An experimental study was conducted to examine the impact of activated collagen/DDR2 signaling on the migration and proliferation of kidney fibroblasts in renal interstitial fibrosis

doi:10.3877/cma.j.issn.2095-1221.2024.05.005

Abstract

Abstract:

Objective

To investigate the effect of collagen/DDR2 signaling on the proliferation and migration of renal fibroblasts during renal interstitial fibrosis.

Methods

A renal tubulointerstitial fibrosis model was established in SPF male C57BL/6 mice via unilateral ureteral obstruction （UUO）， and pathological samples and fresh tissues were obtained for Western blotting and immunohistochemistry. Human kidney fibroblasts were cultivated， followed by the transfection of KFB cells with shDDR2 and its negative control lentivirus， OE-DDR2 and its negative control lentivirus， and screening with 5 μg/mL puromycin. A stable transfection cell line was then created and exposed to 10 μg/mL collagen for 48 hours. Control Group 1： shCtrl + Collagen group （shDDR2 negative control， without downregulation of DDR2 expression but with collagen treatment）； Control Group 2： Control + Collagen group （OE-DDR2 negative control， without overexpression of DDR2 but with collagen treatment）； Experimental Group 1： shDDR2 + Collagen group （DDR2 gene knockdown with collagen treatment）； Experimental Group 2： OE-DDR2 + Collagen group （DDR2 gene overexpression with collagen treatment）； Experimental Group 3： Based on Experimental Group 2， treated with 1 μmol/L dasatinib， with all groups being treated for 48 hours. The expression level of DDR2 mRNA was detected by RT-qPCR， and the level of DDR2 protein was assessed by Western blot. Cell proliferation was evaluated using the CCK-8 assay and the BrdU assay. Cell localization and pathological changes were observed by HE staining and immunohistochemical staining methods. Comparisons between two groups were performed using independent samples t-test， and the t' test was used when variances were unequal. For comparisons among multiple groups，one-way ANOVA was applied， with LSD-t test used for pairwise comparisons when variances were homogeneous， and Tamhane's T2 test was used when variances were heterogeneous.

Results

DDR2 is localized in renal stromal fibroblasts in mouse kidney tissue， and has a colocalization relationship with collagen. Compared with the shCtrl+collagen group， the cell proliferation rate （0.28 ± 0.00 vs 0.38 ± 0.01） in the sh-DDR2+ collagen group slowed down （P＜ 0.05）， and the transwell transfer rate （6.00 ± 1.00 vs 21.66 ± 0.57） decreased （P＜ 0.05）. Compared with the Control+collagen group， the cell proliferation rate of the OE-DDR2+ collagen group （0.43 ± 0.01 vs 0.39 ± 0.01） was accelerated （P＜ 0.05）， and the transwell mobility rate （32.66 ± 0.57 vs 22.33 ± 0.57） was increased（P＜ 0.05）. After the addition of dasatinib， the cell proliferation rate （0.36 ± 0.00 vs 0.39 ± 0.01）was significantly reduced.

Conclusion

Collagen/DDR2 signaling activation significantly promoted the proliferation of KFB cells and inhibited apoptosis， ultimately promoting the occurrence and development of renal interstitial fibrosis.

Key words: Renal interstitial fibrosis, Myofibroblast, Collagen, DDR2, Therapeutic target

Huiyan Chen, Yao Wu, Zongxuan Huang, Xin Bo, Qinghui Wang, Huitao Ji, Yinzhen Chen, Hu Zhao. An experimental study was conducted to examine the impact of activated collagen/DDR2 signaling on the migration and proliferation of kidney fibroblasts in renal interstitial fibrosis[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2024, 14(05): 294-302.

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URL: https://zhxbygxbzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.2095-1221.2024.05.005

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Figures/Tables 9

References 32

1	Wang L， Xu X， Zhang M， et al. Prevalence of chronic kidney disease in China： results from the sixth China chronic disease and risk factor surveillance［J］. J AMA Intern Med， 2023， 183（4）：298-310.
2	Li R，Guo Y， Zhang Y， et al. Salidroside ameliorates renal interstitial fibrosis by inhibiting theTLR4/NF-κB and MAPK signaling pathways［J］.Int J Mol Sci， 2019， 20（5）：1103. doi：10.3390/ijms20051103.
3	Cui D， Liu S， Tang M， et al. Phloretin ameliorates hyperuricemiainduced chronie renal dysfunetion through inhibiting NLRP3 inflammasome and urie acid reabsorption［J］ Phytomedicine， 2020，66：153111.
4	Li Q， Ming Y， Jia H， et al. Poricoie acid a suppresses TGF-B1-induced renal fibrosis and proliferation via the PDGF-C， Smad3 and MAPK pathways［J］.Exp Ther Med， 2021， 21（4）：289. doi：10.3892/etm.2021.9720.
5	雷迪，郑楠薪，隋明星. 自噬细胞在肾脏移植中的研究进展［J/OL］.中华细胞与干细胞杂志（电子版）， 2019， 9（5）：314-318.
6	Tang PM， Nikolic-Paterson DJ， Lan HY. Macrophages： versatile players in renal inflammation and fibrosis［J］. Nat Rev Nephrol， 2019，15（3）：144-158.
7	van Raalte DH， Bjornstad P， Persson F， et al. The impact of sotagliflozin on renal function， albuminuria， blood pressure， and hematocrit in adults with type 1 diabetes［J］. Diabetes Care， 2019， 42（10）：1921-1929.
8	Carafoli F， Hohenester E. Collagen recognition and transmembrane signalling by discoidin domain receptors［J］. Biochim Biophys Acta，2013， 1834（10）：2187-2194.
9	Borza CM， Bolas G， Zhang X， et al. The collagen receptor discoidin domain receptor 1b enhances integrin β1-mediated cell migration by interacting with talin and promoting rac1 activation［J］. Front Cell Dev Biol， 2022， 10：836797. doi：10.3389/fcell.2022.836797.
10	Xiao L， Liu C， Wang B， et al. Targeting discoidin domain receptor 2 for the development of disease-modifying osteoarthritis drugs［J］. Cartilage，2021， 13（2 Suppl）：1285S-1291S.
11	Zhao H， Bian H， Bu X， et al. Targeting of discoidin domain receptor 2 （DDR2） prevents myofibroblast activation and neovessel formation during pulmonary fibrosis［J］. Mol Ther， 2016， 24（10）：1734-1744.
12	Day E， Waters B， Spiegel K， et al. Inhibition of collagen-induced discoidin domain receptor 1 and 2 activation by imatinib， nilotinib and dasatinib［J］. Eur J Pharmacol， 2008， 599（1-3）：44-53.
13	Hammerman PS， Sos ML， Ramos AH，et al. Mutations in the DDR2 kinase gene identify a novel therapeutic target in squamous cell lung cancer［J］. Cancer Discov， 2011， 1（1）：78-89.
14	Kolb EA， Gorlick R， Houghton PJ， et al. Initial testing of dasatinib by the pediatric preclinical testing program［J］. Pediatr Blood Cancer，2008， 50（6）：1198-206.
15	Weber EW， Lynn RC， Sotillo E， et al. Pharmacologic control of CAR-T cell function using dasatinib［J］. Blood Adv， 2019， 3（5）：711-717.
16	李倩倩，李小丽，王变丽，等.盘状结构域受体2 在肿瘤微环境中的作用研究进展［J］.中国肿瘤， 2023， 32（10）：773-779.
17	Luczynski MT， Harrison PT， Lima N， et al. Spatial localisation of Discoidin Domain Receptor 2 （DDR2） signalling is dependent on its collagen binding and kinase activity［J］. Biochem Biophys Res Commun， 2018， 501（1）：124-130.
18	马园园，刘成海，陶艳艳. 肾纤维化动物模型特点与研究进展［J］.中国实验动物学报， 2018， 26（3）：398-403.
19	薛翔，李世军.急性肾损伤进展为慢性肾脏病的机制［J］.肾脏病与透析肾移植杂志， 2023， 32（1）：74-78.
20	杨志超，杨建军，马军，等.改良地黄散抑制TGF-β1-CTGF 信号通路改善肾间质纤维化的研究［J］.南京中医药大学学报， 2019， 35（1）：47-52.
21	Chen L， Li X， Deng Y， et al. The PI3K-Akt-mTOR pathway mediates renal pericyte-myofibroblast transition by enhancing glycolysis through HKII［J］. J Transl Med， 2023， 21（1）：323. doi： 10.1186/s12967-023-04167-7.
22	Guerrot D， Kerroch M， Placier S， et al. Discoidin domain receptor 1 is a major mediator of inflammation and fibrosis in obstructive nephropathy［J］. Am J Pathol， 2011， 179（1）：83-91.
23	Genovese F， Manresa AA， Leeming DJ， et al. The extracellular matrix in the kidney： a source of novel non-invasive biomarkers of kidney fibrosis?［J］. Fibrogenesis Tissue Repair， 2014，7（1）：4. doi：10.1186/1755-1536-7-4.
24	Zeisberg M， Bonner G， Maeshima Y， et al. Renal fibrosis：collagen composition and assembly regulates epithelial-mesenchymal transdifferentiation［J］. Am J Pathol， 2001， 159（4）：1313-1321.
25	Leitinger B. Discoidin domain receptor functions in physiological and pathological conditions［J］. Int Rev Cell Mol Biol， 2014， 310：39-87.
26	Xie B， Lin W， Ye J， et al. DDR2 facilitates hepatocellular carcinoma invasion and metastasis via activating ERK signaling and stabilizing SNAIL1［J］. J Exp Clin Cancer Res， 2015， 34（1）：101. doi： 10.1186/s13046-015-0218-6.
27	Gonzalez ME， Martin EE， Anwar T， et al. Mesenchymal stem cellinduced DDR2 mediates stromal-breast cancer interactions and metastasis growth［J］. Cell Rep， 2017， 18（5）：1215-1228.
28	Vogel W， Gish GD， Alves F， et al. The discoidin domain receptor tyrosine kinases are activated by collagen［J］. Mol Cell， 1997， 1（1）：13-23.
29	Li T， Liu J， Cai H， et al. Incorporation of DDR2 clusters into collagen matrix via integrin-dependent posterior remnant tethering［J］. Int J Biol Sci， 2018， 14（6）：654-666.
30	Feng Y， Cai H， Huang X， et al. Active MT1-MMP is tethered to collagen fibers in DDR2-containing remnants［J］. Gene， 2021，788：145673. doi： 10.1016/j.gene.2021.145673.
31	Liang Z， Xie WJ， Zhao M， et al. DDR2 facilitates papillary thyroid carcinoma epithelial mesenchymal transition by activating ERK2/Snail1 pathway［J］. Oncol Lett， 2017， 14（6）：8114-8121.
32	Iwai LK， Chang F， Huang PH. Phosphoproteomic analysis identifies insulin enhancement of discoidin domain receptor 2 phosphorylation［J］.Cell Adh Migr， 2013， 7（2）：161-164.

基因	序列号	序列（5'→3'）	预期扩增长度（bp）	溶解温度（℃）
Mouse DDR2	NM_022563.2	上游TCATCCTGTGGAGGCAGTTCTG	143	60
		下游CTGTTCACTTGGTGATGAGGAGC
Mouse GAPDH	NM_001411840.1	上游CATCACTGCCACCCAGAAGACTG	153	60
		下游ATGCCAGTGAGCTTCCCGTTCAG

基因	序列号	序列（5'→3'）	预期扩增长度（bp）	溶解温度（℃）
Mouse DDR2	NM_022563.2	上游TCATCCTGTGGAGGCAGTTCTG	143	60
		下游CTGTTCACTTGGTGATGAGGAGC
Mouse GAPDH	NM_001411840.1	上游CATCACTGCCACCCAGAAGACTG	153	60
		下游ATGCCAGTGAGCTTCCCGTTCAG

分组	实验次数	细胞增殖		细胞迁移（个）
分组	实验次数	CCK-8实验	BrdU实验	细胞迁移（个）
shCtrl+胶原	3	0.38±0.01	0.33±0.01	21.66±0.57
shDDR2+胶原	3	0.28±0.00^a	0.25±0.01^a	6.00±1.00^a
Control+胶原	3	0.39±0.01	0.33±0.01	22.33±0.57
OE-DDR2+胶原	3	0.43±0.01^b	0.36±0.01^b	32.66±0.57^b
OE-DDR2+胶原+达沙替尼	3	0.36±0.01^c	0.29±1.25^c	12.66±0.57^c
F值		115.649	67.921	665.500
P值		＜0.001	＜0.001	＜0.001

分组	实验次数	细胞增殖		细胞迁移（个）
分组	实验次数	CCK-8实验	BrdU实验	细胞迁移（个）
shCtrl+胶原	3	0.38±0.01	0.33±0.01	21.66±0.57
shDDR2+胶原	3	0.28±0.00^a	0.25±0.01^a	6.00±1.00^a
Control+胶原	3	0.39±0.01	0.33±0.01	22.33±0.57
OE-DDR2+胶原	3	0.43±0.01^b	0.36±0.01^b	32.66±0.57^b
OE-DDR2+胶原+达沙替尼	3	0.36±0.01^c	0.29±1.25^c	12.66±0.57^c
F值		115.649	67.921	665.500
P值		＜0.001	＜0.001	＜0.001

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