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Please use this identifier to cite or link to this item: http://hdl.handle.net/1942/2902

Title: 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
Authors: JANS, Frank
VANDENABEELE, Frank
Helbert, M
LAMBRICHTS, Ivo
AMELOOT, Marcel
STEELS, Paul
Issue Date: 2000
Publisher: SPRINGER VERLAG
Citation: PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY, 440(4). p. 643-651
Abstract: We describe a simple method for obtaining functionally and morphologically intact primary cultures of cells from the medullary thick ascending limb of rabbit kidneys. After digesting dissected fragments of the inner stripe of the outer medulla with collagenase, a suspension of tubule fragments is obtained, the vast majority of which are medullary thick ascending limb (MTAL) segments. These are identified individually by their morphological appearance and large amounts are collected with a micropipette mounted on a micromanipulator. This ensures maximal homogeneity of the starting material. Monolayers of cells grow out of these MTAL segments after seeding them onto collagen-coated, permeable filter supports. During the week following confluence, the cultures exhibit an apical side-positive transepithelial potential difference. Electron microscopic examination shows a monolayer of polarised cells with characteristics of distal tubular cells. The primary cultures express Tamm-Horsfall protein at their apical surface. Additional evidence for their differentiation and polarisation is the net ammonium influx, which occurs at very high rates across the apical membrane and is much slower across the basolateral membrane, as judged by measurements of intracellular pH, Adenosine 3',5'-cyclic monophosphate (cAMP) production is stimulated by arginine-vasopressin, calcitonin or isoproterenol (all 1 mu mol/l). Intracellular calcium signalling is observed af-ter stimulation with 1 mu mol/l adenosine, adenosine 5'-triphosphate (ATP) and bradykinin. In addition, we compared these characteristics with those of TALH-SVE cell monolayers, an established immortalised cell line of the same origin.
Notes: Limburgs Univ Ctr, Physiol Lab, Biomed Onderzoeksinst DWI, B-3590 Diepenbeek, Belgium. Limburgs Univ Ctr, Histol Lab, Biomed Onderzoeksinst DWI, B-3590 Diepenbeek, Belgium. Univ Antwerp Hosp, Dept Nephrol Hypertens, B-2650 Edegem, Belgium.Steels, P, Limburgs Univ Ctr, Physiol Lab, Biomed Onderzoeksinst DWI, Univ Campus,Gebouw D, B-3590 Diepenbeek, Belgium.
URI: http://hdl.handle.net/1942/2902
DOI: 10.1007/s004240000304
ISI #: 000088351200016
ISSN: 0031-6768
Category: A1
Type: Journal Contribution
Validation: ecoom, 2001
Appears in Collections: Research publications

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