Isolated Kidney Cells Research Articles

Role of Cytochrome P450s in 2,4,5‐Trichlorophenol Nephrotoxicity in Isolated Kidney Cells from Male Fischer 344 Rats

Trichlorophenols (TCPs) are used as chemical intermediates in the manufacture of a wide range of agricultural and industrial products. Previous studies have shown that 2,4,5‐TCP is the most potent nephrotoxicant in vitro of the six TCP isomers in isolated kidney cells (IKCs) from male Fischer 344 rats. It was also determined that 2,4,5‐TCP nephrotoxicity was attenuated by piperonyl butoxide, a non‐specific cytochrome P450 (CYP) inhibitor. The purpose of this study was to determine which CYPs might play a role in 2,4,5‐TCP nephrotoxicity in vitro in IKCs from male Fischer 344 rats. IKCs (~4 million/ml, 3 ml) were incubated with a selective CYP inhibitor or vehicle prior to addition of 2,4,5‐TCP (0.5 mM) or dimethyl sulfoxide (DMSO, 2,4,5‐TCP vehicle) for 60 min with shaking at 37oC under a 95% oxygen/5% carbon dioxide atmosphere. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release at the end of the incubation period. 2,4,5‐TCP‐induced cytotoxicity was completely attenuated by CYP inhibitors sulfaphenazole (CYP2C11) and thiotepa (CYP2B6). 2,4,5‐TCP cytotoxicity was also reduced by pretreatment with omeprazole (CYP2C19), DEDTCA (CYP2C/E), or isoniazid (CYP2E1). The least effective CYP inhibitor at reducing 2,4,5‐TCP cytotoxicity was oleandomycin (CYP3A). These results suggest that CYPs play a role in 2,4,5‐TCP nephrotoxicity in vitro with CYP2C11 and CYP2B6 being major contributors to promoting 2,4,5‐TCP cytotoxicity. The mechanism by which these and other CYPs promote 2,4,5‐TCP nephrotoxicity remains to be determined.

Nephrotoxic Potential of the Three Dichlorobenzene Isomers in Isolated Kidney Cells from Male Fischer 344 Rats

Dichlorobenzenes (DCBs) are commonly used as chemical intermediates in the manufacture of a wide range of agricultural, industrial and pharmaceutical products. Previous studies have shown that 1,4-DCB can induce nephrotoxicity in male rats characterized as α2u-globulin nephropathy. Initially, 1,4-DCB binds to α2u-globulin at extra-renal sites. The 1,4-DCB-protein complex then accumulates in kidney and causes protein droplet formation, ultimately leading to cell death and/or eventually tumor formation. The purpose of this study was to explore the direct cytotoxic effects of the three DCB isomers in isolated kidney cells (IKCs) from male Fischer 344 rats. IKCs (~4 million/ml, 3 ml) were incubated with a DCB (0.25-1.0 mM) or dimethyl sulfoxide (DMSO, vehicle) for 30 or 60 min with shaking at 37oC under a 95% oxygen/5% carbon dioxide atmosphere. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release. In separate experiments, IKCs were pretreated with an antioxidant (ascorbate, 1.0 mM; N-acetyl-L-cysteine, 2.0 mM; glutathione, 1.0 mM) prior to the addition of 1,4-DCB (0.5 mM) and followed by a 60 min co-incubation. 1,4-DCB increased LDH release at all times and concentrations tested. 1,3-DCB increased LDH release at 0.5 mM or greater at both time points, while 1,2-DCB only increased LDH release at 0.5 mM or greater at 60 min. Thus, the order of decreasing nephrotoxic potential was 1,4-DCB > 1,3- DCB > 1,2-DCB. Pretreatment with any of the antioxidants tested attenuated 1,4-DCB nephrotoxicity. These results indicate that DCBs can induce nephrotoxicity in vitro without the presence of extra-renal α2u-globulin, 1,4-DCB is the most potent nephrotoxicant among the three DCB isomers in IKCs, and free radicals play a role in DCB nephrotoxicity.

Nephrotoxic Potential of Putative 3,5-Dichloroaniline (3,5-DCA) Metabolites and Biotransformation of 3,5-DCA in Isolated Kidney Cells from Fischer 344 Rats.

The current study was designed to explore the in vitro nephrotoxic potential of four 3,5-dichloroaniline (3,5-DCA) metabolites (3,5-dichloroacetanilide, 3,5-DCAA; 3,5-dichlorophenylhydroxylamine, 3,5-DCPHA; 2-amino-4,6-dichlorophenol, 2-A-4,6-DCP; 3,5-dichloronitrobenzene, 3,5-DCNB) and to determine the renal metabolism of 3,5-DCA in vitro. In cytotoxicity testing, isolated kidney cells (IKC) from male Fischer 344 rats (~4 million/mL, 3 mL) were exposed to a metabolite (0–1.5 mM; up to 90 min) or vehicle. Of these metabolites, 3,5-DCPHA was the most potent nephrotoxicant, with 3,5-DCNB intermediate in nephrotoxic potential. 2-A-4,6-DCP and 3,5-DCAA were not cytotoxic. In separate experiments, 3,5-DCNB cytotoxicity was reduced by pretreating IKC with antioxidants and cytochrome P450, flavin monooxygenase and peroxidase inhibitors, while 3,5-DCPHA cytotoxicity was attenuated by two nucleophilic antioxidants (glutathione and N-acetyl-L-cysteine). Incubation of IKC with 3,5-DCA (0.5–1.0 mM, 90 min) produced only 3,5-DCAA and 3,5-DCNB as detectable metabolites. These data suggest that 3,5-DCNB and 3,5-DCPHA are potential nephrotoxic metabolites and may contribute to 3,5-DCA induced nephrotoxicity in vivo. In addition, the kidney can bioactivate 3,5-DCNB to toxic metabolites, and 3,5-DCPHA appears to generate reactive metabolites to contribute to 3,5-DCA nephrotoxicity. In vitro, N-oxidation of 3,5-DCA appears to be the primary mechanism of bioactivation of 3,5-DCA to nephrotoxic metabolites.

Time Course of Immune Cell Infiltration and Cytokine Production in the Kidneys of Dahl Salt‐Sensitive (SS) Rats

Inflammation and immunity has been implicated in hypertension and renal disease in animals and humans. Previous studies from our lab have demonstrated that pharmacological or genetic inhibition of immune cell infiltration in the kidney attenuates salt‐sensitive hypertension and kidney damage in SS rats. The present studies were performed to assess the temporal infiltration of different immune cell subsets into the kidneys of Dahl SS rats when maintained on a low salt (0.4% NaCl, Day 0) diet and after 7, 14 and 21 days of high salt (4.0% NaCl) intake. Using a flow cytometry‐based approach, a progressive increase of infiltrating cells in the kidneys of Dahl SS was observed with time on the high NaCl diet. The total number of CD45+ cells (9.3±1.96 vs 35.2±6.8 x 105 cells/kidney), CD3+CD8+ cells (.69±.13 vs 1.9±0.2 x 105 cells/kidney) and CD3+CD4+ cells (.36±.08 vs 1.78±0.37 x 105 cells/kidney) significantly increased from Day 0 to 21 of a high salt diet (n=9‐17 rats/group). Stimulation of isolated kidney cells with PMA and ionomycin followed by intracellular cytokine staining revealed an increase in the number of IFN‐g producing CD8+ T cells and also in TNF, IFN‐g and IL‐17 producing CD4+ T cells (e.g. CD3+CD4+IL‐17+ cells increased from .05±.01 to .56±.16 x 105 cells/kidney from Day 0 to Day 21). IFN‐g and IL‐17 production by CD4+ T cells positively correlated with the number of regulatory T cells (increased from .09±.02 to .43±.01 x 105 cells/kidney from Day 0 to 21) and the total number of activated CD4+ T cells (increased from .14±.02 to .84±.02 x 105 cells/kidney from Day 0 to 21). These data indicate that the time course of immune cells infiltration and cytokine production by T cells parallels hypertension and kidney damage in Dahl SS rats fed a high salt diet.This work is supported by DK‐96859 and HL116264.

Purification and biological effects of a C-type lectin isolated from Bothrops moojeni

Snake venom proteins from the C-type lectin family have very distinct biological activities despite their highly conserved primary structure, which is homologous to the carbohydrate recognition region of true C-type lectins. We purified a lectin-like protein (BmLec) from Bothrops moojeni venom and investigated its effect on platelet aggregation, insulin secretion, antibacterial activity, and isolated kidney cells. The BmLec was purified using two chromatographic steps: affinity chromatography and reverse phase high performance liquid chromatography (HPLC). BmLec showed a dose-dependent platelet aggregation and significantly decreased the bacterial growth rate in approximately 15%. During scanning electron microscopy, the profile of Xanthomonas axonopodis pv. passiflorae treated with lectin disclosed a high vesiculation and membrane rupture. BmLec induced a strong and significant increase in insulin secretion at 2.8 and 16.7 mM glucose concentrations, and this effect was seen in the presence of EGTA in both experiments. BmLec (10 µg/mL) increased the perfusion pressure, renal vascular resistance and urinary flow. The glomerular filtration rate and percentages of sodium, potassium and chloride tubular transport were reduced at 60 minutes of perfusion. Renal alterations caused by BmLec were completely inhibited by indomethacin in all evaluated parameters. In conclusion, the C-type lectin isolated from Bothrops moojeni affected platelet aggregation, insulin secretion, antibacterial activity and isolated kidney function.

Acute renal failure during sepsis: Potential role of cell cycle regulation

This study aimed to evaluate cell cycle regulation in acute kidney injury after intraperitoneal sepsis in rats. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in rats. At 0, 6, 12, 24, 48, and 72 h after CLP, serum creatinine was evaluated. DNA content of isolated kidney cells was analyzed using flow cytometer. Furthermore, the expression of p21, p53, cyclin D1, cyclin E, CDK2, CDK4 and P-pRb was also measured by western blot. After sepsis-induced by CLP, kidney injury of rat was associated with G1 cell cycle arrest, however, recovery of renal function related to cell cycle progression 48h after CLP. Results also showed that the upregulation of p53 and p21 was correlated with G1 cell arrest in 48h after CLP. Nevertheless, upregulation of cyclin D1/CDK4 and cyclin E/CDK2 induced pRb phosphorylation, which resulted in the G1/S transition 48 h after CLP. The data suggest that G1 cell cycle arrest may play a role in the initiation of kidney injury, whereas, through regulating cell cycle, p53, p21, CDKs, cyclins and P-pRb may be involved in the injury or recovery of renal function after intraperitoneal sepsis.

Characterization of the genotoxicity of nitrilotriacetic acid

The chelating agent nitrilotriacetic acid (NTA), classified as an epigenetic rodent carcinogen, was assessed in the in vivo rodent Comet assay on isolated kidney cells. Unexpected potent increases in DNA damage were obtained in both the short (3-6 hr) and long-term (22-26 hr) expression times after a single oral treatment at 1,000-2,000 mg/kg bw. NTA was assayed in the Ames test using TA1537, TA98, TA100, and TA102 tester strains, and in the in vitro micronucleus assay on L5178Y mouse lymphoma cells and on CTLL2 and CTLL2/Bcl2 cells coupled to the apoptosis measurement, both with and without metabolic activation by aroclor 1254-induced liver or kidney rat S9-mix. Whatever the S9 origin, neither genotoxicity nor apoptosis was detected, while a strong increase in the micronuclei formation was observed without S9 without any apoptosis induction. The direct genotoxicity of NTA was confirmed in the mouse lymphoma tk+/- gene mutation assay and in the chromosomal aberrations test on human lymphocytes. When tested in combination with an excess of Ca2+, NTA gave negative results on L5178Y mouse lymphoma cells in the in vitro Comet and micronucleus assays but still induced DNA damage on rat primary kidney cells. The higher sensitivity of renal cells to Ca2+ variations could explained the positive response observed in vivo. The carcinogenicity of NTA could be a consequence of the survival of kidney cells to intracellular variations of Ca2+, leading to a local and indirect genotoxicity. This suggests that threshold dose exists beyond which tumor-generating events will be displayed.

In vivo Comet assay on isolated kidney cells to distinguish genotoxic carcinogens from epigenetic carcinogens or cytotoxic compounds

The objective of this study was to determine the ability of the alkaline in vivo Comet assay (pH > 13) to distinguish genotoxic carcinogens from epigenetic carcinogens when performed on freshly isolated kidney cells and to determine the possible interference of cytotoxicity by assessing DNA damage induced by renal genotoxic, epigenetic or toxic compounds after enzymatic isolation of kidney cells from OFA Sprague–Dawley male rats. The ability of the Comet assay to distinguish (1) genotoxicity versus cytotoxicity and (2) genotoxic versus non-genotoxic (epigenetic) carcinogens, was thus investigated by studying five known genotoxic renal carcinogens acting through diverse mechanisms of action, i.e. streptozotocin, aristolochic acids, 2-nitroanisole, potassium bromate and cisplatin, two rodent renal epigenetic carcinogens: d-limonene and ciclosporine and two nephrotoxic compounds: streptomycin and indomethacin. Animals were treated once with the test compound by the appropriate route of administration and genotoxic effects were measured at the two sampling times of 3–6 and 22–26 h after treatment. Regarding the tissue processing, the limited background level of DNA migration observed in the negative control groups throughout all experiments demonstrated that the enzymatic isolation method implemented in the current study is appropriate. On the other hand, streptozotocin, 20 mg/kg, used as positive reference control concurrently to each assay, caused a clear increase in the mean Olive Tail Moment median value, which allows validating the current methodology. Under these experimental conditions, the in vivo rodent Comet assay demonstrated good sensitivity and good specificity: all the five renal genotoxic carcinogens were clearly detected in at least one expression period either directly or indirectly, as in the case of cisplatin: for this cross-linking agent, the significant decrease in DNA migration observed under standard electrophoresis conditions was clearly amplified when the duration of electrophoresis was increased up to 40 min. In contrast, epigenetic and nephrotoxic compounds failed to induce any signifcant increase in DNA migration. In conclusion, the in vivo rodent Comet assay performed on isolated kidney cells could be used as a tool to investigate the genotoxic potential of a test compound if neoplasic/preneoplasic changes occur after subchronic or chronic treatments, in order to determine the role of genotoxicity in tumor induction. Moreover, the epigenetic carcinogens and cytotoxic compounds displayed clearly negative responses in this study. These results allow excluding a DNA direct-acting mechanism of action and can thus suggest that a threshold exists. Therefore, the current in vivo rodent Comet assay could contribute to elucidate an epigenetic mechanism and thus, to undertake a risk assessment associated with human use, depending on the exposure level.

Modulation of hepatic and renal metabolism and toxicity of trichloroethylene and perchloroethylene by alterations in status of cytochrome P450 and glutathione

The relative importance of metabolism of trichloroethylene (Tri) and perchloroethylene (Perc) by the cytochrome P450 (P450) and glutathione (GSH) conjugation pathways in their acute renal and hepatic toxicity was studied in isolated cells and microsomes from rat kidney and liver after various treatments to modulate P450 activity/expression or GSH status. Inhibitors of P450 stimulated GSH conjugation of Tri and, to a lesser extent, Perc, in both kidney cells and hepatocytes. Perc was a more potent, acute cytotoxic agent in isolated kidney cells than Tri but Perc-induced toxicity was less responsive than Tri-induced toxicity to modulation of P450 status. These observations are consistent with P450-dependent bioactivation being more important for Tri than for Perc. Incubation of isolated rat hepatocytes with Tri produced no acute cytotoxicity in isolated hepatocytes while Perc produced comparable cytotoxicity as in kidney cells. Modulation of P450 status in hepatocytes produced larger changes in Tri- and Perc-induced cytotoxicity than in kidney cells, with non-selective P450 inhibitors increasing toxicity. Induction of CYP2E1 with pyridine also markedly increased sensitivity of hepatocytes to Tri but had little effect on Perc-induced cytotoxicity. Increases in cellular GSH concentrations increased Tri- and Perc-induced cytotoxicity in kidney cells but not in hepatocytes, consistent with the role of GSH conjugation in Tri- and Perc-induced nephrotoxicity. In contrast, depletion of cellular GSH concentrations moderately decreased Tri- and Perc-induced cytotoxicity in kidney cells but increased cytotoxicity in hepatocytes, again pointing to the importance of different bioactivation pathways and modes of action in kidney and liver.

Renal Toxicity of Perchloroethylene and S-(1,2,2-Trichlorovinyl)glutathione in Rats and Mice: Sex- and Species-Dependent Differences

Suspensions of renal cells from rats and renal mitochondria from rats and mice were used to assess the sex and species dependence of acute toxicity due to perchloroethylene (Perc) and its glutathione conjugate S-(1,2,2-trichlorovinyl)glutathione (TCVG). A marked sex dependence in the acute cytotoxicity of both Perc and TCVG was observed: Perc caused significant release of lactate dehydrogenase (LDH) in isolated kidney cells from male but not female rats, and TCVG caused much more LDH release from male than female rat kidney cells. Assessment of toxicity in suspensions of isolated mitochondria from kidneys of male and female rats revealed a generally similar pattern of sensitivity, with mitochondria from males exhibiting significantly more inhibition of State 3 respiration and decrease of respiratory control ratio than mitochondria from females. Respiratory function in mitochondria from male and female mice, however, was also significantly inhibited by Perc or TCVG but exhibited little sex dependence in the degree of inhibition. Comparison with results from similar studies using the congener trichloroethylene and its glutathione conjugate suggested that Perc and TCVG are more potent nephrotoxicants. Neither Perc nor TCVG produced any significant effects on cytotoxicity or mitochondrial function in isolated hepatocytes from rats or in isolated liver mitochondria from rats or mice, suggesting that the liver is not a major acute target for Perc or its glutathione conjugate. Thus, many of the species-, sex-, and tissue-dependent differences in toxicity of Perc and TCVG that are observed in vivo are also observed in these in vitro models.

Desaturation and esterification of fatty acids in kidney cells from spontaneously hypertensive rats

In previous studies, several alterations in lipid metabolism have been related to hypertension, but the mechanisms explaining this relationship have not been elucidated. None of the previous works has focused on the lipid metabolism in kidney, which is a key organ in the overall regulation of blood pressure. The aim of the present work was to study the metabolism of polyunsaturated fatty acids, and the possible compositional changes in kidney from hypertensive rats. Radiolabelled linoleic acid (18:2,n-6) and dihomogammalinolenic acid (20:3, n-6) were incubated with isolated kidney cells from spontaneously hypertensive rats (SHR) or the parent normotensive strain (Wistar Kyoto, WKY). The rats were divided into groups of age 9 (young) and 17 (adult) weeks. Cellular uptake, desaturation, chain-elongation, oxidation and distribution into phospholipids and triacylglycerols were measured. Additionally, the lipid composition of kidney was characterized. With each of the labelled fatty acid substrates the uptake in cells from the SHR rats, compared to the WKY rats, was numerically lower in the young group and higher in the adult group. The incorporation of labelled fatty acids into phospholipids was increased and concomitantly decreased in triacylglycerols in cells from adult SHR rats. The Delta 6-desaturation, measured as the conversion of labelled 18:2(n-6) to 18:3(n-6) was between two and three times increased in cells from the adult rats compared to the young ones, while no difference was found in hypertensives compared to normotensives. Concomitantly, no difference in conversion of labelled 20:3(n-6) to 20:4(n-6) was observed in relation to blood pressure, but, different from Delta 6-desaturation, the Delta 5-desaturation was significantly decreased by age. Taken together, this study demonstrates for the first time desaturation of long-chain polyunsaturated fatty acids in isolated kidney cells in suspension and that, contrary to what has been observed in liver, the desaturase activity is unaffected by hypertension. Also different from what has been observed in liver, no blood-pressure-related changes in lipid composition of kidney were found.

Glutathione Conjugation of Perchloroethylene in Rats and Micein Vitro:Sex-, Species-, and Tissue-Dependent Differences

Perchloroethylene (Per)-induced nephrotoxicity and nephrocarcinogenicity have been associated with metabolism by the glutathione (GSH) conjugation pathway to formS-(1,2,2-trichlorovinyl)glutathione (TCVG). Formation of TCVG was determined in incubations of Per and GSH with isolated renal cortical cells and hepatocytes from male and female Fischer 344 rats and with renal and hepatic cytosol and microsomes from male and female Fischer 344 rats and B6C3F1 mice. The goal was to assess the role of metabolism in the sex and species dependence of susceptibility to Per-induced toxicity. A key finding was that GSH conjugation of Per occurs in kidney as well as in liver. Although amounts of TCVG formation in isolated kidney cells and hepatocytes from male and female rats were generally similar, TCVG formation in subcellular fractions showed marked sex, species, and tissue dependence. This may be due to the presence of multiple pathways for metabolism in intact cells, whereas only the GSH conjugation pathway is active in the subcellular fractions under the present assay conditions. TCVG formation in kidney and liver subcellular fractions from both male rats and mice were invariably higher than corresponding values in female rats and mice. Amounts of TCVG formation in rat liver subcellular fractions were approximately 10-fold higher than in corresponding fractions from rat kidney. Although rats are more susceptible to Per-induced renal tumors than mice, amounts of TCVG formation were 7- to 10-fold higher in mouse kidney subcellular fractions and 2- to 5-fold higher in mouse liver subcellular fractions of both sexes compared to corresponding fractions from the rat. Hence, although the higher amounts of TCVG formation in liver and kidney from male rats correspond to their higher susceptibility to Per-induced renal tumors compared with female rats, the markedly higher amounts of TCVG formation in mice compared with rats suggest that other enzymatic or transport steps in the handling of Per in mice contribute to their relatively low susceptibility to Per-induced renal tumors.

PRO-INFLAMMATORY CYTOKINES AND INTERLEUKIN 6 IN THE RENAL RESPONSE TO BACTERIAL ENDOTOXIN

Pro-inflammatory cytokines, including tumour necrosis factor α (TNF-α), interleukin (IL)-1 and IL-6 are thought to play important roles in the pathophysiology of chronic kidney disorders, including glomerulonephritis. In particular, IL-6 has received considerable attention as it appears at high concentrations to promote the progression of renal disease while at lower levels may be involved in regulating repair mechanisms. As such, cytokine profiles have been examined in the kidney by either examining secretion from isolated kidney cells or quantitating plasma and urinary levels in experimental models of glomerulonephritis. To examine the cytokine responses within the kidney, without the contribution of other organ systems, we used semi-quantitative polymerase chain reaction (RT-PCR) analysis and a recently developed kidney slice culture model from tissues of mice treated with combinations of endotoxin and neutralizing antibodies against TNF-α. The expression of IL-6, in addition to other pro-inflammatory cytokine genes, was increased by endotoxin treatment and reduced by pretreatment with neutralizing antibodies to TNF-α. Immunohistochemical staining revealed that IL-6 was expressed primarily in mesangial cells. Urinary IL-6 was also increased in endotoxin-treated mice and was inhibited by treatment with neutralizing TNF-α antibodies. Kinetics of the kidney-specific cytokine responses indicated that increase in TNF-α occurred initially, followed by IL-1β and finally IL-6. Furthermore, addition of TNF-α to glomerular mesangial cells induces IL-6 secretion. Taken together, these studies indicate that, like in the liver, a cytokine response occurs in the kidney from bacterial endotoxin and that TNF-α acts as a primary cytokine capable of stimulating additional cytokines, including IL-6.

An in vivo micronucleus assay for detecting the clastogenic effect in rat kidney cells

A micronucleus assay in vivo has been developed that is based on the use of freshly isolated kidney cells from mononephrectomized rats. In this validation study, a statistically significant increase in the frequency of micronucleated cells was detected in rats given i.p. a single dose of four kidney carcinogens, N-nitrosodimethylamine, N-nitrosodiethylamine, N-ethyl- N-hydroxyethylnitrosamine and N-nitroso- N-methylurea. The clastogenic effect was more marked when the same dose was injected for 3 successive days. As compared to controls, treated rats displayed a reduction in the frequency of binucleated cells, presumably due to a toxicity-induced inhibition of cellular proliferation. The proposed method should be suitable for the detection of the clastogenic effect of procarcinogens biotransformed into reactive species in the kidney.

Potential metabolism and cytotoxicity of N-(3,5-dichlorophenyl)succinimide and its hepatic metabolites in isolated rat renal cortical tubule cells

N-(3,5-Dichlorophenyl)succinimide (NDPS) is an agricultural fungicide and antimicrobial agent that produces nephrotoxicity in rats. The contribution of the kidney, if any, to the mechanism of toxicity of NDPS is not known. Therefore, the ability of isolated renal cortical tubule cells to metabolize NDPS and some of its known hepatic metabolites was studied. The cytotoxic potential of these compounds was also assessed. Renal cortical tubule cells were isolated by collagenase digestion and were incubated with the test compounds (2 mM) for 3 h. Metabolite formation was monitored by reversed phase HPLC and cell viability was assessed using trypan blue exclusion. The isolated kidney cells do not appear to metabolize NDPS or any of its known hepatic metabolites. In addition, none of these compounds were directly cytotoxic to the renal cells. However, the cells were susceptible to mercuric chloride (1 mM) and chloroform (125 or 200 mM). Intracellular glutathione levels were unaltered by the presence of NDPS in the incubations. These results suggest that NDPS and its metabolites are not directly toxic to the kidney and are not converted into the ultimate nephrotoxic species by the kidney. Extrarenal metabolism may, therefore, be critical to the expression of NDPS-induced nephrotoxicity.

Differential effects of various trivalent and pentavalent organic and inorganic arsenic species on glucose metabolism in isolated kidney cells

We have compared the acute toxicities of the trivalent arsenic species arsenite, oxophenylarsine (PhAsO), 2-chlorovinyloxoarsine (ClvinAsO), methyloxoarsine (MeAsO), and of the pentavalent arsenic species arsenate, methyl- and phenyl-arsonic acid in rat kidney tubules (RKT) and Madin-Darby canine kidney (MDCK) cells. In RKT, PhAsO (1 μmol I−1, 60 min) almost completely (>90%) blocked gluconeogenesis without affecting cell viability as assessed by dye exclusion. In MDCK cells, PhAsO (2 μmol I−1) markedly inhibited glucose uptake (60% of controls) within 30 min, while cell viability, as assessed by formazan formation, was not affected within 180 min. MeAsO and CIvinAsO were similarly effective to PhAsO in both RKT and MDCK cells. Estimated IC50 values for the inhibition of gluconeogenesis were 0.55 (PhAsO), 0.69 (CIvinAsO) and 0.99 μmol I−1 (MeAsO) and for the inhibition of glucose uptake 1.23 (PhAsO). 2.62 (CIvinAsO) and 6.99 μmol I−1 (MeAsO). At longer storage times, aqueous solutions of MeAsO and of CIvinAsO, but not of PhAsO, gradually lost toxic activity in RKT and MDCK cells, especially at alkaline pH. Concomitantly, a gradual decrease in content as assessed by HPLC was detected. Roughly 10-fold higher concentrations of arsenite than of PhAsO were required for comparable effects on gluconeogenesis in RKT, whereas in MDCK cells about 100-fold higher concentrations were needed for similar inhibition of glucose uptake. Pentavalent arsenate and phenylarsonate were two orders of magnitude less effective than PhAsO in RKT, while methylarsonate had virtually no influence on gluconeogenic activity. In MDCK cells the pentavalent arsenic species showed effects only in the millimolar range. It is concluded (1) that different mechanisms are involved in the acute toxicity of oxoarsines and inorganic arsenic and (2) that PhAsO offers advantages as a model substance for mono-substituted trivalent arsenicals, because it is more stable and more readily detectable.

Role of the kidney in the plasma clearance of angiotensinogen in the rat: Plasma clearance and tissue distribution of 125I-angiotensinogen

We studied the tissue distribution and plasma clearance of angiotensinogen (AGN) in rats following an i.v. injection of 125I-labeled AGN. The plasma clearance rate of [ 125I]AGN fits a two-compartment model with half-lives of 10.2 ± 1.5 min and 4.1 ± 0.5 h in non-treated rats and the half-life of slower phase significantly increased to 10.2 ± 1.1 h following bilateral nephrectomy. Radioactivity was predominantly distributed in the kidneys (4.9%) and to a lesser extent in the liver (1.8%), testis (1.2%), spleen (0.61%), heart (0.35%), lung (0.18%), thymus (0.03%) and brain (0.03%). The subcellular distribution of radioactivity in the kidney was 64% in the soluble fraction and 33% in the crude mitochondrial-lysosomal fraction. Sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed that the radioactivity in the soluble fraction consisted of proteins corresponding to intact [ 125I]AGN, whereas the mitochondrial-lysosomal fraction contained additional radioactive proteins with molecular weights between 18,000 and 29,000. When isolated kidney cells were incubated with [ 125I]AGN at 0 °C, the radioactive binding was saturable and specific with a Kd value of 4.8 × 10 −11M, whereas incubation at 37 °C resulted in the appearance of degraded products of [ 125I]AGN in the medium. These results suggested that circulating AGN is cleared mainly by the kidneys via receptor-mediated endocytosis, which may play an important role in regulating plasma level of AGN.

Energetic status and viability of porcine kidney cells after hypoxic, hypothermic preservation in UW solution and subsequent reoxygenation: the influence of taurine

Taurine (2-aminoethane sulfonic acid) is a physiologic compound with osmoregulative properties and presumbly an inhibitor of lipid peroxidation. The present study evaluates the effects on the viability of isolated kidney cells of the addition of various doses of taurine to the UW preservation solution. Porcine tubular cell monolayers were incubated at 4°C for 9 h in UW solution under hypoxic conditions, and subsequently reoxygenated for 30 min in Hank's balanced salt solution equilibrated with room air. Taurine was added to the preservation solution at final concentrations of 0, 1, 10 ot 20 mmol/1. Cel viability was assessed by exclusion on Trypan blue and by determination of the energetic status reoxygenation. While tuarine had no influence on the post-hypoxic vaibility according to Trypan blue exclusion, a dose-related increases in cellular content of high energy phosphates after post-hypoxic reoxygenation was observed after addition of taurine to the preservation medium. It is concluded that taurine is able to reduce tissue alterations during hypoxia and reoxygenation and that the determination of post-hypoxic recovery of energy metabolism may be a more sensitive indicator of cell viability than the morphological Trypan blue exclusion test.

Mutagenicity and cytotoxicity of two regioisomeric mercapturic acids and cysteine S-conjugates of trichloroethylene

The mutagenicity, cytotoxicity and metabolism of two regioisomic L-cysteine- and N-acetyl-L-cysteine-S-conjugates of trichloroethylene were studied. The 1,2-dichlorovinyl(1,2-DCV) isomers of both the cysteine conjugate and the mercapturate were much stronger mutagens in the Ames test with Salmonella typhimurium TA2638 when compared to the corresponding 2,2-dichlorovinyl (2,2-DCV) isomers. Similarly, the 1,2-DCV isomers were more cytotoxic towards isolated rat kidney proximal tubular cells, as assessed by inhibition of alpha-methylglucose uptake, than the 2,2-DCV isomers. The 3-4-fold higher rate of beta-lyase-dependent activation of S-(1,2-dichlorovinyl)-L-cysteine (1,2-DCV-Cys) when compared to S-(1,2-dichlorovinyl)-L-cysteine (2,2-DCV-Cys) as well as the different nature of the reactive intermediates formed is probably responsible for these structure-dependent effects. The cytotoxicity of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (1,2-DCV-NAc) toward isolated kidney cells showed a delayed time course as compared to that of 1,2-DCV-Cys, probably due to the relatively low rate of deacetylation of 1,2-DCV-NAc. The time course of cytotoxicity of N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine (2,2-DCV-NAc), however, parallelled that of 2,2-DCV-Cys. Due to the relatively high rate of N-acetylation and low rate of beta-lyase activation, for 2,2-DCV-Nac the beta-lyase activation step may be rate limiting. Different rates of cellular uptake also may play a role in time course of toxicity of the cysteine conjugates and the mercapturic acids in the renal cells.

Characterization of Ascorbic Acid Uptake by Isolated Rat Kidney Cells

Isolated kidney cells accumulated L[1-14C]ascorbic acid in a time-dependent manner and reached a steady state after 15 min at 37 degrees C. Initial velocity for uptake was over 300 pmol/mg protein per min when cells were separated from the bathing solution using a density gradient established during centrifugation. The uptake process was saturable with an apparent concentration at half maximal uptake of 36 mumols/L. Ascorbate uptake was reduced by metabolic inhibitors and was temperature dependent. Although ascorbic acid is an acid anion at pH 7.4, uptake did not appear to be inhibited by other acid anions such as p-aminohippurate and probenecid; however, involvement of the ion gradient established by Na+, H(+)-adenosine triphosphatase could not be confirmed. Replacing the sodium ion with other monovalent ions reduced the accumulation of ascorbate significantly. Isoascorbic and dehydroascorbic acids inhibited ascorbate uptake (34 and 13 mmol/L, respectively), whereas high concentrations of glucose showed some stimulation. These findings indicated that ascorbic acid is reabsorbed by the kidney in a sodium-dependent active transport process that is not common to other acid anions and has some specificity for the ascorbic acid structure.