Mary W. Smith

HgCl2-induced changes in cytosolic Ca2+ of cultured rabbit renal tubular cells

Fura 2 was used to measure changes in cytosolic [Ca2+] ([Ca2+]i) in cultured rabbit kidney proximal tubule cells exposed to HgCl2. Treatment with 2.5-10 microM HgCl2 resulted in an extracellular [Ca2+] ([Ca2+]e)-independent 2- to 12-fold increase in [Ca2+]i above resting levels of about 100 nM. Treatment with 25-100 microM HgCl2 caused a rapid [Ca2+]e-independent 10- to 12-fold increase in [Ca2+]i within 1 min followed by a recovery to about 2-fold steady state by 3 min. With 25-100 microM HgCl2, both magnitude and rate of Ca2+ increase were similar, but recovery was greater with increasing doses. A slower, secondary increase in [Ca2+]i followed which varied with HgCl2 concentration and required [Ca2+]e. The first increase in [Ca2+]i represents release from intracellular pools. Calcium channel blockers, calmodulin inhibitors, and mitochondrial inhibitors do not alter the patterns of [Ca2+]i changes due to HgCl2. The recovery response with higher HgCl2 concentrations appears to be triggered by Hg2+ and not by the increased [Ca2+]i. Sulfhydryl modifiers N-ethylmaleimide, PCMB and PCMBS produced [Ca2+]e-independent [Ca2+]i increases similar to those induced by low HgCl2 concentrations. Cell killing with HgCl2 was about 50% greater with normal [Ca2+]e than with low [Ca2+]e, suggesting that [Ca2+]e influx is important in accelerating injury leading to cell death.

Changes in mitochondrial lipids of rat kidney during ischemia

Lipid changes in mitochondria isolated from rat kidney after various periods of ischemia were analysed by thin-layer chromatography and gas-liquid chromatography. Free fatty acids were increased at 30 min and more so thereafter. Total phospholipid fatty acids decreased steadily. The proportion of diphosphatidylglycerol (cardiolipin) in the total phospholipid fraction decreased at 30 min, but the proportion of phosphatidylcholine and phosphatidylethanolamine in the total phospholipid fraction did not change until the irreversible phase of ischemic injury. We have shown that decrease of cardiolipin in mitochondrial membrane occurs early during ischemia, and only during the irreversible phase of ischemia are phosphatidylethanolamine and phosphatidylcholine broken down. It is postulated that these phenomena are due to activation of phospholipase in the mitochondrial membrane.

Oxidative Injury Induces Influx-Dependent Changes in Intracellular Calcium Homeostasis*

Understanding of the mechanisms of cell injury and cell death is fundamental to the understanding of both protection against and initiation of cell injury and cell death. We subjected primary cultures of proximal tubular epithelium (PTE) from adult rats to an exogenous oxidative stress, generated by xanthine/xanthine oxidase (X/XOD), and studied its effect on the concentration of cytosolic ionized calcium ([Ca2+]i) by means of digital imaging fluorescence microscopy (DIFM) using a cytosolic calcium probe, fura-2. Exposure to 25 mU/ml X/XOD caused notable increases in [Ca2+]i detectable within 15 sec and increasing to micromolar levels with time. Experiments with Ca2+-free medium containing ethylene glycol-bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid (EGTA) showed that the increase of [Ca2+]i was due to influx from the extracellular space. Smaller and slower increases in [Ca2+]i were seen after exposure to lower concentrations of X/XOD (5 and 10 mU/ml). PTE injury and killing were assessed by measuring the release of cytosolic lactate dehydrogenase (LDH), exclusion of trypan blue, and observation of morphologic changes. Exposures to the 25 mU/ml concentration of X/XOD caused significant LDH release after 2 hr and correlated with trypan blue staining of exposed cells. Again, lesser concentrations of X/XOD resulted in a slower release of smaller amounts of LDH, and thus delayed trypan blue staining. Cytoplasmic bleb formation was seen by phase microscopy within minutes of exposure to 25 mU/ml, followed by cell rounding, retraction, and disintegration. Transmission electron microscopy revealed a progression of changes characteristic of lethal cell injury, beginning with dilatation of the endoplasmic reticulum, detachment of ribosomes, condensation of mitochondria, and chromatin clumping and terminating with mitochondrial swelling and formation of intramitochondrial flocculent densities. These studies clearly show that notable increases of [Ca2+]i precede both sub-lethal and lethal changes in rat PTE. These results indicate that interventions designed to minimize or to accelerate calcium entry could be of importance in cell preservation or cell killing, respectively, and therefore to therapeutic strategies for myocardial infarction, stroke, or shock in the former instance and for cancers in the latter.

Effects of Ca2+ deregulation on mitochondrial membrane potential and cell viability in nucleated cells following lytic complement attack

We have previously shown [Papadimitriou JC. Ramm LE. Drachenberg CB. Trump BF. Shin ML. (1991) J. Immunol., 147, 212-217] that formation of lytic C5b-9 channels on Ehrlich ascites tumor cells induced rapid depletion of adenine nucleotides associated with prelytic leakage preceding cell death. Extracellular Ca2+ concentration ([Ca2+]e) reduction by chelation markedly delayed the onset of cell death, although the adenine nucleotide leakage was enhanced. In the present study, we examined the temporal relationships between ionized cytosolic Ca2+ ([Ca2+]i), mitochondrial membrane potential (delta psi m) and cell death in individual cells by digital imaging fluorescence microscopy (DIFM), during the earliest phase of C5b-9 attack. The results showed an immediate, > 20-fold rise in [Ca2+]i, rapidly followed by dissipation of delta psi m and subsequent acute cell death. These events were markedly delayed by chelation of Ca2+e, but not by nominally Ca2+ free medium. Differing from previous reports indicating propidium iodide labeling of viable cells bearing C5b-9 channels, with DIFM we observed nuclear fluorescence with that marker only in association with cell death. These findings indicate that Ca2+ influx through lytic C5b-9 channels is responsible for the massive increase in [Ca2+]i, as well as for the rapid loss of delta psi m, followed by acute cell death. When this [Ca2+]i increase is prevented, the cell death is probably related to metabolic depletion.

Characterization of a Renal Epithelial Cell Model of Apoptosis Using Okadaic Acid and the NRK-52E Cell Line

Apoptotic cell death plays an important role in the pathogenesis of renal tubular epithelial damage and repair following tubular injury. Presently, the cellular factors involved in regulating apoptotic pathways in the kidney are unknown. To address the possibility that protein phosphorylation may regulate apoptosis in kidney cells, okadaic acid (OKA), a specific inhibitor of protein phosphatases 1A and 2A, was tested for its morphologic and biochemical effects on normal rat kidney epithelial cells (NRK-52E) in culture. As revealed by the DNA-specific stain DAPI, nuclei of cells treated with 1.0 μM okadaic acid contained irregular clumps of dense chromatin. Additional morphologic alterations typical of apoptosis were apparent within 2 hr after treatment with 1.0 μM OKA, including marked cellular rounding, cytoplasmic condensation, and cytoplasmic blebs. Ultrastructurally, 1.0 μM OKA caused cytoplasmic bleb formation, cellular fragmentation, condensation of heterochromatin into clumps, and segregation of nucleoli. At this stage, the cytoplasmic fragments and blebs contained many normal mitochondria. The attached, rounded cells also effectively excluded propidium iodide, demonstrating maintenance of membrane integrity despite pronounced morphologic alterations. A 2-fold increase in intracellular free Ca2+ was apparent 90 min after treatment with 1.0 μM okadaic acid. Transverse alternating field electrophoresis revealed the appearance of large DNA fragments of approximately 300-kbp. The appearance of these 300-kbp fragments correlated temporally with the observed elevation in intracellular calcium and the onset of morphologic alterations. However, preloading cells with EGTA-AM, an intracellular calcium chelator, obliterated the calcium elevation and had no effect on OKA-induced morphology, DNA fragmentation, or cell death. Detectable internucleosomal fragmentation occurred much later than the onset of morphologic changes (24-hr treatment time) and did not correlate with elevations in cellular calcium. These studies support the hypothesis that during apoptosis, chromatin condensation reflects chromatin cleavage at nuclease-sensitive sites between hexameric rosettes. These results also suggest that morphologic and nuclear alterations in the pathway of OKA-induced apoptosis occur independent of observed increases in intracellular calcium.

Extracellular Ca2+-Dependent Elevation in Cytosolic Ca2+ Potentiates HgCl2-Induced Renal Proximal Tubular Cell Damage

While normal fluctuations of cytosolic Ca 2 + ([Ca 2+] i) occur physiologically, the deregulation of cellular Ca 2 + homeostasis leads to cellular injury. The contribution of [ Ca 2+]i to the process of cellular damage was assessed in a model system where HgCl2 was used to induce plasma membrane damage in renal tubular cells. In the presence of 1.37 mM extracellular Ca 2 +, HgCl2 (10-50 μM) induced a slow, dose-dependent, 4-6 fold increase in [Ca2+]i (as measured by Quin 2) by 10 min of exposure, which could be abolished by prior incubation of the cells with dithiothreitol. Correlates of cellular injury, i.e., decrease in cell viability, change in cellular morphology, such as bleb formation, membrane distortion and mitochondrial swelling, were induced after HgCl2 addition. The rate and dose-responses of these changes were similar to that of [ Ca]i elevation. When cells were exposed to HgCl2 in the absence of added extracellular Ca 2+ , there was no increase in [Ca2+ ]i and both the rate and extent of cell damage were reduced. When Ca 2 + was readded to the extracellular medium after HgCl2, there was a rapid elevation of [Ca 2 + ]i, increased cell killing and bleb formation. The observed correlation between [Ca 2 + ]i elevation, decreased cell viability and morphological aberrations in terms of (i) dose-dependency for HgCl2, (ii) requirement for high extracellular Ca 2 +, and (iii) rate of change, suggests that HgCl2-induced renal cell damage involves the entry of Ca 2 + from the extracellular milieu which potentiates the progression of cellular injury.

Changes in [Ca 2+]i in cultured rat proximal tubular epithelium: an in vitro model for renal ischemia

Two components of ischemia, oxygen deprivation and glycolytic inhibition, were studied in primary cultures of rat proximal tubular epithelial cells (PTE). Changes in cytosolic Ca2+ ([Ca2+]i) and its relationship to loss of mitochondrial membrane potential (delta psi m) and cell killing were characterized in single cells whereas ATP and LDH release were determined in populations of monolayer PTE. (1) Inhibition of mitochondrial respiration with KCN or anoxia resulted in little decrease in ATP or cell killing and slight change in [Ca2+]i over many hours. (2) Inhibition of respiration and glycolysis with anoxic HBSS minus glucose resulted in decreased ATP (54.4%) and cell killing (20%) during 5 h anoxic exposure. In all cases, but at highly variable times (113 +/- 62 min), [Ca2+]i initially rose to > 1 microM. In some cases it immediately dropped, stabilizing at about 500 nM for up to 1 h and rising again just prior to cell death. (3) Inhibition with anoxia + 1 mM IAA resulted in rapid depletion of ATP and cell killing, with increases in [Ca2+]i to > 1 microM by 20 +/- 2 min. (4) Depletion of glycolytic metabolites by depriving cells of substrate for 12 h (in HBSS minus glucose) before subjecting to anoxia minus glucose resulted in increases in [Ca2+]i at 40 +/- 17 min followed by cell killing. (5) Injury with anoxic HBSS minus glucose was reversed by reaeration before or during the initial rise in [Ca2+]i. Later reaeration resulted in rapid cell killing. In all cases, delta psi m was dissipated only after [Ca2+]i was significantly elevated.

Differential effects of cigarette smoke condensate and its fractions on cultured normal and malignant human bronchial epithelial cells.

The differential effects of cigarette smoke condensate (CSC) and its fractions (neutral, basic, and acidic fractions) on proliferation and squamous differentiation of normal human bronchial epithelial (NHBE) cells versus human lung carcinoma cells were investigated. CSC, and the neutral and acidic fractions inhibited cellular proliferation more than the basic fraction. When compared to the acidic and basic fractions, CSC and the neural fraction were more effective in causing squamous differentiation of NHBE cells and inhibiting specific binding of phorbol dibutyrate (PDBU). There were no significant changes in ionized cytosolic calcium concentration when NHBE cells were treated with CSC. In contrast to the normal epithelial cells, neither HUT-292 nor the 3 other carcinoma cell lines examined showed marked squamous morphological changes when exposed to either CSC or its fractions and the carcinoma cells were more resistant to their inhibiting effects on cellular proliferation. These results are consistent with the hypothesis that differential effects of tobacco smoke components on cellular proliferation may allow clonal expansion of preneoplastic and neoplastic human bronchial epithelial cells during lung carcinogenesis.

H-ras transfection of the rat kidney cell line NRK-52E results in increased induction of c-fos, c-jun and hsp70 following sulofenur treatment

The effect of the antineoplastic drug sulofenur on the induction of the immediate-early genes (IEG) c-fos and c-jun and the stress gene hsp70 was compared in the rat kidney epithelial-like cell line NRK-52E and a derivative H-ras-transfected (H/1.2NRK-52E) cell line. Fold induction for each gene after sulofenur (500 microM) treatment was greater in H/1.2NRK-52E. The maximum increases for NRK-2E and H/1.2NRK-52E were as follows: c-fos, approximately 10-fold and approximately 18-fold; c-jun, approximately 2.5-fold and approximately 3.6-fold; hsp70, approximately 13-fold and approximately 30-fold. In cells loaded with EGTA/AM or treated in low or no Ca2+ HBSS, c-fos induction was reduced similarly in both cell types. However, inhibition of protein kinases with staurosporin and calphostin C reduced c-fos by 80% in NRK-52E but by only 10-20% in H/1.2NRK.52E. These results indicate that sulofenur-induced IEG elevation is Ca(2+)-dependent and that the requirement for protein kinase C activation is bypassed in H-ras-transfected cells.

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.