Kathryn A. Elliget

HgCl2-induced alteration of actin filaments in cultured primary rat proximal tubule epithelial cells labelled with fluorescein phalloidin

When proximal tubule epithelial cells are exposed to HgCl2, cytoplasmic blebs are formed. These represent on early, potentially reversible response to injury. These blebs are accompanied by reorganization of cytoskeletal proteins, and pre-sumably by alternations in cytoskeletal-plasma membrane interactions. Ca2+-activated proteinases, such as calpain, are known to affect cytoskeletal proteins and to be involved in diverse cellular processes. However, the role of calpains in cytotoxicity d due to HgCl2 is unknown. To determine the relationship between Factin, calpain, and HgCl2 toxicity, cells were stained with fluorescein phalloidin before and after treatment with HgCl2. Cells were grown on coverslips and exposed to HgCl2 (10 or 25 μM) in the presence or absence of the calpain inhibitor, leupeptin. Untreated cells were flat, polygonal, and contained many fluorescent-stained cables of actin filaments. Generally, cells exposed to HgCl2 became pleomorphic and contracted as the blebs formed. These cells showed fewer actin cables and fluorescence was seen mostly as either compact areas of dense stain or as peripheral rings. In many cells, actin cables and filaments were completely absent. Disappearance of F-actin was initially seen by 2 min after exposure to HgCl2. Thus, disruption of the actin cytoskeleton and blebbing were found to be early events in HgCl2 toxicity. When leupeptin was used with HgCl2 treatment, the actin staining appeared similar to that of untreated cells. These findings clearly illustrate that HgCl2 injury to proximal tubule epithelial cells causes rearrangement and alteration of F-actin which may involve the activation of calpain.

Primary cultures of normal rat kidney proximal tubule epithelial cells for studies of renal cell injury

SummaryNormal rat kidney proximal tubule epithelial cell cultures were obtained by collagenase digestion of cortex and studied for 10 days. To assess the purity of the seeding suspension, we histochemically demonstrated γ-glutamyltranspeptidase in >95% of the starting material. To identify cell types in cultures, we investigated several markers. Cells stained positively for lectinArachis hypogaea (rat proximal tubule) and negatively forLotus tetragonolobus (rat distal tubule). Intermediate filament expression of cytokeratin confirmed the epithelial differentiation of the cultured cells. Using indirect immunofluorescence, we found that cultures were negative for vimentin and Factor VIII. Cells exhibited activities of two brush border enzymes, γ-glutamyltranspeptidase and leucine aminopeptidase, and Na+-dependent glucose transport activity. Multicellular domes were evident in the Week 2 of culture. Proliferation was studied by comparing growth factor-supplemented serum-free medium to cells grown in serum; growth enhancers included insulin, hydrocortisone, transferrin, glucose, bovine albumin, and epidermal growth factor. Cells proliferate best in medium with 5 or 10% serum and in serum-free medium supplemented with insulin, hydrocortisone, transferrin, glucose, and bovine albumin. Proliferation was assessed by determining cell number (population doublings). By light microscopy, the cells were squamous with numerous mitochondria, a central nucleus, and a rather well-defined homogeneous ectoplasm. By electron microscopy, the cells were polarized with microvilli and cell junctions at the upper surface and a thin basal lamina toward the culture dish. These data show that the proximal tubule epithelial cells retain a number of functional characteristics and that they represent an excellent model for studies of normal and abnormal biology of the renal proximal tubule epithelium.

H-ras-transformed NRK-52E renal epithelial cells have altered growth, morphology, and cytoskeletal structure that correlates with renal cell carcinoma in vivo

SummaryWe studied the effect of the ras oncogene on the growth kinetics, morphology, cytoskeletal structure, and tumorigenicity of the widely used NRK-52E rat kidney epithelial cell line and two H-ras oncogene-transformed cell lines, H/1.2-NRK-52E (H/1.2) and H/6.1-NRK-52E (H/6.1). Population doubling times of NRK-52E, H/1.2, and H/6.1 cells were 28, 26, and 24 h, respectively, with the transformed cells reaching higher saturation densities than the parent cells. NRK-52E cells had typical epithelial morphology with growth in colonies. H/1.2 and H/6.1 cell colonies were more closely packed, highly condensed, and had increased plasma membrane ruffling compared to parent cell colonies. NRK-52E cells showed microfilament, microtubule, and intermediate filament networks typical of epithelial cells, while H/1.2 and H/6.1 cells showed altered cytoskeleton architecture, with decreased stress fibers and increased microtubule and intermediate filament staining at the microtubule organizing center. H/1.2 and H/6.1 cells proliferated in an in vitro soft agar transformation assay, indicating anchorage-independence, and rapidly formed tumors in vivo with characteristics of renal cell carcinoma, including mixed populations of sarcomatoid, granular, and clear cells, H/6.1 cells consistently showed more extensive alterations of growth kinetics, morphology, and cytoskeleton than H/1.2 cells, and formed tumors of a more aggressive phenotype. These data suggest that analysis of renal cell characteristics in vitro may have potential in predicting tumor behavior in vivo, and significantly contribute to the utility of these cell lines as in vitro models for examining renal epithelial cell biology and the role of the ras proto-oncogene in signal transduction involving the cytoskeleton.

Cytosolic Ca2+ Elevation and Calpain Inhibitors in HgCl2 Injury to Rat Kidney Proximal Tubule Epithelial Cells

This study assessed HgCl2 injury to proximal tubule epithelial cells as it relates to the concentration of ionized cytosolic Ca2+ ([Ca2+]i) elevation and activation of calpains. Experiments in high and low extracellular Ca2+ concentration ([Ca2+]e) were performed using the calpain inhibitors antipain and leupeptin, and also trypsin inhibitor, methylamine, chloroquine, and ryanodine. Cell killing was time/dose dependent and greater with high [Ca2+]e. After 30 min treatment with 25 microM HgCl2, 19% of cells in low [Ca2+]e were dead while 72% died in high [Ca2+]e. Morphologic changes such as cytoplasmic blebbing were also greater in high [Ca2+]e. Antipain and leupeptin diminished toxicity. Leupeptin did not block Ca2+ entry into cells. Results show that HgCl2 toxicity is correlated with increased [Ca2+]i, and that calpains may mediate the resultant pathological changes.

Long-term culture of human esophageal explants and cells

Human esophageal epithelium obtained from intermediate autopsies (<12 h) was maintained as cell and explant cultures. In order to develop a serum-free, defined media culture model, several medias and additives were evaluated. The viability and differentiation of the epithelial cells cultured with serum-free, Keratinocyte Growth Media (KGM, Clonetics Co., San Diego, CA) was improved over that of esophageal cells and explants cultured in either serum-supplemented CMRL 1066 (OCM), serum-free additive-supplemented CMRL 1066, or cimetidine-supplemented CMRL 1066. The KGM component EGF was determined to be trophic for esophagus cells on the basis of findings of increased 3H-TdR tabelling in KGM cultures when compared to control cells grown in KGM without EGF (KBM). The morphologic pattern of the cytoskeletal proteins actin, keratin, and vimentin were characterized in isolated cell populations. The intermediate filaments, keratin, and vimentin were co-expressed in these epithelial cells. Esophageal explant viability, differentiation, and outgrowth from 15 cases were also evaluated in dishes coated with basement membrane associated proteins. Explants cultured in these dishes were equally well-preserved and differentiated. There were no significant differences in the explant histology when there was protein coating of the culture dishes, although one case showed improved outgrowth with laminin coating. A main advantage for using this culture system is that the same medium (KGM) can be used for both the culture of explants and isolated epithelial cells. Future applications of this model include determining: (1) the effect different concentrations of EGF and calcium in the media will have on esophageal proliferation and differentiation, and (2) the role of different basement membrane associated proteins on the plating efficiency of either isolated or outgrowth epithelial esophageal cells.

Purification of neonatal rat cardiac cells by centrifugal elutriation

A method utilizing centrifugal elutriation to obtain a highly purified (95 ± 2% after 3 d in culture) preparation of beating cardiac myocytes from enzyme-dissociated neonatal rat heart is described. An additional fraction enriched for nonmuscle cells is also obtained. Purity of myocyte cultures is determined by Coomassie blue R250 staining of the cytoskeleton; this procedure is also described.

Relation Between Toxicity and Carcinogenesis in the Kidney: an Heuristic Hypothesis

Cellular toxicity and cellular carcinogenesis are closely linked. In the kidney, this relationship has been emphasized by the recent discovery of a number of putatively non-mutagenic chemicals that result in acute and chronic toxicity and ultimately in carcinogenesis, especially in the male rat. Many, but not all such compounds, result in renal PTE phagolysosomal overload. At the same time, known metabolites of other carcinogens, e.g., HCBD and FBPA, result in acute renal injury and/or necrosis, followed by chronic tubular disease, interstitial nephritis, and ultimately carcinogenesis. A series of cell mechanisms have been suggested that lead from acute cell injury to altered control of cell division. These mechanisms appear to involve ion deregulation, (especially [Ca2+]i) resulting from a variety of continued injuries, (e.g., oxidative stress from inflammatory cells) and ultimately leading to altered gene expression.