Extent Of Cell Injury Research Articles

Sensitive electrochemical detection of von Willebrand factor using an immunosensor comprising 7,7,8,8-tetracyanoquinodimethane and nickel-based metal–organic framework

In this work, we developed an immunosensor for determining von Willebrand factor (VWF) by combining a nickel-based metal–organic framework (NiBTC) with 7,7,8,8-tetracyanoquinodimethane (TCNQ), assembling them on a screen-printed electrode (SPE), and immobilizing VWF antibodies on the surface to obtain Ab/TCNQ-NiBTC/SPE. The capture of VWF antigens caused steric hindrance, which mitigated the electrochemical response. The immunoreaction caused a change in the peak current of Ab/TCNQ-NiBTC/SPE, which was proportional to the logarithm of VWF concentration in the range of 1–200 ng/mL, with limits of detection and quantification of 0.887 ng/mL and 2.958 ng/mL, respectively. The proposed Ab/TCNQ-NiBTC/SPE was used to investigate the release of VWF from H2O2-induced oxidative-injured human umbilical vein endothelial cells. The extent of cell injury and the amount of VWF released increased with an increase in the exposure time to H2O2; however, as the cells died due to excessive injury, the amount of VWF released decreased. The proposed TCNQ-NiBTC-based immunosensor can facilitate the convenient and accurate detection of VWF.

DNA damage in BV-2 cells: An important supplement to the neurotoxicity of CdTe quantum dots.

Microglial cells are resident immune cells in the central nervous system. Activation of microglia as induced by CdTe quantum dots (QDs) can trigger damage to neurons. To quantify the intracellular QDs, we monitored the intracellular Cd concentration in the QD-exposed mouse microglial cells (BV-2 cell line). The extent of cell injury at different times correlated with the Cd concentration in cells at that time. In addition to Cd ion detection, we also monitored the intracellular fluorescence of the QDs. More QDs accumulated in the nucleus than in the cytoplasm. Comet assays confirmed that QDs induce DNA damage. However, DNA cannot interact with QDs, so the DNA damage was not caused by CdTe QDs adducts to DNA but by the increase of the Cd ion concentration and the secondary oxidative damage. In addition to DNA damage, biofilm injury and endogenous reduced glutathione depletion were also apparent in QD-exposed BV-2 cells. These changes can be prevented or even reversed by exogenous reduced glutathione administration.

Intermittent Hypoxia Causes Inflammation and Injury to Human Adult Cardiac Myocytes

Intermittent hypoxia may occur in a number of clinical scenarios, including interruption of myocardial blood flow or breathing disorders such as obstructive sleep apnea. Although intermittent hypoxia has been linked to cardiovascular and cerebrovascular disease, the effect of intermittent hypoxia on the human heart is not fully understood. Therefore, in the present study, we compared the cellular responses of cultured human adult cardiac myocytes (HACMs) exposed to intermittent hypoxia and different conditions of continuous hypoxia and normoxia. HACMs were exposed to intermittent hypoxia (0%-21% O2), constant mild hypoxia (10% O2), constant severe hypoxia (0% O2), or constant normoxia (21% O2), using a novel cell culture bioreactor with gas-permeable membranes. Cell proliferation, lactate dehydrogenase release, vascular endothelial growth factor release, and cytokine (interleukin [IL] and macrophage migration inhibitory factor) release were assessed at baseline and after 8, 24, and 72 hours of exposure. A signal transduction pathway finder array was performed to determine the changes in gene expression. In comparison with constant normoxia and constant mild hypoxia, intermittent hypoxia induced earlier and greater inflammatory response and extent of cell injury as evidenced by lower cell numbers and higher lactate dehydrogenase, vascular endothelial growth factor, and proinflammatory cytokine (IL-1β, IL-6, IL-8, and macrophage migration inhibitory factor) release. Constant severe hypoxia showed more detrimental effects on HACMs at later time points. Pathway analysis demonstrated that intermittent hypoxia primarily altered gene expression in oxidative stress, Wnt, Notch, and hypoxia pathways. Intermittent and constant severe hypoxia, but not constant mild hypoxia or normoxia, induced inflammation and cell injury in HACMs. Cell injury occurred earliest and was greatest after intermittent hypoxia exposure. Our in vitro findings suggest that intermittent hypoxia exposure may produce rapid and substantial damage to the human heart.

Investigating membrane and mitochondrial cryobiological responses of HUVEC using interrupted cooling protocols

The success of cryopreservation protocols is largely based on membrane integrity assessments after thawing, since membrane integrity can be considered to give an upper limit in assessment of cell viability and the plasma membrane is considered to be a primary site of cryoinjury. However, the exposure of cells to conditions associated with low temperatures can induce injury to cellular structure and function that may not be readily identified by membrane integrity alone. Interrupted cooling protocols (including interrupted slow cooling without a hold time (graded freezing), and interrupted rapid cooling with a hold time (two-step freezing)), can yield important information about cryoinjury by separating the damage that occurs upon cooling to (and possibly holding at) a critical intermediate temperature range from the damage that occurs upon plunging to the storage temperature (liquid nitrogen). In this study, we used interrupted cooling protocols in the absence of cryoprotectant to investigate the progression of damage to human umbilical vein endothelial cells (HUVEC), comparing an assessment of membrane integrity with a mitochondrial polarization assay. Additionally, the membrane integrity response of HUVEC to interrupted cooling was investigated as a function of cooling rate (for interrupted slow cooling) and hold time (for interrupted rapid cooling). A key finding of this work was that under slow cooling conditions which resulted in a large number of membrane intact cells immediately post thaw, mitochondria are predominantly in a non-functional depolarized state. This study, the first to look directly at mitochondrial polarization throughout interrupted cooling profiles and a detailed study of HUVEC response, highlights the complexity of the progression of cell damage, as the pattern and extent of cell injury throughout the preservation process differs by injury site.

Protective Effect of Parenteral Vitamin E on Ischemia-reperfusion Injury of Rabbit Kidney

Objectives To evaluate the effect of parenteral vitamin E on renal ischemia-reperfusion injury. Methods Twenty German rabbits weighing 1.5-1.9 kg were selected and divided randomly into study and control groups. Each group contained 5 male and 5 female rabbits. Intravenous vitamin E was administered to the study group 5 minutes before left renal artery clamping and the same volume of normal saline was administered to the control group. Ischemia was maintained for 60 minutes. After 48 hours, nephrectomy was performed and kidneys were sent to the pathology laboratory. Histopathologic sections were evaluated by the pathologist and graded by the extent of tissue injury to normal, mild, and moderate to severe injury. Results Histopathologic evaluation of the sections revealed that in the control group 50% of the sections had signs of moderate to severe injury and 50% were categorized as mild injury, whereas 50% of the study group sections developed no sign of ischemia-reperfusion injury and 50% developed mild injury ( P = .03). No significant correlation was found between sex and the extent of cell injury ( P = .99). Conclusions Parenteral injection of vitamin E significantly protects cells against ischemia-reperfusion injury in German rabbits.

Hypoxia/reoxygenation induces cell injury via different mechanisms in cultured rat cortical neurons and glial cells

Hypoxia/reoxygenation (H/R) causes cell injury/death. We examined the protection by drugs intervening at various stages of the injury cascade in cultured neurons and glia. Primary cultures of rat cortical neurons and mixed glia were subjected to H/R. Measurements of cell death (by lactate dehydrogenase release into the medium) and viability (by MTT reduction) indicated that H/R led to time-dependent injury in both neuronal and mixed glial cultures. The extent of cell injury in neurons was significantly greater than in glia cells. Pretreatment with (+)-MK-801 hydrogen maleate (MK-801) (an N-methyl- d-aspartate antagonist), N ω-nitro- l-arginine methyl ester (L-NAME) (an inhibitor of nitric oxide synthase) or free radical scavengers reduced the extent of the H/R-elicited neuronal damage. MK-801, in contrast, was without effect on glial cells while L-NAME was effective. Our results suggest differential mechanism(s) and susceptibility to injury caused by H/R for neurons and mixed glia.

Role of Viscoelastic Properties of Differentiated SH-SY5Y Human Neuroblastoma Cells in Cyclic Shear Stress Injury

Shear stress and strain lead to neurodegeneration in vivo during head injury, glaucoma, and certain repetitive motion disorders. In vitro, shear stress and strain have been shown to lead to cell injury in a number of models using neurons and neuron-like cells. In the present study we examined the relationship between shear stress, strain, and the extent of cell injury in a cyclic shear stress induced model of cell injury using differentiated SH-SY5Y (human neuroblastoma) cells. Shear stress led to cell strain that increased with increasing stress and diminished upon cessation of shear. Strain rate during cyclic application of shear stress increased by over an order of magnitude from the first to all subsequent cycles, suggesting that the cell and/or its polymer network became more elastic upon cyclic shear stress application. To support this conclusion we measured the degree of cytoskeletal polymerization before and after exposure of cells to cyclic shear stress and found that the fraction of polymerized tubulin in the cell relative to total tubulin decreased by a factor of 2 after six cycles of shear stress. The extent of injury, as indicated by the fraction of cells with fragmented DNA, was three times higher for cyclic shear stress than for steady shear stress and may be related to the change in strain rate and/or cytoskeletal reorganization associated with cyclic stress. These findings may aid in understanding the mechanism by which neurons and neuron-like cells respond to cyclic shear stress and strain and lead to new treatments for disease or injury arising from the exposure of neurons to abnormal cyclic or repetitive stress and strain.

Homocysteine-induced changes in mRNA levels of genes coding for cytoplasmic- and endoplasmic reticulum-resident stress proteins in neuronal cell cultures

Elevated homocysteine levels have been suggested to contribute to various pathological states of the brain. However, the basic mechanisms underlying homocysteine-induced neurotoxicity have not yet been fully elucidated. In the present series of experiments, we investigated the effect of homocysteine on mRNA levels of genes coding for cytoplasmic- or endoplasmic reticulum-resident stress proteins. Primary neuronal cell cultures were exposed to different homocysteine levels for 1–24 h. Cell injury was evaluated using the MTT assay, protein synthesis was studied by measuring the incorporation of l-[4,5- 3H]leucine into proteins, mRNA levels of hsp70, gadd153, grp78, and grp94 were evaluated by quantitative PCR, and changes in protein levels of hsp70, grp78 and grp94 were analyzed by immunoblotting. Exposure of cells to 5 or 10 mM homocysteine for 24 h induced marked cell injury (decrease of viability to 58 or 45% of control respectively). After 6 h treatment, gadd153, grp78 and grp94 mRNA levels increased markedly, but only when cells were exposed to levels of homocysteine high enough to induce cell injury. In addition, hsp70 mRNA levels and protein synthesis were significantly reduced. At earlier (1 or 3 h) or later (12 or 24 h) time intervals, homocysteine exposure induced a marked increase in mRNA levels of all genes studied. GRP78 and GRP94 protein levels were increased in cells exposed to 5 mM homocysteine for 24 h but not in cells exposed to 10 mM homocysteine. HSP70 protein levels, in contrast, were decreased in cells exposed to homocysteine for different periods. The expression of genes coding for ER-resident stress proteins is specifically activated under conditions of ER stress. The close relationship between the extent of cell injury and increase in grp78 mRNA levels suggests that ER dysfunction may contribute to the pathological process. The results imply that the ER is an intracellular target of homocysteine toxicity.

The effect of protein kinase C activation on colonic epithelial cellular integrity

We have investigated whether activation of protein kinase C has a direct cytotoxic effect on colonic mucosal epithelial cells and whether oxidant-induced damage to colonocytes is mediated by activation of cellular protein kinase C. Incubation of freshly harvested cells from rat colon with the protein kinase C activator, phorbol 12-myristate, resulted in a concentration-dependent increase in the extent of cell injury. Phorbol 12-myristate acetate (0.1–10 μM) also increased cellular protein kinase C activity and this was reduced significantly by treating cells with the antagonists staurosporine or 2-[1-(3-dimethylaminopropyl)-indol-3-yl]3-(-indol-3-yl)maleimide (GF 109203X; 10 μM). Phorbol 12-myristate acetate treatment also resulted in increased translocation of proteins for protein kinase C isoforms α, δ and ε from cytosol to membrane particulate fractions. The antagonists reduced the extent of cell damage in response to phorbol 12-myristate acetate. Furthermore, cell injury in response to the phorbol acetate was also inhibited by the addition of the oxidant scavengers, superoxide dismutase or catalase to the cell suspension. Addition of H 2O 2 to the incubation medium (0.1–100 μM) resulted in an increase in cellular protein kinase C activity, an increase in the expression of the α, β and ζ isoforms and a reduction in cell integrity. The cellular damaging actions of H 2O 2 were significantly reduced by the protein kinase C antagonists, staurosporine or 2-[1-(3-dimethylaminopropyl)-indol-3-yl]-3-(-indol-3-yl)maleimide (GF 109203X). These findings suggest that protein kinase C activation results in colonic cellular injury and this damage is mediated, at least in part, by release of reactive oxidants. Furthermore, oxidant-mediated damage to these cells also involves protein kinase C activation.

Mast Cell Degranulation and Parenchymal Cell Injury in the Rat Mesentery

The objective of this study was to explore the degree of parenchymal cell injury after mast cell degranulation by application of compound 48/80 (CMP 48/80) in the absence of adherent leukocytes in the rat mesentery. Rats were rendered leukopenic by injection of an antibody against leukocytes, and the mesentery was superfused with CMP 48/80 during intravital microscopy. The extent of cell injury was determined using a fluorescent cell-viability indicator, propidium iodide (PI). In an additional group, mast cell degranulation with CMP 48/80 was prevented by using the mast cell stabilizer Ketotifen. After a reduction in the number of circulating leukocytes, mast cell degranulation produced a mild increase in parenchymal cell injury. The injury levels significantly increased when individual regions of the mesentery were compared. Stabilization of the mast cells with Ketotifen reduced the injury to below baseline values. In the absence of leukocyte adhesion to the endothelium, mast cell degranulation contributes to parenchymal cell injury in the mesentery.

Modelling the influence of temperature on the recovery ofListeria monocytogenesfrom heat injury

The ability ofListeria monocytogenesScott A to recover from heat injury, as monitored by repair of salt-tolerance, was modelled as a function of temperature and extent of cell injury. A modification to the equation of Hills and Mackey ((1995) Food Microbiology, 12, 333-346), which included parameters for the maximum rate of recovery (Rr), and the decreasing rate of recovery (ar), and eliminated the parameter describing the increasing rate of recovery (br), was used to model the recovery from injury. Lag phase duration (LPD) and Rrwere well predicted by models including both temperature and extent of injury (R2=0.99 and 0.92 for LPD and Rrrespectively), while the growth rate (Rg) was influenced by temperature alone (R2=0.96). The degree to which cells had recovered salt-tolerance at the end of the lag phase (i.e. proportion of cells capable of growth on agar containing 5% ((w/v) NaCl) was found to be related to the initial extent of injury. The results suggest that loss of salt-tolerance may not be the most important site of injury, and that alternative models are needed to adequately account for the contribution of sub-populations of cells to growth after repair of heat injury.

VA-045, a novel apovincamic acid derivative attenuates neuronal injury induced by hypoxia or by excitatory amino acids in cultures of rat cortices

Effects of VA-045, a novel apovincaminic acid derivative, on neuronal damage induced by hypoxia or by excitatory amino acids (glutamate (Glu), N-methyl-D-aspartate and kainate) were examined in cultures of the rat cortices. The extent of cell injury was quantified by measuring lactic dehydrogenase activity released from the damaged cells into the culture medium. VA-045 at concentrations between 1 μM and 30 μM significantly attenuated this neuronal damage and exceeded those of vinpocetine. VA-045 had no significant binding affinity to Glu receptor subtypes. The cytoprotection of VA-045 does not seem to be the result of antagonism at Glu receptors. VA-045 inhibited lipid peroxide production in brain homogenates. Vitamin E also had this antioxidant effect, but did not attenuate the hypoxia-induced neuronal damage. A cAMP analogue and a phosphodiesterase (PDE) inhibitor also attenuated the hypoxia-induced neuronal damage. As VA-045 inhibits the activity of PDE, the effect of VA-045 may possibly relate to cAMP cascade. VA-045 may prove to be efficacious for the treatment of disorders related to cerebral neuronal injury.

Role of endonuclease activity and DNA fragmentation in Ca2+ ionophore A23187-mediated injury to rabbit isolated gastric mucosal cells

In the current study, the role of endonuclease activity in calcium ionophore A23187-induced gastric mucosal cellular disruption was examined using rabbit gastric mucosal cells. Cell integrity was assessed using trypan blue dye exclusion and Alamar blue dye absorbance. Ionophore A23187 (1.6-25 microM) induced a concentration-dependent decrease in dye exclusion and cell metabolism in cells suspended in a medium containing Ca2+ (2 mM), while no such effect was observed in cells incubated in the absence of extracellular Ca2+. Cells that were pretreated with the endonuclease inhibitors aurintricarboxylic acid (ATCA; 0.2 or 0.5 mM or Zn2+; 0.01 and 0.1 mM) exhibited significant reduction in the total extent of cell injury when incubated with A23187 in the presence of Ca2+. DNA fragmentation as assessed by measurement of [3H]thymidine liberation or gel electrophoresis was increased in response to ionophore A23187 (12.5 or 25 microM) treatment. A minimal degree of fragmentation was observed when cells were suspended in a Ca(2+)-free medium or incubated in the presence of ATCA or Zn2+. Addition of ethanol (8% w/v) induced a significant increase in cell injury, which was not affected by either removal of extracellular Ca2+ or ATCA pretreatment. Furthermore, treatment with the antioxidants catalase (50 micrograms/ml) or 2',2'-dipyridyl (2 mM) reduced ionophore-induced cell injury but did not reduce the extent of DNA fragmentation. These data suggest that sustained increases in intracellular Ca2+ result in increased endonuclease activity in gastric mucosal cells, leading to extensive DNA lysis and cell damage. Ethanol-induced cell damage does not involve Ca2+ influx and therefore is not mediated by endonuclease activation. Furthermore, sustained increases in cellular Ca2+ may also mediate their effects via formation of reactive oxygen metabolites, but this mechanism of cell damage does not appear to involve DNA fragmentation.

Cytotoxic activity of T cells and non-T cells from diclofenac-immunized mice against cultured syngeneic hepatocytes exposed to diclofenac

To evaluate whether hepatocellular protein adducts of the nonsteroidal antiinflammatory drug diclofenac could elicit a specific cell-mediated or antibody-dependent immune response that eventually results in liver cell destruction, we developed a murine ex vivo/in vitro mixed lymphocyte hepatocyte culture (MLHC) model. C57BL/6 mice were immunized either with diclofenac conjugated to keyhole limpet hemocyanin (KLH) or with KLH alone. Splenocytes from mice exhibiting hgih antidiclofenac antibody titers were isolated and co-cultured at an effector/target cell ratio of 100:1 with syngeneic murine hepatocytes preexposed to diclofenac. By 48 and 72 hours, extracellular alanine transaminase (ALT) activity had increased 6.4- and 7.6-fold, respectively, versus the 24-hour value. In contrast, there was no significant cytotoxic response after either drug treatment alone or immunization alone. Furthermore, those cellular populations capable of inducing ALT release also showed lymphocyte stimulation as determined by interleukin-2 (IL-2) receptor expression and lymphocyte proliferation analysis. The extent of cell injury was highest in the presence of lymphocytes highly enriched in T cells and was reduced by 40% in the presence of anti-MHC I antibodies. Similarly, albeit to a lesser extent, non-T cell-enriched lymphocyte fractions also induced hepatocyte injury. The addition of co-culture supernatants to hepatocytes had no effect, thus ruling out the possibility that soluble factors alone mediated the cell injury. However, supernatants from diclofenac-stimulated lymphocytes, combined with nonstimulated splenocytes, triggered an immediate (< 1 hour) cytotoxic response, suggesting antibody-dependent cell-mediated mechanisms of target cell injury. These results indicate that diclofenac-treated hepatocytes carried antigenic determinants that were recognized by T cells and non-T cells derived from diclofenac/KLH-immunized mice, resulting in cell-mediated destruction of the target hepatocytes.

Cytotoxic activity of T cells and non-T cells from diclofenac-immunized mice against cultured syngeneic hepatocytes exposed to diclofenac

To evaluate whether hepatocellular protein adducts of the nonsteroidal antiinflammatory drug diclofenac could elicit a specific cell-mediated or antibody-dependent immune response that eventually results in liver cell destruction, we developed a murine ex vivo/in vitro mixed lymphocyte hepatocyte culture (MLHC) model. C57BL/6 mice were immunized either with diclofenac conjugated to keyhole limpet hemocyanin (KLH) or with KLH alone. Splenocytes from mice exhibiting high antidiclofenac antibody titers were isolated and co-cultured at an effector/target cell ratio of 100:1 with syngeneic murine hepatocytes preexposed to diclofenac. By 48 and 72 hours, extracellular alanine transaminase (ALT) activity had increased 6.4- and 7.6-fold, respectively, versus the 24-hour value. In contrast, there was no significant cytotoxic response after either drug treatment alone or immunization alone. Furthermore, those cellular populations capable of inducing ALT release also showed lymphocyte stimulation as determined by interleukin-2 (IL-2) receptor expression and lymphocyte proliferation analysis. The extent of cell injury was highest in the presence of lymphocytes highly enriched in T cells and was reduced by 40% in the presence of anti-MHC I antibodies. Similarly, albeit to a lesser extent, non-T cell-enriched lymphocyte fractions also induced hepatocyte injury. The addition of co-culture supernatants to hepatocytes had no effect, thus ruling out the possibility that soluble factors alone mediated the cell injury. However, supernatants from diclofenac-stimulated lymphocytes, combined with nonstimulated splenocytes, triggered an immediate (<1 hour) cytotoxic response, suggesting antibody-dependent cell-mediated mechanisms of target cell injury. These results indicate that diclofenac-treated hepatocytes carried antigenic determinants that were recognized by T cells and non-T cells derived from diclofenac/KLH-immunized mice, resulting in cell-mediated destruction of the target hepatocytes.

A new model for rapid stretch-induced injury of cells in culture: characterization of the model using astrocytes.

The purpose of this study was to develop a simple, reproducible model for examining the morphologic, physiologic, and biochemical consequences of stretch-induced injury on tissue-cultured cells of brain origin. Rat cortical astrocytes from 1- to 2-day-old rats were cultured to confluency in commercially available 25-mm-diameter tissue culture wells with a 2-mm-thick flexible silastic bottom. A cell injury controller was used to produce a closed system and exert a rapid positive pressure of known amplitude (psi) and duration (msec). The deformation of the membrane, and thus the stretch of the cells growing on the membrane, was proportional to the amplitude and duration of the air pressure pulse. Extent of cell injury was qualitatively assessed by light and electron microscopy and quantitatively assessed by nuclear uptake of the fluorescent dye propidium iodide, which is excluded from cells with intact membranes. Lactate dehydrogenase (LDH) enzyme release was measured spectrophotometrically. Cell injury was found to be proportional to the extent of the silastic membrane deformation. Increasing cell stretch caused mitochondrial swelling and vacuolization as well as disruption of glial filaments. Stretching also caused increased dye uptake, with maximum dye uptake occurring with a 50 msec pressure pulse duration, whereas deformations produced over longer periods of time (seconds) caused little dye uptake. With increasing postinjury survival fewer cells took up dye, implying cell repair. LDH release was also proportional to the amplitude of cell stretch, with maximum release occurring within 2 h of injury. In summary we have developed a simple, reproducible model to produce graded, strain-related injuries in cultured cells. Our continuing experiments suggest that this model can be used to study the biochemistry and physiology of injury as well as serve as a tool to examine the efficacy of therapeutic agents.

Coordinated expression of a 45 kD protein and ozone toxicity in a human bronchial epithelial cell line.

The human bronchial epithelial cell line, BEAS-2B, which was immortalized by transformation with SV40 virus, when grown biphasically between 0.1 and 1.0 ppm of ozone and liquid medium showed increased release of Cr, decreased synthesis of various macromolecules, and decreased cell viability. Cell injury was a function of the concentration of ozone to which the cells were exposed. Furthermore, in proportion to the extent of cell injury, ozone exposure also induced and/or enhanced synthesis of a 45 kD protein but not any of the well-characterized heat shock proteins, e.g., HSP 70. Actinomycin D prevented enhanced synthesis of the 45 kD protein in cells exposed to ozone, suggesting transcriptional regulation of expression of the 45 kD protein. Enhanced synthesis of the 45 kD protein was not observed in cells treated with heat, cigarette smoke condensate, hydrogen peroxide, or bleomycin. High concentrations of glutathione added to the culture medium reduced ozone toxicity and ozone-enhanced synthesis of the 45 kD protein. These results suggest that ozone injury and enhanced expression of a gene encoding a 45 kD protein of as yet unknown function are coordinated in the SV40-immortalized bronchial epithelial cells.

New technique to assess hypoxia-induced cell injury in individual isolated renal tubules

Freshly isolated renal proximal tubules are a valuable model to study mechanisms of cell injury induced by hypoxia [1], The extent of cell injury has previously been quantified by determining the release of lactate dehydrogenase (LDH) and by measuring cellular adenosine triphosphate (ATP) and potassium (K) in suspensions of tubules [1, 21. However, the cellular pattern of hypoxic tubule injury is known to be heterogeneous and differences in cellular damage between tubules, as well as within individual tubules, have been described [1]. The present methods for quantification of cell injury as well as spectofluorometric measurements of intracellular ion concentrations with ion sensitive dyes (such as for Ca2, Na, H, Mg2) provide data which represent averages from all tubules in the suspension. Hence, using tubules in suspension prohibits correlation of cytoplasmic ion changes with cellular injury occurring in individual tubules or cells over time. This limitation of studies using tubular suspensions led us to develop a method for induction of hypoxic injury and assessment of cell injury in individual tubules and individual tubular cells.

Chemical structure and toxicity of diuretics in isolated hepatocytes.

The effects of several diuretics, including tienilic acid and indacrinone, on isolated rat heaptocytes were examined. Addition of tienilic acid and indacrinone at 1 mM to a suspension of freshly isolated cells caused dose-dependent loss of cell viability as judged by the LDH-latency test. Survey of 19 structurally related compounds revealed that the extent of cell injury and chemical structure were correlated, and an intense adverse effect was attributed to the 2-thienylcarbonyl moiety. Several other factors influencing cell viability are also disclosed. Further study revealed that tienilic acid and indacrinone were toxic to the primary culture of hepatocytes at a lower dose than that a freshly isolated hepatocytes. Thus, an isolated hepatocyte system can be used to select compounds displaying low hepatotoxicity, as for example is needed when screening diuretics.

Adenine nucleotide metabolism by isolated kidney tubules during oxygen deprivation

Suspensions enriched in isolated rabbit proximal tubules were subjected to varying degrees of oxygen deprivation-induced injury by incubating them under hypoxic conditions at pH 7.4 or pH 6.6 or under high density pelleted conditions and adenine nucleotide degradation was characterized. The major metabolite was hypoxanthine. Its levels increased with the extent of irreversible injury. It was not further degraded or salvaged. Recovery of cell ATP during reoxygenation was predominantly from the remaining cell nucleotides. Allopurinol did not alter the pattern of purine metabolism or the extent of cell injury. These observations provide information on the intrinsic purine metabolic capacity of renal tubule cells during oxygen deprivation which is relevant to understanding both the salvage mechanisms available in these cells as well as the contribution of purine metabolism to the pathogenesis of oxygen deprivation-induced tubule cell injury.