Experimental study on epithelial-mesenchymal-transition of peritoneal mesothelial cells induced by indoxyl sulfate

doi:10.3877/cma.j.issn.2095-1221.2017.03.006

Abstract

Abstract:

Objective

To induce epithelial mesenchymal transition (EMT) of immortalized human peritoneal mesothelial cell line (HMrSV5) with indoxyl sulfate (IS) and investigate the role of transforming growth factor (TGF-β1) .

Methods

HMrSV5 cells were randomly divided into 3 groups: the control group: with 4.25﹪peritoneal dialysate fluid in culture medium; IS groups: with IS at final concentrations oft 250μmol/L, 500μmol/L, and 1 000μmol/L and 4.25﹪peritoneal dialysate fluid; TGF-β1 inhibitor group: with 2μmol/L LY364947, IS and 4.25﹪peritoneal dialysate fluid. At 0 h, 4 h, 12 h, 24 h, 48 h, and 72 h, the morphological changes were observed by immunofluorescence method, and alpha smooth muscle actin (α-SMA) expression was evaluated with Western Blot. At 48 h, supernatant was collected to determinate fibronectin and laminin5 by ELISA.

Results

After IS induction, cells change from typical cobblestone-like shape to long fusiform shape, which was prevented but TGF-β1 inhibitor. Western Blot showed that: compared with control group the expression level ofα-SMA increased significantly in the IS treatment group. The level ofα-SMA was highest at 48 h with 1 000μmol/L IS (39.929±2.610vs0.996±0.001;P< 0.05) , which was decrease to 7.418±3.075 in the presence of TGF-β1 inhibitor (P< 0.05) .Compared with control group, IS increased the secretion of FN (1.368±0.040vs1.094±0.036, P< 0.05) and laminin 5 (1.031±0.085vs0.860±0.063,P< 0.05) , which were decreased by TGF-βinhibitor (0.925±0.15 and 0.795±0.426 respectively,P< 0.05) .

Conclusion

EMT of PMC could be induced by IS via TGF-β1 signaling pathway and is potential target for the treatment of peritoneal fibrosis.

Key words: Peritoneum, Stromal cells, Sulfuric acids, Indophenol, Epithelial-mesenchymal-transition, Transforming growth factor beta

Qiang Wang, Guoqing Yu, Yaqing Huang, Yunquan Fu, Xin Chen, Yongze Zhuang. Experimental study on epithelial-mesenchymal-transition of peritoneal mesothelial cells induced by indoxyl sulfate[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2017, 07(03): 152-158.

/ / Recommend

Add to citation manager EndNote|Ris|BibTeX

URL: https://zhxbygxbzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.2095-1221.2017.03.006

https://zhxbygxbzz.cma-cmc.com.cn/EN/Y2017/V07/I03/152

Figures/Tables 6

References 22

1	Del Peso G, Jiménez-Heffernan JA, Bajo MA, et al. Epithelial-to-mesenchymal transition of mesothelial cells is an early event during peritoneal dialysis and is associated with high peritoneal transport[J].Kidney Int Suppl, 2008(108):S26-S33.
2	Zhang J, Oh KH, Xu H, et al. Vascular endothelial growth factor expression in peritoneal mesothelial cells undergoing transdifferentiation[J]. Perit Dial Int, 2008, 28(5):497-504.
3	Ha HR, Yu MR, Lee HB. High glucose-induced PKC activation mediates TGF-beta 1 and fibronectin synthesis by peritoneal mesothelial cells[J]. Kidney Int, 2001, 59(2):463-470.
4	Leong SC, Sirich TL. Indoxyl Sulfate-Review of toxicity and therapeutic strategies[J]. Toxins (Basel), 2016, 8(12):358.
5	Lekawanvijit S, Adrahtas A, Kelly DJ, et al. Does indoxyl sulfate, a uraemic toxin, have direct effects on cardiac fibroblasts and myocytes?[J]. Eur Heart J, 2010, 31(14):1771-1779.
6	Dou L, Jourde-Chiche N, Faure V, et al. The uremic solute indoxyl sulfate induces oxidative stress in endothelial cells[J]. J Thromb Haemost, 2007, 5(6):1302-1308.
7	Muteliefu G, Enomoto A, Jiang P, et al. Indoxyl sulphate induces oxidative stress and the expression of osteoblast-specific proteins in vascular smooth muscle cells[J]. Nephrol Dial Transplant, 2009, 24(7):2051-2058.
8	Tumur Z, Niwa T. Indoxyl sulfate inhibits nitric oxide production and cell viability by inducing oxidative stress in vascular endothelial cells[J]. Am J Nephrol, 2009, 29(6):551-557.
9	Shimizu H, Hirose Y, Nishijima F, et al. ROS and PDGF-beta [corrected] receptors are critically involved in indoxyl sulfate actions that promote vascular smooth muscle cell proliferation and migration[J]. Am J Physiol Cell Physiol, 2009, 297(2):C389-C396.
10	Adijiang A, Higuchi Y, Nishijima F, et al. Indoxyl sulfate, a uremic toxin, promotes cell senescence in aorta of hypertensive rats[J]. Biochem Biophys Res Commun, 2010, 399(4):637-641.
11	Niwa T, Ise M. Indoxyl sulfate, a circulating uremic toxin, stimulates the progression of glomerular sclerosis[J]. J Lab Clin Med, 1994, 124(1):96-104.
12	Bolati D, Shimizu H, Higashiyama Y, et al. Indoxyl sulfate induces epithelial-to-mesenchymal transition in rat kidneys and human proximal tubular cells[J]. Am J Nephrol, 2011, 34(4):318-323.
13	Liu Q, Mao H, Nie J, et al. Transforming growth factor{beta}1 induces epithelial-mesenchymal transition by activating the JNK-Smad3 pathway in rat peritoneal mesothelial cells[J].Perit Dial Int, 2008, 28(S3):S88-S95.
14	Nagy JA. Peritoneal membrane morphology and function[J]. Kidney Int Suppl, 1996(56):S2-S11.
15	Lee HB, Ha H. Mechanisms of epithelial-mesenchymal transition of peritoneal mesothelial cells during peritoneal dialysis[J]. J Korean Med Sci, 2007, 22(6):943-945.
16	Viaene L, Meijers BK, Vanrenterghem Y, et al. Serum concentrations of p-Cresyl sulfate and indoxyl sulfate, but not inflammatory markers, increase in incident peritoneal dialysis patients in parallel with loss of residual renal function[J]. Perit Dial Int, 2015, 35(4):492.
17	Lin CJ, Wu V, Wu PC, et al. Meta-Analysis of the associations of p-Cresyl sulfate (PCS) and indoxyl sulfate (IS) with cardiovascular events and All-Cause Mortality in Patients with Chronic Renal Failure[J]. PLoS One, 2015, 10(7):e0132589.
18	Vanholder R, De Smet R, Glorieux G, et al. Review on uremic toxins: classification, concentration, and interindividual variability[J]. Kidney Int, 2003, 63(5):1934-1943.
19	Barisione C, Garibaldi S, Furfaro AL, et al. Moderate increase of indoxyl sulfate promotes monocyte transition into profibrotic macrophages[J]. PLoS One, 2016, 11(2):e0149276.
20	Miyazaki T, Ise M, Seo H, et al. Indoxyl sulfate increases the gene expressions of TGF-beta 1, TIMP-1 and pro-alpha 1(I)collagen in uremic rat kidneys[J]. 1997(62):S15-S22.
21	Lu Z, Zeng Y, Lu F, et al. Rhubarb enema attenuates renal tubulointerstitial fibrosis in 5/6 nephrectomized rats by alleviating indoxyl sulfate overload[J]. PLoS One, 2015, 10(12):e0144726.
22	Margetts PJ, Bonniaud P, Liu L, et al. Transient overexpression of TGF-{beta} 1 induces epithelial mesenchymal transition in the rodent peritoneum[J].J Am Soc Nephrol, 2005, 16(2):425-436.

分组	0 h	4 h	12 h	24 h	48 h	72 h
对照组	1.012±0.001	1.115±0.116	1.066±0.114	1.034±0.066	0.996±0.001	1.115±0.116
250 μmol/LIS	1.035±0.305	2.358±0.310^a	2.725±0.310^a	2.151±0.355	0.873±0.419	25.025±2.332^a
500 μmol/LIS	1.245±0.202	3.491±0.328^a	4.079±0.189^a	4.744±0.199^a	1.831±0.505	36.491±2.227^a
1 000 μmol/LIS	1.176±0.316	2.518±0.180^a	5.317±0.275^a	35.707±2.008^a	39.929±2.610^a	25.518±1.173^a
TGF-β1受体抑制剂组	0.821±0.265	1.871±0.535^b	4.305±0.240^b	14.738±2.364^b	7.418±3.075^b	1.871±0.535^b
F值	1.331	21.377	145.845	321.323	254.515	308.506
P值	0.324	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01

分组	0 h	4 h	12 h	24 h	48 h	72 h
对照组	1.012±0.001	1.115±0.116	1.066±0.114	1.034±0.066	0.996±0.001	1.115±0.116
250 μmol/LIS	1.035±0.305	2.358±0.310^a	2.725±0.310^a	2.151±0.355	0.873±0.419	25.025±2.332^a
500 μmol/LIS	1.245±0.202	3.491±0.328^a	4.079±0.189^a	4.744±0.199^a	1.831±0.505	36.491±2.227^a
1 000 μmol/LIS	1.176±0.316	2.518±0.180^a	5.317±0.275^a	35.707±2.008^a	39.929±2.610^a	25.518±1.173^a
TGF-β1受体抑制剂组	0.821±0.265	1.871±0.535^b	4.305±0.240^b	14.738±2.364^b	7.418±3.075^b	1.871±0.535^b
F值	1.331	21.377	145.845	321.323	254.515	308.506
P值	0.324	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01

分组	FN	LN-5
对照组	1.094±0.036	0.860±0.063
250 μmol/LIS	1.077±0.038	0.829±0.085
500 μmol/LIS	1.225±0.035	1.031±0.085^a
1 000 μmol/LIS	1.368±0.040^a	0.860±0.069
TGF-β1受体抑制剂组	0.925±0.154^c	0.795±0.426^b
F值	14.053	7.146
P值	0.000	0.003

分组	FN	LN-5
对照组	1.094±0.036	0.860±0.063
250 μmol/LIS	1.077±0.038	0.829±0.085
500 μmol/LIS	1.225±0.035	1.031±0.085^a
1 000 μmol/LIS	1.368±0.040^a	0.860±0.069
TGF-β1受体抑制剂组	0.925±0.154^c	0.795±0.426^b
F值	14.053	7.146
P值	0.000	0.003

[1]	Xining Wu, Peipei Zhang, Zhenhong Qi, Jing Zhang, Ruina Zhao, Hua Meng, Jianchu Li. Quality of ultrasonic diagnosis of retroperitoneal schwannomas and its influencing factors [J]. Chinese Journal of Medical Ultrasound (Electronic Edition), 2023, 20(07): 701-704.
[2]	Jianfeng Ouyang, Bingquan Li, Yongheng Ye, Shaoyu Hu, Yang Xiang. Effect of arthroscope combining platelet-rich plasma in treatment of adherent periarthritis of shoulder [J]. Chinese Journal of Joint Surgery(Electronic Edition), 2023, 17(06): 765-772.
[3]	Luyi Wang, Chenguang Du, Congxiao Wang, Yahui Liang, Xueying Lyu, Yao Zhang, Shuyan Qie. Effect of Duhuo-jisheng decoction on wind-cold-dampness Bi type knee osteoarthritis [J]. Chinese Journal of Joint Surgery(Electronic Edition), 2022, 16(06): 715-720.
[4]	Lei Chu, Yi Guo, Xiaowen Tong. Research status of periostin in gynecological malignancies [J]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2022, 18(02): 145-149.
[5]	Kang Yang, Dechen Wang, Zijie Yang, Mingbin Gui, Zengqiang Yang, Lianping Qu, Feng Gao. Current status and strategies of pelvic floor peritoneum reconstruction in radical resection for low rectal cancer [J]. Chinese Archives of General Surgery(Electronic Edition), 2023, 17(04): 316-320.
[6]	Tong Hui, Yinghui Chen, Xu Zhao. Prevention and management of difficulties and complications in transanal total mesorectal excision [J]. Chinese Journal of Operative Procedures of General Surgery(Electronic Edition), 2023, 17(05): 487-490.
[7]	Guanning Li, Junjie Yang, Zhenhuai Yang. Comparative study of two different sigmoidostomy in laparoscopic abdominoperineal resection of rectal cancer [J]. Chinese Journal of Operative Procedures of General Surgery(Electronic Edition), 2021, 15(05): 546-549.
[8]	Yangbai Lu, Wei Li, Yongxin Zhang, Yaqiang Huang, Bin Cao, Gang Luo, Runqiang Yuan, Hongxing Huang. The effectiveness and safety of "one scalpel and one scissors" method to establish laparoscopic observation access for transperitoneal laparoscopic surgery [J]. Chinese Journal of Endourology(Electronic Edition), 2021, 15(03): 192-197.
[9]	Kun Jiao, Xiaoju Chen, Jing Lu. Pathological evaluation of animal experiments with intraperitoneal abdominal hernia repair [J]. Chinese Journal of Hernia and Abdominal Wall Surgery(Electronic Edition), 2022, 16(06): 628-633.
[10]	Binbin Song, Yilin Zhu, Jichao Yang, Jie Chen. Effect of carbon dioxide pneumoperitoneum on electrocardiogram during laparoscopic inguinal hernia repair:A prospective randomized controlled study [J]. Chinese Journal of Hernia and Abdominal Wall Surgery(Electronic Edition), 2021, 15(02): 199-202.
[11]	Yan Gao, Lin Jin. Sensitivity and specificity of color Doppler ultrasound in the differential diagnosis of external abdominal external hernia [J]. Chinese Journal of Hernia and Abdominal Wall Surgery(Electronic Edition), 2021, 15(01): 40-43.
[12]	Liangliang Huang, Weidong Wu, Tingfeng Wang. The application of completely visualized cavity construction technique in posterior sheath single incision inguinal hernia repair [J]. Chinese Journal of Laparoscopic Surgery(Electronic Edition), 2023, 16(04): 216-219.
[13]	Yiheng Xue, Lei Wang, Shaochuan Sun, Yongsheng Jiang. Clinical study of the pelvic peritoneum reconstruction with biological mesh in laparoscopic APR [J]. Chinese Journal of Laparoscopic Surgery(Electronic Edition), 2021, 14(02): 70-74.
[14]	Tingting Qi, Xiaoming Xu, Yunlong Xia, Yongzhen Guo, Miaomiao Fan, Yuan He, Mai Chen, Ling Tao, Wenjun Yan, Yanhong Fan. Research progress of intravenous infusion of mesenchymal stromal cells in the treatment of ischemic heart disease [J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2022, 12(02): 105-109.
[15]	Xiangxing Kong, Yeting Hu, Kefeng Ding, Jianwei Wang. A case of pelvic huge solitary fibrous tumor with rectum and retroperitoneum invasion [J]. Chinese Journal of Colorectal Diseases(Electronic Edition), 2021, 10(03): 324-326.

Please choose a citation manager

Content to export