Pflügers Archiv - European Journal of Physiology

, Volume 466, Issue 2, pp 343–356 | Cite as

A primary culture system of mouse thick ascending limb cells with preserved function and uromodulin processing

  • Bob Glaudemans
  • Sara Terryn
  • Nadine Gölz
  • Martina Brunati
  • Angela Cattaneo
  • Angela Bachi
  • Lama Al-Qusairi
  • Urs Ziegler
  • Olivier Staub
  • Luca Rampoldi
  • Olivier DevuystEmail author
Organ physiology


The epithelial cells lining the thick ascending limb (TAL) of the loop of Henle perform essential transport processes and secrete uromodulin, the most abundant protein in normal urine. The lack of differentiated cell culture systems has hampered studies of TAL functions. Here, we report a method to generate differentiated primary cultures of TAL cells, developed from microdissected tubules obtained in mouse kidneys. The TAL tubules cultured on permeable filters formed polarized confluent monolayers in ∼12 days. The TAL cells remain differentiated and express functional markers such as uromodulin, NKCC2, and ROMK at the apical membrane. Electrophysiological measurements on primary TAL monolayers showed a lumen-positive transepithelial potential (+9.4 ± 0.8 mV/cm2) and transepithelial resistance similar to that recorded in vivo. The transepithelial potential is abolished by apical bumetanide and in primary cultures obtained from ROMK knockout mice. The processing, maturation and apical secretion of uromodulin by primary TAL cells is identical to that observed in vivo. The primary TAL cells respond appropriately to hypoxia, hypertonicity, and stimulation by desmopressin, and they can be transfected. The establishment of this primary culture system will allow the investigation of TAL cells obtained from genetically modified mouse models, providing a critical tool for understanding the role of that segment in health and disease.


Epithelial transport NKCC2 ROMK Loop of Henle TAL 



The authors would like to thank Gery Barmettler, Soline Bourgeois, Huguette Debaix, David Hoogewijs and Klaus Marquardt for assistance and helpful suggestions. Prof. Jan Loffing kindly provided the parvalbumin-EGFP mouse. The uromodulin knockout mouse was kindly provided by Prof. X-R. Wu. These studies were supported in part by the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 246539 (Marie Curie) and grant no. 305608 (EURenOmics); an Action de Recherche Concertée (ARC, Communauté Française de Belgique); the FNRS and FRSM; the Inter-University Attraction Pole (IUAP, Belgium Federal Government); the NCCR Kidney. CH program (Swiss National Science Foundation); the Gebert Rüf Stiftung (Project GRS-038/12); and the Swiss National Science Foundation 31003A-125422/1 (to OS) and 310030–146490 (to OD).

Conflict of interest

The authors declare no competing interests.


  1. 1.
    Allen ML, Nakao A, Sonnenburg WK et al (1988) Immunodissection of cortical and medullary thick ascending limb cells from rabbit kidney. Am J Physiol 255:F704–F710PubMedGoogle Scholar
  2. 2.
    Allen F, Tisher CC (1976) Morphology of the ascending thick limb of Henle. Kidney Int 9:8–22PubMedCrossRefGoogle Scholar
  3. 3.
    Ares GR, Caceres PS, Ortiz PA (2011) Molecular regulation of NKCC2 in the thick ascending limb. Am J Physiol Ren Physiol 301:F1143–F1159CrossRefGoogle Scholar
  4. 4.
    Bates JM, Raffi HM, Prasadan K et al (2004) Tamm–Horsfall protein knockout mice are more prone to urinary tract infection: rapid communication. Kidney Int 65:791–797PubMedCrossRefGoogle Scholar
  5. 5.
    Baudouin-Legros M, Bouthier M, Teulon J (1993) [Arginine]vasopressin hydrolyses phosphoinositides in the medullary thick ascending limb of mouse nephron. Pflugers Arch 425:381–389PubMedCrossRefGoogle Scholar
  6. 6.
    Bourgeois S, Rossignol P, Grelac F et al (2003) Differentiated thick ascending limb (TAL) cultured cells derived from SV40 transgenic mice express functional apical NHE2 isoform: effect of nitric oxide. Pflugers Arch 446:672–683PubMedCrossRefGoogle Scholar
  7. 7.
    Burg MB, Ferraris JD, Dmitrieva NI (2007) Cellular response to hyperosmotic stresses. Physiol Rev 87:1441–1474PubMedCrossRefGoogle Scholar
  8. 8.
    Burg M, Green N, Sohraby S, Steele R, Handler J (1982) Differentiated function in cultured epithelia derived from thick ascending limbs. Am J Physiol 242:C229–C233PubMedGoogle Scholar
  9. 9.
    Chamberlin ME, LeFurgey A, Mandel LJ (1984) Suspension of medullary thick ascending limb tubules from the rabbit kidney. Am J Physiol 247:F955–F964PubMedGoogle Scholar
  10. 10.
    Chang CT, Hung CC, Tian YC, Yang CW, Wu MS (2007) Cyclosporine reduces paracellin-1 expression and magnesium transport in thick ascending limb cells. Nephrol Dial Transplant 22:1033–1040PubMedCrossRefGoogle Scholar
  11. 11.
    Dahan K, Devuyst O, Smaers M et al (2003) A cluster of mutations in the UMOD gene causes familial juvenile hyperuricemic nephropathy with abnormal expression of uromodulin. J Am Soc Nephrol 14:2883–2893PubMedCrossRefGoogle Scholar
  12. 12.
    Devuyst O (2008) Salt wasting and blood pressure. Nat Genet 40:495–496PubMedCrossRefGoogle Scholar
  13. 13.
    Devuyst O, Christie PT, Courtoy PJ et al (1999) Intra-renal and subcellular distribution of the human chloride channel, CLC-5, reveals a pathophysiological basis for Dent's disease. Hum Mol Genet 8:247–257PubMedCrossRefGoogle Scholar
  14. 14.
    Di Stefano A, Jounier S, Wittner M (2001) Evidence supporting a role for KCl cotransporter in the thick ascending limb of Henle's loop. Kidney Int 60:1809–1823PubMedCrossRefGoogle Scholar
  15. 15.
    Di Stefano A, Roinel N, de Rouffignac C et al (1993) Transepithelial Ca2+ and Mg2+ transport in the cortical thick ascending limb of Henle's loop of the mouse is a voltage-dependent process. Ren Physiol Biochem 16:157–166PubMedGoogle Scholar
  16. 16.
    Drugge ED, Carroll MA, McGiff JC (1989) Cells in culture from rabbit medullary thick ascending limb of Henle's loop. Am J Physiol 256:C1070–C1081PubMedGoogle Scholar
  17. 17.
    Dublineau I, Elalouf JM, Pradelles P et al (1989) Independent desensitization of rat renal thick ascending limbs and collecting ducts to ADH. Am J Physiol 256:F656–F663PubMedGoogle Scholar
  18. 18.
    Eckardt KU, Bernhardt WM, Weidemann A et al (2005) Role of hypoxia in the pathogenesis of renal disease. Kidney Int 99:S46–S51CrossRefGoogle Scholar
  19. 19.
    Eng B, Mukhopadhyay S, Vio CP et al (2007) Characterization of a long-term rat mTAL cell line. Am J Physiol Ren Physiol 293:F1413–F1422CrossRefGoogle Scholar
  20. 20.
    Eveloff J, Haase W, Kinne R (1980) Separation of renal medullary cells: isolation of cells from the thick ascending limb of Henle's loop. J Cell Biol 87:672–681PubMedCrossRefGoogle Scholar
  21. 21.
    Gamba G, Friedman PA (2009) Thick ascending limb: the Na(+):K (+):2Cl (−) co-transporter, NKCC2, and the calcium-sensing receptor, CaSR. Pfluegers Arch 458:61–76CrossRefGoogle Scholar
  22. 22.
    Hebert S (1995) An ATP-regulated inwardly rectifying potassium channel from rat kidney. Kidney Int 48:1010–1016PubMedCrossRefGoogle Scholar
  23. 23.
    Hebert SC, Culpepper RM, Andreoli TE (1981) NaCl transport in mouse medullary thick ascending limbs. I. Functional nephron heterogeneity and ADH-stimulated NaCl cotransport. Am J Physiol 241:F412–F431PubMedGoogle Scholar
  24. 24.
    Jans F, Vandenabeele F, Helbert M et al (2000) A simple method for obtaining functionally and morphologically intact primary cultures of the medullary thick ascending limb of Henle's loop (MTAL) from rabbit kidneys. Pflugers Arch 440:643–651PubMedGoogle Scholar
  25. 25.
    Köttgen A (2010) Genome-wide association studies in nephrology research. Am J Kidney Dis 56:743–758PubMedCrossRefGoogle Scholar
  26. 26.
    Kwon MS, Lim SW, Kwon HM (2009) Hypertonic stress in the kidney: a necessary evil. Physiology (Bethesda) 24:186–191CrossRefGoogle Scholar
  27. 27.
    Liu Y, Mo L, Goldfarb DS et al (2010) Progressive renal papillary calcification and ureteral stone formation in mice deficient for Tamm–Horsfall protein. Am J Physiol Ren Physiol 299:F469–F478CrossRefGoogle Scholar
  28. 28.
    Lu M, Wang T, Yan Q et al (2002) Absence of small conductance K+channel (SK) activity in apical membranes of thick ascending limb and cortical collecting duct in ROMK (Bartter's) knockout mice. J Biol Chem 277:37881–37887PubMedCrossRefGoogle Scholar
  29. 29.
    Meyer AH, Katona I, Blatow M et al (2002) In vivo labeling of parvalbumin-positive interneurons and analysis of electrical coupling in identified neurons. J Neurosci 22:7055–7064PubMedGoogle Scholar
  30. 30.
    Mo L, Zhu XH, Huang HY, Shapiro E, Hasty DL, Wu XR (2004) Ablation of the Tamm–Horsfall protein gene increases susceptibility of mice to bladder colonization by type 1-fimbriated Escherichia coli. Am J Physiol Ren Physiol 286:795–802CrossRefGoogle Scholar
  31. 31.
    Mutig K, Kahl T, Saritas T et al (2011) Activation of the bumetanide-sensitive Na+, K+,2Cl− cotransporter (NKCC2) is facilitated by Tamm–Horsfall protein in a chloride-sensitive manner. J Biol Chem 286:30200–30210PubMedCrossRefGoogle Scholar
  32. 32.
    Mutig K, Paliege A, Kahl T, Jöns T, Müller-Esterl W, Bachmann S (2007) Vasopressin V2 receptor expression along rat, mouse, and human renal epithelia with focus on TAL. Am J Physiol Ren Physiol 293:F1166–F1177CrossRefGoogle Scholar
  33. 33.
    Olsen JV, de Godoy LM, Li G et al (2005) Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol Cell Proteomics 12:2010–2021CrossRefGoogle Scholar
  34. 34.
    Pizzonia JH, Gesek FA, Kennedy SM, Coutermarsh BA, Bacskai BJ, Friedman PA (1991) Immunomagnetic separation, primary culture, and characterization of cortical thick ascending limb plus distal convoluted tubule cells from mouse kidney. In Vitro Cell Dev Biol 27A:409–416PubMedCrossRefGoogle Scholar
  35. 35.
    Rampoldi L, Scolari F, Amoroso A et al (2011) The rediscovery of uromodulin (Tamm–Horsfall protein): from tubulointerstitial nephropathy to chronic kidney disease. Kidney Int 80:338–347PubMedCrossRefGoogle Scholar
  36. 36.
    Rappsilber J, Ishihama Y, Mann M (2003) Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal Chem 75:663–670PubMedCrossRefGoogle Scholar
  37. 37.
    Renigunta A, Renigunta V, Saritas T, Decher N, Mutig K, Waldegger S (2011) Tamm–Horsfall glycoprotein interacts with renal outer medullary potassium channel ROMK2 and regulates its function. J Biol Chem 286:2224–2235PubMedCrossRefGoogle Scholar
  38. 38.
    Santambrogio S, Cattaneo A, Bernascone I et al (2008) Urinary uromodulin carries an intact ZP domain generated by a conserved C-terminal proteolytic cleavage. Biochem Biophys Res Commun 370:410–413PubMedCrossRefGoogle Scholar
  39. 39.
    Schley G, Klanke B, Schödel J et al (2011) Hypoxia-inducible transcription factors stabilization in the thick ascending limb protects against ischemic acute kidney injury. J Am Soc Nephrol 22:2004–2015PubMedCrossRefGoogle Scholar
  40. 40.
    Stiehl DP, Wirthner R, Koditz J et al (2006) Increased prolyl 4-hydroxylase domain proteins compensate for decreased oxygen levels. Evidence for an autoregulatory oxygen-sensing system. J Biol Chem 281:23482–23491PubMedCrossRefGoogle Scholar
  41. 41.
    Terryn S, Jouret F, Vandenabeele F et al (2007) A primary culture of mouse proximal tubular cells, established on collagen-coated membranes. Am J Physiol Ren Physiol 293:F476–F485CrossRefGoogle Scholar
  42. 42.
    Valentich JD, Stokols MF (1986) An established cell line from mouse kidney medullary thick ascending limb. I. Cell culture techniques, morphology, and antigenic expression. Am J Physiol 251:C299–C311PubMedGoogle Scholar
  43. 43.
    Wiggins RC (1987) Uromucoid (Tamm–Horsfall glycoprotein) forms different polymeric arrangements on a filter surface under different physicochemical conditions. Clin Chim Acta 162:329–340PubMedCrossRefGoogle Scholar
  44. 44.
    Wu MS, Bens M, Cluzeaud F, Vandewalle A (1994) Role of F-actin in the activation of Na(+)-K(+)-Cl− cotransport by forskolin and vasopressin in mouse kidney cultured thick ascending limb cells. J Membr Biol 142:323–336PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Bob Glaudemans
    • 1
  • Sara Terryn
    • 2
  • Nadine Gölz
    • 1
  • Martina Brunati
    • 3
  • Angela Cattaneo
    • 4
  • Angela Bachi
    • 4
  • Lama Al-Qusairi
    • 6
  • Urs Ziegler
    • 5
  • Olivier Staub
    • 6
  • Luca Rampoldi
    • 3
  • Olivier Devuyst
    • 1
    • 2
    Email author
  1. 1.Institute of Physiology, ZIHPUniversity of ZurichZürichSwitzerland
  2. 2.Division of NephrologyUniversité catholique de Louvain (UCL) Medical SchoolBrusselsBelgium
  3. 3.Dulbecco Telethon Institute, Molecular Genetics of Renal Disorders UnitSan Raffaele Scientific InstituteMilanItaly
  4. 4.Biomolecular Mass Spectrometry Unit, Division of Genetics and Cell BiologySan Raffaele Scientific InstituteMilanItaly
  5. 5.Center for Microscopy and Image Analysis (ZMB)University of ZurichZurichSwitzerland
  6. 6.Department of Pharmacology and ToxicologyUniversity of LausanneLausanneSwitzerland

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