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

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 ob tained, the vast majority of which are medullary thick ascending limb (MTAL ) segments. These are identified individually by their morphological appear ance and large amounts are collected with a micropipette mounted on a micro manipulator. This ensures maximal homogeneity of the starting material. Mon olayers of cells grow out of these MTAL segments after seeding them onto co llagen-coated, permeable filter supports. During the week following conflue nce, the cultures exhibit an apical side-positive transepithelial potential difference. Electron microscopic examination shows a monolayer of polarise d 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, whic h occurs at very high rates across the apical membrane and is much slower a cross 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). Intrac ellular calcium signalling is observed af-ter stimulation with 1 mu mol/l a denosine, adenosine 5'-triphosphate (ATP) and bradykinin. In addition, we c ompared these characteristics with those of TALH-SVE cell monolayers, an es tablished immortalised cell line of the same origin.