Isolated perfused salamander proximal tubule: methods, electrophysiology, and transport

Abstract

Techniques are presented for the isolation and perfusion of renal proximal tubules from the neotenic salamander Ambystoma tigrinum. Methods are described for a determination of normal values for fluid transport and electrophysiological parameters. Stable cellular microelectrode recordings are reported that constitute the first intracellular measurements in an isolated perfused tubule preparation. With identical solutions in lumen and bath, fluid reabsorption averaged 0.28 nl.min-1.mm-1, transepithelial potential difference averaged -4.5 mV, transepithelial resistance was 52.1 omega.cm2, and the transepithelial chloride-to-sodium transference number ratio was 3.4. The basolateral cell membrane potential difference averaged -59.6 mV, and the ratio of apical-to-basolateral cell membrane resistance was between 3.9 and 5. Viability of the isolated perfused salamander proximal tubule preparation is demonstrated by a detailed comparison of the present data with results of in vivo micropuncture experiments on both Necturus and intact Ambystoma kidneys. In addition to being an advantageous preparation for long-term intracellular recordings, the Ambystoma kidney is unique in that proximal tubules can be studied both in isolation and by conventional micropuncture.