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Zebrafish Model to Study Podocyte Function Within the Glomerular Filtration Barrier

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Kidney Research

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2664))

Abstract

The zebrafish model has been used in many different fields of research because of its high homology to the human genome, its easy genetic manipulation, its high fecundity, and its rapid development. For glomerular diseases, zebrafish larvae have proven to be a versatile tool to study the contribution of different genes, because the zebrafish pronephros is very comparable to the human kidney in function and ultrastructure. Here we describe the principle and use of a simple screening assay based on the measurement of the fluorescence in the retinal vessel plexus of the Tg(l-fabp:DBP:eGFP) zebrafish line (“eye assay”) to indirectly determine proteinuria as a hallmark of podocyte dysfunction. Furthermore, we illustrate how to analyze the obtained data and outline methods to attribute the findings to podocyte impairment.

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References

  1. Chang WJ, Hwang PP (2011) Development of zebrafish epidermis. Birth Defects Res C Embryo Today 93(3):205–214. https://doi.org/10.1002/bdrc.20215

    Article  CAS  PubMed  Google Scholar 

  2. Drummond IA, Davidson AJ (2016) Zebrafish kidney development. Methods Cell Biol 134:391–429. https://doi.org/10.1016/bs.mcb.2016.03.041

    Article  CAS  PubMed  Google Scholar 

  3. Goessling W, Sadler KC (2015) Zebrafish: an important tool for liver disease research. Gastroenterology 149(6):1361–1377. https://doi.org/10.1053/j.gastro.2015.08.034

    Article  PubMed  Google Scholar 

  4. Staudt D, Stainier D (2012) Uncovering the molecular and cellular mechanisms of heart development using the zebrafish. Annu Rev Genet 46:397–418. https://doi.org/10.1146/annurev-genet-110711-155646

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Tiso N, Moro E, Argenton F (2009) Zebrafish pancreas development. Mol Cell Endocrinol 312(1–2):24–30. https://doi.org/10.1016/j.mce.2009.04.018

    Article  CAS  PubMed  Google Scholar 

  6. Marques IJ, Lupi E, Mercader N (2019) Model systems for regeneration: zebrafish. Development 146(18):dev167692. https://doi.org/10.1242/dev.167692

    Article  CAS  PubMed  Google Scholar 

  7. Gore AV, Pillay LM, Venero Galanternik M, Weinstein BM (2018) The zebrafish: a fintastic model for hematopoietic development and disease. Wiley Interdiscip Rev Dev Biol 7(3):e312. https://doi.org/10.1002/wdev.312

    Article  PubMed  PubMed Central  Google Scholar 

  8. Gut P, Reischauer S, Stainier DYR, Arnaout R (2017) Little fish, big data: zebrafish as a model for cardiovascular and metabolic disease. Physiol Rev 97(3):889–938. https://doi.org/10.1152/physrev.00038.2016

    Article  PubMed  PubMed Central  Google Scholar 

  9. Morales EE, Wingert RA (2017) Zebrafish as a model of kidney disease. Results Probl Cell Differ 60:55–75. https://doi.org/10.1007/978-3-319-51436-9_3

    Article  CAS  PubMed  Google Scholar 

  10. MacRae CA, Peterson RT (2015) Zebrafish as tools for drug discovery. Nat Rev Drug Discov 14(10):721–731. https://doi.org/10.1038/nrd4627

    Article  CAS  PubMed  Google Scholar 

  11. Schenk H, Muller-Deile J, Kinast M, Schiffer M (2017) Disease modeling in genetic kidney diseases: zebrafish. Cell Tissue Res 369(1):127–141. https://doi.org/10.1007/s00441-017-2593-0

    Article  CAS  PubMed  Google Scholar 

  12. Howe K, Clark MD, Torroja CF et al (2013) The zebrafish reference genome sequence and its relationship to the human genome. Nature 496(7446):498–503. https://doi.org/10.1038/nature12111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Kawakami K, Takeda H, Kawakami N, Kobayashi M, Matsuda N, Mishina M (2004) A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Dev Cell 7(1):133–144. https://doi.org/10.1016/j.devcel.2004.06.005

    Article  CAS  PubMed  Google Scholar 

  14. Langheinrich U, Hennen E, Stott G, Vacun G (2002) Zebrafish as a model organism for the identification and characterization of drugs and genes affecting p53 signaling. Curr Biol 12(23):2023–2028. https://doi.org/10.1016/s0960-9822(02)01319-2

    Article  CAS  PubMed  Google Scholar 

  15. Li Q, Sadowski S, Frank M, Chai C, Varadi A, Ho SY, Lou H, Dean M, Thisse C, Thisse B, Uitto J (2010) The abcc6a gene expression is required for normal zebrafish development. J Invest Dermatol 130(11):2561–2568. https://doi.org/10.1038/jid.2010.174

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Müller-Deile J, Dannenberg J, Schroder P, Lin MH, Miner JH, Chen R, Bräsen JH, Thum T, Nyström J, Staggs LB, Haller H, Fiedler J, Lorenzen JM, Schiffer M (2017) Podocytes regulate the glomerular basement membrane protein nephronectin by means of miR-378a-3p in glomerular diseases. Kidney Int 92(4):836–849. https://doi.org/10.1016/j.kint.2017.03.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Muller-Deile J, Gellrich F, Schenk H, Schroder P, Nystrom J, Lorenzen J, Haller H, Schiffer M (2016) Overexpression of TGF-beta inducible microRNA-143 in zebrafish leads to impairment of the glomerular filtration barrier by targeting proteoglycans. Cell Physiol Biochem 40(5):819–830. https://doi.org/10.1159/000453142

    Article  CAS  PubMed  Google Scholar 

  18. Muller-Deile J, Schroder P, Beverly-Staggs L, Hiss R, Fiedler J, Nystrom J, Thum T, Haller H, Schiffer M (2018) Overexpression of preeclampsia induced microRNA-26a-5p leads to proteinuria in zebrafish. Sci Rep 8(1):3621. https://doi.org/10.1038/s41598-018-22070-w

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Patra C, Diehl F, Ferrazzi F, van Amerongen MJ, Novoyatleva T, Schaefer L, Muhlfeld C, Jungblut B, Engel FB (2011) Nephronectin regulates atrioventricular canal differentiation via Bmp4-Has2 signaling in zebrafish. Development 138(20):4499–4509. https://doi.org/10.1242/dev.067454

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Yoruk B, Gillers BS, Chi NC, Scott IC (2012) Ccm3 functions in a manner distinct from Ccm1 and Ccm2 in a zebrafish model of CCM vascular disease. Dev Biol 362(2):121–131. https://doi.org/10.1016/j.ydbio.2011.12.006

    Article  CAS  PubMed  Google Scholar 

  21. Lafont AG, Wang YF, Chen GD, Liao BK, Tseng YC, Huang CJ, Hwang PP (2011) Involvement of calcitonin and its receptor in the control of calcium-regulating genes and calcium homeostasis in zebrafish (Danio rerio). J Bone Miner Res 26(5):1072–1083. https://doi.org/10.1002/jbmr.301

    Article  CAS  PubMed  Google Scholar 

  22. Eisen JS, Smith JC (2008) Controlling morpholino experiments: don’t stop making antisense. Development 135(10):1735–1743. https://doi.org/10.1242/dev.001115

    Article  CAS  PubMed  Google Scholar 

  23. Stainier DYR, Raz E, Lawson ND, Ekker SC, Burdine RD, Eisen JS, Ingham PW, Schulte-Merker S, Yelon D, Weinstein BM, Mullins MC, Wilson SW, Ramakrishnan L, Amacher SL, Neuhauss SCF, Meng A, Mochizuki N, Panula P, Moens CB (2017) Guidelines for morpholino use in zebrafish. PLoS Genet 13(10):e1007000. https://doi.org/10.1371/journal.pgen.1007000

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK (2013) Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31(3):227–229. https://doi.org/10.1038/nbt.2501

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Moore FE, Reyon D, Sander JD, Martinez SA, Blackburn JS, Khayter C, Ramirez CL, Joung JK, Langenau DM (2012) Improved somatic mutagenesis in zebrafish using transcription activator-like effector nucleases (TALENs). PLoS One 7(5):e37877. https://doi.org/10.1371/journal.pone.0037877

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Naylor RW, Qubisi SS, Davidson AJ (2017) Zebrafish pronephros development. Results Probl Cell Differ 60:27–53. https://doi.org/10.1007/978-3-319-51436-9_2

    Article  CAS  PubMed  Google Scholar 

  27. Wingert RA, Davidson AJ (2008) The zebrafish pronephros: a model to study nephron segmentation. Kidney Int 73(10):1120–1127. https://doi.org/10.1038/ki.2008.37

    Article  CAS  PubMed  Google Scholar 

  28. Schenk H, Masseli A, Schroder P, Bolanos-Palmieri P, Beese M, Hegermann J, Brasen JH, Haller H (2019) Sulfatases, in particular Sulf1, are important for the integrity of the glomerular filtration barrier in zebrafish. Biomed Res Int 2019:4508048. https://doi.org/10.1155/2019/4508048

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Drummond IA, Majumdar A, Hentschel H, Elger M, Solnica-Krezel L, Schier AF, Neuhauss SC, Stemple DL, Zwartkruis F, Rangini Z, Driever W, Fishman MC (1998) Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function. Development 125(23):4655–4667

    Article  CAS  PubMed  Google Scholar 

  30. Hanke N, King BL, Vaske B, Haller H, Schiffer M (2015) A fluorescence-based assay for proteinuria screening in larval zebrafish (Danio rerio). Zebrafish 12(5):372–376. https://doi.org/10.1089/zeb.2015.1093

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Schlehr FJ, Limbird TA, Swiontkowski MF, Keller TS (1987) The use of laser Doppler flowmetry to evaluate anterior cruciate blood flow. J Orthop Res 5(1):150–153. https://doi.org/10.1002/jor.1100050120

    Article  CAS  PubMed  Google Scholar 

  32. Hentschel DM, Park KM, Cilenti L, Zervos AS, Drummond I, Bonventre JV (2005) Acute renal failure in zebrafish: a novel system to study a complex disease. Am J Physiol Renal Physiol 288(5):F923–F929. https://doi.org/10.1152/ajprenal.00386.2004

    Article  CAS  PubMed  Google Scholar 

  33. Kolatsi-Joannou M, Osborn D (2020) A technique for studying glomerular filtration integrity in the zebrafish pronephros. Methods Mol Biol 2067:25–39. https://doi.org/10.1007/978-1-4939-9841-8_3

    Article  CAS  PubMed  Google Scholar 

  34. Mahmood F, Mozere M, Zdebik AA, Stanescu HC, Tobin J, Beales PL, Kleta R, Bockenhauer D, Russell C (2013) Generation and validation of a zebrafish model of EAST (epilepsy, ataxia, sensorineural deafness and tubulopathy) syndrome. Dis Model Mech 6(3):652–660. https://doi.org/10.1242/dmm.009480

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Outtandy P, Russell C, Kleta R, Bockenhauer D (2019) Zebrafish as a model for kidney function and disease. Pediatr Nephrol 34(5):751–762. https://doi.org/10.1007/s00467-018-3921-7

    Article  PubMed  Google Scholar 

  36. Gorgulho R, Jacinto R, Lopes SS, Pereira SA, Tranfield EM, Martins GG, Gualda EJ, Derks RJE, Correia AC, Steenvoorden E, Pintado P, Mayboroda OA, Monteiro EC, Morello J (2018) Usefulness of zebrafish larvae to evaluate drug-induced functional and morphological renal tubular alterations. Arch Toxicol 92(1):411–423. https://doi.org/10.1007/s00204-017-2063-1

    Article  CAS  PubMed  Google Scholar 

  37. Oltrabella F, Pietka G, Ramirez IB, Mironov A, Starborg T, Drummond IA, Hinchliffe KA, Lowe M (2015) The Lowe syndrome protein OCRL1 is required for endocytosis in the zebrafish pronephric tubule. PLoS Genet 11(4):e1005058. https://doi.org/10.1371/journal.pgen.1005058

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Hentschel DM, Mengel M, Boehme L, Liebsch F, Albertin C, Bonventre JV, Haller H, Schiffer M (2007) Rapid screening of glomerular slit diaphragm integrity in larval zebrafish. Am J Physiol Renal Physiol 293(5):F1746–F1750. https://doi.org/10.1152/ajprenal.00009.2007

    Article  CAS  PubMed  Google Scholar 

  39. Schenk H, Muller-Deile J, Schroder P, Bolanos-Palmieri P, Beverly-Staggs L, White R, Brasen JH, Haller H, Schiffer M (2019) Characterizing renal involvement in Hermansky-Pudlak syndrome in a zebrafish model. Sci Rep 9(1):17718. https://doi.org/10.1038/s41598-019-54058-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Teng B, Schroder P, Muller-Deile J, Schenk H, Staggs L, Tossidou I, Dikic I, Haller H, Schiffer M (2016) CIN85 deficiency prevents nephrin endocytosis and proteinuria in diabetes. Diabetes 65(12):3667–3679. https://doi.org/10.2337/db16-0081

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Xie J, Farage E, Sugimoto M, Anand-Apte B (2010) A novel transgenic zebrafish model for blood-brain and blood-retinal barrier development. BMC Dev Biol 10:76. https://doi.org/10.1186/1471-213X-10-76

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Arif E, Rathore YS, Kumari B, Ashish F, Wong HN, Holzman LB, Nihalani D (2014) Slit diaphragm protein Neph1 and its signaling: a novel therapeutic target for protection of podocytes against glomerular injury. J Biol Chem 289(14):9502–9518. https://doi.org/10.1074/jbc.M113.505743

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Muller-Deile J, Schenk H, Niggemann P, Bolanos-Palmieri P, Teng B, Higgs A, Staggs L, Haller H, Schroder P, Schiffer M (2019) Mutation of microphthalmia-associated transcription factor (mitf) in zebrafish sensitizes for glomerulopathy. Biol Open 8(3):bio040253. https://doi.org/10.1242/bio.040253

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Kotb AM, Simon O, Blumenthal A, Vogelgesang S, Dombrowski F, Amann K, Zimmermann U, Endlich K, Endlich N (2016) Knockdown of ApoL1 in zebrafish larvae affects the glomerular filtration barrier and the expression of nephrin. PLoS One 11(5):e0153768. https://doi.org/10.1371/journal.pone.0153768

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

The authors thank lab members Dr. Patricia Bolaños-Palmieri, Johanna Sonntag, and Franz Tiefenböck for fruitful and constructive discussion.

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Authors and Affiliations

  1. Department of Medicine 4 – Nephrology and Hypertension, Universitätsklinikum Erlangen, Erlangen, Germany

    Nina Sopel

  2. Friedrich-Alexander Universiät Erlangen-Nuremberg, Erlangen, Germany

    Nina Sopel & Janina Müller-Deile

Authors
  1. Nina Sopel
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  2. Janina Müller-Deile
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Corresponding author

Correspondence to Janina Müller-Deile .

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Editors and Affiliations

  1. Department of Nephrology, The Royal Melbourne Hospital, Parkville, Melbourne, VIC, Australia

    Tim D. Hewitson

  2. Department of Nephrology, The Royal Melbourne Hospital, Parkville, Melbourne, VIC, Australia

    Nigel D. Toussaint

  3. Department of Nephrology, The Royal Melbourne Hospital, Parkville, Melbourne, VIC, Australia

    Edward R. Smith

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Sopel, N., Müller-Deile, J. (2023). Zebrafish Model to Study Podocyte Function Within the Glomerular Filtration Barrier. In: Hewitson, T.D., Toussaint, N.D., Smith, E.R. (eds) Kidney Research. Methods in Molecular Biology, vol 2664. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3179-9_11

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  • DOI: https://doi.org/10.1007/978-1-0716-3179-9_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3178-2

  • Online ISBN: 978-1-0716-3179-9

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