The effect of lethal acid stress on Na+/H+ exchanger isoforms in cultured inner medullary collecting duct cells: deletion of NHE-2 and over expression of NHE-1.

Cultured inner medullary collecting duct (mIMCD-3) cells express Na+/H+ exchanger isoforms NHE-2 and NHE-1 (Soleimani et al. (1994) J. Biol. Chem. 269, 27973-27978). In the present studies we examined the effect of lethal acid stress on Na+/H+ exchanger activity and isoform expression in mIMCD-3 cells. mIMCD-3 cells were incubated for 10 min with 20 mM ammonium, and exposed to an ammonium-free acidic solution (pH 6.0) for 120 min. Thereafter, cells were recovered and grown in normal culture media. The surviving clones were isolated and subjected to two additional cycles of acid stress. A mutant clone was isolated and characterized for Na+/H+ exchange activity and isoform expression. The mutant mIMCD-3 clone demonstrated significant over-expression of Na+/H+ exchange activity as assessed by acid-stimulated 22Na influx (11.56 nmol/mg protein in mutant vs. 4.06 nmol/mg in parent cells, P < 0.001, n = 4) and sodium-dependent pHi recovery from an acid load (0.55 pH/min in mutant vs. 0.28 pH/min in parent cells, P < 0.01, n = 6). A dose-response inhibition of the exchanger showed that the mutant cells were very sensitive to dimethylamiloride (IC50 158 nM in mutant vs. 889 nM in parent mIMCD-3 cells, P < 0.001). To compare the Na+/H+ exchanger isoforms in mutant and parent mIMCD-3 cells, poly(A)+ RNA was isolated from each group and probed with radiolabeled NHE-1 or NHE-2 cDNA. The expression of NHE-1 mRNA was increased by approximately 100% in mutant cells. The NHE-2 mRNA, on the other hand, was found to be absent in mutant mIMCD-3 cells. Examination of the regulatory mechanisms of the Na+/H+ exchanger isoforms in parent mIMCD-3 cells, which express NHE-2 and NHE-1, and mutant mIMCD-3 cells, which only express NHE-1, would be helpful in elucidating the roles of NHE-2 and NHE-1 in inner medullary collecting duct cells.

Cholera toxin action on rabbit renal brush-border membranes inhibits phosphate transport.

Cholera toxin was used to enhance ADP-ribosylation of rabbit renal brush-border membranes. Treatment of brush-border membrane sheets with cholera toxin in the presence of NAD resulted in a specific inhibition of the initial phase of Na+-dependent Pi uptake, compared to controls incubated with NAD alone. The Pi uptake was determined after conversion of the membrane sheets to vesicles. The equilibrium uptake of Pi, the Na+-independent uptake of Pi, the Na+-dependent uptake of L-proline and the activities of several brush-border membrane enzymes were not changed. The inhibition of Pi transport was dependent on the presence of both NAD and cholera toxin. Incubation of membrane sheets with [3H]NAD produced acid-stable binding of radioactivity to the membranes and the binding was increased 5-fold by the presence of cholera toxin. The use of [32P]NAD and autoradiography confirmed that the bound radioactivity was associated with several different membrane proteins, and that cholera toxin increased binding to these proteins including three that were not labelled in the absence of the toxin. The specific inhibitory action of cholera toxin on Na+/Pi cotransport is probably mediated by ADP-ribosylation of membrane proteins, suggesting that the Pi transport system can be regulated by ADP-ribosylation, at least in vitro.

Parathyroid hormone inhibits plasma membrane Pi transport without changing endocytic activity in opossum kidney cells.

Parathyroid hormone (PTH) inhibits Na(+)-dependent Pi uptake in renal epithelial cells from opossum kidney (OK). This requires an intact endocytic pathway, suggesting that one action of PTH may be to promote endocytic removal of Na+/Pi cotransporters from the cell membrane. The present study tested if PTH, at a dose that inhibited membrane Pi transport, also produced an increase in endocytic activity. Pi transport was measured in isolated plasma membrane vesicles. Endocytosis was measured by allowing cells to take up horseradish peroxidase (HRP) followed by assay of triton-sensitive (latent) HRP activity in subcellular fractions isolated by density gradient centrifugation. Incubation of OK cells with 10(-7) M PTH for 3 h decreased Na+/Pi cotransport by membrane vesicles to 328 +/- 54 pmol/mg/min compared to 448 +/- 67 pmol/mg/min (mean +/- S.E., P < 0.03) in controls. Latent HRP content of endosomal fractions was dependent on the time and temperature used to load cells with HRP and on the concentration of HRP. However, incubation of OK cells with 10(-7) M PTH for either 1 or 3 h produced no change in latent HRP activity. Thus the action of PTH on the Na+/Pi cotransporter in the plasma membrane of OK cells does not require a change in the rate of endocytosis.

Prostaglandin production by type II alveolar epithelial cells.

Prostaglandin production was studied in fetal and adult type II alveolar epithelial cells. Two culture systems were employed, fetal rat lung organotypic cultures consisting of fetal type II cells and monolayer cultures of adult lung type II cells. Dexamethasone, thyroxine, prolactin and insulin, hormones which influence lung development, each reduced the production of prostaglandin E and F alpha by the organotypic cultures. The fetal cultures produced relatively large quantities of prostaglandin E and F alpha and smaller quantities of 6-keto-prostaglandin F1 alpha and thromboxane B2. However, prostaglandin E2 production was predominant. In contrast, the adult type II cells in monolayer culture produced predominantly prostacyclin (6-keto-prostaglandin F1 alpha) along with smaller quantities of prostaglandin E2 and F2 alpha. The type II cells were relatively unresponsive to prostaglandins. Exogenously added prostaglandin E, had no effect on cell growth, and only a minimal effect on cyclic AMP levels in the monolayer cultures.

Strategies for improved shock wave lithotripsy.

Research in lithotripsy that started with the effort to characterize acute shock wave damage to the kidney has led to advances on several fronts, including discovery of strategies that have improved clinical treatment. It is appreciated now that shock wave trauma is primarily a vascular lesion, that injury is dose dependent, and that hemorrhage can be severe and can lead to a permanent loss of functional renal mass. Studies of the renal functional response to lithotripsy have shown that shock wave treatment triggers vasoconstriction in the kidney. This finding has been turned to advantage, and it is now known that when treatment is begun using low amplitude pulses, subsequent high amplitude shock waves are far less damaging. Thus, when shock waves are delivered judiciously, treatment can have a protective effect. The finding that cavitation is a key mechanism in vessel rupture has led to the development of novel experimental methods of shock wave delivery that can suppress bubble expansion and minimize tissue damage. Progress has also been made in understanding the physical mechanisms involved in stone comminution, and it is seen that the forces generated by cavitation, shear stress and circumferential squeezing act synergistically to fragment stones. Recent work suggests that a broad focal zone may be an advantage, allowing stones to be broken with lower amplitude pulses. Cavitation has been shown to play a critical role in reducing stone fragments to a size that can be voided. Cavitation is also the factor that limits the rate at which treatment can be performed, as stones break significantly better at slow rate than at fast ratean observation from basic research that is now appreciated in clinical practice. The current environment in lithotripsy research is encouraging. There is great interest in developing new technology, and in finding ways to improve how lithotripsy is performed.

Beta-adrenergic stimulation of aldosterone production by rat adrenal capsular explants.

Adrenal glomerulosa was examined for the presence of an adrenergic influence on aldosterone production. Cultured rat adrenal capsular explants were transferred to a perifusion system where the effect of exposure to catecholamines on aldosterone production was assessed. At 10(-6) M, isoproterenol greater than epinephrine greater than norepinephrine significantly stimulated aldosterone production, whereas at 10(-8) M only isoproterenol showed significant stimulation. Propranolol, a beta-adrenoreceptor antagonist, inhibited stimulation by epinephrine, and the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine, enhanced stimulation by a submaximal dose of epinephrine. Epinephrine and norepinephrine were found by radioenzymatic assay to be present in fresh as well as cultured capsular tissue, although levels were considerably lower in tissue that had been in culture (about one tenth that of fresh tissue). The epinephrine-norepinephrine ratio was similar in capsule and medulla, suggesting a medullary source of capsular catecholamines. Whether catecholamines in the capsule arose from the in vitro manipulation of adrenal tissue or existed in vivo is unclear. In summary, beta-agonists stimulate aldosterone production in cultured rat capsular explants.

Bioeffects of extracorporeal shock-wave lithotripsy. Strategy for research and treatment.

Available information on the bioeffects of ESWL is insufficient to characterize the tissue injury induced by shock waves. The cellular mechanisms have not been elucidated, nor are there enough data to establish objective criteria for treatment.

Beta-adrenergic enhancement of angiotensin II-stimulated aldosterone secretion.

Beta-adrenergic agonists have been shown to stimulate aldosterone secretion. Angiotensin II (AII) is one of the important stimuli of aldosterone secretion; conceivably beta-adrenergic influences affect the stimulatory potential of AII. Using cultured rat adrenal capsules, we found that 10(-7) M epinephrine and 10(-7) M isoproterenol enhanced 10(-7) M AII-stimulated aldosterone production. Propranolol (10(-7) M) completely inhibited the ability of epinephrine to augment the stimulatory actions of AII. In conclusion, beta-adrenergic agonists promote stimulation of aldosterone secretion by AII.

Mechanical haemolysis in shock wave lithotripsy (SWL): II. In vitro cell lysis due to shear.

In this work we report injury to isolated red blood cells (RBCs) due to focused shock waves in a cavitation-free environment. The lithotripter-generated shock wave was refocused by a parabolic reflector. This refocused wave field had a tighter focus (smaller beam width and a higher amplitude) than the lithotripter wave field, as characterized by a membrane hydrophone. Cavitation was eliminated by applying overpressure to the fluid. A novel passive cavitation detector (HP-PCD) operating at high overpressure (up to 7 MPa) was used to measure acoustic emission due to bubble activity. The typical 'double-bang' emission measured in the lithotripter free-field was replaced by a continuum of weak signals when the fluid was enclosed in a pressure chamber. No acoustic emissions were measured above an overpressure of 5.5 MPa. Aluminium foils were used to study shock wave damage and had distinct deformation features corresponding to exposure conditions, i.e. pitting and denting accompanied by wrinkling. Pitting was eliminated by high overpressure and so was due to cavitation bubble collapse, whereas denting and wrinkling were caused by the reflected shock wave refocused by the parabolic reflector. RBCs suspended in phosphate-buffered saline (PBS) were exposed to the reflected wave field from a parabolic reflector and also from a flat reflector. Exposure to the wave field from the parabolic reflector increased haemolysis four-fold compared with untreated controls and was twice that of cell lysis with the flat reflector. Recently we analysed deformation and rupture of RBCs when subjected to a flow field set up by a focused shock. The cell lysis results presented here are in qualitative agreement with our theoretical prediction that haemolysis is directly related to the gradient of shock strength and validates shearing as a cell lysis mechanism in SWL.

Cell damage by lithotripter shock waves at high pressure to preclude cavitation.

Acoustic cavitation has been implicated as a cause of cell damage by lithotripter shock waves, particularly under in vitro conditions. When red blood cells were exposed to shock waves (from an electrohydraulic lithotripter) while under high hydrostatic pressure (> 80 atm), cell lysis was dramatically reduced over that seen at atmospheric pressure, which is consistent with damage due to acoustic cavitation. However, even at > 120 atm of pressure, lysis was still 97% above that of cells not exposed to shock waves, revealing significant damage that apparently was due to mechanisms other than cavitation. Hydrostatic pressure alone did not cause cell lysis, and shock-wave-dependent damage occurred when the cells were in fluid suspension, or when they were centrifuged to the end of the vial. Shock-wave damage at high pressure increased with increasing shock-wave number, and was seen at 24 and 20 kV, but not at 16 kV. This shock-wave damage at high pressure makes up a noteworthy portion of the total cell injury seen at atmospheric pressure (about 10% at 24 kV), suggesting significant noncavitational injury to cells in vitro. Because cavitation occurs far more readily in vitro than in vivo, the noncavitational damage seen in the present study could represent a substantial portion of cell injury seen in vivo with shock-wave lithotripsy.

The effect of polypropylene vials on lithotripter shock waves.

In studies to understand the mechanisms responsible for shock wave lithotripsy (SWL) cell injury, we observed that shock waves (SWs) are influenced by the shape of the specimen vial. Lytic injury to kidney cells treated in a Dornier HM3 lithotripter was higher (p < 0.0001) when SWs entered the vial through the flat end (cap end) compared to the round end. Measurements of the acoustic field within polypropylene vials were carried out using both lithotripter SWs and pulsed ultrasound (US) in the megahertz frequency range. We compared pressure amplitudes inside the round and flat vials and found significant differences. When SWs entered through the round end, the average peak positive pressure was 40% of free-field pressure, due mostly to a dramatic reduction in pressure off axis. The average peak pressure inside the flat vial was twice that of the round vial. Experiments with US demonstrated that sound field focusing was induced by the curved interface of the round vial. Ray analysis for the round vial indicates the presence of "hot spots" on axis and "cold spots" off axis, in qualitative agreement with pressure profiles. We conclude that the shape of the specimen vial is an important factor that should be considered in model systems of SWL cell injury.

Quantitation of shock wave cavitation damage in vitro.

Acoustic cavitation damage was quantitated using aluminum foil targets placed within 2-mL polypropylene cryovials. The vials contained various media tested for their potential to support cavitation and were exposed to shock waves using an unmodified Dornier HM3 lithotripter. Foil damage, expressed in terms of a "damage index," was measured from digitized light microscopy images by quantitating the spread of gray-scale histograms. Target sensitivity was demonstrated by reproducible dose-response curves over the range (1-200 shock waves) commonly used for in vitro cell injury studies. Increased shock wave repetition rate reduced the damage index. Untreated foils showed a very low damage index (0.001% +/- 0.001%), while treated foils submerged in Ringer buffer yielded significant damage (2.2% +/- 0.3%, p < 0.001). Degassing the buffer reduced damage to 0.3% +/- 0.1% (p < 0.001). Foils submerged in castor oil showed virtually no damage. These results implicate acoustic cavitation in target damage. Targets immersed in biological fluids (blood and urine) had significantly less damage than in Ringer. The effect of degassing was also evaluated in a red blood cell lysis assay. Hemoglobin release in degassed preparations was significantly reduced compared to nondegassed controls (p < 0.001) and correlated with reduced foil damage index in cell-free vials. These findings characterize a sensitive method to quantitate acoustic cavitation and implicate a role for cavitation in shock wave lithotripsy-induced cell lysis.

Effect of macroscopic air bubbles on cell lysis by shock wave lithotripsy in vitro.

In studies of cells or stones in vitro, the material to be exposed to shock waves (SWs) is commonly contained in plastic vials. It is difficult to remove all air bubbles from such vials. Because SWs reflect at an air-fluid interface, and because existing gas bubbles can serve as nuclei for cavitation events, we sought to determine in our system whether the inclusion of small, visible bubbles in the specimen vial has an effect on SW-induced cell lysis. We found that even small bubbles led to increased lysis of red blood cells (1- to 3-mm diameter bubbles, 9.8+/-0.5% lysis, n = 7; no bubbles, 4.4+/-0.8%, n = 4), and that the degree of lysis increased with bubble size. Damage could not be reduced by centrifuging the cells to the opposite end of the vial, away from the bubble. B-scan ultrasound imaging of blood in polypropylene pipette bulbs showed that, with each SW, bubbles were recruited from the air interface, mixing throughout the fluid volume, and these appeared to serve as nuclei for increased echogenicity during impact by subsequent SWs; thus, bubble effects in vials could involve the proliferation of cavitation nuclei from existing bubbles. Whereas injury to red blood cells was greatly increased by the presence of bubbles in vials, lytic injury to cultured epithelial cells (LLC-PK1, which have a more complex cytoarchitecture than red blood cells) was not increased by the presence of small air bubbles. This suggests different susceptibility to SW damage for different types of cells. Thus, the presence of even a small air bubble can increase SW-induced cell damage, perhaps by increasing the number of cavitation nuclei throughout the vial, but this effect is variable with cell type.

Correction of helical CT attenuation values with wide beam collimation: in vitro test with urinary calculi.

RATIONALE AND OBJECTIVES: Urinary calculi are now commonly detected with helical computed tomography (CT), and it has been proposed that stone composition can be determined from CT attenuation values. However, typical scans are made with a beam collimation of 5 mm or more, resulting in volume averaging and reduction in accuracy of attenuation measurement. The authors tested a model for correction of errors in attenuation values, even at section widths larger than the width of the object. MATERIALS AND METHODS: Human urinary stones were scanned with helical CT at different beam collimation widths. A computer model was used to predict the effect of beam width and stone size on accuracy of measured attenuation. RESULTS: At 3-mm collimation, the model corrected the attenuation readings with an underestimation of 12% +/- 1 (compared with values at 1-mm collimation; 127 stones; diameters of 1.7-11.3 mm). With attenuation measured at 10-mm collimation, the model underestimated the true value by 34% +/- 3 (103 stones), with a significant negative correlation with stone diameter on magnitude of error (diameters of 3.0-11.3 mm). Correlation of data from patient scans with subsequent in vitro scanning of the same stones confirmed the validity of the model, but corrected in vivo scans consistently yielded lower values for the stones than in vitro. CONCLUSION: Volume averaging effects on attenuation in helical CT are predictable in vitro for urinary calculi--and presumably for other roughly spherical structures--as long as section width does not excessively exceed the diameter of the structure.

Effect of radiographic contrast material exposure on spiral CT attenuation of renal calculi.

RATIONALE AND OBJECTIVES: The authors performed this study to determine whether exposure of renal calculi to radiographic contrast material has an effect on the attenuation values at computed tomography (CT) performed with varying collimation widths. MATERIALS AND METHODS: Renal calculi (23 stones of various composition) were scanned with 1-, 3-, and 10-mm collimation. Stones were then exposed to a solution of radiographic contrast material for 5 minutes, washed with water, and rescanned 36 hours later. The reproducibility of the CT attenuation measurements on different days was evaluated by obtaining measurements in a subset of 16 renal stones on 4 different days. RESULTS: There was no statistically significant change in attenuation after contrast material exposure at narrow collimation. At wider collimation, statistically significant increases were noted in both attenuation and standard deviation. A small amount of variability between readings was noted on different days, with a minimal increase in attenuation each day. Correlation between readings remained very high. CONCLUSION: Exposure of stones to a radiographic contrast material had a statistically significant effect on CT attenuation values only at wide collimation. This may be related to technical factors including volume averaging. Absence of an effect at narrow collimation suggests that the attenuation values of renal stones do not significantly change after exposure to contrast material.

Calcium stone fragility is predicted by helical CT attenuation values.

BACKGROUND AND PURPOSE: Helical CT has become the preferred method for imaging urinary calculi, and so it would be useful if data from helical CT could also be used to predict the number of shockwaves (SWs) needed to break a given stone. METHODS AND MATERIALS: We measured the number of SWs required to comminute calcium stones in vitro. RESULTS: The SW requirement correlated with stone size (volume, weight, diameter) and with helical CT attenuation values when the scans were performed at 3-mm collimation. When CT scans were performed at 1-mm collimation, the number of SWs needed for comminution did not correlate with helical CT attenuation values. This result indicates that the correlation with 3-mm scans was attributable to volume-averaging effects, in which smaller stones yield smaller attenuation values. That is, attenuation values from helical CT at larger beam collimation widths contain information about stone size that can be exploited to predict the fragility of calcium stones. We observed that for calcium stones, the number of SWs to comminution was generally less than half the stone CT attenuation value in Hounsfield units. This "half-attenuation rule" predicted the number of SWs needed to complete fragmentation for 95% of calcium stones (24/24 calcium oxalate monohydrate, 13/13 hydroxyapatite, 8/10 brushite stones). CONCLUSION: This in vitro study suggests that it may be possible to predict effective SW dose using helical CT prior to lithotripsy.

Epidermal growth factor accelerates recovery of LLC-PK1 cells following oxidant injury.

In renal tubular epithelial cells, oxidant injury results in several metabolic alterations including ATP depletion, decreased Na+K+ATPase activity, and altered intracellular sodium and potassium content. To investigate the recovery of LLC-PK1 cells following oxidant injury and to determine if recovery can be accelerated, we induced oxidant stress in LLC-PK1 cells with 500 microM hydrogen peroxide for 60 min. Identical cohorts of oxidant-stressed cells were incubated in recovery medium without epidermal growth factor (EGF) or recovery medium containing 25 ng EGF per ml. ATP levels, Na+K+ATPase activity in whole cells, Na+K+ATPase activity in disrupted cells, and intracellular sodium and potassium ion content were determined at 0, 5, 24, 48, and 72 h following oxidant injury in each cohort of cells. In oxidant-stressed cells recovering in medium without EGF, ATP levels, Na+K+ATPase activity, and intracellular ion content improved but continued to remain substantially lower than control values at all time points following oxidant stress. In cells recovering in medium with EGF, ATP levels, Na+K+ATPase activity, and the intracellular potassium-to-sodium ratio were significantly higher at nearly all time points than values in cells recovering in medium alone. In cells recovering with added EGF, Na+K+ATPase activity had improved to control levels, whereas ATP levels and intracellular ion content approached control values by 72 h following oxidant stress. We conclude that oxidant-mediated ATP depletion, altered Na+K+ATPase activity, and intracellular ion content remain depressed for several d following oxidant stress and that EGF accelerated recovery of LLC-PK1 cells from oxidant injury.

Tracheal morphogenesis and fetal development of the mucociliary epithelium of the rat.

In the rat, tracheal development begins at mid-gestation (Day 11) with the formation of the tracheal groove, a longitudinal diverticulum of endodermal epithelium that evaginates from the floor of the pharynx and tubular foregut. At this stage the tracheal groove and developing foregut share a common lumen. Paired primary bronchial buds (lung buds), surrounded by lung bud mesenchyme (splanchnic mesoderm), arise from the caudal end of the tracheal groove. The formation of a longitudinal tracheoesophageal septum divides the combined tracheal groove and developing foregut into two structures, the trachea and esophagus. The trachea and esophagus grow apart, surrounded by independently organized populations of mesenchymal cells. Near this time (Day 12-13), the primary bronchial buds give rise to secondary (lobar) buds of pulmonary epithelium. This establishes the lobar pattern of the right and left lungs and marks the formation of extrapulmonary bronchi. The development of smooth muscle and cartilage within the tracheal mesenchyme precedes the differentiation of the mucociliary epithelium. Smooth muscle forms transversely oriented fascicles in the dorsal tracheal wall (pars membranacea), while pre-cartilage rings surround the remaining ventral and lateral walls (pars cartilagina). Epithelial differentiation is first evident at Day 17, with the formation of ciliated cells in the epithelium of the pars membranacea. Differentiation in the pars cartilagina trails development in the dorsal epithelium. Cell surface characteristics of the tracheal epithelium indicate that secretory cells differentiate about Day 19-20. The precise time and sequence of differentiation of mature cell types of the tracheal epithelium is yet to be determined. It is clear, however, that the development of the mucociliary epithelium in the rat is not completed at birth, but continues into the neonatal period.

Reactive oxygen molecule-mediated injury in endothelial and renal tubular epithelial cells in vitro.

To investigate renal tubular epithelial cell injury mediated by reactive oxygen molecules and to explore the relative susceptibility of epithelial cells and endothelial cells to oxidant injury, we determined cell injury in human umbilical vein endothelial cells and in four renal tubular epithelial cell lines including LLC-PK1, MDCK, OK and normal human kidney cortical epithelial cells (NHK-C). Cells were exposed to reactive oxygen molecules including superoxide anion, hydrogen peroxide and hydroxyl radical generated by xanthine oxidase and hypoxanthine. We determined early sublethal injury with efflux of 3H-adenine metabolites and a decline in ATP levels, while late lytic injury and cell detachment were determined by release of 51chromium. When the cells were exposed to 25, 50, and 100 mU/ml xanthine oxidase with 5.0 mM hypoxanthine, ATP levels were significantly lower (P less than 0.001) in LLC-PK1, NHK-C and OK cells compared to MDCK cells while ATP levels were significantly lower (P less than 0.01) in endothelial cells compared to all tubular cell lines. A similar pattern of injury was seen with efflux of 3H-adenine metabolites. When the cells were exposed to 50 mU/ml xanthine oxidase with 5.0 mM hypoxanthine for five hours, total 51chromium release was significantly (P less than 0.001) greater in LLC-PK1, NHK-C and OK cells compared to MDCK cells, while total 51chromium release was significantly (P less than 0.001) greater in endothelial cells compared to all tubular cells. However, lytic injury was the greatest in LLC-PK1 cells and NHK-C cells while cell detachment was the greatest in endothelial cells. MDCK cells were remarkably resistant to oxidant-mediated cell detachment and cell lysis. In addition, we determined ATP levels, 3H-adenine release and 51chromium release in LLC-PK1, NHK-C and endothelial cells in the presence of superoxide dismutase to dismute superoxide anion, catalase to metabolize hydrogen peroxide, DMPO to trap hydroxyl radical and DMTU to scavenge hydrogen peroxide and hydroxyl radical. We found that catalase and DMTU (scavengers of hydrogen peroxide) provided significant protection from ATP depletion, prevented efflux of 3H-adenine metabolites and cell detachment while DMPO (scavenger of hydroxyl radical) prevented lytic injury. In addition, we found that the membrane-permeable iron chelator, phenanthroline, and preincubation with deferoxamine prevented cell detachment and cell lysis, confirming the role of hydroxyl radical in cell injury.(ABSTRACT TRUNCATED AT 400 WORDS)