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Microscopic observation of NRK-52E rat renal epithelial cells after uranium acute exposure: morphological changes and cellular distribution
M. Carrière, L. Avoscan, H. Khodja, B. Gouget
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Microscopic observation of NRK-52E rat renal epithelial cells after uranium acute exposure: morphological changes and cellular distribution

Fig. 1. Phase contrast appearance of NRK-52E cells. A: control cells, B: cells exposed 24 h to 700 µM uranyl-bicarbonate, C: cells exposed 24 h to 20 µM cadmium chloride.

Uranium is known as a nephrotoxicant that exert most of its toxicity on proximal tubule epithelial cells. Although renal damages induced by uranium acute intoxication have been largely studied in vivo, few in vitro data are available concerning the mechanisms implicated in its cytotoxicity. The aim of this study was to characterize cell morphological changes consecutive to uranium acute exposure and to confirm that uranium cytotoxicity was correlated with its cellular intake. Phase contrast microscopic observation of NRK-52E rat kidney proximal tubule epithelial cells exposed to uranium and cadmium, the latter being considered as the reference nephrotoxicant, revealed very different morphological changes even if cellular repartition of these toxics, analyzed by nuclear microprobe imaging, is quite similar. Transmission and scanning electron microscopy images are reported, allowing a first step towards the comprehension of mechanisms of uranium intake and cell effects. Control cells, observed in phase contrast microscopy at 100% confluency, are polyhedral and exhibit a compact cobblestone appearance (figure 1A) as previously described. Uranium toxicity appeared after 15 h uranyl-bicarbonate exposure. From this time, some of the cells appeared to have exploded, whereas others just nearby appeared to be insensitive to uranium exposure (figure 1B). The fields containing exploded cells expanded continuously with exposure time. The whole monolayer was reduced to fragments after 24 h exposure. These fragments remained attached to the semi-permeable culture membrane. In the case of cadmium exposure, the links between the cells rapidly and almost simultaneously broke (figure 1C); cells then detached from the culture membrane.  
These patterns were confirmed by SEM observation. Control cells appeared like a compact monolayer, covered with extracellular matrix (figure 2A). In the case of uranium exposed cells, extracellular matrix disappeared and a part of the monolayer appeared totally disrupted into cell fragments (figure 2B). Cadmium treated cells had a rounded appearance and were partly detached from the culture membrane; the extracellular matrix still covered the cells (Figure 2C). These very different cell aspects after exposure to uranium or cadmium are certainly associated with very different metabolisms of these toxicants. In the case of cadmium, the observations are consistent with previously published data. It is well known cadmium interferes with adhesion molecules cadherin-catenin complex. Cadmium exposure thus causes the cells to detach from one another and then detach from their culture membrane. Furthermore, cadmium is known to enter cells via receptors and then associate with intracellular thiols such as GSH and metallothionein (MT); this explains the absence of cadmium precipitates in the cell cytoplasms. Molecular causes of uranium toxicity are still under investigation. This work shows that uranium toxicity can be correlated to its cellular accumulation. After uranium exposure, cells are reduced to small fragments looking like apoptotic bodies; it is thus possible that programmed cell death appears after uranium contamination of renal epithelial cells in vitro, this hypothesis has to be confirmed. Uranium precipitates are detected in cell cytoplasms, they can either form inside cells after uranium internalization in a soluble chemical form, or precipitate outside the cells and then be internalized. Detailed study of cell penetration pathway and intracellular traffic of uranium is necessary to answer these questions.  
Microscopic observation of NRK-52E rat renal epithelial cells after uranium acute exposure: morphological changes and cellular distribution

Fig. 2. Scanning Electron Microscopy of NRK-52E cells. A: control cells, B. cells exposed 24 h to 700 µM uranyl-bicarbonate, C: cells exposed 24 h to 20 µM cadmium chloride.

#427 - Màj : 25/03/2005

 

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