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Functional analysis reveals calcium-sensing receptor gene regulating cell–cell junction in renal tubular epithelial cells

  • Urology - Original Paper
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Abstract

Purpose

Calcium-sensing receptor (CASR) influences the expression pattern of multiple genes in renal tubular epithelial cells. The objective of this inquiry was to explore the molecular mechanisms of CASR in renal tubular epithelial cells and nephrolithiasis.

Methods

HK-2 cells were transfected with lentiviruses carrying either CASR (named CASR) or an empty vector negative control (named NC), as well as shRNA intended to target CASR (named shCASR) or its corresponding negative control (named shNC). CCK-8 assay was used to detect the effect of CASR on the proliferation of HK-2 cells. RNA-Sequencing was applied to explore potential pathways regulated by CASR in HK-2 cells.

Results

PCR and western blot results showed that CASR expression was significantly increased in CASR cells and was decreased in shCASR cells when compared to their corresponding negative control, respectively. CCK-8 assay revealed that CASR inhibited the proliferation of HK-2 cells. RNA-Sequencing results suggested that the shCASR HK-2 cells exhibited a significant up-regulation of 345 genes and a down-regulation of 366 genes. These differentially expressed genes (DEGs) were related to cell apoptosis and cell development. In CASR HK-2 cells, 1103 DEGs primarily functioned in mitochondrial energy metabolism, and amino acid metabolism. With the Venn diagram, 4 DEGs (Clorf116, ENPP3, IL20RB, and CLDN2) were selected as the hub genes regulated by CASR. Enrichment analysis revealed that these hub genes were involved in cell–cell junction, and epithelial cell development.

Conclusions

In summary, our investigation has the potential to offer novel perspectives on CASR regulating cell–cell junction in HK-2 cells.

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Data availability

All data experimental data are available upon request.

References

  1. Lieske JC, Toback FG (1996) Interaction of urinary crystals with renal epithelial cells in the pathogenesis of nephrolithiasis. Semin Nephrol 16:458–473

    CAS  PubMed  Google Scholar 

  2. Ding T, Zhao T, Li Y, Liu Z, Ding J, Ji B, Wang Y, Guo Z (2021) Vitexin exerts protective effects against calcium oxalate crystal-induced kidney pyroptosis in vivo and in vitro. Phytomedicine 86:153562

    Article  CAS  PubMed  Google Scholar 

  3. Riccardi D, Valenti G (2016) Localization and function of the renal calcium-sensing receptor. Nat Rev Nephrol 12:414–425

    Article  CAS  PubMed  Google Scholar 

  4. Riccardi D, Brown EM (2010) Physiology and pathophysiology of the calcium-sensing receptor in the kidney. Am J Physiol Renal Physiol 298:F485-499

    Article  CAS  PubMed  Google Scholar 

  5. Vezzoli G, Terranegra A, Soldati L (2012) Calcium-sensing receptor gene polymorphisms in patients with calcium nephrolithiasis. Curr Opin Nephrol Hypertens 21:355–361

    Article  CAS  PubMed  Google Scholar 

  6. Vezzoli G, Macrina L, Magni G, Arcidiacono T (2019) Calcium-sensing receptor: evidence and hypothesis for its role in nephrolithiasis. Urolithiasis 47:23–33

    Article  CAS  PubMed  Google Scholar 

  7. Li X, Chen S, Feng D, Fu Y, Wu H, Lu J, Bao J (2021) Calcium-sensing receptor promotes calcium oxalate crystal adhesion and renal injury in Wistar rats by promoting ROS production and subsequent regulation of PS ectropion, OPN, KIM-1, and ERK expression. Ren Fail 43:465–476

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Ibeh CL, Yiu AJ, Kanaras YL, Paal E, Birnbaumer L, Jose PA, Bandyopadhyay BC (2019) Evidence for a regulated Ca(2+) entry in proximal tubular cells and its implication in calcium stone formation. J Cell Sci 132:jcs225268

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Renkema KY, Bindels RJ, Hoenderop JG (2011) Role of the calcium-sensing receptor in reducing the risk for calcium stones. Clin J Am Soc Nephrol 6:2076–2082

    Article  CAS  PubMed  Google Scholar 

  10. Renkema KY, Velic A, Dijkman HB, Verkaart S, van der Kemp AW, Nowik M, Timmermans K, Doucet A, Wagner CA, Bindels RJ, Hoenderop JG (2009) The calcium-sensing receptor promotes urinary acidification to prevent nephrolithiasis. J Am Soc Nephrol 20:1705–1713

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Vezzoli G, Terranegra A, Rainone F, Arcidiacono T, Cozzolino M, Aloia A, Dogliotti E, Cusi D, Soldati L (2011) Calcium-sensing receptor and calcium kidney stones. J Transl Med 9:201

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Zhao J, Wu Y, Zhou K, Huang M, Sun Y, Kang J, Su Q, Zhao Y, Liu Q, Li C (2023) Ferroptosis in calcium oxalate kidney stone formation and the possible regulatory mechanism of ANKRD1. Biochim Biophys Acta Mol Cell Res 1870:119452

    Article  CAS  PubMed  Google Scholar 

  13. Dong C, Song C, He Z, Song Q, Song T, Liu J, Xiong Y, Su X, Zhou J, Yang S, Liao W (2023) Protective efficacy of Schizandrin B on ameliorating nephrolithiasis via regulating GSK3β/Nrf2 signaling-mediated ferroptosis in vivo and in vitro. Int Immunopharmacol 117:110042

    Article  CAS  PubMed  Google Scholar 

  14. He Z, Liao W, Song Q, Li B, Liu J, Xiong Y, Song C, Yang S (2021) Role of ferroptosis induced by a high concentration of calcium oxalate in the formation and development of urolithiasis. Int J Mol Med 47:289–301

    Article  PubMed  Google Scholar 

  15. Hill F, Sayer JA (2019) Precision medicine in renal stone-formers. Urolithiasis 47:99–105

    Article  PubMed  Google Scholar 

  16. Magno AL, Leatherbarrow KM, Brown SJ, Wilson SG, Walsh JP, Ward BK (2020) Functional analysis of calcium-sensing receptor variants identified in families provisionally diagnosed with familial hypocalciuric hypercalcaemia. Calcif Tissue Int 107:230–239

    Article  CAS  PubMed  Google Scholar 

  17. Wang L, Zhou Z, Yang Y, Gao P, Lin X, Wu Z (2022) A genetic polymorphism in the WDR72 gene is associated with calcium nephrolithiasis in the Chinese han population. Front Genet 13:897051

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Howles SA, Wiberg A, Goldsworthy M, Bayliss AL, Gluck AK, Ng M, Grout E, Tanikawa C, Kamatani Y, Terao C et al (2019) Genetic variants of calcium and vitamin D metabolism in kidney stone disease. Nat Commun 10:5175

    Article  PubMed  ADS  PubMed Central  Google Scholar 

  19. Peng Y, Yang C, Shi X, Li L, Dong H, Liu C, Fang Z, Wang Z, Ming S, Liu M et al (2019) Sirt3 suppresses calcium oxalate-induced renal tubular epithelial cell injury via modification of FoxO3a-mediated autophagy. Cell Death Dis 10:34

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Peng Y, Fang Z, Liu M, Wang Z, Li L, Ming S, Lu C, Dong H, Zhang W, Wang Q et al (2019) Testosterone induces renal tubular epithelial cell death through the HIF-1α/BNIP3 pathway. J Transl Med 17:62

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Gu Y, Shen Y, Chen W, He H, Ma Y, Mei X, Ju D, Liu H (2022) Protective effects of interleukin-22 on oxalate-induced crystalline renal injury via alleviating mitochondrial damage and inflammatory response. Appl Microbiol Biotechnol 106:2637–2649

    Article  CAS  PubMed  Google Scholar 

  22. Qiao X, Rao P, Zhang Y, Liu L, Pang M, Wang H, Hu M, Tian X, Zhang J, Zhao Y et al (2018) Redirecting TGF-β signaling through the β-catenin/foxo complex prevents kidney fibrosis. J Am Soc Nephrol 29:557–570

    Article  CAS  PubMed  Google Scholar 

  23. Yuan Q, Tang B, Zhang C (2022) Signaling pathways of chronic kidney diseases, implications for therapeutics. Signal Transduct Target Ther 7:182

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Parsana P, Amend SR, Hernandez J, Pienta KJ, Battle A (2017) Identifying global expression patterns and key regulators in epithelial to mesenchymal transition through multi-study integration. BMC Cancer 17:447

    Article  PubMed  PubMed Central  Google Scholar 

  25. Li S, Lan Y, Wu W, Duan X, Kong Z, Wu W, Zeng G (2019) Peroxisome proliferator-activated receptor γ modulates renal crystal retention associated with high oxalate concentration by regulating tubular epithelial cellular transdifferentiation. J Cell Physiol 234:2837–2850

    Article  CAS  PubMed  Google Scholar 

  26. Tsai SH, Kinoshita M, Kusu T, Kayama H, Okumura R, Ikeda K, Shimada Y, Takeda A, Yoshikawa S, Obata-Ninomiya K et al (2015) The ectoenzyme E-NPP3 negatively regulates ATP-dependent chronic allergic responses by basophils and mast cells. Immunity 42:279–293

    Article  CAS  PubMed  Google Scholar 

  27. Letavernier E, Kauffenstein G, Huguet L, Navasiolava N, Bouderlique E, Tang E, Delaitre L, Bazin D, de Frutos M, Gay C et al (2018) ABCC6 deficiency promotes development of randall plaque. J Am Soc Nephrol 29:2337–2347

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Sundararaman SS, van der Vorst EPC (2021) Calcium-sensing receptor (CaSR), its impact on inflammation and the consequences on cardiovascular health. Int J Mol Sci 22:2478

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Raju P, Shashikanth N, Tsai PY, Pongkorpsakol P, Chanez-Paredes S, Steinhagen PR, Kuo WT, Singh G, Tsukita S, Turner JR (2020) Inactivation of paracellular cation-selective claudin-2 channels attenuates immune-mediated experimental colitis in mice. J Clin Invest 130:5197–5208

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Gamero-Estevez E, Andonian S, Jean-Claude B, Gupta I, Ryan AK (2019) Temporal effects of quercetin on tight junction barrier properties and claudin expression and localization in MDCK II cells. Int J Mol Sci 20:4889

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Curry JN, Saurette M, Askari M, Pei L, Filla MB, Beggs MR, Rowe PS, Fields T, Sommer AJ, Tanikawa C et al (2020) Claudin-2 deficiency associates with hypercalciuria in mice and human kidney stone disease. J Clin Invest 130:1948–1960

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Wiener SV, Chen L, Shimotake AR, Kang M, Stoller ML, Ho SP (2018) Novel insights into renal mineralization and stone formation through advanced imaging modalities. Connect Tissue Res 59:102–110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Sherer BA, Chen L, Kang M, Shimotake AR, Wiener SV, Chi T, Stoller ML, Ho SP (2018) A continuum of mineralization from human renal pyramid to stones on stems. Acta Biomater 71:72–85

    Article  PubMed  PubMed Central  Google Scholar 

  34. Mandel N, Riese R (1991) Crystal-cell interactions: crystal binding to rat renal papillary tip collecting duct cells in culture. Am J Kidney Dis 17:402–406

    Article  CAS  PubMed  Google Scholar 

  35. Peerapen P, Thongboonkerd V (2011) Effects of calcium oxalate monohydrate crystals on expression and function of tight junction of renal tubular epithelial cells. Lab Invest 91:97–105

    Article  CAS  PubMed  Google Scholar 

  36. Hadpech S, Peerapen P, Thongboonkerd V (2022) Alpha-tubulin relocalization is involved in calcium oxalate-induced tight junction disruption in renal epithelial cells. Chem Biol Interact 368:110236

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This research was funded by National Natural Science Foundation of China (No. 81970603 and No. 82100807).

Author information

Author notes

  1. Zijian Zhou, Peng Gao, and Tongtong Zhang have contributed equally to this work.

Authors and Affiliations

  1. Department of Urology, Huashan Hospital, Fudan University, Shanghai, 200040, People’s Republic of China

    Zijian Zhou, Peng Gao, Yuanyuan Yang, Qiang Ding, Zhong Wu & Lujia Wang

  2. Clinical Research Center of Urolithiasis, Shanghai Medical College, Fudan University, Shanghai, 200040, People’s Republic of China

    Zijian Zhou, Peng Gao, Yuanyuan Yang, Qiang Ding, Zhong Wu & Lujia Wang

  3. Urology Centre, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, 200040, People’s Republic of China

    Tongtong Zhang

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  1. Zijian Zhou
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Contributions

ZZJ, WLJ, and WZ conceived and designed the experiments; ZZJ, YYY, and GP performed the experiments; WLJ and DQ analyzed the data; ZZJ wrote the manuscript; ZTT, WZ and WLJ reviewed the manuscript; ZZJ and ZTT revised the manuscript.

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Correspondence to Zhong Wu or Lujia Wang.

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Zhou, Z., Gao, P., Zhang, T. et al. Functional analysis reveals calcium-sensing receptor gene regulating cell–cell junction in renal tubular epithelial cells. Int Urol Nephrol (2024). https://doi.org/10.1007/s11255-024-03948-3

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