Effect of aldosterone on the regulation of the volume of principal cells of rat cortical collecting duct epithelium in early postnatal development

Abstract

A rapid nongenomic regulatory effect of aldosterone on the volume of principal cells in the cortical segment of cortical collecting ducts (CCDs) of rat kidney was studied using the fluorescent dye calcein under hypoosmotic shock conditions (280‐140 mOsm/kg). In the presence of aldosterone (10 nM), the amplitude of cell volume growth significantly decreased ( p < 0.05) and the cell volume recovery time increased twice both in adult and ten-day-old animals. Age differences were detected when the time required for the cell volume recovery under hypoosmotic conditions was determined: in the adult animals, the cell volume recovered much more rapidly (by an order of magnitude) than in the ten-day-old animals both in the control and in the presence of aldosterone ( 0.09 ± 0.02 vs 2.8 ± 0.3 and 0.21 ± 0.03 vs 4.4 ± 0.5 , respectively, p < 0.001). This was the first study to demonstrate the involvement of aldosterone in the regulation of amplitude and dynamics of changes in the volume of CCD principal cells in early postnatal ontogeny. One of the key aspects of development of the osmoregulatory function of the kidney is the development of the molecular mechanisms of regulation of sodium transport in nephron epithelial cells by aldosterone. It is known that, in early ontogeny, kidneys of altricial animals including rats are insensitive to the antinatriuretic effect of aldosterone. Regulation of sodium transport in postnatal ontogeny of the rat is detected starting from the tenth day of life, when the transition from milk feeding to independent feeding is ended. The molecular mechanisms underlying the development of hormonal sensitivity of target cells of an immature kidney have been studied insufficiently well. It is known that the main proteins involved in the transmembrane sodium transport in the principal cells of the CCDs of rat nephron are the proteins of the apical epithelial of the sodium channel (ENaC) and the basolateral sodium pump ( Na + ,a + -ATPase) [6, 7]. The intracellular sodium concentration ([ ]) in principal cells is primarily determined by the dynamic equilibrium between the sodium influx through ENaC and its efflux form the cell into the intersticium against the concentration gradient by ATP-driven Na + ,a + -ATPase. Earlier, we showed that the expression of the genes encoding these proteins is suppressed and the intracellular sodium concentration in CCD principal cells is decreased in kidneys of tenday-old animals [1, 4]. At the same time, we discovered that aldosterone had a rapid nongenomic effect expressed in maintaining an increased [ ] in CCD principal cells at a low (14 mM) sodium concentration in the ambient medium both in adult and ten-day-old rats [4]. These data led us to assume that, along with the well-known mineralocorticoid effect, aldosterone is also involved in the regulation of the volume of CCD cells in rat nephron in hypoosmotic medium and that this mechanism functions even in the preweaning period. However, the rapid nongenomic effects of aldosterone on the regulation of the volume of CCD principal cells have been studied insufficiently comprehensively. In view of above, the goal of this study was to investigate the rapid nongenomic effect of aldosterone at a physiological concentration on the volume of CCD principal cells in kidneys of ten-day-old and adult rats under hypoosmotic shock conditions. Experiments were performed with CCDs of ten- and sixty-day-old Wistar rats, which were obtained by microdissection. The changes in the cell volume were assessed by the changes in the fluorescence intensity of the intracellular dye calcein and expressed in relative units. Fluorescence intensity was measured in the cells located at the open end of a CCD fragment; the basolateral and apical surfaces of these cells are in contact with the ambient solution. Continuous fluorescence recording during cell volume changes in response to osmotic shock was performed in our modification [11]. Renal tubule fragments contained in 100 µ l of suspension in aOa culture medium were loaded on a polylysinecoated slide and loaded with the fluorescent dye calcein