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Expression Analysis of GDNF/RET Signaling Pathway in Human AD-MSCs Grown in HEK 293 Conditioned Medium (HEK293-CM)

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Abstract

Mesenchymal stem cells have been considered as the suitable source for the repair of kidney lesions. The study and identification of novel approaches could improve the efficiency of these cells in the recovery of kidney. In the present study, the effect of HEK 293 conditioned medium (HEK293-CM) was evaluated on the expression of GDNF/RET signaling pathway and their downstream genes in the human adipose-derived mesenchymal stem cells (AD-MSCs). For this purpose, the human AD-MSCs were cultured in the medium containing HEK293-CM. After the RNA extraction and cDNA synthesis, the expression level of GFRA1, GDNF, SPRY1, ETV4, ETV5, and CRLF1 genes were determined by SYBR Green Real time PCR. The obtained results indicated that the GDNF and GFRA1 expression enhanced in the AD-MSCs following treatment with 10% HEK293-CM-5%FBS as compared to the untreated AD-MSCs. These results were consistent with the decreased expression of SPRY1. The significant increased expression of ETV4, ETV5, and CRLF1 genes also showed that HEK293-CM activated the GDNF/RET signaling pathway in the AD-MSCs (P < 0.05). The obtained data suggested that the treatment with HEK293-CM activated the GDNF/RET signaling pathway in the human AD-MSCs.

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References

  1. Neuen, B. L., Chadban, S. J., Demaio, A. R., Johnson, D. W. & Perkovic, V. (2017). Chronic kidney disease and the global NCDs agenda. BMJ Global Health, 2(2), e000380.

    PubMed  PubMed Central  Google Scholar 

  2. Perlman, R. L., Finkelstein, F. O., Liu, L., Roys, E., Kiser, M., Eisele, G. et al. (2005). Quality of life in chronic kidney disease (CKD): a cross-sectional analysis in the Renal Research Institute-CKD study. American Journal of Kidney Disease 45(4), 658–666.

    Google Scholar 

  3. Gansevoort, R. T., Correa-Rotter, R., Hemmelgarn, B. R., Jafar, T. H., Heerspink, H. J., Mann, J. F. et al. (2013). Chronic kidney disease and cardiovascular risk: epidemiology, mechanisms, and prevention. Lancet, 382(9889), 339–352.

    PubMed  Google Scholar 

  4. Leung, J. C. (2014). Inherited renal diseases. Current Pediatric Reviews, 10(2), 95–100.

    CAS  PubMed  Google Scholar 

  5. Chronic Kidney Disease Prognosis Consortium, Matsushita, K., van der Velde, M., Astor, B. C., Woodward, M., Levey, A. S., de Jong, P. E. et al. (2010). Association of estimated glomerular filtration rate and albuminuria with all-cause and cardiovascular mortality in general population cohorts: a collaborative meta-analysis. Lancet, 375(9731), 2073–2081.

    Google Scholar 

  6. Turner, J. M., Bauer, C., Abramowitz, M. K., Melamed, M. L. & Hostetter, T. H. (2012). Treatment of chronic kidney disease. Kidney International, 81(4), 351–362.

    CAS  PubMed  Google Scholar 

  7. Fraser, S. D. & Blakeman, T. (2016). Chronic kidney disease: identification and management in primary care. Pragmatic Observational Research, 7, 21–32.

    PubMed  Google Scholar 

  8. Bruno, S., Grange, C., Deregibus, M. C., Calogero, R. A., Saviozzi, S., Collino, F. et al. (2009). Mesenchymal stem cell-derived microvesicles protect against acute tubular injury. Journal of the American Socity Nephrology, 20(5), 1053–1067.

    CAS  Google Scholar 

  9. Monsel, A., Zhu, Y. G., Gennai, S., Hao, Q., Liu, J. & Lee, J. W. (2014). Cell-based therapy for acute organ injury: preclinical evidence and ongoing clinical trials using mesenchymal stem cells. Anesthesiology, 121(5), 1099–1121.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Ranghino, A., Bruno, S., Bussolati, B., Moggio, A., Dimuccio, V., Tapparo, M. et al. (2017). The effects of glomerular and tubular renal progenitors and derived extracellular vesicles on recovery from acute kidney injury. Stem Cell Research & Therapy, 8(1), 24.

    Google Scholar 

  11. Barachini, S., Trombi, L., Danti, S., D’Alessandro, D., Battolla, B., Legitimo, A. et al. (2009). Morpho-functional characterization of human mesenchymal stem cells from umbilical cord blood for potential uses in regenerative medicine. Stem Cells and Development, 18(2), 293–305.

    PubMed  Google Scholar 

  12. Friedman, R., Betancur, M., Boissel, L., Tuncer, H., Cetrulo, C. & Klingemann, H. (2007). Umbilical cord mesenchymal stem cells: adjuvants for human cell transplantation. Biology of Blood and Marrow Transplantation, 13(12), 1477–1486.

    PubMed  Google Scholar 

  13. Tsuchiya, A., Kojima, Y., Ikarashi, S., Seino, S., Watanabe, Y., Kawata, Y. et al. (2017). Clinical trials using mesenchymal stem cells in liver diseases and inflammatory bowel diseases. Inflammation Regeneration, 37, 16.

    PubMed  Google Scholar 

  14. D’Ippolito, G., Diabira, S., Howard, G. A., Menei, P., Roos, B. A. & Schiller, P. C. (2004). Marrow-isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential. Journal of Cell Science, 117(Pt 14), 2971–2981.

    PubMed  Google Scholar 

  15. Tan, K., Zheng, K., Li, D., Lu, H., Wang, S. & Sun, X. (2017). Impact of adipose tissue or umbilical cord derived mesenchymal stem cells on the immunogenicity of human cord blood derived endothelial progenitor cells. PLoS ONE, 12(5), e0178624.

    PubMed  PubMed Central  Google Scholar 

  16. Lu, H., Wang, F., Mei, H., Wang, S. & Cheng, L. (2018). Human adipose mesenchymal stem cells show more efficient angiogenesis promotion on endothelial colony-forming cells than umbilical cord and endometrium. Stem Cells International, 2018, 7537589.

    PubMed  PubMed Central  Google Scholar 

  17. Kim, Y., Kim, H., Cho, H., Bae, Y., Suh, K. & Jung, J. (2007). Direct comparison of human mesenchymal stem cells derived from adipose tissues and bone marrow in mediating neovascularization in response to vascular ischemia. Cellular Physiology Biochemistry, 20(6), 867–876.

    CAS  PubMed  Google Scholar 

  18. Ahmadian Kia, N., Bahrami, A. R., Ebrahimi, M., Matin, M. M., Neshati, Z., Almohaddesin, M. R. et al. (2011). Comparative analysis of chemokine receptor’s expression in mesenchymal stem cells derived from human bone marrow and adipose tissue. Journal of Molecular Neuroscience, 44(3), 178–185.

    PubMed  Google Scholar 

  19. Fang, Y., Tian, X., Bai, S., Fan, J., Hou, W., Tong, H. et al. (2012). Autologous transplantation of adipose-derived mesenchymal stem cells ameliorates streptozotocin-induced diabetic nephropathy in rats by inhibiting oxidative stress, pro-inflammatory cytokines and the p38 MAPK signaling pathway. International Journal of Molecular Medicine, 30(1), 85–92.

    CAS  PubMed  Google Scholar 

  20. Chen, Y. T., Sun, C. K., Lin, Y. C., Chang, L. T., Chen, Y. L., Tsai, T. H. et al. (2011). Adipose-derived mesenchymal stem cell protects kidneys against ischemia-reperfusion injury through suppressing oxidative stress and inflammatory reaction. Journal of Translation Medicine, 9, 51.

    CAS  Google Scholar 

  21. Lee, S. R., Lee, S. H., Moon, J. Y., Park, J. Y., Lee, D., Lim, S. J. et al. (2010). Repeated administration of bone marrow-derived mesenchymal stem cells improved the protective effects on a remnant kidney model. Renal Failure, 32(7), 840–848.

    CAS  PubMed  Google Scholar 

  22. van Koppen, A., Joles, J. A., Bongartz, L. G., van den Brandt, J., Reichardt, H. M., Goldschmeding, R. et al. (2012). Healthy bone marrow cells reduce progression of kidney failure better than CKD bone marrow cells in rats with established chronic kidney disease. Cell Transplantation, 21(10), 2299–2312.

    PubMed  Google Scholar 

  23. Fan, M., Zhang, J., Xin, H., He, X. & Zhang, X. (2018). Current perspectives on role of MSC in renal pathophysiology. Frontiers in Physiology, 9, 1323.

    PubMed  PubMed Central  Google Scholar 

  24. Wang, B., Yao, K., Huuskes, B. M., Shen, H. H., Zhuang, J., Godson, C. et al. (2016). Mesenchymal stem cells deliver exogenous microRNA-let7c via exosomes to attenuate renal fibrosis. Molecular Therapy, 24(7), 1290–1301.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Sariola, H. & Saarma, M. (2003). Novel functions and signalling pathways for GDNF. Journal of Cell Science, 116(Pt 19), 3855–3862.

    CAS  PubMed  Google Scholar 

  26. Costantini, F. & Shakya, R. (2006). GDNF/Ret signaling and the development of the kidney. Bioessays, 28(2), 117–127.

    CAS  PubMed  Google Scholar 

  27. Schuchardt, A., D’Agati, V., Larsson-Blomberg, L., Costantini, F. & Pachnis, V. (1994). Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature, 367(6461), 380–383.

    CAS  PubMed  Google Scholar 

  28. Moore, M. W., Klein, R. D., Fariñas, I., Sauer, H., Armanin, M., Phillips, H. et al. (1996). Renal and neuronal abnormalities in mice lacking GDNF. Nature, 382(6586), 76–79.

    CAS  PubMed  Google Scholar 

  29. Enomoto, H., Araki, T., Jackman, A., Heuckeroth, R. O., Snider, W. D., Johnson, Jr, E. M. et al. (1998). GFR alpha1-deficient mice have deficits in the enteric nervous system and kidneys. Neuron, 21(2), 317–324.

    CAS  PubMed  Google Scholar 

  30. Kurtzeborn, K., Cebrian, C. & Kuure, S. (2018). Regulation of renal differentiation by trophic factors. Frontiers in Physiology, 9, 1588.

    PubMed  PubMed Central  Google Scholar 

  31. Orth, S. R., Ritz, E. & Suter-Crazzolara, C. (2000). Glial cell line-derived neurotrophic factor (GDNF) is expressed in the human kidney and is a growth factor for human mesangial cells. Nephrology Dialysis Transplantation, 15(5), 589–595.

    CAS  Google Scholar 

  32. Shakya, R., Watanabe, T. & Costantini, F. (2005). The role of GDNF/Ret signaling in ureteric bud cell fate and branching morphogenesis. Developmental Cell, 8(1), 65–74.

    CAS  PubMed  Google Scholar 

  33. Basson, M. A., Watson-Johnson, J., Shakya, R., Akbulut, S., Hyink, D., Costantini, F. D. et al. (2006). Branching morphogenesis of the ureteric epithelium during kidney development is coordinated by the opposing functions of GDNF and Sprouty1. Developmental Biology, 299(2), 466–477.

    CAS  PubMed  Google Scholar 

  34. Cortés, D., Carballo-Molina, O. A., Castellanos-Montiel, M. J. & Velasco, I. (2017). The non-survival effects of glial cell line-derived neurotrophic factor on neural cells. Frontiers in Molecular Neuroscience, 10, 258.

    PubMed  PubMed Central  Google Scholar 

  35. Pichel, J. G., Shen, L., Sheng, H. Z., Granholm, A. C., Drago, J., Grinberg, A. et al. (1996). Defects in enteric innervation and kidney development in mice lacking GDNF. Nature, 382(6586), 73–76.

    CAS  PubMed  Google Scholar 

  36. Takemura, T., Hino, S., Kuwajima, H., Yanagida, H., Okada, M., Nagata, M. et al. (2001). Induction of collecting duct morphogenesis in vitro by heparin-binding epidermal growth factor-like growth factor. Journal of the American Society of Nephrology, 12(5), 964–972.

    CAS  PubMed  Google Scholar 

  37. Qiao, J., Uzzo, R., Obara-Ishihara, T., Degenstein, L., Fuchs, E. & Herzlinger, D. (1999). FGF-7 modulates ureteric bud growth and nephron number in the developing kidney. Development, 126(3), 547–554.

    CAS  PubMed  Google Scholar 

  38. Carev, D., Saraga, M. & Saraga-Babic, M. (2008). Expression of intermediate filaments, EGF and TGF-alpha in early human kidney development. Journal of Molecular Histology, 39(2), 227–235.

    CAS  PubMed  Google Scholar 

  39. Kobayashi, H., Kawakami, K., Asashima, M. & Nishinakamura, R. (2007). Six1 and Six4 are essential for Gdnf expression in the metanephric mesenchyme and ureteric bud formation, while Six1 deficiency alone causes mesonephric-tubule defects. Mechanisms of Development, 124(4), 290–303.

    CAS  PubMed  Google Scholar 

  40. Esquela, A. F. & Lee, S. J. (2003). Regulation of metanephric kidney development by growth/differentiation factor 11. Developmental Biology, 257(2), 356–370.

    CAS  PubMed  Google Scholar 

  41. Huang, Z. Y., Hong, L. Q., Na, N., Luo, Y., Miao, B. & Chen, J. (2012). Infusion of mesenchymal stem cells overexpressing GDNF ameliorates renal function in nephrotoxic serum nephritis. Cell Biochemistry and Function, 30(2), 139–144.

    PubMed  Google Scholar 

  42. Li, S., Zhao, Y., Wang, Z., Wang, J., Liu, C. & Sun, D. (2019). Transplantation of amniotic fluid-derived stem cells preconditioned with glial cell line-derived neurotrophic factor gene alleviates renal fibrosis. Cell Transplantation, 28(1), 65–78.

    PubMed  Google Scholar 

  43. Garrett, E., Miller, A. R., Goldman, J. M., Apperley, J. F. & Melo, J. V. (2000). Characterization of recombination events leading to the production of an ecotropic replication-competent retrovirus in a GP+envAM12-derived producer cell line. Virology, 266(1), 170–179.

    CAS  PubMed  Google Scholar 

  44. Chong, H., Starkey, W. & Vile, R. G. (1998). A replication-competent retrovirus arising from a split-function packaging cell line was generated by recombination events between the vector, one of the packaging constructs, and endogenous retroviral sequences. Journal of Virology, 72(4), 2663–2670.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Vannucci, L., Lai, M., Chiuppesi, F., Ceccherini-Nelli, L. & Pistello, M. (2013). Viral vectors: a look back and ahead on gene transfer technology. New Microbiologica, 36(1), 1–22.

    CAS  PubMed  Google Scholar 

  46. Noverina, R., Widowati, W., Ayuningtyas, W., Kurniawan, D., Afifah, E., Laksmitawati, D. R. et al. (2019). Growth factors profile in conditioned medium human adipose tissue-derived mesenchymal stem cells (CM-hATMSCs). Clinical Nutrition Experimental, 24, 34–44.

    Google Scholar 

  47. Bhardwaj, R., Ansari, M. M., Parmar, M. S., Chandra, V. & Sharma, G. T. (2016). Stem cell conditioned media contains important growth factors and improves in vitro buffalo embryo production. Animal Biotechnology, 27(2), 118–125.

    CAS  PubMed  Google Scholar 

  48. Rozen, E. J., Schmidt, H., Dolcet, X., Basson, M. A., Jain, S. & Encinas, M. (2009). Loss of Sprouty1 rescues renal agenesis caused by Ret mutation. Journal of the American Society of Nephrology, 20(2), 255–259.

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Basson, M. A., Akbulut, S., Watson-Johnson, J., Simon, R., Carroll, T. J., Shakya, R. et al. (2005). Sprouty1 is a critical regulator of GDNF/RET-mediated kidney induction. Developmental Cell, 8(2), 229–239.

    CAS  PubMed  Google Scholar 

  50. Lu, B. C., Cebrian, C., Chi, X., Kuure, S., Kuo, R., Bates, C. M. et al. (2009). Etv4 and Etv5 are required downstream of GDNF and Ret for kidney branching morphogenesis. Nature Genetics, 41(12), 1295–1302.

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Schmidt-Ott, K. M., Yang, J., Chen, X., Wang, H., Paragas, N., Mori, K. et al. (2005). Novel regulators of kidney development from the tips of the ureteric bud. Journal of the American Society of Nephrology, 16(7), 1993–2002.

    CAS  PubMed  Google Scholar 

  52. Costantini, F. (2010). GDNF/Ret signaling and renal branching morphogenesis: from mesenchymal signals to epithelial cell behaviors. Organogenesis, 6(4), 252–262.

    PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The present study was approved by the Ethics Committee of the School of Medicine Shahid Beheshti University of Medical Sciences (Tehran, Iran; Ethical code: IR.SBMU.MSP.REC.1398.562). The present study was financially supported by “Deputy of Research and Technology of Shahid Beheshti University of Medical Sciences” (Grant no.: 17470).

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Authors and Affiliations

  1. Department of Medical Genetics, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Zahra Esmaeilizadeh, Bahar Mohammadi, Sayyed Mohammad Hossein Ghaderian, Mir Davood Omrani & Zahra Fazeli

  2. Department of Clinical Biochemistry, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Masoumeh Rajabibazl

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  1. Zahra Esmaeilizadeh
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Esmaeilizadeh, Z., Mohammadi, B., Rajabibazl, M. et al. Expression Analysis of GDNF/RET Signaling Pathway in Human AD-MSCs Grown in HEK 293 Conditioned Medium (HEK293-CM). Cell Biochem Biophys 78, 531–539 (2020). https://doi.org/10.1007/s12013-020-00936-z

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