Renal Slices Research Articles

Cephaloridinein VitroToxicity and Accumulation in Renal Slices from Normoglycemic and Diabetic Rats

Cephaloridinein VitroToxicity and Accumulation in Renal Slices from Normoglycemic and Diabetic Rats

The nuclear microprobe at the University of Arizona

A nuclear microprobe has been operational at the University of Arizona since early 1994. It utilizes a magnetic quadrupole doublet (model QL-300 from Microscope Associates, Inc.) with an 11 mm diameter aperture and lens lengths of 6 cm each. The magnetic pole tips are electrically insulated to enable electric rotational alignment and beam rastering by application of varying voltages to the pole tips. Ion beams are obtained from a 5.5 MV model CN, High Voltage Engineering Corp. Van de Graaff accelerator with a Penning type ion source. Present mininum beam spot size is about 2 μm obtained with a 4 MeV H 2 + beam with a current of about 40 pA. To date, the instrument has been successfully used to map concentrations of Hg, Cr, and As in rabbit renal slices using PIXE.

Lipid profiling in renal and hepatic tissue slices following exposure to atractyloside.

The diterpene glycoside, atractyloside (ATR) is present in a number of plants used as popular medicines throughout Africa and along the Mediterranean. These plants cause fatal intoxication in a significant number of individuals [ 1-31. especially children. Clinical data from humans poisoned by these plants and experimental studies in animals treated with ATR have shown an acute cellular necrosis, largely confined to the proximal tubule of the kidney [4] and centrilobular portion of the liver [5]. The main target of ATR is thought to be the mitochondria, where it inhibits the adenine nucleotide translocase and thus cellular energy production [6]. ATR also alters catabolic and anabolic furictions in vivo [2], and although several clinical abnormalities have been reported details of the underlying metabolic changes have not been investigated or studied in virro. The present study is part of a long-term interest in using in virro systems to help understand the toxic effects and alterations in intermediary metabolism caused by diterpenoids. We have examined renal and liver tissue slices exposed to ATR (for relatively short periods) and used high performance thin layer chromatography (HPTLC) with densitometry to study changes in cholesterol, total phospholipid and triacyglycerol profiles. Precision-cut renal cortical and liver slices (200 pm thick) were made from cylindrical cores of rat kidney (8 mm diameter) and liver (10 mm diameter) tissues using a Krumdieck tissue slicer [7] containing pH 7.4, 150 mM-phosphate buffered saline at 4°C. Slices were preincubated in media to allow the tissue to stabilise and then used for investigation. Individual slices were placed in a 24-well plate (Linbro, UK) containing ATR (0.2-2.0 mM) dissolved in 1 ml serum-free, phenol-red-free Dulbecco Modified Eagles nutrient mixture: F-12 Ham. This was incubated for 3 h at 37°C on an orbital shaking platform (70 cycldmin). At the end of the incubation period, slices were removed, blotted, weighed, homogenized and extracted with 1 ml chloroformxnethanol (2:l, containing 0.1% w/v butylated hydroxytoluene). The lipid profile was assessed in 5 p1 of tissue extract, delivered under N, by a Camag Linomat IV applicator to Merck silica gel HPTLC plates (10 x 20 cm). The plates were developed with petroleum ether:diethyl ether:acetic acid, 80:20:3 by volume in a Camag horizontal developing chamber. Separated bands were stained using the manganese chloride-sulphuric acid reagent [8] heated in an oven at 110°C for 40 min. The pinkish coloured bands appeared on a whitish background and were quantified using a Desaga CD-60 densitometer with a Hg lamp, scanned at an optimized wavelength of 250 nm, and using a slit width of 0.02 mm and 3 mm in height. Commercially available cholesterol, phospholipid and triacyglycerol standards (Sigma, Poole) were processed and used to calibrate the system. ATR caused a significant and dose-related decrease of triacylglycerol in liver slices, but not in kidney slices (Table 1). By contrast ATR increased phospholipids and cholesterol in renal, but had no effects in hepatic tissue. These data show that intermediary metabolism can be affected by ATR in both kidney and liver slices within a relatively short period. The decrease in liver triacylglycerol by ATR suggests a depletion in one of the vital cellular energy sources, as much of the energy to be derived from lipids relates to the metabolism of the fatty acid chains of triacylglycerol. This, in addition to the usual hypoglycaemic effect of ATR [2], could account for the associated decline . . Table 1. Effect of v : in rat kidnev and li ver slices i n c u b a d for 3 hou rs.

Reduced susceptibility of renal slices to oxidative damage.

Reduced susceptibility of renal slices to oxidative damage.

Cephaloridine in Vitro Toxicity and Accumulation in Renal Slices from Normoglycemic and Diabetic Rats

Previous work has shown a reduction in cephaloridine nephrotoxicity in a diabetic rat model. The following studies examined in vitro cephaloridine toxicity in renal slices from normoglycemic and diabetic Fischer 344 rats. Diabetes was induced by acute intraperitoneal injection of 35 mg/kg streptozotocin. Renal cortical slices were isolated from normoglycemic and diabetic animals. Tissues were exposed to 0-5 mM cephaloridine for 15-120 min. Pyruvate-directed gluconeogenesis was diminished in all groups exposed to 2-5 mM cephaloridine for 60-120 min. Leakage of lactate dehydrogenase (LDH) was apparent only in the normoglycemic group in the presence of 4-5 mM cephaloridine for 120 min. LDH leakage was not increased at any cephaloridine concentration in the diabetic tissue. Total glutathione levels were compared in renal cortical slices exposed to cephaloridine for 30-120 min. Baseline values for glutathione were comparable between normoglycemic and diabetic tissue suggesting that the mechanism for reduced toxicity was not due to higher glutathione levels in diabetic tissue. Total glutathione levels were diminished more rapidly in normoglycemic than diabetic tissue by incubation with 5 mM cephaloridine. Comparison of cephaloridine accumulation indicated that diabetic tissue accumulated less cephaloridine than the normoglycemic group when tissues were incubated with 0-2 mM cephaloridine. However, renal slice accumulation was similar between normoglycemic and diabetic groups following in vitro incubation with 4-5 mM cephaloridine. These results suggest that the mechanism for reduced in vitro cephaloridine toxicity in diabetic tissue cannot be limited to differences in accumulation and must include an unidentified cellular component.

Utilization of renal slices to evaluate the efficacy of chelating agents for removing mercury from the kidney

Mercury is an environmental contaminant that preferentially accumulates in the kidney. It has been previously shown using proton-induced X-ray emission analysis that mercury (HgCl2) accumulated in precision-cut rabbit renal cortical slices. In this study, the efficacy of seven chelating agents for the removal of Hg from renal slices has been examined. Rabbits were injected with HgCl2 (10 mg/kg) and 3 h later kidneys were sliced, or renal slices were exposed in vitro to a mildly toxic concentration of HgCl2 (5 × 10−5 M, 4 h). The slices were then treated in vitro with 10 mM concentrations of EDTA, lipoic acid (LA), penicillamine (PA), glutathione (GSH), 1,4-dithiothreitol (DTT), DMSA, or DMPS. DMPS proved to be the most effective in mobilizing Hg from in vivo or in vitro HgCl2-exposed renal tissue (> 85% of control after 3 h incubation). Relative efficacies for the seven agents were DMPS > DMSA, PA > DTT, GSH > LA, EDTA. The use of renal slices appears to be a useful in vitro tool for assessing the efficacy of chelating agents on mobilizing accumulated Hg from renal tissue. Copyright

New group of lipid mediators containing omega-hydroxyarachadonic acid (20-HETE).

20-HETE is a biologically active product of arachidonic acid and cytochrome P450 ω-hydroxylase of the 4A gene family and is an abundant microsomal eicosanoid in the rat kidney.1 However, very little is known about factors controlling the biosynthesis and metabolic fate of 20-HETE in the kidney. Cyclooxygenase metabolizes 20-HETE into ω-OH-PGs, formation of which was proposed to explain the vasoconstrictor activity of 20-HETE in the kidney.2 Human platelets metabolize 20-HETE into a complex mixture of products of which 11,20-dihydroxyeicosatetraenoic acid (11,20-DiHETE), and 12,20-DiHETE are the major metabolites.3. We observed that the stimulation of the rat kidney with hormones or calcium ionophore results in an accumulation of 20-HETE, which is readily detectable by GC/MS.4, 5 The release of 20-HETE from renal slices stimulated by calcium ionophore cannot be inhibited with ETYA, which suggested to us that calcium influx is required for the release of 20-HETE. The isolated perfused kidney, challenged with angiotensin II (ang II) or endothelin-1, rapidly releases 20-HETE and often other CYP450 subterminal HETEs, into the perfusion fluid in amounts 5 to 10-fold higher relative to control levels. Brief stimulation of mTAL cells with ang II leads to a selective release of 20-HETE.6 These results suggested that 20-HETE could be released from renal tissue phospholipids via a receptor-mediated activation of phospholipase (PL)A2. In this study we obtained evidence that the release of 20-HETE from renal phospholipids is possible and represents a new component of ang II-dependent effects on renal function.

In Vitro toxicity of 2- and 4-chloroaniline: comparisons with 4-amino-3-chlorophenol, 2-amino-5-chlorophenol and aminophenols

Chloroanilines have been associated with renal and hepatic toxicity. This study (a) examined the in vitro hepatic and renal toxicity of 2-chloroaniline and 4-chloroaniline, (b) further examined whether aromatic ring hydroxylation would increase toxicity of the parent compound and (c) compared toxicity between respective aminochlorophenol and aminophenol. Renal and hepatic slices were exposed to varying concentrations of 2-chloroaniline, 4-chloroaniline. 4-amino-3-chlorophenol, 2-amino-5-chlorophenol, 2-aminophenol or 4-aminophenol. Toxicity was monitored by measurement of pyruvate-directed gluconeogenesis and leakage of lactate dehydrogenase (LDH). Hepatic tissue was less susceptible to toxicity than kidney tissue for all compounds since LDH leakage was elevated only in renal tissue. Gluconeogenesis was reduced in renal cortical slices exposed to 0.1 μm aminochlorophenols or 4-aminophenol, whereas a concentration of 0.5 μm was necessary for the chloroanilines and 2-aminophenol. LDH release was increased in renal slices by aminochlorophenols and aminophenols but not by the chloroanilines. The nephrotoxic potential in renal cortical slices was 4-aminophenol> 2-amino-5-chlorophenol> 4-amino-3-chlorophenol> 2-aminophenol> 2-chloroaniline= 4-chloroaniline. These results suggest that aromatic ring hydroxylation increased in vitro toxicity of the chloroanilines. Comparison of aminophenols with aminochlorophenols indicated that addition of a halogen can have variable effects on toxicity.

Tiopronin Protects against the Nephrotoxicity of Cisplatin in Rat Renal Cortical Slicesin Vitro

The protective effect ofN-(2-mercaptopropionyl)-glycine (tiopronin), a clinically used sulfhydryl-containing compound, on cisplatin-induced toxicity to rat renal cortical slices was investigated. Exposure of the slices to cisplatin (2 mM) resulted in toxicity, as shown by an increase in leakage of the two enzymes aspartate aminotransferase and lactate dehydrogenase into the incubation medium and a time-dependent decrease in the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by the slices. Tiopronin (2 mM) completely prevented the cisplatin-induced increase in enzyme leakage and substantially blocked the decrease of MTT reduction caused by cisplatin. These protective effects were concentration-dependent and furthermore, the depletion of ATP, glutathione and induction of lipid peroxidation in the slices by cisplatin (2 mM) were reversed by 2 mMtiopronin. Pretreatment of slices with tiopronin for 60 min also significantly protected the renal slices from cisplatin-induced toxic effects. These protective effects, however, were abolished byp-aminohippuric acid, a compound with some structural similarity to tiopronin, which both undergoes and inhibits active transport in the cells of the proximal convoluted tubule. Cisplatin (1 mM) also depleted the free sulfhydryls of tiopronin (1 mM) in a second incubation medium system and PAH (2 mM) diminished the extent of this depletion somewhat. These observations suggest that tiopronin protects against cisplatin-induced nephrotoxicity by acting as an alternative target for cisplatin both intra- and extracellularly and thus protects against cisplatin-induced depletion of glutathione in the kidney cell.

Protein arylation precedes acetaminophen toxicity in a dynamic organ slice culture of mouse kidney.

Acetaminophen (APAP) is an analgesic and antipyretic agent which may cause hepatotoxicity and nephrotoxicity with overdose in man and laboratory animals. In vivo studies suggest that in situ activation of APAP contributes to the development of nephrotoxicity. Associated with target organ toxicity is selective arylation of proteins, with a 58-kDa acetaminophen binding protein (58-ABP) being the most prominent cytosolic target. In this study a mouse kidney slice model was developed to further evaluate the contribution of in situ activation of APAP to the development of nephrotoxicity and to determine the selectivity of protein arylation. Precision cut kidney slices from male CD-1 mice were incubated with selected concentrations of APAP (0-25 mM) for 2 to 24 hr. APAP caused a dose- and time-dependent decrease in nonprotein sulfhydryls (NPSH), ATP content, and K+ retention. Preceding toxicity was arylation of cytosolic proteins, the most prominent one being the 58-ABP. The association of 58-ABP arylation with APAP toxicity in this mouse kidney slice model is consistent with earlier, in vivo results and demonstrates the importance of in situ activation of APAP for the development of nephrotoxicity. Precision cut renal slices and dynamic organ culture are a good model for further mechanistic studies of APAP-induced renal toxicity.

Author Index for Volume 33

Previous work has shown a reduction in cephaloridine nephrotoxicity in a diabetic rat model. The following studies examinedin vitrocephaloridine toxicity in renal slices from normoglycemic and diabetic Fischer 344 rats. Diabetes was induced by acute intraperitoneal injection of 35 mg/kg streptozotocin. Renal cortical slices were isolated from normoglycemic and diabetic animals. Tissues were exposed to 0–5 mmcephaloridine for 15–120 min. Pyruvate-directed gluconeogenesis was diminished in all groups exposed to 2–5 mmcephaloridine for 60–120 min. Leakage of lactate dehydrogenase (LDH) was apparent only in the normoglycemic group in the presence of 4–5 mmcephaloridine for 120 min. LDH leakagewas notincreased at any cephaloridine concentration in the diabetic tissue. Total glutathione levels were compared in renal cortical slices exposed to cephaloridine for 30–120 min. Baseline values for glutathione were comparable between normoglycemic and diabetic tissue suggesting that the mechanism for reduced toxicity was not due to higher glutathione levels in diabetic tissue. Total glutathione levels were diminished more rapidly in normoglycemic than diabetic tissue by incubation with 5 mmcephaloridine. Comparison of cephaloridine accumulation indicated that diabetic tissue accumulated less cephaloridine than the normoglycemic group when tissues were incubated with 0–2 mmcephaloridine. However, renal slice accumulation was similar between normoglycemic and diabetic groups followingin vitroincubation with 4–5 mmcephaloridine. These results suggest that the mechanism for reducedin vitrocephaloridine toxicity in diabetic tissue cannot be limited to differences in accumulation and must include an unidentified cellular component.

Differential effects of cisplatin on the production of NADH-dependent superoxide and the activity of antioxidant enzymes in rat renal cortical slices in vitro.

Cisplatin-induced cytotoxicity has been investigated with rat renal cortical slices in vitro. Incubation of renal slices with cisplatin caused an unexpected decrease in NADH-dependent. lucigenin-enhanced chemiluminescence in the homogenate of the slices in a time- and concentration-dependent fashion. Cisplatin caused a concentration-related (0.2 1 mM) decrease in the formation of NADH-dependent superoxide anion. Cisplatin (2 mM) significantly suppressed the chemiluminescence to 70% of control as early as 15 min, and to 15% after 90 min. of incubation. A decrease was also observed 90 min. after incubation of slices with 0.25 mM cisplatin. In addition, the activities of superoxide dismutase (SOD) and catalase in the slices were significantly increased after 60 min. of exposure to cisplatin (2 mM) while decreases in the activities of glutathione (GSH) peroxidase and GSSG reductase became significant at 90 and 120 min. of incubation respectively. SOD and catalase activities were increased by 1.6 and 1.5 fold respectively after 90 min. of incubation and the activities of GSH peroxidase and GSSG reductase were decreased to 82% and 72% of control, respectively at 120 min. Both dithiothreitol (2 mM), a sulphydryl agent and diphenylphenylenediamine (5 microM), an antioxidant, protected against cisplatin-induced leakage of lactate dehydrogenase, lipid peroxidation and decreases of GSH peroxidase and GSSG reductase but had no effect on the decrease of chemiluminescence caused by cisplatin. The results suggest that neither an increase in the production of NADH-dependent superoxide anion nor a decrease in activity of several antioxidant enzymes were directly responsible for cisplatin-induced lipid peroxidation.

Assessment of the Role of Glutathione Conjugation in the Protection Afforded by Anethol Dithiolthione against Hexachloro- 1,3-butadiene-Induced Nephrotoxicity

Administration of anethol dithiolthione (ADT) to rodents can afford protection against some chemically induced toxicities. The aim of the present study was to assess the effects of ADT on hexachloro-1,3-butadiene (HCBD)-induced nephrotoxicity in the rat and to determine the mechanism of its action. Renal integrity was evaluated by measuring urinary excretion of glucose, protein, and γ-glutamyl transpeptidase and by histological evaluation. A 3-day pretreatment with ADT (300 mg/kg/day) protected against the toxicity of various doses of HCBD (ranging from 15.6 to 62.5 mg/kg). The pretreatment increased (1.4-fold) the nonprotein sulfhydryl content (NPSH) of the liver. However, it did not modify the biliary excretion of radiolabeled materials in [14C]HCBD-treated (20 mg/kg) rats, nor that of the bioactivated HCBD metab- olite,S-(1,2,3,4,4-pentachloro-1,3-butadienyl)-glutathione (PCBG). Moreover, ADT pretreatment protected rats against the nephrotoxicity induced by PCBG (20 mg/kg) itself. The extent of covalent binding to kidney proteins of [14C]HCBD-derived metabolites was not modified by pretreatment with ADT. Incubation of rat kidney cortical slices in a medium containing 0.1 mMof the nephrotoxic glutathione (PCBG) or cysteine (PCBC,S-(1,2,3,4,4-pentachloro-1,3-butadienyl)-L-cysteine) conjugates of HCBD for 30 min resulted in a 75% reduction in the slice/medium ratio ofp-aminohipurate (PAH) compared to that seen in controls. When the cortical slices were incubated with ADT (30 min, 0.2 mM) prior to incubation with the nephrotoxic conjugates, the reduction was only 33%. Neither thein vitronor thein vivotreatments did modify the activity of renal cytosolic β-lyase; however, the latter treatment caused an increase in NPSH content. A 15-min incubation of kidney cortical slices with glutathione (10 mM) resulted in a 5-fold increase of NPSH, but failed to prevent the reduction in PAH uptake caused by PCBG and PCBC. Altogether, thein vivoand renal slice data suggest that ADT protects rats against HCBD-induced nephrotoxicity by a mechanism that does not involve the modulation of HCBD conjugation with liver GSH, nor the modulation of the kidney NPSH level and β-lyase activity. The mechanism of protection conferred to rats by an ADT pretreatment against HCBD-induced nephrotoxicity appears to take place in the kidney at a step beyond the generation of ultimate toxic metabolites derived from PCBC.

Renal function and organic anion and cation transport during dehydration and/or food restriction in chickens.

The effect of dehydration in the presence or absence of continued food intake on renal function was evaluated in chickens. In addition, renal transport of organic anions and cations under these conditions was assessed in vitro by uptake of 14C-para-aminohippuric acid and 14C-tetraethylammonium bromide by renal slices. Water restriction with continued food intake resulted in increases in serum osmolality and serum concentrations of sodium, uric acid, calcium and total protein. If food was restricted in addition to water, only serum osmolality and sodium concentration were significantly increased after 48 hours. Dehydration with continued access to food resulted in marked decreases in extracellular fluid volume, glomerular filtration rate and effective renal plasma flow. If food was restricted during dehydration, the decrease in effective renal plasma flow was attenuated despite reductions in glomerular filtration rate and extracellular fluid comparable to that seen in dehydrated birds allowed free access to food. Transport of organic anions was significantly increased after 24 and 48 hours of water restriction, regardless of whether food was withheld. Enhanced transport of organic anions in the presence of decreased glomerular filtration rate and effective renal plasma flow during dehydration may promote precipitation of urates and nephrosis in chickens.

Tonic inhibition of renin secretion by the 12 lipoxygenase pathway: augmentation by high salt intake.

Recent evidence suggests that lipoxygenase (LO) metabolites inhibit renin production in vitro. However, the physiological significance of this effect has not been determined. This study examined the role of the LO pathway in the regulation of plasma renin concentration (PRC) in vivo. The acute administration of two structurally unrelated LO inhibitors, phenidone (30 and 60 mg/kg) and esculetin (60 mg/kg), resulted in suppression of platelet 12 hydroxyeicosatetraenoic acid (12HETE) production, reduction in systemic arterial pressure and a 2- to 3-fold increase in PRC. To determine whether the esculetin-induced increase in PRC was secondary to hypotension, esculetin was also administered to rats preinfused with a pressor dose of norepinephrine. In these acutely hypertensive rats, esculetin still induced a 2.5-fold increase in PRC, whereas blood pressure remained over 40 mm Hg above basal levels. Further, esculetin (10(-6)M) increased renin release in renal slices from 150 +/- 10 to 310 +/- 20 ng/ml.h (P < 0.05) and this rise was entirely blocked in the presence of 12HETE (10(-7)M; 130 +/- 40 ng/ml.h). In rats placed on high salt intake, 12HETE concentration in renal slices from the outer cortex was considerably higher than in renal slices from salt-restricted rats (116.5 +/- 15.7 vs. 65 +/- 12 pg/mg protein; P < 0.05). Chronic administration of the LO inhibitor phenidone also resulted in an increase of PRC, which was independent of changes in blood pressure. On either high salt (3.15%0 or low salt (0.05%) diet phenidone-treated rats had higher PRC levels than the respective control groups [high salt 9.7 +/- 3.5 vs. 1.9 +/- 1.4 ng/ml.h; P < 0.05; low salt 33.2 +/- 5.3 vs. 19.4 +/- 3.10 ng/ml.h; P < 0.05]. The finding that LO blockers are potent stimulators of PRC in vivo suggests the existence of a physiological tonic inhibition of renin secretion by LO products that is operative under a wide range of salt intake. High salt intake enhances this inhibitory tone by increasing renal cortical 12 LO activity and, in fact, normal suppression of PRC during high salt diet does not occur in LO-blocked animals. Thus, the LO pathway exerts a tonic inhibitory effect on renin release, which appears particularly important for renin suppression during high salt intake.

Demethylation of 3-O-methyldopa in the kidney: a possible source for dopamine in urine

The possibility that demethylation of 3-O-methyldopa (OM-dopa) in the kidney could provide a source for dopamine in the urine was explored in male Wistar rats aged 60-90 days, using in vivo and in vitro approaches. The results showed that endogenous OM-dopa is filtered, reabsorbed and extensively metabolized in the kidney. Infusion of OM-dopa into anesthetized rats increased significantly urinary excretion of Na+, dopa, dopamine, and 3,4 dihydroxyphenylacetic acid. Whole kidney homogenates, slices from renal cortex, and microdissected proximal tubules produced significant amounts of both dopa and dopamine when incubated with OM-dopa. Renal cortex slices produced dose-dependent amounts of dopa and dopa-mine when incubated with 1-100 microM OM-dopa. Incubation of microdissected proximal tubule segments with 1 microM OM-dopa produced a fourfold (P < 0.025) increment in dopa and a twofold (P < 0.05) increment in dopamine (an effect similar to that observed with 1 microM L-dopa). One micromolar OM-dopa or 1 microM L-dopa decreased (P < 0.05) Na(+)-K(+)-adenosinetriphosphatase activity measured at maximal velocity condition in proximal tubules. In conclusion, these experiments show that in vitro the kidney is able to produce dopamine by demethylation of OM-dopa, while the results of the OM-dopa infusion suggest that this conversion may also occur in vivo.

Protein Arylation Precedes Acetaminophen Toxicity in a Dynamic Organ Slice Culture of Mouse Kidney

Acetaminophen (APAP) is an analgesic and antipyretic agent which may cause hepatotoxicity and nephrotoxicity with overdose in man and laboratory animals. In vivo studies suggest that in situ activation of APAP contributes to the development of nephrotoxicity. Associated with target organ toxicity is selective arylation of proteins, with a 58-kDa acetaminophen binding protein (58-ABP) being the most prominent cytosolic target. In this study a mouse kidney slice model was developed to further evaluate the contribution of in situ activation of APAP to the development of nephrotoxicity and to determine the selectivity of protein arylation. Precision cut kidney slices from male CD-1 mice were incubated with selected concentrations of APAP (0–25 mM) for 2 to 24 hr. APAP caused a dose- and time-dependent decrease in nonprotein sulfhy-dryls (NPSH), ATP content, and K+ retention. Preceding toxicity was arylation of cytosolic proteins, the most prominent one being the 58-ABP. The association of 58-ABP arylation with APAP toxicity in this mouse kidney slice model is consistent with earlier, in vivo results and demonstrates the importance of in situ activation of APAP for the development of nephrotoxicity. Precision cut renal slices and dynamic organ culture are a good model for further mechanistic studies of APAP-induced renal toxicity.

The Promoting Action of Magnesium Lithospermate B on the Kinin‐Prostaglandin E2 System in the Kidney

The effect of magnesium lithospermate B purified from Salviae Miltiorrhizae Radix on the kinin-prostaglandin E2 system was investigated using kidney slices and isolated kidney microsomes. Magnesium lithospermate B markedly increased the amount of thiobarbituric acid-reactive substances produced by incubation of arachidonic acid in renal slices. Enhancement of arachidonic acid-mediated oxygenation of 1,3-diphenylisobenzofuran was also observed in renal microsomes from rats treated with magnesium lithospermate B, indicating an increase of prostaglandin biosynthesis. Furthermore, magnesium lithospermate B significantly increased the synthesis of prostaglandin E2 in renal slices, but the magnesium lithospermate B-mediated response was blunted with kinin antagonist. On the basis of the above results, it is apparent that magnesium lithospermate B exerts an influence on the prostaglandin system via kinin receptors.

Comparison of the in vitro toxicity of dichloroaniline structural isomers

Acute exposure to certain dichloroaniline (DCA) isomers results in renal and hepatic toxicity in vivo. In the present study we examined whether dichloroaniline structural isomers were cytotoxic to liver and kidney slices in vitro and compared the toxicities of the different structural isomers. These studies were necessary in order to validate the use of an in vitro slice system for examination of the cellular mechanisms for toxicity. Renal cortical and hepatic slices were incubated for 90 min with 2,3-DCA, 2,4-DCA, 2,5-DCA, 2,6-DCA, 3,4-DCA or 3,5-DCA at a final concentration of 0–1 m m. Pyruvate-directed gluconeogenesis was measured following an additional 30-min incubation with 10 m M pyruvate. Cytotoxicity was also determined by measurement of lactate dehydrogenase (LDH) release 120 min after the addition of dichloroaniline isomers at a final concentration of 0, 0.5, 1 or 2 m M. Gluconeogenesis in renal cortical slices was inhibited by all of the isomers beginning at a concentration of 0.5 m M. Renal slice LDH leakage was elevated above control levels by 1–2 m M 3,4-DCA or 3,5-DCA. A final concentration of 2 m M was needed for 2,3-DCA, 2,4-DCA, 2,5-DCA or 2,6-DCA in order to detect a significant ( P < 0.05) increase in renal slice LDH leakage. Hepatic slices incubated with 0.5–2 m M 2,3-DCA or 2 m M 2,5-DCA exhibited diminished pyruvate-directed gluconeogenesis. After exposure to 2,4-DCA, 2,6-DCA, 3,4-DCA or 3,5-DCA, pyruvate-directed gluconeogenesis was similar to that in the controls. LDH leakage was increased significantly ( P < 0.05) above control values by exposure to 2 m M 3,4-DCA or 3,5-DCA. In conclusion, DCA structural isomers were toxic in vitro to liver and kidney slices. These results indicated that the kidney was more sensitive than the liver to DCA isomers, and that the most toxic isomer was 3,5-DCA. These results are similar to those previously observed in vivo.

Comparison of ?1A- and ?1B-adrenoceptor coupling to inositol phosphate formation in rat kidney

We have compared the coupling mechanisms of rat renal alpha 1A- and alpha 1B-like adrenoceptors to inositol phosphate formation. The experiments were performed in parallel in native renal tissue preparations and in those where alpha 1B-adrenoceptors had been inactivated by treatment with 10 mumol/l chloroethylclonidine for 30 min at 37 degrees C; renal slices were used in most experiments but isolated renal cells were also used in some cases. The Ca2+ chelating agent, EGTA (5 mmol/l), reduced noradrenaline-stimulated inositol phosphate formation in native but enhanced it in chloroethylclonidine-treated renal slices. The inhibitory effect of EGTA was not mimicked by 100 nmol/l nifedipine. Inactivation of 87% of cellular Gi by 16-20 h treatment with 500 ng/ml pertussis toxin did not significantly affect noradrenaline-stimulated inositol phosphate formation in isolated renal cells but abolished the inhibitory effect of chloroethylclonidine. The adenylate cyclase activator, forskolin (20 mumol/l), inhibited noradrenaline-stimulated inositol phosphate formation in native and chloroethylclonidine-treated slices, and the inhibitory effects of chloroethylclonidine treatment and forskolin were additive. We conclude that in rat kidney inositol phosphate formation via alpha 1B-like adrenoceptors may involve the influx of extracellular Ca2+ and a pertussis toxin-sensitive G-protein but is insensitive to inhibition by forskolin. In contrast alpha 1A-like adrenoceptor-mediated inositol phosphate formation does not require the presence of extracellular Ca2+ or of Gi and is sensitive to inhibition by forskolin. In comparison to published data from other model systems we further conclude that the signaling mechanisms of alpha 1-adrenoceptor subtypes may depend on their cellular environment.