Development and application of fluorescence polarization assays in drug discovery.

Fluorescence polarization technology has been used in basic research and commercial diagnostic assays for many decades, but has begun to be widely used in drug discovery only in the past six years. Originally, FP assays for drug discovery were developed for single-tube analytical instruments, but the technology was rapidly converted to high-throughput screening assays when commercial plate readers with equivalent sensitivity became available. This review will discuss fluorescence polarization assays in current use in drug discovery research as well as those in development that will likely be used in the near future. These assays include targets such as kinases, phosphatases, proteases, G-protein coupled receptors, and nuclear receptors.

Effects of enhanced oxygen release from hemoglobin by RSR13 in an acute renal failure model.

BACKGROUND: Acute renal failure is believed to be caused, in some circumstances, by impaired oxygen delivery to the outer medulla. This study examined the effect of RSR13, a synthetic allosteric modifier of hemoglobin oxygen-binding affinity, on renal function in a setting of acute renal failure in rats. METHODS: An in vivo model of acute renal failure in the rat produced by reduced renal mass, salt restriction, volume depletion, prostaglandin inhibition, and radiocontrast administration was used. A computer-based simulation of oxygen tensions along the nephron was utilized to interpret the findings. Mechanistic studies were subsequently performed using oxygen-sensitive electrodes and 31P nuclear magnetic resonance (NMR) spectroscopy to define the effect of RSR13 on renal function in the setting of compromised acute renal failure. RESULTS: RSR13 did not attenuate acute renal failure in this model; rather, serum creatinine increased to a greater degree in the RSR13-treated rats than in rats receiving saline vehicle as the control (P < 0.05). Simulations explained this finding under conditions of severe medullary hypoxia. Mechanistic studies demonstrated marked worsening of medullary hypoxia following RSR13 under conditions similar to our experimental model. Furosemide pretreatment to reduce the imbalance between oxygen supply and demand markedly attenuated the basal-medullary hypoxia produced in the presence of indomethacin and RSR13 (P < 0.01). Additionally, 31P NMR studies demonstrated renal adenosine 5'-triphosphate (ATP) depletion in rats with acute renal failure treated with RSR13 (45% decrease, P < 0.01); again, this effect of RSR13 was completely prevented by pretreatment with furosemide. CONCLUSIONS: Under conditions of severe renal medullary hypoxia, induced in part by indomethacin-mediated reductions in outer medullary blood flow, the administration of RSR13 can exacerbate acute renal dysfunction. However, reducing the rate of oxygen consumption by inhibiting sodium transport with furosemide pretreatment or post-treatment appears to be functionally protective.

Inhibition of DNA replication and induction of S phase cell cycle arrest by G-rich oligonucleotides.

The discovery of G-rich oligonucleotides (GROs) that have non-antisense antiproliferative activity against a number of cancer cell lines has been recently described. This biological activity of GROs was found to be associated with their ability to form stable G-quartet-containing structures and their binding to a specific cellular protein, most likely nucleolin (Bates, P. J., Kahlon, J. B., Thomas, S. D., Trent, J. O., and Miller, D. M. (1999) J. Biol. Chem. 274, 26369-26377). In this report, we further investigate the novel mechanism of GRO activity by examining their effects on cell cycle progression and on nucleic acid and protein biosynthesis. Cell cycle analysis of several tumor cell lines showed that cells accumulate in S phase in response to treatment with an active GRO. Analysis of 5-bromodeoxyuridine incorporation by these cells indicated the absence of de novo DNA synthesis, suggesting an arrest of the cell cycle predominantly in S phase. At the same time point, RNA and protein synthesis were found to be ongoing, indicating that arrest of DNA replication is a primary event in GRO-mediated inhibition of proliferation. This specific blockade of DNA replication eventually resulted in altered cell morphology and induction of apoptosis. To characterize further GRO-mediated inhibition of DNA replication, we used an in vitro assay based on replication of SV40 DNA. GROs were found to be capable of inhibiting DNA replication in the in vitro assay, and this activity was correlated to their antiproliferative effects. Furthermore, the effect of GROs on DNA replication in this assay was related to their inhibition of SV40 large T antigen helicase activity. The data presented suggest that the antiproliferative activity of GROs is a direct result of their inhibition of DNA replication, which may result from modulation of a replicative helicase activity.

Nonenteric Escherichia coli isolates from dogs: 674 cases (1990-1998).

OBJECTIVE: To determine nonenteric sites associated with Escherichia coli isolates in dogs and the antimicrobial susceptibilities of the isolates. DESIGN: Retrospective study. SAMPLE POPULATION: 17,000 canine specimens. PROCEDURE: Medical records of 17,000 canine specimens submitted for bacteriologic culture were examined and the number of isolations of E coli was determined. For these cases, records were further examined with respect to body system involvement, sex, concurrent infection with other species of bacteria, and antimicrobial susceptibility. RESULTS: 674 E coli isolates (424 from urine, 62 from the skin, 52 from the respiratory tract, 45 from the ear, 43 from the female reproductive tract, 25 from the male reproductive tract, and 23 from other organ systems) were identified. There was a significantly higher proportion of isolates from urine specimens from spayed females than from sexually intact females or males. Escherichia coli was isolated in pure culture from 65.9% of the specimens. Most E coli isolates were susceptible to norfloxacin (90%), enrofloxacin (87.5%), gentamicin (90.7%), and amikacin (85.9%). CONCLUSIONS AND CLINICAL RELEVANCE: Most nonenteric E coli infections in dogs involve the urinary tract. Amikacin, gentamicin, norfloxacin, and enrofloxacin have the highest efficacy against canine E coli isolates. For E coli isolates from dogs, in vitro susceptibility to commonly used antimicrobial agents has remained fairly stable during the past decade.

Human red blood cell hemolysate is a potent mitogen for renal tubular epithelial cells.

Conditions associated with intravascular hemolysis are often complicated by acute renal failure which is characterized by renal vasoconstriction and tubular injury. However, the effects of human red blood cell (RBC) hemolysate on renal tubular epithelial cell function have not been well characterized. We therefore measured the effect of distilled water-lysed human RBCs on cultured LLC-PK1 cell function. We found that human RBC hemolysate produced a marked effect to promote LLC-PK1 3H-thymidine uptake that was several-fold higher than that produced by maximal concentrations of several known growth factors. Partial purification of human RBC hemolysate by sequential centrifugation and passage over a column that removes low molecular weight substances each significantly reduced, but did not totally eliminate, the effect of human RBC hemolysate to promote 3H-thymidine uptake. Exposure of LLC-PK1 cells to horse myoglobin also stimulated 3H-thymidine incorporation in LLC-PK1 cells. A reducing agent (ascorbic acid) decreased the effect of horse myoglobin and of human RBC hemolysate to promote LLC-PK1 mitogenesis. Ascorbic acid also decreased the methemoglobin content of human RBC hemolysate. The effect of human RBC hemolysate to increase LLC-PK1 incorporation of 3H-thymidine could also be significantly decreased by either of two inhibitors of tyrosine kinase. These results suggest that there are several components of human RBC hemolysate that promote LLC-PK1 3H-thymidine incorporation. One of these components appears to be methemoglobin that exerts its effect via a tyrosine kinase signal transduction pathway.

Small for gestational age: a new insight?

Acidosis reduces the ability of nitric oxide synthase to generate nitric oxide (NO) from L-arginine (L-arg), even if dietary intake or circulating plasma levels of L-arg are normal. During systemic acidemia, therefore, vascular perfusion in one or more organs may be compromised. Arginine is also a powerful anabolic amino acid. If dietary sources of L-arg are lower than normal, or if the production of NO is reduced even without frank acidemia, then vascular perfusion, and growth, and tissue repair are likely to be compromised. Two conditions in which acidemia is reported to occur, namely slow fetal growth in utero (acidemia is severe) and loss of bone and muscle in microgravity (acidemia is modest), are compared with respect to the accompanying alteration in the balance between acidemia and NO production.

Factors maintaining a pH gradient within the kidney: role of the vasculature architecture.

BACKGROUND: The architecture of the vasa rectae produces significant oxygen (O2) "shunting" and marked decreases in renal medullary pO2 values. We hypothesized that carbon dioxide (CO2) trapping and increases in medullary pCO2 along with decreases in medullary pH values should also accompany this O2 shunting. METHODS: We developed computer simulations employing a model of gas exchange through the countercurrent vasculature that predicted trapping of CO2 along with O2 shunting. To test the validity of this model directly, medullary pH was measured by using needle electrodes in the in situ kidney before and after the administration of mannitol or furosemide, or by decreasing blood flow with a transient decrease of renal perfusion pressure with a suprarenal clamp. Data are expressed as mean +/- SD. RESULTS: Medullary pH was lower than cortical pH (7.20 +/- 0.09 vs. 7.39 +/- 0.08, P < 0.01). Mannitol caused a decrease in medullary pH to 7.02 +/- 0.07 (P < 0.01), whereas furosemide increased medullary pH to 7. 31 +/- 0.09 (P < 0.01). Brief periods of severe hypotension decreased medullary pH to 6.90 +/- 0.09 (P < 0.01). CONCLUSIONS: These data demonstrate that a significant pH gradient exists within the kidney parenchyma. This gradient is related to the metabolic activity of the thick ascending limb of Henle and the countercurrent vascular architecture, and may be relevant to a variety of physiological phenomena involved in volume, electrolyte, and acid-based homeostasis.

Augmentation of wound healing using monochromatic infrared energy. Exploration of a new technology for wound management.

The results presented in this paper document healing of different types of extremity wounds with 890 nanometer (nm) monochromatic infrared energy. Recalcitrant dermal lesions, including venous ulcers, diabetic ulcers, and a wound related to scleroderma, were treated with a Food and Drug Administration-cleared infrared device. The infrared protocol was instituted after conventional management protocols were shown to be ineffective. The rate and quality of healing of these previously refractory wounds, following use of monochromatic infrared energy, may be related to local increases in nitric oxide concentration. Increases in nitric oxide previously have been demonstrated to correlate with vasodilatory and anabolic responses. Further research is needed to confirm the results found in these patients.

cGMP and cAMP cause pulmonary vasoconstriction in the presence of hemolysate.

We recently reported that addition of a small amount of hemolysate to the salt solution that perfused isolated rat lungs hypersensitized the vasculature to subsequent additions of ANG II or exposure to hypoxia, and addition of NO gas (. NO) to the perfusate that contained hemolysate caused a strong vasoconstrictor rather than a vasodilator response. In the present study, we demonstrate that CO and the secondary messengers cGMP and cAMP (usually associated with vasodilation) exert similar effects in hemolysate-perfused lungs. Analogs of the cyclic nucleotides cGMP or cAMP (8-bromo-cGMP and dibutyryl-cAMP, respectively) caused profound vasoconstriction in the isolated rat lung perfused with a salt solution that contained hemolysate. The cGMP- or cAMP-analog-induced vasoconstriction was inhibited by chemically dissimilar Ca2+ antagonists, by the protein phosphatase inhibitor okadaic acid, and, to a lesser degree, by protein kinase inhibitor H-7. Antiphosphothreonine immunoblotting demonstrated that lungs perfused with hemolysate exhibit increased phosphorylation of several proteins. These data indicate that, in the presence of hemolysate, pulmonary vasculature responds to nominally vasodilatory stimuli, including analogs of cGMP and cAMP, with vasoconstriction rather than vasodilation. The importance of our finding is the paradoxical nature of the response to (analogs of) cyclic nucleotides because, to our knowledge, cyclic nucleotide-induced vasoconstriction has not been previously reported.

Ischemic preconditioning attenuates functional, metabolic, and morphologic injury from ischemic acute renal failure in the rat.

Ischemic preconditioning has been shown to ameliorate injury due to subsequent ischemia in several organs. However, relatively little is known about preconditioning and the kidney. To address this, rats were randomized to control (C, N = 14), 2 min of ischemic preconditioning (P2 N = 10), 3 periods of 2 min of ischemia separated by 5 min periods of reflow (P2,3 N = 7), or three 5 min periods of ischemia separated by 5 min of reflow (P5,3 N = 6) prior to 45 min of bilateral renal ischemia followed by 24 hours of reperfusion. We observed a lower serum creatinine after 24 hours of reflow in P2, P2, 3 but not P5, 3 rats compared with C. Histology was examined in the C and P2, 3 groups and demonstrated less severe injury in the P2, 3 group. To gain insight into the mechanism by which preconditioning ameliorated ischemic injury, we performed near IR spectroscopy and 31P NMR spectroscopy. Based on near IR spectroscopy, the P2, 3 group had closer coupling of cytochrome aa3 redox state with that of hemoglobin during reflow. In the 31P NMR studies, the changes in ATP and pHi were similar during ischemia, but the P2, 3 group recovered ATP and pHi faster than C. These data suggest that ischemic preconditioning may ameliorate ischemic renal injury as assessed by functional, metabolic and morphological methods. The mechanism(s) by which this occurs requires additional study.

Hemolysate-mediated renal vasoconstriction and hypersensitization.

The present studies measured vessel diameter, before and after addition of hemolysate, in isolated afferent arterioles (AA) and efferent arterioles (EA) obtained from the rat kidney. Human red blood cells (RBC) were hemolyzed in distilled water and membranes were discarded after centrifugation. Hemolysate added to the bath solution caused vigorous AA and EA contraction and, after washout, hypersensitized the AA and EA to doses of angiotensin II (AII) which would normally only elicit 50% contraction (EC50). Neither the contraction nor the hypersensitization were mimicked by pure human hemoglobin. The vasoconstrictive responses in the AA and EA were accompanied by increased cytosolic-free calcium concentration. Further purification (desalting) of the hemolysate to remove substance of < or = 1000 Da (which include ATP) did not eliminate the vasoconstrictive component from the hemolysate. Finally, cultured rat aortic vascular smooth muscle cells also demonstrated a rapid increase in (Ca2+i) when exposed to hemolysate. This increase in (Ca2+i) was, in part, dependent on Ca2+ influx since it could be attenuated with diltiazem (10(-5) M). In conclusion, hemolysate contains a factor which induces contractions of the isolated rat kidney AA and EA and rapid elevations in (Ca2+i). This factor, from hemolyzed RBC, is not hemoglobin itself.

Arachidonic acid protects against hypoxic injury in rat proximal tubules.

Free fatty acids (FFA) and lysophospholipids accumulate during hypoxia (H) in rat proximal tubular epithelial cells partly as a result of increased phospholipase A2 (PLA2) activity. The role of FFA in mediating hypoxic injury and modulating PLA2 activity is not clear. In the present study, the effect of several FFA including arachidonic acid (AA, 20:4) on hypoxia-induced injury and PLA2 activity was assessed in freshly isolated rat proximal tubules. Hypoxia (H) was induced in the presence of either an unsaturated free fatty acid (uFFA) [AA or linoleic acid (LA, 18:2)] or a saturated FFA (sFFA) [palmitic (PA, 16:0) or myristic acid (MA, 14:0)]. Cell membrane injury was assessed by measuring lactate dehydrogenase release (LDH). AA markedly reduced LDH release during hypoxia in a dose dependent manner, while sFFA had no protective effect. LA showed similar protection to that observed with AA. AA did not affect buffer calcium concentration, buffer pH, intracellular pH or intracellular calcium concentration. Neither inhibiting the cyclooxygenase pathway with indomethacin, nor the lipoxygenase pathway with nordihydroguaiaretic acid (NDGA) had any effect on the AA observed cytoprotection. In vitro PLA2 activity in the control tubular extracts was compared to that following addition of AA or PA. PLA2 activity decreased significantly with AA but not with PA in a dose dependent manner. These data suggest that: (1) AA protects against hypoxic injury in rat proximal tubules. (2) This cytoprotection is not specific for AA and other uFFA have a similar effect. (3) AA significantly inhibits PLA2 activity, (4) AA induced cytoprotection may be related to a negative feedback inhibition of PLA2 activity.

Induced alterations in calcium uptake rate in normoxic rate proximal tubules.

This study is well-oxygenated, freshly isolated rat proximal tubules (RPT), examined the effects of several drugs that alter the transmembrane K+ and Na+ gradients across cell membranes, including valinomycin (VAL), amphotericin B (AMPHO), and ouabain (OUAB). The effects of high extracellular potassium chloride (KCl) concentrations (45 mM) and low extracellular sodium concentration (100mM) were also studied. After 10 min of drug exposure Ca2+ uptake rate (nmol/mg/min) increased from 2.7 to 3.8 with VAL (p < .02), from 2.9 to 3.7 with AMPHO (p < .05), from 3.6 to 4.1 with OUAB (p < .05), and from 3.2 to 4.8 with 45 mM KCl (p < .001). Ca2+ uptake rate was sustained at these high levels at 20 min in all treated RPT except those exposed to OUAB. LDH release averaged less than 15% in control tubules and did not increase significantly except in RPT treated with VAL, where LDH release at 10 min was 48% and at 20 min was 57% (both p < .001). Of importance, only in VAL-treated RPT did ATP decrease to low levels (6.7 nmol/mg in control to 2.0 +/- 0.3 nmol/mg in VAL, p < .001). Treatment with verapamil reduced Ca2+ uptake rates at 10 min in VAL-treated RPT (from 3.8 to 3.1, p < .02, in AMPHO-treated RPT (from 3.8 to 3.1 p < .001), in OUAB-treated tubules (from 4.0 to 3.4, p < .01), and in KCl-treated RPT (from 3.7 to 3.2, p < .01). These results indicate that acute changes in the transmembrane ion gradient in RPT are accompanied by increased Ca2+ uptake rates. Ca2+ uptake rates are also increased during O2 deprivation in RPT, a situation in which the transmembrane ion gradient is likewise altered. The increased Ca2+ uptake rate observed in the present study and during hypoxia may have a common basis, that is, altered transmembrane ion gradients or some function thereof.

The role of cysteine proteases in hypoxia-induced rat renal proximal tubular injury.

The role of the lysosomal proteases cathepsins B and L and the calcium-dependent cytosolic protease calpain in hypoxia-induced renal proximal tubular injury was investigated. As compared to normoxic tubules, cathepsin B and L activity, evaluated by the specific fluorescent substrate benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin, was not increased in hypoxic tubules or the medium used for incubation of hypoxic tubules in spite of high lactate dehydrogenase (LDH) release into the medium during hypoxia. These data in rat proximal tubules suggest that cathepsins are not released from lysosomes and do not gain access to the medium during hypoxia. An assay for calpain activity in isolated proximal tubules using the fluorescent substrate N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin was developed. The calcium ionophore ionomycin induced a dose-dependent increase in calpain activity. This increase in calpain activity occurred prior to cell membrane damage as assessed by LDH release. Tubular calpain activity increased significantly by 7.5 min of hypoxia, before there was significant LDH release, and further increased during 20 min of hypoxia. The cysteine protease inhibitor N-benzyloxycarbonyl-Val-Phe methyl ester (CBZ) markedly decreased LDH release after 20 min of hypoxia and completely prevented the increase in calpain activity during hypoxia. The increase in calpain activity during hypoxia and the inhibitor studies with CBZ therefore supported a role for calpain as a mediator of hypoxia-induced proximal tubular injury.

Nitric oxide synthase inhibition and acute renal ischemia: effect on systemic hemodynamics and mortality.

This study was designed to examine if acute systemic blockade of nitric oxide (NO) production by inhibition of nitric oxide synthase (NOS) with N-omega-nitro-L-arginine methyl ester (L-NAME) would worsen the severity of ischemic acute renal failure (ARF). Initially three groups of rats, were studied: 45 min of bilateral renal ischemia (I) alone, Group I; L-NAME (L; 10 mg/kg BW, i.v.) alone, Group L; and L-NAME administered 15 min before renal ischemia, Group L+I. We observed, however, a 60% mortality in Group I+L during the first 4 h of reflow. Captopril, administered acutely 15 min before L-NAME in an attempt to offset any detrimental effects of increased angiotensin II generation in response to renal ischemia, failed to obviate the mortality because 67% of rats in this group (Group C+L+I) also died. Therefore, additional studies were performed in rats instrumented for cardiovascular studies to evaluate the acute hemodynamic responses during the first 90 min of reperfusion following renal ischemia in rats pretreated with L-NAME. As expected, L-NAME injection was accompanied by a 25-30 mm Hg increase in mean systemic arterial pressure (SAP) (p < 0.05), a bradycardia (p < 0.02), and a decrease in cardiac output (CO) (p < 0.02). The increase in SAP was due exclusively to an increase in systemic vascular resistance (SVR) (p < 0.01). Ischemia and reflow in the L-NAME-treated rats were attended by a progressive increase in SVR and a progressive decrease in CO such that by the end of 45 min of reperfusion SVR had increased 10-fold and CO had decreased to one third of its initial rate (both p < 0.02). Pulmonary artery pressure (PAP) increased promptly following L-NAME injection. Total pulmonary resistance (PRT) increased significantly by the end of reperfusion. L-NAME in combination with renal ischemia and reflow induces a large increase in both SVR and PRT, and is accompanied by a 70% reduction in CO and substantial mortality.

Verapamil attenuates calcium-induced mitochondrial swelling and respiratory dysfunction.

A protective effect of verapamil against hypoxic renal proximal tubule injury has been demonstrated in vitro. In contrast to other cytoprotective agents such as glycine or alanine, the protective effect of verapamil is associated with better maintenance of cellular ATP and potassium levels. These findings suggested a possible direct effect of verapamil on the mitochondria in addition to known effects of verapamil on membrane Ca channels. In the present study, the direct effects of verapamil on Ca-induced swelling, respiratory dysfunction, Ca uptake rate and phospholipase activity of renal cortical mitochondria were determined. Verapamil (100 microM) significantly inhibited Ca-induced mitochondrial swelling and partially prevented the associated reduction in respiratory control ratio (State 3/State 4: Ca + verapamil: 2.8 +/- 0.1 vs. Ca alone, 2.0 +/- 0.2; P < .01). A phospholipase A2 inhibitor, dibucaine (100 microM), significantly inhibited Ca-induced mitochondrial swelling and attenuated the decrease in respiratory control ratio (Ca + dibucaine: 2.9 +/- 0.1 vs. Ca alone, 2.0 +/- 0.2; P < .001). Neither agent, either alone or combined, completely prevented the respiratory dysfunction. Either verapamil or dibucaine attenuated the mitochondrial Ca uptake rate and reduced the rate of Ca-stimulated polyunsaturated free fatty acid accumulation; verapamil treatment also was associated with diminished net release of saturated and monounsaturated free fatty acids. These findings demonstrate that verapamil exerts a protective effect against Ca-induced mitochondrial damage which may be mediated in part by its effect to suppress mitochondrial Ca uptake and mitochondrial phospholipase activity.

Nitric oxide-related vasoconstriction in lungs perfused with red cell lysate.

The present study in isolated rat lungs demonstrates that nitric oxide gas (.NO, 70 nM) added to the perfusate containing a small amount of hemolysate [175 microliters of lysed red blood cells (RBC) per 50 ml of Earle's balanced salt solution (EBSS)] triggered profound and sustained vasoconstriction. Vasoconstriction was not observed when .NO was added to lungs perfused with washed intact rat or human RBC or with oxyhemoglobin (Hgb 20 microM). The presence of hemolysate in the perfusate also caused vasoconstriction in response to n-acetylcysteine (50 microM), glutathione (10(-4) M), or ascorbic acid (10(-4) M) and potentiated greatly the vasoconstrictor response to 5 mM KCl. Not only .NO, but also nitroprusside (SNP) or L-arginine and paradoxically three .NO synthesis inhibitors, including N-monomethyl L-arginine, L-NAME, and nitroblue tetrazolium, which have different mechanisms of action, each caused in the presence of hemolysate large vasoconstrictive responses. Hemolysate itself enhanced O2 consumption by slices of lung; no effects of this dose of .NO on lung slice respiration were seen in the absence of hemolysate. Both Hgb and hemolysate lowered perfusate cGMP levels to the same degree suggesting that the vasoconstrictive response was not due to unique effects of hemolysate on guanylyl cyclase. Addition of superoxide dismutase (SOD) and catalase (CAT) to the hemolysate containing perfusate, or addition of a cyclooxygenase or 5-lipoxygenase inhibitor, virtually abolished the .NO induced vasoconstriction. The latter data are consistent with the concept that exposure of the vasculature to hemolysate may result in the formation of peroxynitrite. However, SOD and CAT did not abolish the pulmonary vasoconstriction induced by L-arginine or by NAC. Our data indicate that hemolysate has profound effects on lung vessel tone regulation and on lung tissue mitochondrial function, yet the precise molecular mechanisms responsible for the action of hemolysate are likely to be very complex.

Evidence for role of cytosolic free calcium in hypoxia-induced proximal tubule injury.

The role of cytosolic free Ca2+ ([Ca2+]i) in hypoxic injury was investigated in rat proximal tubules. [Ca2+]i was measured using fura-2 and cell injury was estimated with propidium iodide (PI) in individual tubules using video imaging fluorescence microscopy. [Ca2+]i increased from approximately 170 to approximately 390 nM during 5 min of hypoxia. This increase preceded detectable cell injury as assessed by PI and was reversible with reoxygenation. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; 100 microM) reduced [Ca2+]i under basal conditions (approximately 80 nM) and during hypoxia (approximately 120 nM) and significantly attenuated hypoxic injury. When [Ca2+]i and hypoxic cell injury were studied concurrently in the same individual tubules, the 10 min [Ca2+]i rise correlated significantly with subsequent cell damage observed at 20 min. 2 mM glycine did not block the rise in [Ca2+]i, yet protected the tubules from hypoxic injury. These results indicate that in rat proximal tubules, hypoxia induces an increase of [Ca2+]i which occurs before cell damage. The protective effect of BAPTA supports a role for [Ca2+]i in the initiation of hypoxic proximal tubule injury. The glycine results, however, implicate calcium-independent mechanisms of injury and/or blockade of calcium-mediated processes of injury such as activation of phospholipases or proteases.