Characterization of primary cultured rabbit MTAL cells and effects of metabolic inhibition on intracellular pH

Abstract

Since existing methods for obtaining primary cell cultures from MTAL segments have the disadvantage of yielding a mixture of different cell types, the first goal of this study is to develop a new method for obtaining 100% pure primary cell cultures from MT AL segments of rabbit kidneys. This will allow us to evaluate whether cells within the same monolayer behave homogeneously. A second goal is to characterize these primary cell cultures with respect to their phenotype as MT AL cells. This will include the investigation of their morphology, electrophysiological characteristics and hormone responsiveness. These characteristics can then be compared with those from an immortalized cell line of the same origin (TALH-SVE) (Scott et al., 1986), which will be used in parallel studies. Since changes in intra- and extracellular pH play a crucial role in IRI, a third goal of this work consists of the extensive characterization of the pH regulatory mechanisms of these primary cell cultures. Apart from Na+/H+ exchangers and H+ pumps, NH4+ transport pathways will be investigated since NH/ reabsorption by the MTAL has a profound impact on its intracellular pH. The fourth goal of this study is to investigate the cellular pathophysiology of metabolic inhibition in these primary cultured MT AL cells. The discovery of the important role of glycine as a protective agent during ischemia, emphasizes the importance of working in physiologically relevant conditions. Therefore all experiments will be performed at 37° C and a balanced salt solution containing phosphate, magnesium and small amino acids will be used. The focus will be on sublethal injury to the cells. Therefore, first the ischemia and reperfusion time interval that leads to sublethal injury in these cells will be determined. To further validate this model of sublethal ischemic damage, changes in ATP during this time interval will be measured. For reasons outlined in the introduction, a detailed description of the parallel changes in [Ca2+]i and pHi during ischemia and reperfusion will be realized. By using digitized microfluorescence, the correlations between different parameters (for example pH; and cell death) can be investigated at the single cell level. Possible protective mechanisms and/or interventions, such as NHE inhibition, also belong to the scope of this study. A final goal of this study is to compare the results obtained during IRI in these primary cell cultures to the results that were obtained on immortalized cell lines of the distal nephron such as A6, MOCK and TALH-SVE cells.