Advertisement

Epithelial Function in the Drosophila Malpighian Tubule: An In Vivo Renal Model

  • Shireen-A. DaviesEmail author
  • Pablo Cabrero
  • Richard Marley
  • Guillermo Martinez Corrales
  • Saurav Ghimire
  • Anthony J. Dornan
  • Julian A. T. DowEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1926)

Abstract

The insect renal (Malpighian) tubule has long been a model system for the study of fluid secretion and its neurohormonal control, as well as studies on ion transport mechanisms. To extend these studies beyond the boundaries of classical physiology, a molecular genetic approach together with the ‘omics technologies is required. To achieve this in any vertebrate transporting epithelium remains a daunting task, as the genetic tools available are still relatively unsophisticated. Drosophila melanogaster, however, is an outstanding model organism for molecular genetics. Here we describe a technique for fluid secretion assays in the D. melanogaster equivalent of the kidney nephron. The development of this first physiological assay for a Drosophila epithelium, allowing combined approaches of integrative physiology and functional genomics, has now provided biologists with an entirely new model system, the Drosophila Malpighian tubule, which is utilized in multiple fields as diverse as kidney disease research and development of new modes of pest insect control.

Key words

Drosophila melanogaster Epithelia Renal Malpighian tubules Fluid secretion assays Kidney disease model Functional genomics 

Notes

Acknowledgments

Extensive support by the Biotechnology and Biological Sciences Research Council UK to the corresponding authors has been instrumental in developing the new fields of D. melanogaster integrative physiology and functional genomics.

References

  1. 1.
    Morgan TH (1910) Sex limited inheritance in Drosophila. Science 32:120–122PubMedCrossRefPubMedCentralGoogle Scholar
  2. 2.
    Rubin GM, Lewis EB (2000) A brief history of Drosophila’s contributions to genome research. Science 287:2216–2218PubMedCrossRefPubMedCentralGoogle Scholar
  3. 3.
    Rubin GM, Spradling AC (1983) Vectors for P element-mediated gene transfer in Drosophila. Nucleic Acids Res 11:6341–6351PubMedPubMedCentralCrossRefGoogle Scholar
  4. 4.
    Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118:401–415PubMedPubMedCentralGoogle Scholar
  5. 5.
    Rosay P et al (1997) Cell-type specific calcium signalling in a Drosophila epithelium. J Cell Sci 110(Pt 15):1683–1692PubMedPubMedCentralGoogle Scholar
  6. 6.
    Davies SA, Terhzaz S (2009) Organellar calcium signalling mechanisms in Drosophila epithelial function. J Exp Biol 212:387–400PubMedCrossRefPubMedCentralGoogle Scholar
  7. 7.
    Tian L et al (2009) Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat Methods 6:875–881PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Shafer OT et al (2008) Widespread receptivity to neuropeptide PDF throughout the neuronal circadian clock network of Drosophila revealed by real-time cyclic AMP imaging. Neuron 58:223–237PubMedPubMedCentralCrossRefGoogle Scholar
  9. 9.
    Cabrero P et al (2014) Chloride channels in stellate cells are essential for uniquely high secretion rates in neuropeptide-stimulated Drosophila diuresis. Proc Natl Acad Sci U S A 111:14301–14306PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Akerboom J et al (2013) Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Front Mol Neurosci 6:2PubMedPubMedCentralCrossRefGoogle Scholar
  11. 11.
    Efetova M et al (2013) Separate roles of PKA and EPAC in renal function unraveled by the optogenetic control of cAMP levels in vivo. J Cell Sci 126:778–788PubMedPubMedCentralCrossRefGoogle Scholar
  12. 12.
    Dietzl G et al (2007) A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448:151–156PubMedCrossRefPubMedCentralGoogle Scholar
  13. 13.
    Gramates L et al (2017) FlyBase at 25: looking to the future. Nucleic Acids Res 45:D663–D671PubMedCrossRefPubMedCentralGoogle Scholar
  14. 14.
    Chintapalli VR, Wang J, Dow JA (2007) Using FlyAtlas to identify better Drosophila melanogaster models of human disease. Nat Genet 39:715–720PubMedCrossRefPubMedCentralGoogle Scholar
  15. 15.
    Leader DP, Krause SA, Pandit A, Davies SA, Dow JAT (2018) FlyAtlas 2: a new version of the Drosophila melanogaster expression atlas with RNA-Seq, miRNA-Seq and sex-specific data. Nucleic Acids Res 46:D809–D815PubMedCrossRefPubMedCentralGoogle Scholar
  16. 16.
    Dow JT, Davies SA (2003) Integrative physiology and functional genomics of epithelial function in a genetic model organism. Physiol Rev 83:687–729PubMedCrossRefPubMedCentralGoogle Scholar
  17. 17.
    Dow JAT (2012) Drosophila as an experimental organism for functional genomics. In: eLS. John Wiley & Sons Ltd, ChichesterGoogle Scholar
  18. 18.
    Ugur B, Chen K, Bellen HJ (2016) Drosophila tools and assays for the study of human diseases. Dis Model Mech 9:235–244PubMedPubMedCentralCrossRefGoogle Scholar
  19. 19.
    Dow JA, Romero MF (2010) Drosophila provides rapid modeling of renal development, function, and disease. Am J Physiol Renal Physiol 299:F1237–F1244PubMedPubMedCentralCrossRefGoogle Scholar
  20. 20.
    Strange K (2016) Drug discovery in fish, flies, and worms. ILAR J 57:133–143PubMedPubMedCentralCrossRefGoogle Scholar
  21. 21.
    Sonoshita M, Cagan RL (2017) Modeling human cancers in Drosophila. Curr Top Dev Biol 121:287–309PubMedCrossRefPubMedCentralGoogle Scholar
  22. 22.
    Krench M, Littleton JT (2017) Neurotoxicity pathways in Drosophila models of the polyglutamine disorders. Curr Top Dev Biol 121:201–223PubMedCrossRefPubMedCentralGoogle Scholar
  23. 23.
    Berridge MJ, Oschman JL (1969) A structural basis for fluid secretion by malpighian tubules. Tissue Cell 1:247–272PubMedCrossRefPubMedCentralGoogle Scholar
  24. 24.
    Maddrell SHP (1971) The mechanisms of insect excretory systems. Adv Insect Physiol 8:199–331CrossRefGoogle Scholar
  25. 25.
    Dow JAT (2013) In: Chapman RF, Simpson SJ, Douglas AE (eds) The insects, structure and function. Cambridge University Press, CambridgeGoogle Scholar
  26. 26.
    Marcelli Malpighii Philosophii & Medici Bononiensis Dissertatio epistolica De Bombyce: Societati Regiae, Londini ad Scientiam Naturalem promovendam institutae, dicata Malpighi, Marcello, 1628–1694. https://encore.lib.gla.ac.uk/iii/encore/record/C_Rb2671014;jsessionid=DD1890B70359EE4BC74ADB74D5041C5B?lang=eng
  27. 27.
    Maddrell S (2009) Insect homeostasis: past and future. J Exp Biol 212:446–451PubMedCrossRefPubMedCentralGoogle Scholar
  28. 28.
    Wessing A, Eichelberg D (1978) The genetics and biology of Drosophila, vol 2c. Academic Press, LondonGoogle Scholar
  29. 29.
    Denholm B (2013) Shaping up for action: the path to physiological maturation in the renal tubules of Drosophila. Organogenesis 9:40–54PubMedPubMedCentralCrossRefGoogle Scholar
  30. 30.
    Beyenbach KW, Skaer H, Dow JA (2010) The developmental, molecular, and transport biology of Malpighian tubules. Annu Rev Entomol 55:351–374PubMedCrossRefPubMedCentralGoogle Scholar
  31. 31.
    Sozen MA, Armstrong JD, Yang M, Kaiser K, Dow JA (1997) Functional domains are specified to single-cell resolution in a Drosophila epithelium. Proc Natl Acad Sci U S A 94:5207–5212PubMedPubMedCentralCrossRefGoogle Scholar
  32. 32.
    Dow JA et al (1994) The Malpighian tubules of Drosophila melanogaster: a novel phenotype for studies of fluid secretion and its control. J Exp Biol 197:421–428PubMedPubMedCentralGoogle Scholar
  33. 33.
    Dube K, McDonald DG, O'Donnell MJ (2000) Calcium transport by isolated anterior and posterior Malpighian tubules of Drosophila melanogaster: roles of sequestration and secretion. J Insect Physiol 46:1449–1460PubMedCrossRefPubMedCentralGoogle Scholar
  34. 34.
    Dube KA, McDonald DG, O'Donnell MJ (2000) Calcium homeostasis in larval and adult Drosophila melanogaster. Arch Insect Biochem Physiol 44:27–39PubMedCrossRefPubMedCentralGoogle Scholar
  35. 35.
    O’Donnell MJ, Maddrell SH (1995) Fluid reabsorption and ion transport by the lower Malpighian tubules of adult female Drosophila. J Exp Biol 198:1647–1653PubMedPubMedCentralGoogle Scholar
  36. 36.
    Dow JA (2009) Insights into the Malpighian tubule from functional genomics. J Exp Biol 212:435–445PubMedCrossRefPubMedCentralGoogle Scholar
  37. 37.
    Dow JA (1999) The multifunctional Drosophila melanogaster V-ATPase is encoded by a multigene family. J Bioenerg Biomembr 31:75–83PubMedCrossRefPubMedCentralGoogle Scholar
  38. 38.
    Allan AK, Du J, Davies SA, Dow JAT (2005) Genome-wide survey of V-ATPase genes in Drosophila reveals a conserved renal phenotype for lethal alleles. Physiol Genomics 22:128–138PubMedCrossRefPubMedCentralGoogle Scholar
  39. 39.
    Torrie LS et al (2004) Resolution of the insect ouabain paradox. Proc Natl Acad Sci U S A 101:13689–13693PubMedPubMedCentralCrossRefGoogle Scholar
  40. 40.
    Kaufmann N et al (2005) Developmental expression and biophysical characterization of a Drosophila melanogaster aquaporin. Am J Physiol Cell Physiol 289:C397–C407PubMedCrossRefPubMedCentralGoogle Scholar
  41. 41.
    Kerr M, Davies SA, Dow JA (2004) Cell-specific manipulation of second messengers; a toolbox for integrative physiology in Drosophila. Curr Biol 14:1468–1474PubMedCrossRefPubMedCentralGoogle Scholar
  42. 42.
    Wang J et al (2004) Function-informed transcriptome analysis of Drosophila renal tubule. Genome Biol 5:R69PubMedPubMedCentralCrossRefGoogle Scholar
  43. 43.
    Chintapalli VR et al (2012) Functional correlates of positional and gender-specific renal asymmetry in Drosophila. PLoS One 7:e32577PubMedPubMedCentralCrossRefGoogle Scholar
  44. 44.
    Davies SA et al (2012) Immune and stress response ‘cross-talk’ in the Drosophila Malpighian tubule. J Insect Physiol 58:488–497PubMedCrossRefPubMedCentralGoogle Scholar
  45. 45.
    Davies SA et al (2014) Cell signalling mechanisms in stress tolerance. J Exp Biol 217:119–128PubMedCrossRefPubMedCentralGoogle Scholar
  46. 46.
    Terhzaz S et al (2010) Cell-specific inositol 1,4,5 trisphosphate 3-kinase mediates epithelial cell apoptosis in response to oxidative stress in Drosophila. Cell Signal 22:737–748PubMedCrossRefPubMedCentralGoogle Scholar
  47. 47.
    Piermarini PM, Esquivel CJ, Denton S (2017) Malpighian tubules as novel targets for mosquito control. Int J Environ Res Public Health 14. https://doi.org/10.3390/ijerph14020111PubMedCentralCrossRefGoogle Scholar
  48. 48.
    Dow JA, Davies SA (2006) The Malpighian tubule: rapid insights from post-genomic biology. J Insect Physiol 52:365–378PubMedCrossRefPubMedCentralGoogle Scholar
  49. 49.
    Ianowski JP, O’Donnell MJ (2004) Basolateral ion transport mechanisms during fluid secretion by Drosophila Malpighian tubules: Na+ recycling, Na+:K+:2Cl− cotransport and Cl− conductance. J Exp Biol 207:2599–2609PubMedCrossRefPubMedCentralGoogle Scholar
  50. 50.
    Linton SM, O’Donnell MJ (1999) Contributions of K+:Cl− cotransport and Na+/K+-ATPase to basolateral ion transport in malpighian tubules of Drosophila melanogaster. J Exp Biol 202:1561–1570PubMedPubMedCentralGoogle Scholar
  51. 51.
    Maddrell SH, Overton JA (1988) Stimulation of sodium transport and fluid secretion by ouabain in an insect malpighian tubule. J Exp Biol 137:265–276PubMedPubMedCentralGoogle Scholar
  52. 52.
    Davies SA et al (1996) Analysis and inactivation of vha55, the gene encoding the vacuolar ATPase B-subunit in Drosophila melanogaster reveals a larval lethal phenotype. J Biol Chem 271:30677–30684PubMedCrossRefPubMedCentralGoogle Scholar
  53. 53.
    Karet FE et al (1999) Mutations in the gene encoding B1 subunit of H+-ATPase cause renal tubular acidosis with sensorineural deafness. Nat Genet 21:84–90PubMedCrossRefPubMedCentralGoogle Scholar
  54. 54.
    Kamleh MA, Hobani Y, Dow JA, Zheng L, Watson DG (2009) Towards a platform for the metabonomic profiling of different strains of Drosophila melanogaster using liquid chromatography-Fourier transform mass spectrometry. FEBS J 276:6798–6809PubMedCrossRefPubMedCentralGoogle Scholar
  55. 55.
    O’Donnell MJ (2009) Too much of a good thing: how insects cope with excess ions or toxins in the diet. J Exp Biol 212:363–372PubMedCrossRefPubMedCentralGoogle Scholar
  56. 56.
    Dow JA (2007) Integrative physiology, functional genomics and the phenotype gap: a guide for comparative physiologists. J Exp Biol 210:1632–1640PubMedCrossRefPubMedCentralGoogle Scholar
  57. 57.
    Tardif G, Murnik M (1975) Frequency-dependent sexual selection among wild-type strains of Drosophila melanogaster. Behav Genet 5:373–379PubMedCrossRefPubMedCentralGoogle Scholar
  58. 58.
    Ramsay J (1954) Active transport of water by the Malpighian tubules of the stick insect, Dixippus morosus (Orthoptera, Phasmidae). J Exp Biol 31:104–113Google Scholar
  59. 59.
    Chintapalli VR, Wang J, Herzyk P, Davies SA, Dow JA (2013) Data-mining the FlyAtlas online resource to identify core functional motifs across transporting epithelia. BMC Genomics 14:518PubMedPubMedCentralCrossRefGoogle Scholar
  60. 60.
    Du J et al (2006) The SzA mutations of the B subunit of the Drosophila vacuolar H+ ATPase identify conserved residues essential for function in fly and yeast. J Cell Sci 119:2542–2551PubMedCrossRefPubMedCentralGoogle Scholar
  61. 61.
    Maddrell SH, O’Donnell MJ (1992) Insect Malpighian tubules: v-ATPase action in ion and fluid transport. J Exp Biol 172:417–429PubMedPubMedCentralGoogle Scholar
  62. 62.
    Coast GM, Webster SG, Schegg KM, Tobe SS, Schooley DA (2001) The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules. J Exp Biol 204:1795–1804PubMedPubMedCentralGoogle Scholar
  63. 63.
    Giannakou ME, Dow JA (2001) Characterization of the Drosophila melanogaster alkali-metal/proton exchanger (NHE) gene family. J Exp Biol 204:3703–3716PubMedPubMedCentralGoogle Scholar
  64. 64.
    Davies SA, Day JP (2006) cGMP signalling in a transporting epithelium. Biochem Soc Trans 34:512–514PubMedCrossRefPubMedCentralGoogle Scholar
  65. 65.
    Coast G (2007) The endocrine control of salt balance in insects. Gen Comp Endocrinol 152:332–338PubMedCrossRefPubMedCentralGoogle Scholar
  66. 66.
    Blumenthal EM (2003) Regulation of chloride permeability by endogenously produced tyramine in the Drosophila Malpighian tubule. Am J Physiol Cell Physiol 284:C718–C728PubMedCrossRefPubMedCentralGoogle Scholar
  67. 67.
    Wu Y, Schellinger JN, Huang CL, Rodan AR (2014) Hypotonicity stimulates potassium flux through the WNK-SPAK/OSR1 kinase cascade and the Ncc69 sodium-potassium-2-chloride cotransporter in the Drosophila renal tubule. J Biol Chem 289:26131–26142PubMedPubMedCentralCrossRefGoogle Scholar
  68. 68.
    O’Donnell MJ, Ianowski JP, Linton SM, Rheault MR (2003) Inorganic and organic anion transport by insect renal epithelia. Biochim Biophys Acta 1618:194–206PubMedCrossRefPubMedCentralGoogle Scholar
  69. 69.
    Halberg KA et al (2016) The cell adhesion molecule Fasciclin2 regulates brush border length and organization in Drosophila renal tubules. Nat Commun 7:11266PubMedPubMedCentralCrossRefGoogle Scholar
  70. 70.
    Wu Y, Baum M, Huang CL, Rodan AR (2015) Two inwardly rectifying potassium channels, Irk1 and Irk2, play redundant roles in Drosophila renal tubule function. Am J Physiol Regul Integr Comp Physiol 309:R747–R756PubMedPubMedCentralCrossRefGoogle Scholar
  71. 71.
    Rodan AR, Baum M, Huang CL (2012) The Drosophila NKCC Ncc69 is required for normal renal tubule function. Am J Physiol Cell Physiol 303:C883–C894PubMedPubMedCentralCrossRefGoogle Scholar
  72. 72.
    Day JP et al (2008) Identification of two partners from the bacterial kef exchanger family for the apical plasma membrane V-ATPase of Metazoa. J Cell Sci 121:2612–2619PubMedCrossRefPubMedCentralGoogle Scholar
  73. 73.
    Terhzaz S, Cabrero P, Chintapalli VR, Davies SA, Dow JAT (2010) Mislocalization of mitochondria and compromised renal function and oxidative stress resistance in Drosophila SesB mutants. Physiol Genomics 41:33–41PubMedCrossRefPubMedCentralGoogle Scholar
  74. 74.
    MacPherson MR, Lohmann SM, Davies SA (2004) Analysis of Drosophila cGMP-dependent protein kinases and assessment of their in vivo roles by targeted expression in a renal transporting epithelium. J Biol Chem 279:40026–40034PubMedCrossRefPubMedCentralGoogle Scholar
  75. 75.
    MacPherson MR et al (2004) The dg2 (for) gene confers a renal phenotype in Drosophila by modulation of cGMP-specific phosphodiesterase. J Exp Biol 207:2769–2776PubMedCrossRefPubMedCentralGoogle Scholar
  76. 76.
    Ruka KA, Miller AP, Blumenthal EM (2013) Inhibition of diuretic stimulation of an insect secretory epithelium by a cGMP-dependent protein kinase. Am J Physiol Renal Physiol 304:F1210–F1216PubMedPubMedCentralCrossRefGoogle Scholar
  77. 77.
    Broderick KE et al (2003) Interactions between epithelial nitric oxide signaling and phosphodiesterase activity in Drosophila. Am J Physiol Cell Physiol 285:C1207–C1218PubMedCrossRefPubMedCentralGoogle Scholar
  78. 78.
    Pollock VP et al (2003) NorpA and itpr mutants reveal roles for phospholipase C and inositol (1,4,5)- trisphosphate receptor in Drosophila melanogaster renal function. J Exp Biol 206:901–911PubMedCrossRefPubMedCentralGoogle Scholar
  79. 79.
    MacPherson MR et al (2005) Transient receptor potential-like channels are essential for calcium signaling and fluid transport in a Drosophila epithelium. Genetics 169:1541–1552PubMedPubMedCentralCrossRefGoogle Scholar
  80. 80.
    Terhzaz S et al (2012) Mechanism and function of Drosophila capa GPCR: a desiccation stress-responsive receptor with functional homology to human neuromedinU receptor. PLoS One 7:e29897PubMedPubMedCentralCrossRefGoogle Scholar
  81. 81.
    Cannell E et al (2016) The corticotropin-releasing factor-like diuretic hormone 44 (DH) and kinin neuropeptides modulate desiccation and starvation tolerance in Drosophila melanogaster. Peptides 80:96–107PubMedPubMedCentralCrossRefGoogle Scholar
  82. 82.
    Zandawala M, Marley R, Davies SA, Nassel DR (2018) Characterization of a set of abdominal neuroendocrine cells that regulate stress physiology using colocalized diuretic peptides in Drosophila. Cell Mol Life Sci 75:1099–1115PubMedCrossRefPubMedCentralGoogle Scholar
  83. 83.
    Blumenthal EM (2009) Isoform- and cell-specific function of tyrosine decarboxylase in the Drosophila Malpighian tubule. J Exp Biol 212:3802–3809PubMedCrossRefPubMedCentralGoogle Scholar
  84. 84.
    Ashburner M (1989) Drosophila: A laboratory handbook. Cold Spring Harbor Laboratory: 1331Google Scholar
  85. 85.
    Maddrell SHP (1991) BioEssays 13(7):357CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Shireen-A. Davies
    • 1
    Email author
  • Pablo Cabrero
    • 1
  • Richard Marley
    • 1
  • Guillermo Martinez Corrales
    • 1
  • Saurav Ghimire
    • 1
  • Anthony J. Dornan
    • 1
  • Julian A. T. Dow
    • 1
    Email author
  1. 1.Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life SciencesUniversity of GlasgowGlasgowUK

Personalised recommendations