Cyclosporin A tubular effects contribute to nephrotoxicity: role for Ca2+ and Mg2+ ions.

BACKGROUND: Cyclosporin A (CsA) nephrotoxicity has been attributed primarily to renal haemodynamic alterations caused by afferent arteriolar vasoconstriction. However, CsA nephropathy is also characterized by CsA-induced pre-glomerular disturbances and interstitial injury that may occur independently of haemodynamic changes. Given the high lipophilic activity of CsA, we hypothesized that direct tubular injury is likely to contribute to nephrotoxicity. METHODS: To investigate tubular toxicity of CsA, increasing concentrations of CsA (1, 2.5, 10, 25, 50 and 100 micro g/ml) and its vehicle (cremophor) were added to isolated rat proximal tubules (PT). Cell injury was assessed by lactate dehydrogenase (LDH) release. The role of Ca(2+) ions in tubular toxicity and the effect of calcium channel blockers on CsA toxicity were evaluated by measuring intracellular calcium using the fluorescent dye Fura-2 AM. The role of Mg(2+) ions was assessed using high extracellular Mg(2+) medium (2 mM). RESULTS: Whereas cremophor alone was not toxic to PT, CsA caused PT injury but only at the highest concentration (100 micro g/ml). After 90 min incubation, LDH was 22.5% in control PT and 41.9% in PT treated with 100 micro g/ml CsA (P < 0.001, n = 11). There was a transient increase in intracellular calcium ([Ca(2+)](i)) after CsA administration. A low calcium medium (100 nM) prevented CsA injury to renal tubules. However, verapamil, but not nifedipine, enhanced cell damage. Only nifedipine completely prevented [Ca(2+)](i) increases following CsA. Finally, a high Mg(2+) medium attenuated CsA-induced injury. CONCLUSION: We found that high CsA concentrations caused Ca(2+)- and Mg(2+)-dependent PT injury. Thus, low extracellular Ca(2+) and high Mg(2+) media attenuated CsA-induced tubular injury. Verapamil, but not nifedipine, enhanced CsA tubular toxicity. Therefore, CsA-induced tubular injury may contribute to CsA nephrotoxicity independently of haemodynamic disturbances.     

P glycoprotein-mediated cholesterol cycling determines proximal tubular cell viability

BACKGROUND: MDR P glycoproteins may help transport plasma membrane free cholesterol (FC) to the endoplasmic reticulum (ER), where it undergoes acylation, forming cholesterol esters (CE). This study assessed whether P glycoprotein inhibitors alter renal tubular FC/CE expression, thereby altering cell integrity. METHODS: Mouse proximal tubule segments (PTS) were exposed to chemically dissimilar P glycoprotein inhibitors [progesterone (prog), trifluoperazine (TFP), or cyclosporine A (CsA)]. Their effects on FC/CE and adenosine 5'-triphosphate (ATP) levels, phospholipid expression, lipid peroxidation, and cell viability (lactate dehydrogenase release; LDH) were assessed. P glycoprotein inhibitor effects on cultured proximal tubular (HK-2) cell viability and susceptibility to Fe-induced oxidant stress were also addressed. RESULTS: When applied to PTS, prog, TFP, and, to lesser extent, CsA induced dose-dependent ATP reductions (< or =90%), CE decrements (approximately 40%), and LDH release (< or =60%). No concomitant changes in lipid peroxidation or phospholipid profiles were observed. Ouabain did not preserve tubular ATP, suggesting that decreased ATP production, rather than increased consumption, was operative. Mechanisms leading to cell lysis were not identical, as glycine and arachidonic acid blocked prog- but not TFP-mediated cell death. When prog-driven CE reductions were attenuated in PTS with a procycling agent (cholesterol oxidase), decreased cell death resulted. P glycoprotein inhibitors also caused dose-dependent HK-2 cell death. Blocking Fe-mediated CE formation ( approximately x10) with sublethal CsA doses led to a marked increase in Fe-mediated cell death. CONCLUSIONS: P glycoproteins may be critical to tubule cholesterol transport. If blocked with pharmacologic agents, decreased ATP production, overt cell lysis, and/or a marked propensity to superimposed tubular cell injury can result.     

Acute phosphate depletion and in vitro rat proximal tubule injury: protection by glycine and acidosis.

The effects of phosphate (PO4) removal from Krebs Henseleit buffer on freshly isolated rat proximal tubules (rPT) were assessed by measuring Ca2+ uptake (nmol/mg protein), cellular adenosine triphosphate (ATP) (nmol/mg), tissue K+ content (nmol/mg) and lactate dehydrogenase (LDH) as an index of cell integrity. Ca2+ uptake increased by 50% in rPT incubated in zero PO4 medium as compared to control (2.6 +/- 0.1 vs. 3.9 +/- 0.19, P less than 0.001) and LDH release increased 2.5-fold from 14.2 +/- 0.6 to 31.6 +/- 1.6%, P less than 0.001. Neither verapamil (200 microM) nor mepacrine (50 microM) reduced Ca2+ uptake or decreased LDH release suggesting that the increased Ca2+ uptake was not occurring through potential operated channels and that phospholipase-induced cell injury was not the cause of increased LDH release. Either glycine (2 mM) or extracellular fluid acidosis (pH 7.06), however, significantly diminished rPT injury and Ca2+ uptake. Specifically, as compared to the increased LDH released in untreated. PO4-depleted rPT, LDH release was diminished significantly by glycine treatment (31.0 +/- 0.9 vs. 15.5 +/- 1.6%, P less than 0.001) or acidosis (30.3 +/- 0.04 vs. 19.2 +/- 0.9%, P less than 0.01). Ca2+ uptake did not increase in glycine treated tubules (2.6 +/- 0.1 vs. 2.8 +/- 0.2 nmol/mg, NS) or in the presence of acidosis (2.6 +/- 0.1 vs. 2.97 +/- 0.17 nmol/mg, NS). ATP concentrations were markedly reduced by PO4 depletion (2.8 +/- 0.2 vs. 4.8 +/- 0.3 nmol/mg, P less than 0.001) and remained at low levels during either acidosis or glycine-induced protection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence against increased hydroxyl radical production during oxygen deprivation-reoxygenation proximal tubular injury.

The purpose of this study was to assess whether proximal renal tubules generate excess hydroxyl radical (.OH) during hypoxia/reoxygenation or ischemia/reperfusion injury, thereby supporting the hypothesis that reactive oxygen species contribute to the pathogenesis of postischemic acute renal failure. In the first phase of the study, rat isolated proximal tubular segments (PTS) were subjected to hypoxia (95% N2- 5% CO2) for 15, 30, or 45 min, followed by 15 to 30 min of reoxygenation in the presence of sodium salicylate, a stable .OH trap. Cellular injury after hypoxia and reoxygenation was assessed by lactate dehydrogenase release; .OH production was gauged by hydroxylated salicylate by-product generation (2,3-, 2,5-dihydroxybenzoic acids (DHBA); quantified by HPLC/electrochemical detection). Continuously oxygenated PTS served as controls. Despite substantial lactate dehydrogenase release during hypoxia (8 to 46%) and reoxygenation (8 to 11%), DHBA production did not exceed that of the coincubated, continuously oxygenated control PTS. In the second phase of the study, salicylate-treated rats were subjected to 25 or 40 min of renal arterial occlusion +/- 15 min of reperfusion. No increase in renal DHBA concentrations occurred during ischemia or reperfusion, compared with that in sham-operated controls. To validate the salicylate trap method, PTS were incubated with a known .OH-generating system (Fe2+/Fe3+); in addition, rats were treated with antioxidant interventions (oxypurinol plus dimethylthiourea). Fe caused marked DHBA production, and the antioxidants halved in vivo DHBA generation. In conclusion, these results suggest that exaggerated .OH production is not a consequence of O2 deprivation/reoxygenation tubular injury.     

Radiographic contrast media-induced tubular injury: evaluation of oxidant stress and plasma membrane integrity

BACKGROUND: Experimental and clinical investigations suggest that oxidant stress is a critical determinant of radiocontrast nephropathy (RCN), and that N acetyl cysteine (NAC) can prevent this damage. This study addresses these issues directly at the tubular cell level. Potential alternative mechanisms for RCN have also been sought. METHODS: Isolated mouse proximal tubule segments (PTS), or cultured proximal tubule (HK-2) cells, were subjected to radiocontrast media (RCM) (Ioversol, Optiray 320) exposure, followed by assessments of cellular viability [% lactate dehydrogenase (LDH) release, tetrazolium dye (MTT), uptake] and lipid peroxidation. These experiments were conducted in the absence or presence of a variety of antioxidants [NAC, glutathione (GSH), superoxide dismutase, catalase] or pro-oxidant (GSH depletion, heme oxygenase inhibition) strategies. RCM effects on mitochondrial and plasma membrane integrity were also assessed. RESULTS: RCM exposure did not induce PTS lipid peroxidation. Neither antioxidant nor pro-oxidant interventions mitigated or exacerbated RCM-induced tubular cell injury, respectively. RCM impaired mitochondrial integrity, as assessed by ouabain-resistant ATP reductions, and by cytochrome c release (before cell death). RCM also induced plasma membrane damage, as indicated by loss of key resident proteins (NaK-ATPase, caveolin) and by increased susceptibility to phospholipase A2 (PLA2) attack (increase of >/=2 times in free fatty acid and NaK-ATPase release). Hyperosmolality could not account for RCM's toxic effects. CONCLUSION: RCM toxicity can be dissociated from tubular cell oxidant stress. Alternative mechanisms may include mitochondrial injury/cytochrome c release and plasma membrane damage. The latter results in critical protein loss, as well as a marked increase in plasma membrane susceptibility to exogenous/endogenous PLA2 attack.     

Plasma membrane cholesterol: a critical determinant of cellular energetics and tubular resistance to attack

BACKGROUND: Cholesterol is a major component of plasma membranes, forming membrane microdomains ("rafts" or "caveolae") via hydrophobic interactions with sphingolipids. We have recently demonstrated that tubule cholesterol levels rise by 18 hours following diverse forms of injury, and this change helps to protect kidneys from further damage (so-called acquired cytoresistance). The present study was undertaken to better define the effects of membrane cholesterol/microdomains on tubule homeostasis and cell susceptibility to superimposed attack. METHODS: Plasma membrane cholesterol was perturbed in normal mouse proximal tubular segments with either cholesterol esterase (CE) or cholesterol oxidase (CO). Alternatively, cholesterol-sphingomyelin complexes were altered by sphingomyelinase (SMase) treatment. Changes in cell energetics (ATP/ADP ratios + ouabain), viability [lactate dehydrogenase (LDH) release], phospholipid profiles, and susceptibility to injury (Fe-induced oxidant stress, PLA2, Ca2+ ionophore) were determined. The impacts of selected cytoprotectants were also assessed. RESULTS: Within 15 minutes, CE and CO each induced approximately 90% ATP/ADP ratio suppressions. These were seen prior to lethal cell injury (LDH release), and it was ouabain resistant (suggesting decreased ATP production, not increased consumption). SMase also depressed ATP without inducing cell death. After 45 minutes, CE and CO each caused marked cytotoxicity (up to 70% LDH release). However, different injury mechanisms were operative since (1) CE, but not CO, toxicity significantly altered cell phospholipid profiles, and (2) 2 mmol/L glycine completely blocked CE- but not CO-mediated cell death. Antioxidants also failed to attenuate CO cytotoxicity. Disturbing cholesterol/microdomains with a sublytic CE dose dramatically increased tubule susceptibility to Fe-mediated oxidative stress and Ca2+ overload, but not PLA2-mediated damage. CONCLUSION: Intact plasma membrane cholesterol/microdomains are critical for maintaining cell viability both under basal conditions and during superimposed attack. When perturbed, complex injury pathways can be impacted, with potential implications for both the induction of acute tubular damage and the emergence of the postinjury cytoresistance state.     

Alleviation of cyclosporine nephrotoxicity with verapamil and ATP-MgCl2. Mitochondrial respiratory and calcium studies.

Although recent studies have shown that combined treatment with verapamil and ATP-MgCl2 (ATP) prevents cyclosporine (CyA)-induced nephrotoxicity, the mechanism of these effects remains unknown. To study this, rat kidneys were perfused at 100 mmHg for 100 minutes with Krebs buffer containing 7.5 g/dL of albumin and substrates. After an equilibration period of 30 minutes, 500 ng/mL CyA was added. In some experiments 1 microgram/mL verapamil was added 10 minutes prior to CyA and in others 2 mM ATP was added to CyA. At the end of the perfusion, cortical mitochondria (mito) were isolated and mito Ca2+ and Mg2+ (mumoles/g protein) and respiratory control ratios (RCR) were measured. In addition, total tissue Ca2+ and Mg2+ levels were measured. The results indicate that CyA treatment leads to an accumulation of mito Ca2+ and a decrease in ADP/O ratio. Simultaneous administration of ATP with CyA led to an increased mito Ca2+ accumulation and depressed RCR, which were corrected by verapamil pretreatment. The combination of verapamil pretreatment and ATP cotreatment with CyA increased tissue ATP levels from 0.8 +/- 0.4 (control) to 1.4 +/- 0.1 mumol/g. This pharmacologic regimen may prevent CyA-induced nephrotoxicity by preventing mito Ca2+ accumulation and by preserving mitochondrial respiratory function. This allows a more efficient generation of ATP and consequently preservation of renal function.     

Parenteral iron nephrotoxicity: potential mechanisms and consequences

BACKGROUND: Parenteral iron administration is a mainstay of anemia management in renal disease patients. However, concerns of potential iron toxicity persist. Thus, this study was conducted to more fully gauge iron toxicologic profiles and potential determinants thereof. METHODS: Isolated mouse proximal tubule segments (PTS) or cultured proximal tubular [human kidney (HK-2)] cells were exposed to four representative iron preparations [iron sucrose (FeS), iron dextran (FeD), iron gluconate (FeG), or iron oligosaccharide (FeOS)] over a broad dosage range (0, 30 to 1000 microg iron/mL). Cell injury was assessed by lactate deyhdrogenase (LDH) release, adenosine triphosphate (ATP) reductions, cell cytochrome c efflux, and/or electron microscopy. In vivo toxicity (after 2 mg intravenous iron injections) was assessed by plasma/renal/cardiac lipid peroxidation [malondialdehyde (MDA)], renal ferritin (protein)/heme oxygenase-1 (HO-1) (mRNA) expression, electron microscopy, or postiron injection PTS susceptibility to attack. RESULTS: In each test, iron evoked in vitro toxicity, but up to 30x differences in severity (e.g., ATP declines) were observed (FeS > FeG > FeD = FeOS). The in vitro differences paralleled degrees of cell (HK-2) iron uptake. In vivo correlates of iron toxicity included variable increases in renal MDA, ferritin, and HO-1 mRNA levels. Again, these changes appeared to parallel in vivo (glomerular) iron uptake (seen with FeS and FeG, but not with FeD or FeOS). Iron also effected in vivo alterations in proximal tubule cell homeostasis, as reflected by the "downstream" emergence of tubule resistance to in vitro oxidant attack. CONCLUSION: Parenteral iron formulations have potent, but highly variable, cytotoxic potentials which appear to parallel degrees of cell iron uptake (FeS > FeG > FeD or FeOS). That in vitro injury can be expressed at clinically relevant iron concentrations, and that in vivo glomerular iron deposition/injury may result, suggest caution is warranted if these agents are to be administered to patients with active renal disease.     

The effect of halothane, isoflurane, and verapamil on ischemic-isolated rabbit renal tubules

The effects of the volatile anesthetics halothane and isoflurane, and the calcium entry blocker verapamil, were studied in isolated rabbit renal tubules under nonischemic and simulated ischemic conditions. Isolated rabbit renal tubules were subjected to zero (control), 30 (I-30), or 60 (I-60) minutes of simulated ischemia following the method of Weinberg. Following the ischemic period, tubules were reoxygenated in a Gilson respirometer (simulated reperfusion) and treated with either halothane (1%) or isoflurane (1%) in the controls and at I-30, or halothane (1%, 2%, 4%) or verapamil (5 microM, 15 microM, 30 microM) at I-60. Tubules were analyzed for lactate dehydrogenase (LDH) release (measuring cell membrane integrity), intracellular potassium and adenosine triphosphate (ATP), and oxygen consumption (cellular respiratory rate). In nonischemic tubules, exposure to 1% isoflurane caused significantly reduced LDH release compared with that released by controls, indicating cell membrane protection, whereas 1% halothane had no effect on these cells. With 30 min of ischemia, 1% isoflurane was associated with significantly higher cellular LDH release and lower ATP concentration, suggesting increased cellular damage. Halothane (1%) was associated with only an increased ATP concentration in tubules exposed to 30 min of ischemia. Following 60 min of ischemia, halothane (4%) decreased LDH release by 45% (29.2 +/- 2.3% vs. 47.0 +/- 9.6% without halothane). Tubules exposed to halothane also had higher intracellular potassium and ATP concentrations, and increased respiratory rates. Halothane (2%) was less protective and only increased the ATP concentration. The release of LDH was not statistically different with or without 2% halothane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric oxide—mediated renal epithelial cell injury during hypoxia and reoxygenation

The potent endothelial-derived vasodilator nitric oxide (NO) has been identified as a protective agent in acute renal failure. However, some recent studies have suggested a detrimental effect of NO on rat proximal tubules exposed to hypoxia and reoxygenation. We determined whether NO metabolites cause intracellular oxidation during hypoxia and reoxygenation and whether this oxidative stress is linked to irreversible cell injury. Primary cultures of rat proximal tubular epithelial cells were studied in a subconfluent stage and subjected to 60 min hypoxia and 30 min reoxygenation. Intracellular oxidation was assessed by monitoring the conversion of nonfluorescent dihydrorhodamine 123 (DHR) to fluorescent rhodamine 123 as a probe for the long-lived oxidant peroxvnitrite. Hypoxia and reoxygenation produced a marked increase in cellular generation of oxidant species. Intracellular oxidation of DHR was reduced by approsimately 40% when cells were also exposed to the NO svnthase inhibitor L-NAME. Oxidation of DHR following hypoxia and reoxygenation was not affected by SOD or DATTU. A combination of SOD and L-NAME was no more effective than L-NAME alone. Hypoxia and reoxygenation produced substantial injury (as LDH release). There was a 40% reduction in LDH release when cells were pretreated with a NO synthase inhibitor. In summary, increased generation of NO capable of inducing intracellular oxidizing reactions and cell death occurred during renal hypoxia and reoxygenation.     

In vitro cyclosporine toxicity. The effect of verapamil

The epithelial cell line LLC-PK1, which expresses many proximal tubular characteristics, was used to investigate the relationship between calcium, the calcium channel blocker verapamil, and cyclosporine toxicity. The LLC-PK1 cells took up cyclosporine when this was added in a concentration of 2 micrograms/ml, and this uptake was maximal at 30 min (112 +/- 3 ng cyclosporine/mg cell protein). At 12 micrograms/ml it inhibited the sodium glucose cotransporter, as assessed by phlorizin-inhibitable 14C-alpha-methyl glucopyranoside (alpha-MG) uptake (control 37.2 +/- 6.3, 12 micrograms/ml 21.2 +/- 1.1 mumol/hr/mg protein). Cyclosporine at 2 micrograms/ml did not affect cell growth after 5 days (control 945 +/- 60 micrograms cell protein per 25 cm2 flask, 2 micrograms/ml cyclosporine/ml 1046 +/- 32 micrograms protein/flask), even in the presence of 7.6 mM ionized calcium (862 +/- 37 micrograms protein/flask). Cyclosporine at 12 micrograms/ml inhibited cell growth (286 +/- 27 micrograms protein/flask), and raising the ambient ionized calcium concentration to 7.6 mM reduced cell growth further (91 +/- 6 micrograms protein/flask). Cyclosporine at concentrations of 2 and 12 micrograms/ml produced increasing cell vacuolation, as seen in vivo. Short-term uptake of 2 micrograms/ml cyclosporine could be inhibited by 1.0 mM and 0.5 mM verapamil (49 +/- 9.5 and 71 +/- 6.4 ng cyclosporine/mg cell protein, respectively, at 30 min). However, in the presence of 2 micrograms/ml cyclosporine 0.1 mM verapamil was toxic to the cells grown over five days (44 +/- 5 micrograms protein/flask). At 0.01 mM verapamil was not toxic to cell growth (921 +/- 29 micrograms protein/flask), but raising the medium calcium to 7.6 mM reduced cell growth (652 +/- 96 micrograms/ml). Inhibition of cyclosporine uptake did not occur with 0.01 mm verapamil (control 145.6 +/- 12.3 vs. 0.01 mM verapamil 150.4 +/- 3.8 ng cyclosporine/mg cell protein). The LLC-PK1 cell line represents a good in vitro model for cyclosporine renal tubular toxicity, as the in vivo observation of glycosuria and proximal tubular cell vacuolation in cyclosporine nephrotoxicity can be reproduced. In vitro this is shown to be associated with inhibition of sodium-dependent glucose cotransport. Verapamil inhibited cyclosporine uptake, but only at concentrations that were toxic to the cells. Verapamil potentiated rather than reduced the increased cyclosporine toxicity produced by increasing the medium calcium concentration. The suggested protective effect of verapamil against cyclosporine nephrotoxicity is therefore unlikely to be due to inhibition of cyclosporine uptake or of calcium entry into proximal tubular cells.     

Plasma membrane phospholipid integrity and orientation during hypoxic and toxic proximal tubular attack.

BACKGROUND: Acute cell injury can activate intracellular phospholipase A2 (PLA2) and can inhibit plasma membrane aminophospholipid translocase(s). The latter maintains inner/outer plasma membrane phospholipid (PL) asymmetry. The mechanistic importance of PLA2-mediated PL breakdown and possible PL redistribution ("flip flop") to lethal tubule injury has not been well defined. This study was performed to help clarify these issues. METHODS: Proximal tubule segments (PTS) from normal CD-1 mice were subjected to either 30 minutes of hypoxia, Ca2+ ionophore (50 microM A23187), or oxidant attack (50 microM Fe). Lethal cell injury [the percentage of lactate dehydrogenase (LDH) release], plasma membrane PL expression [two-dimensional thin layer chromatography (TLC)], and free fatty acid (FFA) levels were then assessed. "Flip flop" was gauged by preferential decrements in phosphatidylserine (PS) versus phosphatidylcholine (PC; PS/PC ratios) in response to extracellular (Naja) PLA2 exposure. RESULTS: Hypoxia induced approximately 60% LDH release, but no PL losses were observed. FFA increments suggested, at most 3% or less PL hydrolysis. Naja PLA2 reduced PLs in hypoxic tubules, but paradoxically, mild cytoprotection resulted. In contrast to hypoxia, Ca2+ ionophore and Fe each induced significant PL losses (6 to 15%) despite minimal FFA accumulation or cell death (26 to 27% LDH release). Arachidonic acid markedly inhibited PLA2 activity, potentially explaining an inverse correlation (r = -0.91) between tubule FFA accumulation and PL decrements. No evidence for plasma membrane "flip flop" was observed. In vivo ischemia reperfusion and oxidant injury (myohemoglobinuria) induced 0 and 24% cortical PL depletion, respectively, validating these in vitro data. CONCLUSIONS: (a) Plasma membrane PLs are well preserved during acute hypoxic/ischemic injury, possibly because FFA accumulation (caused by mitochondrial inhibition) creates a negative feedback loop, inhibiting intracellular PLA2. (b) Exogenous PLA2 induces PL losses during hypoxia, but decreased cell injury can result. Together these findings suggest that PL loss may not be essential to hypoxic cell death. (c) Oxidant/Ca2+ overload injury induces early PL losses, perhaps facilitated by ongoing mitochondrial FFA metabolism, and (d) membrane "flip flop" does not appear to be an immediate mediator of acute necrotic tubular cell death.     

Breakdown of hepatic tight junctions during reoxygenation injury.

We investigated whether reoxygenation following anoxia increased biliary permeability and whether or not allopurinol had a protective effect. Isolated rat livers were perfused for 30 min in a one-pass system with buffer equilibrated with 100% nitrogen after stabilization, and then for 60 min with the oxygenated buffer. Hepatic tight junction permeability was assessed by quantifying the early appearance in the bile of horseradish peroxidase (HRP) injected with the perfusate. This early peak represents paracellular passage of HRP, whereas a later second peak results from transcellular passage. In the control livers, 7% of the total HRP passage (93 +/- 50 pg/g liver) was paracellular and 93% was transcellular. After 30 min of reoxygenation following anoxia, however, 516 +/- 20 pg/g liver of HRP passed paracellularly. Addition of allopurinol (5 micrograms/ml) to the perfusate from the start of perfusion reduced paracellular passage of HRP to 219 +/- 49 pg/g liver after anoxia and reperfusion (P less than 0.01). Allopurinol also reduced the cumulative lactate dehydrogenase (LDH) release during the first 30 min of reoxygenation from 2.1 +/- 0.3 x 10(4) to 1.4 +/- 0.4 x 10(4) units/g liver (P less than 0.01). Reduction of the anoxic period from 30 min to 25 min significantly reduced the change in tight junction permeability and the extent of cellular injury: Paracellular passage of HRP was 336 +/- 20 pg/g and LDH release was 0.7 +/- 0.1 x 10(4) units/g liver, both significantly lower than those at 30 min (P less than 0.01). No significant difference in hepatic ATP levels after 60 min of reoxygenation was noted among the experimental groups, but all had lower levels than the control group. The protective effect of allopurinol suggests that the mechanism of biliary reoxygenation injury involves free radical generation. Susceptibility of tight junctions suggests a pattern of injury similar to that involved in anoxic damage of the vascular endothelium.     

Possible mechanism of efferent arteriole (Ef-Art) tubuloglomerular feedback

Adenosine triphosphate (ATP) is liberated from macula densa cells in response to increased tubular NaCl delivery. However, it is not known whether ATP from the macula densa is broken down to adenosine, or whether this adenosine mediates efferent arteriole (Ef-Art) tubuloglomerular feedback (TGF). We hypothesized that increased macula densa Ca(2+), release of ATP and degradation of ATP to adenosine are necessary for Ef-Art TGF. Rabbit Ef-Arts and adherent tubular segments (with the macula densa) were simultaneously microperfused in vitro while changing the NaCl concentration at the macula densa. The Ef-Art was perfused orthograde through the end of the afferent arteriole (Af-Art). In Ef-Arts preconstricted with norepinephrine (NE), increasing NaCl concentration from 10 to 80 mM at the macula densa dilated Ef-Arts from 7.5+/-0.7 to 11.1+/-0.3 microm. Buffering increases in macula densa Ca(2+) with the cell-permeant Ca(2+) chelator BAPTA-AM diminished Ef-Art TGF from 3.1+/-0.3 to 0.1+/-0.2 microm. Blocking adenosine formation by adding alpha-beta-methyleneadenosine 5'-diphosphate (MADP) blocked Ef-Art TGF from 2.9+/-0.5 to 0.1+/-0.2 microm. Increasing luminal NaCl at the macula densa from 10 to 45 mM caused a moderate Ef-Art TGF response, 1.3+/-0.1 microm. It was potentiated to 4.0+/-0.3 microm by adding hexokinase, which enhances conversion of ATP into adenosine. Our data show that in vitro changes in macula densa Ca(2+) and ATP release are necessary for Ef-Art TGF. ATP is broken down to form adenosine, which mediates signal transmission of Ef-Art TGF.     

Role of reactive oxygen metabolites in organophosphate-bidrin-induced renal tubular cytotoxicity.

Due to low toxicity to nontarget species and rapid degradation after its application, organophosphate (OP) remains a widely used class of pesticide. Suicidal or accidental overdose of OP can result in acute tubular necrosis. Experimental evidence shows little correlation between the renal tubular necrosis and the degree of OP-induced acetylcholinesterase inhibition, the main mechanism of OP's toxicity, suggesting the involvement of alternate mechanisms. Since reactive oxygen species (ROS) are known mediators of many toxin-induced renal injuries, this study was conducted to investigate whether ROS play a role in Bidrin (BD)-induced renal tubular epithelial cell (LLC-PK1) toxicity. BD is an OP insecticide formulation with dicrotophos as the active ingredient. LLC-PK1 cell death, determined by lactate dehydrogenase (LDH) release (% of total), rose concentration- and time-dependently after exposure of the cells to 1000, 1250, 1500, 1750, and 2000 ppm of BD for 6, 12, 24, and 48 h. Antioxidants 2-methylaminochroman (2-MAC; 0.3 to 2.5 microM) and desferrioxamine (DFO; 0.25 to 2 mM) reduced cell damage induced by 1250 ppm of BD over a 24-h incubation in a concentration-related manner. The greatest reductions in % LDH were produced by DFO 2 mM and 2-MAC 2.5 microM, both significantly lower than BD alone. H2O2 levels (micromol/mg protein per h) were significantly elevated after exposure to 1250 ppm of BD. Significantly increased malondialdehyde formation (nmol/mg protein) compared with control was also found in BD-exposed cells indicating enhanced lipid peroxidation. Malondialdehyde generation was significantly suppressed by 2-MAC and DFO. These results demonstrate that the organophosphate BD can cause direct tubular cytotoxicity, and implicate, at least in part, a role for ROS and accompanying lipid peroxidation in cytotoxicity. Based on these direct in vitro findings, it is hypothesized that, besides hypotension that often accompanies OP intoxication, OP-induced oxidative stress at the tubular level may play a role in the pathogenesis of acute tubular necrosis.     

Reoxygenation results in cell death of human alveolar epithelial cells.

BACKGROUND: The functional response of isolated alveolar epithelial cells (AECs) to ischemia/reperfusion injury (I/R) is incompletely understood. Using a cell culture model, we investigated the tolerance of human type II alveolar cells (ATII) to hypoxia and subsequent reoxygenation. METHODS: Cell cultures of A549 cells (human lung adenocarcinoma) and primary ATII were incubated in 95% N(2)/5% CO(2) saturated medium at 37 degrees C for 48 hours or 72 hours. The hypoxic medium was subsequently exchanged to normoxic medium at 37 degrees C. Lactate dehydrogenase (LDH) release and mitochondrial viability, as assessed by WST-1 metabolism, were determined during both hypoxia and reoxygenation. A549 cells and ATII maintained under normoxic conditions served as controls. RESULTS: Before reoxygenation, after 48 or 72 hours of hypoxia, WST-1 metabolism in A549 cells was significantly reduced (p < 0.05), but LDH release remained low in both cell types. Reoxygenation after 48 h of hypoxia was associated with recovery of WST-1 metabolism and an only minimal increase in LDH release. Reoxygenation after 72 hours of hypoxia, in contrast, induced marked injury in both A549 cells and primary ATII as indicated by significantly reduced WST-1 metabolism and a dramatic increase of LDH release compared with normoxic controls (p < 0.05). CONCLUSIONS: Viability of alveolar cell lines and primary ATII is maintained during hypoxia for up to 72 hours. Reoxygenation after 72 hours of hypoxia results in rapid development of injury and cell death in both cell types.     

A synergistic effect of extracellular hypocalcemic condition for hyperoxic reoxygenation injury in rat hepatocytes

BACKGROUND: Calcium accumulation of cells and mitochondria during reperfusion or reoxygenation has been implicated as a potential factor in cell injury as the result of mitochondrial damage. The objective of this study was to disclose whether or not low extracellular calcium ion concentration ([Ca2+]ex) in the medium at the time of reoxygenation might prevent calcium accumulation and attenuate hepatocytes injury after severe hypoxia. METHODS: Isolated rat hepatocytes were incubated under a hyperoxic or hypoxic atmosphere for 60 min. During the ensuing 60-min hyperoxic reoxygenation, medium [Ca2+]ex was varied from 0.6 microM to 2.0 mM by altering total calcium and addition of chelators. RESULTS: Incubation in low [Ca2+]ex reduced total cellular calcium and mitochondrial calcium in both the hyperoxic and hypoxic group. Under hyperoxic/hyperoxic incubation (control), hepatocytes were able to maintain potassium balance when [Ca2+]ex was >3.0 microM (pCa=5.5) and cellular viability (% lactate dehydrogenase release) at all levels of extracellular calcium. Under hypoxic/hyperoxic incubation (reoxygenation), however, loss of the ability to restore potassium balance as well as apparent increase in lactate dehydrogenase release were observed at severely low [Ca2+]ex (<30 microM; pCa=4.5). This low [Ca2+]ex-induced exacerbation of hepatocytes viability could not be generated under mild reoxygenation such as normoxia. CONCLUSIONS: In normal isolated hepatocytes, very low [Ca2+]ex levels produce only very subtle changes in membrane permeability of isolated hepatocytes. After hypoxia, however, hypocalcemia acts synergistically with hyperoxic reoxygenation to produce more severe damage. These results suggested that [Ca2+]ex should be maintained on the physiological level to attenuate hepatocytes injury after severe hypoxia.     

氯离子通道阻断剂对氧化剂诱导的肾小管上皮细胞损伤的保护作用

目的研究氯离子（Cl-）通道阻断剂在氧化剂诱导的肾小管上皮细胞损伤中的作用。方法以H2O2诱导肾小管上皮细胞株（LLC-PK1）损伤，观察Cl-通道阻断剂对受损细胞LDH释放量、ATP含量和DNA降解程度的影响。结果Cl-通道阻断剂可使受损细胞的LDH释放量下降、ATP含量回升和DNA降解程度减轻。结论Cl-通道参与了氧化剂对细胞损伤的病理过程，Cl-通道阻断剂对肾小管上皮细胞具有保护作用。     

Oxalate and calcium oxalate mediated free radical toxicity in renal epithelial cells: effect of antioxidants

In a previous study we demonstrated that oxalate induced free radical injury can promote calcium oxalate stone formation. In the present study, we tested whether the antioxidants vitamin E, superoxide dismutase (SOD), catalase and desferoxamine (DFO) can provide protection against oxalate toxicity in LLC-PK(1) cells. LLC-PK(1) cells were exposed to oxalate (1.0 mM) or oxalate+calcium oxalate monohydrate crystals (COM, 500 microg) for 3, 6, and 9 h. Cellular injury was assessed by lactate dehydrogenase (LDH) release. Malondialdehyde (MDA) content, catalase and glutathione peroxidase activities were also measured. The effect of vitamin E (200 microM), DFO (1.0 mM), SOD (400 U), and catalase (400 U) on oxalate-exposed cells was tested. LLC-PK(1) cells exposed to oxalate showed a significant increase in LDH release and MDA content, which was further elevated when COM crystals were added. Cellular glutathione peroxidase and catalase activities were decreased on exposure to oxalate. The addition of vitamin E, SOD, catalase and DFO significantly reduced the release of LDH and restored glutathione peroxidase and catalase activities towards the control level. The increased formation of MDA on oxalate or oxalate+COM toxicity was restored towards normalization by antioxidants and antioxidant enzymes. The protection rendered by vitamin E was greater than that of SOD, catalase and DFO. We conclude that oxalate associated free radical injury may promote stone formation by providing cellular debris for crystal nucleation and aggregation and augment crystal attachment to other tubular cells. Antioxidant administration may prevent calcium oxalate nucleation and retention in the renal tubules by preventing oxalate mediated peroxidative injury.     

Improvement of renal preservation by verapamil with 24-hour cold perfusion in the isolated rat kidney

There is substantial evidence that increased cellular calcium may activate processes that lead to cellular injury and death, and calcium entry blockers (CEB) have been shown to protect against renal ischemic injury. This approach has been used experimentally to enhance kidney preservation during both warm and cold ischemia. In the present study, the effect of the CEB verapamil on kidney function after 24 hr of hypothermic (4-7 degrees C) perfusion was examined and compared with simple cold storage with Eurocollins' solution (4 hr), 4 or 24 hr cold perfusion, without the addition of verapamil. The cold perfusion media consisted of 3% albumin in phosphate-free Krebs-Henseleit saline supplemented with 5 mM glucose. Cold perfusion was performed at 40 mmHg perfusion pressure with either 0 (C) or 5 microM verapamil (V) added to the cold perfusion media. Renal functional parameters of plasma flow (RPF), inulin clearance (Cin), fractional (FRNa+) and net sodium reabsorption (TNa+) were assessed during 60 min of reperfusion at 37 degrees C using 6.7% albumin in Krebs-Henseleit saline supplemented with glucose, inulin, and 20 amino acids. There was no increase in RPF with V (33 +/- 1 vs. 32 +/- 2 ml/min/g,NS) but Cin was significantly higher (271 +/- 30 vs. 168 +/- 20 microliter/min/g P less than 0.01) with V. Preservation of tubular function by V was demonstrated by an increase in FRNa+ (84 +/- 5 vs. 57 +/- 8%, P less than .01), TNa+ (32 +/- 6 vs. 15 +/- 3 mumol/min/g, P less than .01) and renal adenosine triphosphate (ATP) concentration (8.0 +/- 5 vs. 4.7 +/- 1.0 mumol/g dry tissue, P less than .01). Thus, V appears not only to enhance kidney preservation with warm and cold ischemia but also improves renal function, as assessed by glomerular filtration rate (GFR) tubular function, and tissue ATP concentration with 24-hr cold perfusion.