Calcium transport in rabbit distal cells

Abstract

In normal conditions the kidney reabsorbs almost all the calcium filtered at the glomerulus. The major part of the reabsorption is achieved passively along the proximal tubule through the paracellular pathway. Calcium transport continues in the loop of Henle where about 20% of the filtered load is reabsorbed by both passive and active transport pathways. The remaining calcium reabsorption occurs mainly along the distal tubule. The high transepithelial resistance of this structure excludes the passage of calcium by the paracellular shunt and implies the predominance of transcellular mechanisms. Supporting this assumption several workers have demonstrated that calcium transport in these segments is hormonally controlled. In the rabbit, the distal tubule consists of three different segments: the early distal convoluted bright tubule (DCTb), the connecting tubule and the collecting duct. The early distal consists of only one cell type and the transition with the following segment is sharp, so that this segment is very well delimited. It possesses in its basolateral membrane an adenylate cyclase sensitive only to calcitonin [1]. Although Shimizu et al [2] have clearly established an overall active calcium transport in this segment, the cellular mechanisms involved remain to be clarified. Of course, according to the classical two-step process the ultrafiltrable calcium enters the cell through the apical membrane and is extruded across the basolateral one. Due to the very low cytoplasmic free calcium concentration and the negative membrane potential, there is a large electrochemical gradient which favors the passive diffusion of calcium across the apical membrane. On the other hand, the efflux across the basolateral membrane is the result of primary or secondary active coupling mechanisms. To clarify the mechanisms underlying the calcium transport in both membranes of DCTb we used primary cultures of microdis-sected well-defined nephron segments. The resulting epithelia have been characterized in previous studies [3]. They retain most of the morphological, biochemical and immunological properties of the original segment. The simple geometry and free access to the apical membrane of cultured monolayers are features which make these cultures well adapted to studies using electrophysiological techniques. We therefore investigated the existence of calcium permeable channels by using the patch clamp technique on the apical membrane. To determine the intracellular calcium concentration, we also performed fluorescence experiments after loading the cells with calcium sensitive fluorescent probes (Fura 2 or Indo 1). The fluorescence intensity inside the cells was analyzed by either video microscopy or laser scan cytometry. The high sensitivity of these methods made it possible to study cultures growing on semi-transparent permeable supports. By means of a custom-made perfusion chamber, the basolateral and apical media could be changed independently providing an indirect method of estimating calcium movement through the monolayer. By comparing the data from the fluorescence and patch clamp techniques, a model of calcium reabsorption across the distal cell was established.