Isolated Rat Kidney Research Articles

Protein localization of SLC26A2 (DTDST) in rat kidney

The SLC26 family represents a group of integral membrane anion transport proteins. Mutations in one member of this protein family, SLC26A2 (DTDST or diastrophic dysplasia sulfate transporter), result in various chondrodysplasias due to undersulfation of proteoglycans in chondrocytes, a major site of DTDST protein expression. DTDST mRNA has been detected in the kidney, but protein expression has not been characterized. Our objective for this study was to determine the protein localization of this sulfate transporter in the kidney. We used immunofluorescence (IMF) techniques with an anti-DTDST monoclonal antibody to examine kidneys harvested from adult rats. Double labeling was performed with antibodies directed against megalin, which is found in the microvillus membrane and coated pits of the proximal tubule. IMF analysis indicated that DTDST protein expression was limited to the microvillus membrane of proximal tubule cells in the renal cortex but absent in glomeruli and other nephron segments. DTDST was also detected in isolated rat kidney proximal tubule microvillus membranes by Western blot analysis, confirming the immunofluorescent localization of the DTDST transporter to this nephron segment. The functional role of the DTDST protein in the kidney is unknown, but it may play a role in proximal tubule sulfate transport.

MitoQ protects against cold ischemic injury in renal cells and rat kidneys

Cold preservation is a beneficial and necessary procedure used to preserve deceased donor kidneys prior to transplantation. However, extended preservation periods result in injury that can lead to poor graft function following transplantation. Thus, this study's purpose was to evaluate the early effects of cold storage (CS) alone and to determine if MitoQ, a mitochondrial targeted antioxidant, could offer protection during this stage. We hypothesize that CS increases oxidant production and mitochondrial dysfunction, and that adding MitoQ to cold preservation solutions will attenuate injury. Renal cells and rat kidneys were exposed to CS (UW‐Viaspan solution; 4°C) with or without MitoQ (1μM; 100μM respectively) and monitored for oxidants (O2·−, NO, ONOO−), mitochondrial function (respiratory complexes, mitochondrial membrane potential), and cell viability. Our cell model results suggest that CS first causes mitochondrial membrane depolarization, followed by oxidant production, then mitochondrial dysfunction, and modest cell death. MitoQ reduced oxidant production and protected against mitochondrial complex inactivation using both cellular and isolated rat kidney models. Excitingly, we have identified a potential therapeutic reagent that could be added to existing preservation solutions to reduce CS damage and possibly improve renal function following transplantation.

Novel polymyxin derivatives are effective in treating experimental Escherichia coli peritoneal infection in mice

Novel synthetic polymyxin derivatives including NAB737 and NAB739 are as effective as polymyxin B in vitro against the common opportunistic pathogen Escherichia coli. Another derivative, NAB7061, lacks direct antibacterial action but sensitizes E. coli to several other antibacterial agents including macrolides. The renal handling of NAB739 and NAB7061 in rats differs from that of polymyxin B. Furthermore, the affinities of NAB739 and NAB7061 for isolated rat kidney brush border membrane are significantly lower than that of polymyxin B. Here we investigate the in vivo antibacterial effect of these compounds. The polymyxin derivatives were evaluated in an experimental murine peritonitis model. Immunocompetent mice were infected intraperitoneally with E. coli IH3080 and were subcutaneously treated with NAB737, NAB739 or NAB7061. A >4.0 log(10) reduction in bacterial load compared with saline control was achieved 6 h after initiation of treatment with 1 mg/kg of NAB739 twice or 4 mg/kg of NAB737 twice. Combination therapy with NAB7061 (5 mg/kg) twice and erythromycin (10 mg/kg) resulted during the same time course in a >2.0 log(10) reduction in bacterial load compared with saline control. Neither NAB7061 nor erythromycin was effective as monotherapy. Together with the ability to reduce bacterial load, the NAB compounds also improved the clinical status of the mice. We found that the three novel synthetic polymyxin B derivatives had a potent in vivo bactericidal effect against E. coli.

Effect of Amlodipine in Comparison to Nifedipine on Vascular Perfusion Pressure of Isolated Rat Kidney

Objective(s) This study aimed to investigate and to compare the effects of nifedipine and amlodipine, dihydropyridine (DHP) calcium channel blockers (CCBs) on perfusion pressure of isolated perfused rat kidney. Materials and Methods Following the establishment of renal perfusion with a constant baseline pressure of 85-95 mmHg, the renal vasculature was constricted by phenylephrine (PE) injection. Changes in the baseline perfusion pressure were recorded. Then nifedipine and amlodipine prepared in perfusion medium was fed to the kidney for 30 min. Finally alterations in the baseline pressure arising from PE administrations in the presence of CCBs were recorded and data analyses were done. Results PE-induced increases in perfusion pressure attenuated significantly in the presence of 5 and 10 µM of nifedipine and 1, 5, and 10 µM of amlodipine. Increases in perfusion pressure arising from PE (100 and 200 µM) in the presence of amlodipine (1, 5, and 10 µM) was significantly less than that in the presence of nifedipine (1, 5, and 10 µM). Calculated EC50 value of amlodipine for inhibition was significantly lower than that of nifedipine. Based on the EC50 values, the potency of amlodipine in inhibiting PE-induced responses is significantly higher compared to nifedipine. Conclusion

Extracellular 2′,3′-cAMP Is a Source of Adenosine

We discovered that renal injury releases 2',3'-cAMP (positional isomer of 3',5'-cAMP) into the interstitium. This finding motivated a novel hypothesis: renal injury leads to activation of an extracellular 2',3'-cAMP-adenosine pathway (i.e. metabolism of extracellular 2',3'-cAMP to 3'-AMP and 2'-AMP, which are metabolized to adenosine, a retaliatory metabolite). In isolated rat kidneys, arterial infusions of 2',3'-cAMP (30 mumol/liter) increased the mean venous secretion of 3'-AMP (3,400-fold), 2'-AMP (26,000-fold), adenosine (53-fold), and inosine (adenosine metabolite, 30-fold). Renal injury with metabolic inhibitors increased the mean secretion of 2',3'-cAMP (29-fold), 3'-AMP (16-fold), 2'-AMP (10-fold), adenosine (4.2-fold), and inosine (6.1-fold) while slightly increasing 5'-AMP (2.4-fold). Arterial infusions of 2'-AMP and 3'-AMP increased secretion of adenosine and inosine similar to that achieved by 5'-AMP. Renal artery infusions of 2',3'-cAMP in vivo increased urinary excretion of 2'-AMP, 3'-AMP and adenosine, and infusions of 2'-AMP and 3'-AMP increased urinary excretion of adenosine as efficiently as 5'-AMP. The implications are that 1) in intact organs, 2'-AMP and 3'-AMP are converted to adenosine as efficiently as 5'-AMP (previously considered the most important adenosine precursor) and 2) because 2',3'-cAMP opens mitochondrial permeability transition pores, a pro-apoptotic/pro-necrotic process, conversion of 2',3'-cAMP to adenosine by the extracellular 2',3'-cAMP-adenosine pathway would protect tissues by reducing a pro-death factor (2',3'-cAMP) while increasing a retaliatory metabolite (adenosine).

Activation of glutamate dehydrogenase increases renal ammoniagenesis: implications for the HI/HA syndrome

Children with gain‐of‐function mutations in Glutamate Dehydrogenase (GDH) display hyperinsulinemia (HI) and hyperammonemia (HA). The HI has been clearly linked to an effect of increased GDH flux on insulin secretion. However, the origin of the HA is uncertain, particularly because hepatic GDH is considered to be close to thermodynamic equilibrium in vivo. We found increased ammonia release into the renal vein of rats infused with β‐2‐aminobicyclo[2.2.1]heptane‐2‐carboxylic acid (BCH), an activator of GDH. BCH infusion had no effect on the hepatic handling of ammonia as portal venous ammonia was effectively removed by the liver. BCH also caused increased ammonia production by isolated rat proximal tubules incubated with glutamine. BCH also increased ammonia production and flux through GDH in isolated rat kidney mitochondria incubated with glutamine. We conclude that activation of renal GDH may be responsible, at least in part, for the HA of the HI/HA Syndrome. (Supported by IGTC and CIHR).

Antivenom action on renal effects induced by Thalassophryne nattereri venom

Thalassophryne nattereri (niquim) is a venomous fish responsible for numerous accidents involving fishermen in northern and northeastern Brazil. The aim of the present investigation was to evaluate the action of antivenom on renal effects caused by Thalassophryne nattereri venom. Isolated kidneys of Wistar rats were perfused with a previously dialyzed Krebs-Henseleit solution containing 6 g% bovine serum albumin. The antivenom action was studied through perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). The niquim venom (1 µg/mL), the antivenom alone (1 µg/mL) or the venom incubated with antivenom were added to the system 30 minutes after the beginning of each perfusion. Previous works have shown venom induced-alterations of renal function parameters. In the isolated rat kidney, T. nattereri venom (1 µg/mL) increased the perfusion pressure and renal vascular resistance at 60, 90 and 120 minutes. UF and GFR also increased at 60, 90 and 120 minutes when compared with the control group; however, no effects were observed on the percent of sodium (%TNa+control = 81.1 ±0.86; %TNa+60 = 78.04 ±1.18; %TNa+90 = 76.16 ±3.34; %TNa+120 = 79.49 ±0.87) and potassium (%TK+control = 72.29 ±1.12; %TK+60 = 75.41 ±0.65; %TK+90 = 71.23 ±2.55; %TK+120 = 76.62 ±1.04) tubular transport. The administration of the antivenom (1 µg/mL) incubated with venom (1 µg/mL) reduced the changes in PP, RVR, UF and GFR provoked by Thalassophryne nattereri venom. The group perfused with venom alone showed a moderate deposit of a proteinaceous material in the tubules and urinary space. The group perfused with the antivenom presented similar results to the control group. In conclusion, the antivenom was able to decrease the effects induced by T. nattereri venom in isolated rat kidney.

Effect of the Two New Calcium Channel Blockers Mebudipine and Dibudipine in Comparison to Nifedipine on Vascular Flow of Isolated Rat Kidney

In previous researches mebudipine and dibudipine two newly synthesized calcium channel blockers (CCBs) showed considerable relaxant effects on vascular and atrial smooth muscle cells. In this study we investigated the effects of these new drugs on vascular flow of isolated rat kidney and compare their potencies to nifedipine. It is concluded that mebudipine and dibudipine (5 μM, 10 μM) caused concentration-dependent inhibition of increases in perfusion pressure induced by phenylephrine. Mebudipine (10 μM) inhibited more greatly increases in perfusion pressure induced by phenylephrine compared to (10 μM) nifedipine.

Identification of Novel Endogenous Cytochrome P450 Arachidonate Metabolites with High Affinity for Cannabinoid Receptors

Arachidonic acid is an essential constituent of cell membranes that is esterified to the sn-2-position of glycerophospholipids and is released from selected lipid pools by phospholipase cleavage. The released arachidonic acid can be metabolized by three enzymatic pathways: the cyclooxygenase pathway forming prostaglandins and thromboxanes, the lipoxygenase pathway generating leukotrienes and lipoxins, and the cytochrome P450 (cP450) pathway producing epoxyeicosatrienoic acids and hydroxyeicosatetraenoic acids. The present study describes a novel group of cP450 epoxygenase-dependent metabolites of arachidonic acid, termed 2-epoxyeicosatrienoylglycerols (2-EG), including two regioisomers, 2-(11,12-epoxyeicosatrienoyl)glycerol (2-11,12-EG) and 2-(14,15-epoxyeicosatrienoyl)glycerol (2-14,15-EG), which are both produced in the kidney and spleen, whereas 2-11,12-EG is also detected in the brain. Both 2-11,12-EG and 2-14,15-EG activated the two cannabinoid (CB) receptor subtypes, CB1 and CB2, with high affinity and elicited biological responses in cultured cells expressing CB receptors and in intact animals. In contrast, the parental arachidonic acid and epoxyeicosatrienoic acids failed to activate CB1 or CB2 receptors. Thus, these cP450 epoxygenase-dependent metabolites are a novel class of endogenously produced, biologically active lipid mediators with the characteristics of endocannabinoids. This is the first evidence of a cytochrome P450-dependent arachidonate metabolite that can activate G-protein-coupled cell membrane receptors and suggests a functional link between the cytochrome P450 enzyme system and the endocannabinoid system.

Development and validation of a direct enantiomeric separation of pregabalin to support isolated perfused rat kidney studies

Pregabalin (Lyrica™) is the first compound approved to treat the neural pain associated with fibromyalgia. Pregabalin is the S-enantiomer of a γ-amino acid analogue and chiral separation from its R-enantiomer must be achieved to support metabolic studies. The direct chiral separation of pregabalin from its R-enantiomer has been developed and HPLC/MS/MS assays have been validated to support isolated perfused rat kidney studies. The separation was developed through serial coupling of various macrocyclic glycopeptide stationary phases until partial separation of the enantiomers was achieved. Identification of the resolving stationary phase followed by optimization of the mobile phase enabled the baseline resolution of the enantiomers using mass spectrometry compatible solvents and modifiers. Assays were developed and validated for quantitation of the enantiomers from rat urine, isolated rat kidney perfusate, and isolated rat kidney perfusate ultrafiltrate to support pregabalin metabolic studies.

Novel Polymyxin Derivatives Carrying Only Three Positive Charges Are Effective Antibacterial Agents

The lack of novel antibiotics against gram-negative bacteria has reinstated polymyxins as the drugs of last resort to treat serious infections caused by extremely multiresistant gram-negative organisms. However, polymyxins are nephrotoxic, and this feature may complicate therapy or even require its discontinuation. Like that of aminoglycosides, the nephrotoxicity of polymyxins might be related to the highly cationic nature of the molecule. Colistin and polymyxin B carry five positive charges. Here we show that novel polymyxin derivatives carrying only three positive charges are effective antibacterial agents. NAB739 has a cyclic peptide portion identical to that of polymyxin B, but in the linear portion of the peptide, it carries the threonyl-D-serinyl residue (no cationic charges) instead of the diaminobutyryl-threonyl-diaminobutyryl residue (two cationic charges). The MICs of NAB739 for 17 strains of Escherichia coli were identical, or very close, to those of polymyxin B. Furthermore, NAB739 was effective against other polymyxin-susceptible strains of Enterobacteriaceae and against Acinetobacter baumannii. At subinhibitory concentrations, it dramatically sensitized A. baumannii to low concentrations of antibiotics such as rifampin, clarithromycin, vancomycin, fusidic acid, and meropenem. NAB739 methanesulfonate was a prodrug analogous to colistin methanesulfonate. NAB740 was the most active derivative against Pseudomonas aeruginosa. NAB7061 (linear portion of the peptide, threonyl-aminobutyryl) lacked direct antibacterial activity but sensitized the targets to hydrophobic antibiotics by factors up to 2,000. The affinities of the NAB compounds for isolated rat kidney brush border membrane were significantly lower than that of polymyxin B.

A possible mechanism for the hypotonicity-induced change in [Ca2+]i and BK channel activity in isolated rat kidney CCD

A possible mechanism for the hypotonicity-induced change in [Ca2+]i and BK channel activity in isolated rat kidney CCD

Adenosine A2B receptor mediates an increase on VEGF-A production in rat kidney glomeruli

Up-regulation of the glomerular expression and the activity of vascular endothelial growth factor-A (VEGF) have been identified as an early pathogenic event for the progression of diabetic nephropathy. Currently, however the mediators are not yet clearly recognized. In this study we identified all four adenosine receptor (AR) subtypes, i.e. A1, A2A, A2B and A3 in isolated rat kidney glomeruli. We localized the expression of A2BAR in podocytes, the primary VEGF producing cells. The ex vivo treatment of kidney glomeruli with adenosine or a general AR agonist NECA, increases VEGF protein content. In addition, NECA treatment elicits VEGF release. These effects were blocked by the A2BAR selective antagonist MRS1754 supplementation. Furthermore, we showed that A2BAR activation was necessary to promote a higher expression of VEGF in kidney glomeruli upon exposure to high d-glucose concentration, a pathogenic condition like those observed in diabetic nephropathy.

The renal effects of alginates isolated from brown seaweed Sargassum vulgare.

Alginates isolated from Sargassum vulgare, present a strong antitumor activity, associated with kidney reversible damage, as analysed by histopathology of treated animals. In the present study, the renal alteration mechanisms of S. vulgare alginates were investigated using the isolated perfused rat kidney and the isolated perfused rat mesenteric blood vessel methods. The results showed that the effects of Sargassum vulgare low viscosity (SVLV) alginate were more potent than those of Sargassum vulgare high viscosity (SVHV) alginate in the isolated rat kidney. The SVLV alginate caused considerable changes in renal physiology, as shown by an increase in parameters such as perfusion pressure, renal vascular resistance, glomerular filtration rate, urinary flow and sodium, potassium and chloride excretion and by reduction of chloride tubular transport. The effects of SVHV were weaker than those of SVLV. The effects of SVLV on kidney could be related to direct vascular action as demonstrated with SVLV alginate on mesenteric blood vessels. In conclusion, the Sargassum vulgare alginate altered the renal function parameters evaluated. S. vulgare low viscosity alginate renal effects were more potent than S. vulgare high viscosity alginate. It is suggested that physicochemical differences between SVHV and SVLV could explain the differences found in the results.

Evaluation of preservation solutions by ESR-spectroscopy: Superior effects of University of Wisconsin over Histidine–Tryptophan–Ketoglutarate in reducing renal reactive oxygen species

Despite the causative role of oxidative stress in renal ischemia-reperfusion (I-R) injury effects of preservation solutions on reactive oxygen species (ROS) release have not been sufficiently evaluated. We compared the effects of most common solutions in kidney transplantation, University of Wisconsin (UW) and Histidine-Tryptophan-Ketoglutarate (HTK). ROS formation in isolated perfused rat kidney was detected by electron spin resonance spectroscopy using spin label 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine. Donor kidneys from Lewis rats were pretreated with saline (controls), in therapeutic groups, kidneys underwent 18 h of cold storage (CS) preserved by HTK or UW solution. Experimental protocol included a stabilization period followed by additional I-R. Kidneys preserved by HTK produced highest ROS values in the control period after CS, whereas levels in UW and control group did not vary significantly. A peak release induced by additional I-R was also significantly highest in HTK kidneys, and UW did not differ from controls. During reperfusion, levels in HTK exceeded control and UW values. Renal vascular resistance, caspase-3-activity, and tissue hydration were enhanced in HTK compared with UW group, whereas ATP concentration was less reduced in UW-preserved tissue. These data show the greater antioxidative potential of UW solution, which also attenuated organ impairment after CS in the early reperfusion period.

Protein localization of SLC26A2 (DTDST) in rat kidneys

The SLC26 family represents a group of integral membrane anion transport proteins. Mutations in one member of this protein family, SLC26A2 (DTDST or diastrophic dysplasia sulfate transporter), result in a range of chondrodysplasias due to undersulfation of proteoglycans in chondrocytes, a major site of DTDST protein expression. DTDST mRNA had previously been detected in the kidney, but protein expression had not been characterized. Our objective for this study was to determine the protein localization of this sulfate transporter in the kidney. We used immunofluorescence (IMF) techniques with an anti-DTDST monoclonal antibody to examine kidneys harvested from adult rats. Double labeling was also performed with antibodies directed against megalin, which is found in the brush border membrane and coated pits of the proximal tubule. IMF analysis indicates that DTDST protein expression is limited to the brush border membrane of proximal tubule cells in the renal cortex. DTDST expression was not detected in glomeruli or other nephron segments. DTDST was also detected in isolated rat kidney proximal tubule microvillus membranes by Western blot analysis, confirming the immunofluorescent localization of the DTDST transporter to this nephron segment. The functional role of the DTDST protein in the kidney is unknown, but it may play a role in proximal tubule sulfate transport.

Purification and biological activity of the thrombin-like substance isolated from Bothrops insularis venom

The venom of Bothrops insularis snake, known in Brazil as jararaca ilhoa, contains a variety of proteolytic enzymes such as a thrombin-like substance that is responsible for various pharmacological effects. B. insularis venom chromatography profile showed an elution of seven main fractions. The thrombin-like activity was detected in fractions I and III, the latter being subjected to two other chromatographic procedures, so to say DEAE and Hi Trap Benzamidine. The purity degree of this fraction was confirmed by analytical reverse phase HPLC, which displayed only one main fraction confirmed by SDS-PAGE constituting fraction III. About 5 μg of fraction III protein potentiated the secretion of insulin induced by 2.8 mM of glucose in rats isolated pancreatic β-cells treated; the increase being around 3-fold higher than its respective control. B. insularis lectin (BiLec; 10 μg/mL) was also studied as to its effect on the renal function of isolated perfused rat kidneys with the use of six Wistar rats. BiLec increased perfusion pressure (PP), renal vascular resistence (RVR), urinary flow (UF) and glomerular filtration rate (GFR). Sodium (%TNa +) and chloride tubular reabsorption (%TCl −) decreased at 120 min, without alteration in potassium transport. In conclusion, the thrombin-like substance isolated from B. insularis venom induced an increase in insulin secretion, in vitro, and transiently altered vascular, glomerular and tubular parameters in the isolated rat kidney.

Action of diclofenac on kidney mitochondria and cells

The mitochondrial membrane potential measured in isolated rat kidney mitochondria and in digitonin-permeabilized MDCK type II cells pre-energized with succinate, glutamate, and/or malate was reduced by micromolar diclofenac dose-dependently. However, ATP biosynthesis from glutamate/malate was significantly more compromised compared to that from succinate. Inhibition of the malate–aspartate shuttle by diclofenac with a resultant decrease in the ability of mitochondria to generate NAD(P)H was demonstrated. Diclofenac however had no effect on the activities of NADH dehydrogenase, glutamate dehydrogenase, and malate dehydrogenase. In conclusion, decreased NAD(P)H production due to an inhibition of the entry of malate and glutamate via the malate–aspartate shuttle explained the more pronounced decreased rate of ATP biosynthesis from glutamate and malate by diclofenac. This drug, therefore affects the bioavailability of two major respiratory complex I substrates which would normally contribute substantially to supplying the reducing equivalents for mitochondrial electron transport for generation of ATP in the renal cell.

Effects of Crotalus durissus collilineatus venom in the isolated rat kidney

Ophidian accidents caused by the subspecies Crotalus durissus are responsible for high morbity and mortality rates. Acute renal failure is a common complication observed in these accidents. The aim of the present study was to investigate the renal effects promoted by the venom of C. d. collilineatus and its fractions, crotoxin and phospholipase A 2. C. d. collilineatus (Cdc; 30 μg mL −1), crotoxin (CTX; 10 μg mL −1) and phospholipase A 2 (PLA 2; 10 μg mL −1) were tested in isolated rat kidney. The first 30 min of each experiment were used as an internal control and Cdc or its fractions, CTX and PLA 2 were added to the system after this period. All experiments lasted 120 min. The venom of Cdc decreased perfusion pressure (PP; control 120=110.3±3.69 mmHg; Cdc 120=96.7±8.1 mmHg), renal vascular resistance (RVR; control 120=6.42±0.78 mmHg mL g −1 min −1; Cdc 120=4.8±0.56 mmHg/mL g −1 min −1), urinary flow (UF; control 120=0.19±0.03 mL g −1 min −1; Cdc 120=0.12±0.01 mL g −1 min −1), and glomerular filtration rate (GFR; control 120=0.79±0.07 mL g −1 min −1; Cdc 120=0.53±0.09 mL g −1 min −1), but had no effect on the percent of sodium tubular transport (%TNa +), percent of chloride tubular transport (%TK +) and percent of potassium tubular transport (%TCl −). CTX and PLA 2 reduced the GFR, while UF, PP and RVR remained stable during the full 120 min of perfusion. Crotoxin administration also diminished the %TK + (control 120=69.94±6.49; CTX 120=33.28±4.78) and %TCl − (control 120=79.53±2.67; CTX 120=64.62±6.93). PLA 2 reduced the %TK +, but exerted no effect on the %TNa + or on that of TCl −. In conclusion, the C. d. collilineatus venom altered the renal functional parameters evaluated. We suggest that crotoxin and phospholipase A 2 were involved in this process, since the renal effects observed would be due to the synergistic action of the components of the venom.

Effects of co‐supplementation of vitamins E and C on gentamicin‐induced nephrotoxicity in rat

Gentamicin (GM) is an effective antibiotic against severe gram-negative infections. However it can produce nephrotoxicity in human. Reactive oxygen species (ROS) have been proposed as the causative factors of the renal side effects the drug. This study was performed to investigate the protective role of antioxidant vitamins against GM-mediated nephropathy in an in situ model of isolated rat kidney. Male Sprague-Dawley rats were randomly assigned to one of the following groups of seven rats: group 1 (Control) was perfused with Tyrode solution; group 2 (GM), 200 microg ml(-1) GM was added to the perfusate; group 3 (GM + Vit C), as group 2 with vitamin C added to the drinking water for 3 days (200 mg l(-1)) and to the perfusate (100 mg l(-1)); group 4 (GM + Vit E), as group 2 with vitamin E (100 mg (100 g body weight)(-1), i.m.) injected 12 h before the start of the experiment; group 5 (GM + Vit C + Vit E) as group 2 with vitamin E and C co-administered (concentrations and conditions as in groups 3 and 4). To compare the groups, urinary lactate dehydrogenase (LDH), N-acetyle-beta-D-glucosaminidase (NAG) and alkaline phosphatase (ALP) activities, inulin clearance (glomerular filtration rate, GFR) and renal tissue glutathione (GSH) content were measured. GM caused a significant nephrotoxicity demonstrated by an increase in urinary LDH, NAG and ALP activities. Reduction in GSH content and a marked decrease in GFR were observed compared to controls. Vitamin C inhibited the GM-induced increase in urinary enzyme activities but did not show a significant effect on the GSH content or GFR. Vitamin E prevented the GM-induced reduction in GSH level without a significant improvement in GFR. Co-administration of vitamins C and E significantly prevented the GM-induced nephrotoxicity demonstrating by preservation of GFR and GSH levels and prevention of increase in urinary enzyme activities. We conclude that co-administration of moderate doses of vitamins C and E has beneficial effects on renal preservation in GM-induced nephrotoxicity.