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Verapamil inhibited the development of ureteral stricture by blocking CaMK II-mediated STAT3 and Smad3/JunD pathways

  • Urology - Original Paper
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Abstract

Background

Ureteral stricture (US) is a fibrotic process that leads to urinary tract obstruction and even kidney damage, with the characteristic of reduced extracellular matrix (ECM) degradation and increased collagen synthesis. Verapamil, as a calcium channel blocker, was reported to prevent scar formation. Our work aimed to investigate the biological effects and mechanism of verapamil in US.

Methods

Fibroblasts were subjected to transforming growth factor-beta 1 (TGF-β1) to stimulate collagen synthesis, and the messenger ribonucleic acid (mRNA) and protein expressions in fibroblasts were assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The location of phosphorylation-signal transducer and activator of transcription 3 (p-STAT3) and Jund proto-oncogene subunit (JunD) in fibroblasts were determined by immunofluorescence (IF). The binding relationship between signal transducer and activator of transcription 3 (STAT3) and collagen type I alpha1 (COL1A1)/collagen type III alpha 1 chain (COL3A1) and the binding relationship between JunD and tissue inhibitor of metalloproteinases-1 (TIMP-1) were verified by dual luciferase reporter gene and chromatin Immunoprecipitation (ChIP) assays.

Results

Herein, we found that verapamil could inhibit TGF-β1/Ca2 + ⁄calmodulin-dependent protein kinase II (CaMK II)-mediated STAT3 activation in fibroblasts, and STAT3 inhibition repressed collagen production. In addition, verapamil could inhibit TGF-β1/CaMK II-mediated Mothers against DPP homolog 3 (Smad3)/JunD pathway activation in fibroblasts, and JunD silencing inhibited TIMP1 (a matrix metalloproteinase inhibitor) expression. Our subsequent experiments revealed that STAT3 bound with COL1A1 promoter and COL3A1 promoter and activated their transcription, and JunD bound with TIMP1 promoter and activated its transcription. Moreover, as expected, STAT3 activation could eliminate the inhibitory effect of verapamil treatment on TGF-β1-induced collagen production in fibroblasts, and JunD overexpression reversed the inhibitory effect of verapamil treatment on TGF-β1-induced TIMP1 expression in fibroblasts.

Conclusion

Verapamil inhibited collagen production and TIMP-1 expression in US by blocking CaMK II-mediated STAT3 and Smad3/JunD pathways.

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Abbreviations

US:

Ureteral stricture

mRNA:

Messenger ribonucleic acid

ECM:

Extracellular matrix

TGF-β1:

Transforming growth factor-beta 1

CaMK II:

Ca2+⁄calmodulin-dependent protein kinase II

p-STAT3:

Phosphorylation-signal transducer and activator of transcription 3

STAT:

Signal transducers and activators of transcription

JunD:

Jund proto-oncogene subunit

Smad3:

Mothers against DPP homolog 3

COL1A1:

Collagen type I alpha1

COL3A1:

Collagen type III alpha 1 chain

TIMP-1:

Tissue inhibitor of metalloproteinases-1

MMP1:

Matrix metallopeptidase 1

Ang II:

Angiotensin II

DMEM:

Dulbecco’s modified Eagle’s medium

FBS:

Fetal bovine serum

ATCC:

American type culture collection

IF:

Immunofluorescence

PVDF:

Polyvinylidene fluoride

BCA:

Bicinchoninic acid

ChIP:

Chromatin immunoprecipitation

DAPI:

4′,6-Diamidino-2-phenylindole

DMSO:

Dimethyl sulfoxide

cDNA:

Complementary DNA

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

qRT-PCR:

Quantitative real-time polymerase chain reaction

RIPA:

Radioimmunoprecipitation assay buffer

SD:

Standard deviation

ANOVA:

Analysis of variance

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Acknowledgements

We would like to give our sincere gratitude to the reviewers for their constructive comments.

Funding

This work was supported by Doctoral Fund Project of Hunan Provincial People’s Hospital (BSJJ202114), and the Research Project from Hunan Provincial Health Commission (202104051787).

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Authors and Affiliations

  1. Department of Urology, Hunan Provincial People’s Hospital, The First Affiliated Hospital of Hunan Normal University, No. 61, Jiefang Road, Changsha, 410005, Hunan province, People’s Republic of China

    Zhibiao Qing, Wuxiong Yuan, Jiansong Wang, Wei Song, Jianjun Luo, Xuecheng Wu, Qiang Lu, Yuanwei Li & Mingqiang Zeng

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Qing, Z., Yuan, W., Wang, J. et al. Verapamil inhibited the development of ureteral stricture by blocking CaMK II-mediated STAT3 and Smad3/JunD pathways. Int Urol Nephrol 54, 2855–2866 (2022). https://doi.org/10.1007/s11255-022-03284-4

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