.01) gene relative expression levels were decreased after adding PDF+TRI culture for 48 h. The relative expression level of E-cadherin (1.80±0.03 vs 0.88±0.02) was increased, and the difference was statistically significant (P < 0.05) .

Conclusion

Indoxyl sulfate can promote EMT through upregulation of TGF-β1 gene expression, and Twist1 was involved in the process induced by indoxyl sulfate in peritoneal mesenchymal cells.

Key words: Epithelial-mesenchymal transition, Indoxol sulfate, Transforming growth factor-β1, Twist1

Qiang Wang, Xin Chen, Xiaoxue Weng, Yongze Zhuang, Guoqing Yu. Indoxyl sulfate induces transdifferentiation of peritoneal mesenchymal cells through TGF-β1[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2022, 12(06): 329-334.

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URL: https://zhxbygxbzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.2095-1221.2022.06.002

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Figures/Tables 6

References 16

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基因	序列（5'→3'）	预期扩增产物长度（bp）	退火温度（℃）
Twist1	上游GTCCGCGTCCCACTAGC	90	59.72
	下游TCCATTTTCTCCTTCTCTGGAA		52.79
E-cadherin	上游GCCGAGAGCTACACGTTCAC	117	58.91
	下游GTCGAGGGAAAAATAGGCTG		53.27
TGF-β1	上游CCCTGGACACCAACTATTGC	92	56.27
	下游CTTCCAGCCGAGGTCCTT		57.57
hGAPDH	上游GGCTGAGAACGGGAAGCTTGTCAT	110	62.05
	下游CAGCCTTCTCCATGGTGGTGAAGA		61.51

基因	序列（5'→3'）	预期扩增产物长度（bp）	退火温度（℃）
Twist1	上游GTCCGCGTCCCACTAGC	90	59.72
	下游TCCATTTTCTCCTTCTCTGGAA		52.79
E-cadherin	上游GCCGAGAGCTACACGTTCAC	117	58.91
	下游GTCGAGGGAAAAATAGGCTG		53.27
TGF-β1	上游CCCTGGACACCAACTATTGC	92	56.27
	下游CTTCCAGCCGAGGTCCTT		57.57
hGAPDH	上游GGCTGAGAACGGGAAGCTTGTCAT	110	62.05
	下游CAGCCTTCTCCATGGTGGTGAAGA		61.51

分组	0 h	4 h	24 h	48 h	72 h
对照	0.10±0.00	0.22±0.01	0.32±0.00	0.34±0.00	0.48±0.01
硫酸吲哚酚	0.10±0.01	0.42±0.01^a	0.51±0.01^a	1.42±0.02^a	1.26±0.02^a
PDF	0.11±0.00	0.42±0.01^a	0.51±0.01^a	1.60±0.03^a	1.54±0.02^a
硫酸吲哚酚+TRI	0.10±0.00	0.21±0.00^bc	0.22±0.00^abc	0.51±0.01^abc	0.50±0.01^b
PDF+TRI	0.10±0.00	0.22±0.01^bc	0.23±0.00^abc	0.48±0.01^abc	0.46±0.01^bc
F值	3.094	1081.438	3023.632	5010.075	4881.937
P值	0.067	< 0.001	< 0.001	< 0.001	< 0.001

分组	0 h	4 h	24 h	48 h	72 h
对照	0.10±0.00	0.22±0.01	0.32±0.00	0.34±0.00	0.48±0.01
硫酸吲哚酚	0.10±0.01	0.42±0.01^a	0.51±0.01^a	1.42±0.02^a	1.26±0.02^a
PDF	0.11±0.00	0.42±0.01^a	0.51±0.01^a	1.60±0.03^a	1.54±0.02^a
硫酸吲哚酚+TRI	0.10±0.00	0.21±0.00^bc	0.22±0.00^abc	0.51±0.01^abc	0.50±0.01^b
PDF+TRI	0.10±0.00	0.22±0.01^bc	0.23±0.00^abc	0.48±0.01^abc	0.46±0.01^bc
F值	3.094	1081.438	3023.632	5010.075	4881.937
P值	0.067	< 0.001	< 0.001	< 0.001	< 0.001

分组	0 h	4 h	24 h	48 h	72 h
对照	0.08±0.01	0.09±0.00	0.15±0.00	0.28±0.01	0.68±0.01
硫酸吲哚酚	0.07±0.00	0.16±0.00^a	0.20±0.00^a	1.03±0.01^a	0.98±0.01^a
PDF	0.08±0.00	0.17±0.00^a	0.19±0.00^a	1.07±0.01^a	1.00±0.04^a
硫酸吲哚酚+TRI	0.07±0.00	0.08±0.01^bc	0.15±0.00^bc	0.14±0.00^abc	0.14±0.00^abc
PDF+TRI	0.07±0.00	0.08±0.00^bc	0.14±0.00^bc	0.14±0.00^abc	0.14±0.00^abc
F值	2.860	274.909	617.471	12058.420	1886.263
P值	0.081	< 0.001	< 0.001	< 0.001	< 0.001

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