Cellular defence mechanisms in renal distal tubular A6 cells facing metabolic inhibition

Abstract

In view of the high resistance to ischemic injury of distal tubular cells (see section 1.5.1), a distal tubular cell line is used in this study as an in vitro model to examine some of the cellular defence processes occurring in surviving renal cells during energy depletion. To allow a detailed study of some cellular events and to investigate the relationships between them, a cell line with a low rate metabolism was chosen: the A6 cell line. Moreover, this cell line is a well-characterised model tight epithelium, which resembles the principal cells in mammalian collecting tubules, and that allows electrophysiological measurements to follow transepithelial transport characteristics. To enable us to understand the survival of distal tubular cells in ischemic conditions with threatening high apical sodium levels in the distal tubular lumen, we first characterised the dependency of Na· transport characteristics on the apical Na· concentration (Chapter 3) in normal conditions. Moreover, since there is convincing evidence in literature that intracellular acidification in ischemic conditions might be cytoprotective (see section 1.5.2.2), we determined first the effects of intracellular pH-shifts on Na· transport in control conditions (Chapter 4). A second objective was to investigate the time course of changes in morphology and to determine when irreversible cell death features, such as plasma membrane damage occur in A6 cells facing metabolic inhibition (MI) (Chapter 5). This allowed us to establish a 45 min protocol of MI, since in this time period no detectable cell death occurred and hence the epithelial cells were expected to be sub lethally injured at most. Since A6 cells, like mammalian distal tubular epithelia, are active Na· transporting epithelia, we then focused on the suppression of Na· transport in energy-depleted conditions. The third aim of this study was to look for possible cellular defence strategies related with mechanisms underlying Na· transport suppression during MI. Moreover, it was examined whether transcellular Na· transport and cellular energy levels recovered after MI (Chapter 6). One of the nefast consequences of renal ischemia is excessive tubular epithelial cell swelling, which contributes to renal dysfunction by inducing obstruction of the tubular lumen and vascular congestion in the outer medulla (Mason et al., 1989; Lang et al., 1995). Therefore, the last aim of this thesis was to examine in our experimental model the extent of cell swelling during MI, and to investigate whether metabolically challenged cells still possess the capacity to regulate their cell volume in iso- as well as in anisotonic conditions (Chapter 7).