Endoplasmic reticulum Ca(2+) signaling and calpains mediate renal cell death.

The goal of the current study was to determine the roles of ATP content, endoplasmic reticulum (ER) Ca(2+) stores, cytosolic free Ca(2+) (Ca(2+)(f)) and calpain activity in the signaling of rabbit renal proximal tubular (RPT) cell death (oncosis). Increasing concentrations (0.3-10 microM) of the mitochondrial inhibitor antimycin A produced rapid ATP depletion that correlated to a rapid and sustained increase in Ca(2+)(f), but not phospholipase C activation. The ER Ca(2+)-ATPase inhibitors thapsigargin (5 microM) or cyclopiazonic acid (100 microM) alone produced similar but transient increases in Ca(2+)(f). Pretreatment with thapsigargin prevented antimycin A-induced increases in Ca(2+)(f) and antimycin A pretreatment prevented thapsigargin-induced increases in Ca(2+)(f). Calpain activity increased in conjunction with ER Ca(2+) release. Pretreatment, but not post-treatment, with thapsigargin or cyclopiazonic acid prevented antimycin A-induced cell death. These data demonstrate that extensive ATP depletion signals oncosis through ER Ca(2+) release, a sustained increase in Ca(2+)(f) and calpain activation. Depletion of ER Ca(2+) stores prior to toxicant exposure prevents increases in Ca(2+)(f) and oncosis.

The use of explant lens culture to assess cataractogenic potential.

Explanted cultures of crystalline lenses have been used to investigate mechanisms of xenobiotic-induced cataract formation. However, very few studies have utilized mechanistic information to predict the cataractogenic potential of structurally diverse xenobiotics. The present investigation outlines how visual assessment of lens clarity, biochemical endpoints of toxicity, and mechanisms of lenticular opacity formation can be used to select compounds with a lower probability of causing cataract formation in vivo. The rat lens explant culture system has been used to screen thiazolidinediones against ciglitazone for their direct cataractogenic potential in vitro. The two compounds that were selected as development candidates (englitazone and darglitazone) did not produce cataracts in rats exposed daily for 3 months. The culture system has also been used to illustrate that the lens is capable of metabolizing compounds to reactive intermediates. In this example, the toxicity of S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a model cataractogen, was attenuated by inhibiting lenticular cysteine conjugate beta-lyase metabolism using aminooxyacetic acid. Finally, this model was used retrospectively to investigate the cataractogenic potential of CJ-12,918 and CJ-13,454 in rats. These compounds showed differences in the incidence of cataract formation in vivo based on differences in hepatic metabolism and penetration of parent drug and metabolites into the lens. The rank order of cataractogenic potential in vitro correlated better with in vivo results when an induced S9 microsomal fraction was added to the culture media. However, the model did not correctly predict the cataractogenic potential of ZD2138, a structurally similar compound. These studies illustrate the use of explant culture to assess mechanisms of cataract formation and outline its use and limitations for predicting cataractogenic potential in vivo.

Recovery of cellular functions following oxidant injury.

This study investigated the recovery of renal proximal tubule cellular (RPTC) functions following oxidant-induced sublethal injury. tert-Butylhydroperoxide (TBHP) treatment resulted in 24% cell death and loss 4 h following the exposure. The remaining sublethally injured RPTC proliferated, and monolayer DNA content returned to control values on day 4 following TBHP exposure. Basal oxygen consumption (Qo2) and ATP content in sublethally injured RPTC were decreased 64 and 63%, respectively, at 4 h and returned to control values on day 6. Net lactate consumption decreased 71% at 4 h and returned to control values on day 4. In contrast, net glutamine consumption increased 2.7-fold at 4 h and returned to control values on day 6. Ouabain-sensitive Qo2, Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity, and Na(+)-coupled glucose transport were inhibited 77, 88, and 83%, respectively, at 4 h and recovered to control values on day 6. These data show that 1) mitochondrial function, Na(+)-K(+)-ATPase activity, active Na+ transport, and Na(+)-coupled glucose transport are decreased in sublethally injured RPTC following oxidant exposure and are repaired over time; 2) monolayer regeneration precedes the recovery of mitochondrial and transport functions, and 3) sublethal injury and subsequent regeneration are associated with alterations in metabolic substrate utilization. These results suggest that oxidant-induced sublethal injury to RPTC may contribute to renal dysfunction and that RPTC can repair and regain cellular functions following oxidant injury.

Mechanistic analysis of S-(1,2-dichlorovinyl)-L-cysteine-induced cataractogenesis in vitro.

Chronic exposure to low concentrations of the nephrotoxic cysteine conjugate S-(1,2-dichlorovinyl)-l-cysteine (DCVC) causes cataracts in mice. This study explored mechanisms of DCVC-induced cataractogenesis using explanted lenses from male Sprague-Dawley rats. Lenses placed in organ culture were exposed to 2.5 microM-1 mM DCVC for 24 hr. DCVC caused concentration and time-dependent changes in biochemical markers of toxicity (lenticular adenosine 5'-triphosphate (ATP) content, mitochondrial reduction of the tetrazolium dye MTT, and glutathione (GSH) content) at concentrations >/=25 microM. Lens clarity was adversely affected at concentrations >/=50 microM. Within 24 hr, 1 mM DCVC altered lens ATP content (-77 +/- 2%), mitochondrial MTT reduction (-40 +/- 3%), and GSH content (-19 +/- 4%) (percent difference from controls, p < 0.05). ATP was the most sensitive index of DCVC exposure in this model, while lens weight was not altered. The role of lenticular DCVC metabolism was investigated using the beta-lyase inhibitor aminooxyacetic acid (AOA) and the flavin monooxygenase (FMO) inhibitor methimazole (MAZ). AOA (1 mM) provided nearly complete protection from changes in biochemical parameters and lens transparency caused by DCVC, while MAZ (1 mM) provided only partial protection. The mitochondrial Ca2+ uniport inhibitor ruthenium red (30 microM) and the poly(ADP ribosyl)transferase inhibitor 3-aminobenzamide (3 mM) were only partially protective, whereas adverse changes in lens transparency and biochemical markers were not prevented by an antioxidant (2 mM dithiothreitol) or nontoxic transport substrates (200 microM probenecid or 10 mm phenylalanine, S-benzyl-L-cysteine or para-aminohippuric acid). Calpain inhibitors E64d (100 microM) and calpain inhibitor II (1 mM) were ineffective in preventing opacity formation caused by DCVC. In a small separate study, DCVC toxicity to explanted lenses from cynomologus monkeys was also ameliorated by coincubation with AOA. These results indicate that opacity formation by DCVC in rodent and primate lenses in vitro is primarily mediated via lenticular beta-lyase metabolism of DCVC to a reactive metabolite. Metabolism of DCVC by FMO and perturbations in mitochondrial calcium (Ca2+) homeostasis and increased poly(ADP-ribosylation) of nuclear proteins may play a limited role in opacity formation in vitro. However, opacity formation does not appear to be the result of oxidative stress or calpain activation. DCVC toxicity to the lens was not blocked with competitive inhibitors of the amino acid and organic anion transporters of DCVC as is found in the kidney.

Neurosteroid inhibition of cell death.

Diverse gamma-aminobutyric acid (GABAA) receptor modulators exhibited novel cytoprotective effects and mechanisms of action in rabbit renal proximal tubules subjected to mitochondrial inhibition (antimycin A) or hypoxia. Cytoprotective potencies (50% effective concentration, EC50) were 0.3 nM allopregnanolone (AP) > 0.4 nM 17 alpha-OH-allopregnanolone (17 alpha-OH-AP) > 30 nM dehydroepiandrosterone sulfate (DHEAS) = 30 nM pregnenolone sulfate (PS) > 500 nM pregnenolone (PREG) > 30 microM muscimol > 10 mM GABA following antimycin A exposure. Maximal protection with AP and 17 alpha-OH-AP was 70%, whereas DHEAS, PS, PREG, and muscimol produced 100% cytoprotection. Experiments with AP, PS, and muscimol revealed the return of mitochondrial function and active Na+ transport following hypoxia/reoxygenation. Muscimol inhibited the antimycin A-induced influx of both extracellular Ca2+ and Cl- that occurs during the late phase of cell injury, whereas the neurosteroids only inhibited influx of Cl-. Radioligand binding studies with AP and PS did not reveal a specific binding site; however, structural requirements were observed for cytoprotective potency and efficacy. In conclusion, we suggest that the GABAA receptor modulators muscimol and neurosteroids are cytoprotective at different cellular sites in the late phase of cell injury; muscimol inhibits Ca2+ and subsequent Cl- influx, whereas the neurosteroids inhibit Cl- influx.

Model development and analysis of tenidap-induced proteinuria in the rat.

Tenidap is a novel antirheumatic agent that causes a mild, reversible proteinuria in human clinical trials. In order to achieve a mechanistic understanding and safety perspective of the proteinuric effects of tenidap observed in clinical trials, female Sprague-Dawley rats were treated with up to 100 mg/kg/day of tenidap in the diet for 4 to 6 weeks followed by a 1- to 6-week reversal period. Pharmacokinetics and measurements of renal function and histology were assessed during the study. Sustained high plasma concentrations of tenidap [area under the plasma concentration curve (0-24 hr) of 941-1021 micrograms. hr/ml and peak plasma concentration of 61-67 micrograms/ml] increased urinary protein, albumin and phosphate excretion (2- to 8-fold) in rats. These renal effects were reversible within 9 days after removal of the drug. These effects preceded later occurring changes in renal morphology (papillary degeneration and necrosis). There was no evidence of glomerular damage, proximal tubule degeneration or necrosis or tubulointerstitial nephritis at the light microscopic level. Other indices of overall renal function (glomerular filtration rate, electrolyte and glucose excretion) were unaffected. Examination in situ of microperfused proximal tubules from treated rats revealed a 68% decrease in the rate of proximal tubule albumin absorption compared to controls (19 +/- 4 vs. 59 +/- 7 pg/min/mm, respectively). Fluid absorption rate and bicarbonate handling by the proximal tubule, along with blood bicarbonate concentrations, pH, PCO2 and PO2, were unaffected by treatment. It was concluded that tenidap caused a rapid, stable and reversible phosphaturia, microalbuminuria and proteinuria in the rat. The proteinuric effects were due to impaired proximal tubule albumin reabsorption that were not associated with other signs of impaired renal function or histological evidence of tubulointerstitial nephritis or proximal tubule/glomerular damage.

The role of short chain fatty acid substrates in aerobic and glycolytic metabolism in primary cultures of renal proximal tubule cells.

This study examined the role of odd and even short-chain fatty acid substrates on aerobic and glycolytic metabolism in well-aerated primary cultures of rabbit renal proximal tubule cells (RPTC). Increasing oxygen delivery to primary cultures of RPTC by shaking the dishes (SHAKE) reduced total lactate levels and lactate dehydrogenase (LDH) activity and reduced net glucose consumption compared to RPTC cultured under standard conditions (STILL). The addition of butyrate, valerate, heptanoate, or octanoate to SHAKE RPTC produced variable effects on glycolytic metabolism. Although butyrate and heptanoate further reduced total lactate levels and net glucose consumption during short-term culture (< 24 h), no fatty acid tested further reduced total lactate levels, net glucose consumption, or LDH activity during long-term culture (7 days). During the first 12 h of culture, maintenance of aerobic metabolism in SHAKE RPTC was dependent on medium supplementation with fatty acid substrates (2 mM). However, by 24 h, SHAKE RPTC did not require fatty acid substrates to maintain levels of aerobic metabolism equivalent to freshly isolated proximal tubules and greater than STILL RPTC. This suggests that SHAKE RPTC undergo adaptive changes between 12 and 24 h of culture, which give RPTC the ability to utilize other substrates for mitochondrial oxidation, therefore allowing greater expression of mitochondrial oxidative potential in SHAKE RPTC than in STILL RPTC.

Primary cultures of rabbit renal proximal tubule cells. III. Comparative cytotoxicity of inorganic and organic mercury.

The present study further developed primary cultures of rabbit renal proximal tubule cells (RPTC) as an in vitro model to study chemical-induced toxicity by investigating the comparative cytotoxicity of mercuric chloride (HgCl2) and methyl mercury chloride (CH3HgCl) to RPTC. Confluent monolayer cultures of RPTC exposed to HgCl2 and CH3HgCl for 24 hr exhibited a concentration-dependent loss in cell viability at culture medium concentrations greater than 25 and 2.5 microM, respectively. Vital dye exclusion was a more sensitive indicator of cytotoxicity than the amount of lactate dehydrogenase activity, alkaline phosphatase activity, N-acetylglucosaminidase activity, and protein content remaining on the culture dish. On the basis of vital dye exclusion, HgCl2 was less toxic to proximal tubule cells in culture than CH3HgCl after 24 hr of exposure, whether cytotoxicity was based on LC50 values (34.2 microM HgCl2 vs 6.1 microM CH3HgCl) or total cellular mercury uptake (4.6 nmol Hg2+/10(5) cells vs 1.25 nmol CH3Hg+/10(5) cells). Differences in the extent and rate of metal uptake were also evident. Maximum cellular uptake of Hg2+ occurred within 6-24 hr after exposure and was not concentration-dependent, whereas maximum uptake of CH3Hg+ occurred within 3 hr of exposure and was concentration-dependent. The intracellular distribution of both mercurials between acid-soluble and acid-insoluble binding sites also differed. At noncytotoxic concentrations of HgCl2 (0.04-5 microM), intracellular Hg2+ bound increasingly to acid-soluble binding sites as a function of time, from 15-30% after 6 hr of exposure to 40-60% after 72 hr of exposure. However, at subcytotoxic (25 microM) and cytotoxic (34.2 microM) concentrations, Hg2+ binding to acid-soluble binding sites remained constant at approximately 30-40% for 6, 12, 24, and 72 hr after exposure. In contrast, only 20% of total cellular CH3Hg+ was bound to acid-soluble binding sites after exposure to 0.039 to 6.1 microM CH3HgCl for 6, 12, and 24 hr. Total cellular glutathione content was unaffected after exposure to 0.04-5 microM HgCl2 and 0.039-6.1 microM CH3HgCl, but was depleted 6 hr after exposure to 25 and 34.2 microM HgCl2. These results indicate that CH3HgCl was a more potent cytotoxicant to RPTC in primary culture than HgCl2. Furthermore, compared to Hg2+, the low binding of CH3Hg+ to acid-soluble binding sites and the absence of a redistribution of CH3Hg+ from acid-insoluble to acid-soluble binding sites appeared to contribute to its more potent toxicity to cultured cells.(ABSTRACT TRUNCATED AT 400 WORDS)

The neurotoxicants strychnine and bicuculline protect renal proximal tubules from mitochondrial inhibitor-induced cell death.

Glycine-induced cytoprotection of renal proximal tubules exposed to chemical- or hypoxic/anoxic-induced cell death is shared by a few amino acid agonists of the neuronal strychnine-sensitive glycine receptor. The goal of this study was to determine if antagonists of the strychnine-sensitive glycine receptor attenuated the cytoprotective effects of glycine. Strychnine did not antagonize the cytoprotective effects of glycine in proximal tubules exposed to antimycin A. In contrast, strychnine was cytoprotective, was equipotent as glycine (EC50 = 0.4 mM), and the combination of strychnine and glycine was additive. Likewise, bicuculline and norharmane were cytoprotective but 20-50% less potent than glycine. These results suggest that glycine and strychnine act as a common site to produce proximal tubule cytoprotection, but this site does not share the same potency and agonist/antagonist properties as the neuronal strychnine-sensitive glycine receptor.

Regulation of glycolytic metabolism during long-term primary culture of renal proximal tubule cells.

Adequate oxygenation was a major factor regulating the induction of glycolytic metabolism in primary cultures of rabbit renal proximal tubule cells during short-term (less than 1 day) and long-term (1-7 day) culture. As measured by cellular lactate content, glucose consumption, and lactate dehydrogenase activity, less glycolytic metabolism was induced in cultured cells that were constantly aerated than in cells that were held stationary. When oxidative metabolism is supported by providing 2-10 mM heptanoate (HEP) as a metabolic substrate glycolytic metabolism further decreased during short-term, but not long-term culture. Cellular proliferation did not play a major role in regulating the induction of glycolytic metabolism, since glycolytic metabolism increased before cell growth had occurred, did not decline once logarithmic cell growth had ceased, and was stimulated less by cell growth than by inadequate oxygenation. Fructose-1,6-bisphosphatase and alkaline phosphatase, representative markers of gluconeogenic and brush-border membrane enzyme activities, respectively, declined during culture regardless of culture conditions or the presence of HEP. Therefore, glycolytic metabolism can be effectively minimized by constantly aerating cultured proximal tubule cells and can be further reduced by the addition of HEP during short-term culture.

The role of altered mitochondrial function in citrinin-induced toxicity to rat renal proximal tubule suspensions.

Citrinin (CTN), a mycotoxin produced by several species of Penicillium and Aspergillus, causes renal proximal tubule (RPT) cell injury and death by an unknown mechanism of action. Using suspensions of rat RPT, the cellular events preceding CTN-induced cytotoxicity were investigated. Tubule viability decreased in a concentration- and time-dependent manner after CTN exposure, with cell death beginning 1, 2, and 4 hr after exposure to 500, 125-250, and 63 microM, respectively. Basal oxygen consumption (QO2) of RPT increased from 41 to 53 nmol O2.mg protein-1.min-1 30 min after exposure to 250 microM CTN and returned to control values 1 hr after exposure. A similar concentration- and time-dependent transitory rise in basal QO2 occurred at all concentrations of CTN tested (63-500 microM). Nystatin-stimulated QO2, an indirect measure of mitochondrial state 3 respiration in RPT, decreased 11% at 0.5 and 1 hr after exposure to 500 and 250 microM CTN, respectively, but was not affected after exposure to 63 and 125 microM CTN. Adenosine triphosphate content declined 22% to 48% in RPT at 0.5 and 1.5 hr after exposure to 500 and 125-250 microM CTN, respectively. Although lipid peroxidation occurred concurrently with RPT cell death, iron-mediated oxidative stress was not a causative factor in the development of toxicity since pretreatment with 1 mM deferoxamine prevented iron-mediated lipid peroxidation but did not protect RPT from CTN-induced cell death. Further studies using RPT and isolated renal cortical mitochondria (RCM) showed that CTN had multiple effects on mitochondrial function. Direct probing of mitochondrial function within RPT showed that a 1-hr exposure to 250 microM CTN increased spontaneous respiration 55% in RPT respiring on the site I respiratory substrates glutamate/malate while state 3 respiration decreased 34%. CTN also decreased succinate supported respiration but had no effect on cytochrome c-cytochrome oxidase. With isolated RCM, a 3-min exposure to 125 and 250 microM CTN increased state 4 respiration in the absence of a phosphate acceptor 27 and 67%, respectively, while 250 microM CTN decreased state 3 respiration 23%. Respiration in the presence of a known uncoupler was reduced after CTN exposure (63-250 microM) in a concentration-dependent manner. These results indicate that CTN has multiple effects on mitochondrial function in RPT and isolated RCM which may contribute to the development of cell death in rat RPT.

Mitochondrial dysfunction is an early event in ochratoxin A but not oosporein toxicity to rat renal proximal tubules.

Ochratoxin A (OA) and oosporein (OSN) are two mycotoxins that may cause nephrotoxicity through either mitochondrial dysfunction or lipid peroxidation. Using isolated rat renal proximal tubules in suspension, the cellular events preceding OA- or OSN-induced cytotoxicity were investigated. OA and OSN decreased tubule viability in a concentration (0-1 mM)- and time (0-4 hr)-dependent manner, with initial decreases occurring 1 hr after exposure. Tubule basal and nystatin-stimulated oxygen consumption decreased before cell death after OA (0.5 and 1 mM) and 0.25 mM t-butyl hydroperoxide (TBHP) exposure, but did not decrease after OSN exposure (0.25-1 mM). The oxidant TBHP was used as a positive control in these studies. Direct probing of mitochondrial function within proximal tubules confirmed the toxicity of OA to mitochondria. Respiration was reduced in the absence and presence of a phosphate acceptor using site I (glutamate/malate) and site II (succinate) respiratory substrates 15 and 30 min after exposure to 1 mM OA. Lipid peroxidation preceded cell death after exposure to 1 mM OA and 0.25 mM TBHP, but did not occur after exposure to 1 mM OSN. Deferoxamine (1 mM) pretreatment before the addition of 1 mM OA or OSN prevented OA-induced lipid peroxidation, but did not prevent OA- or OSN-induced cytotoxicity. In contrast, deferoxamine pretreatment prevented lipid peroxidation, mitochondrial dysfunction, and the loss of tubule viability after exposure to 0.25 mM TBHP. This study shows that mitochondrial dysfunction is an early event during the development of OA toxicity, but not in OSN-induced toxicity. Furthermore, iron-mediated lipid peroxidation does not contribute to OA- or OSN-induced proximal tubule cell death.

Toxicity of N-(3,5-dichlorophenyl)succinimide and metabolites to rat renal proximal tubules and mitochondria.

The acute nephrotoxicity caused by N-(3,5-dichlorophenyl) succinimide (NDPS) has been shown to be due to a metabolite(s) of the parent compound. This study examined the toxicity of NDPS, its known metabolites N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS), N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (NDHSA), N-(3,5-dichlorophenyl)malonamic acid (DMA), N-(3,5-dichlorophenyl)succinamic acid (NDPSA), and two postulated metabolites N-(3,5-dichlorophenyl)maleamic acid (NDPMA) and N-(3,5-dichlorophenyl)maleimide (NDPM) to suspensions of renal proximal tubules (RPT) prepared from male Fischer 344 rats. Tubule viability and mitochondrial function were not adversely affected by exposure of RPT to either 1 mM NDPS, NDHS, NDHSA, DMA, NDPSA, or NDPMA for 4 h. However, NDPM caused a concentration-(25-100 microM) and time-dependent (0.25-4 h) loss in basal and nystatin stimulated oxygen consumption and tubule viability. Investigations using isolated renal cortical mitochondria (RCM) showed that NDPM was a potent inhibitor of mitochondrial function. Isolated RCM respiring on pyruvate/malate and exposed to NDPM exhibited a concentration (25-100 microM) dependent decrease in state 3 and state 4 respiration. Inhibition of mitochondrial state 3 respiration by NDPM was mediated through site 1 of the respiratory chain. NDPM did not inhibit cytochrome c-cytochrome oxidase or the electron transport chain. These results indicated that NDPS, its known metabolites, and NDPMA were not directly toxic to rat RPT. However, the postulated metabolite NDPM, was a potent tubule cytotoxicant that inhibited mitochondrial function in isolated RCM and RPT and may produce cell death through this mechanism.

Differences in enzymatic and mechanical isolated rabbit renal proximal tubules: comparison in long-term incubation.

Suspensions of renal proximal tubules (RPT) are the in vitro model for many biochemical and physiologic investigations. Inasmuch as there are numerous procedures for tubule isolation and the more commonly used enzymatic procedures may disrupt the basement membrane, there is a need for information comparing the influence of various isolation methods on RPT viability and function in long-term suspension. Rabbit RPT isolated a) enzymatically (ENZ) by in vitro collagenase digestion and Percoll size and density purification, and b) mechanically (MECH) by in vitro iron oxide perfusion and purification by sieving and magnetic removal of glomeruli were compared for viability, morphology, and functional stability during long-term suspension. RPT isolated by ENZ and MECH methods had excellent viability (less than 15% lactate dehydrogenase release), limited lipid peroxidation (less than 0.2 nmol MDA.mg protein-1), and stable nystatin-stimulated oxygen consumption (QO2) (38 and 36 nmol O2.mg protein-1.min-1) throughout 24 h of incubation. Basal QO2 was higher in ENZ than MECH tubules (27 and 19 nmol O2.mg protein-1.min-1, respectively), and was unchanged over 24 h in each preparation. The higher basal QO2 in ENZ tubules was ouabain-sensitive, suggesting an increased rate of Na+,K(+)-ATPase activity in these tubules. Total glutathione content (oxidized + reduced) in ENZ and MECH tubules increased over the 24-h incubation from 8 to 18 nmol.mg protein-1. gamma-Glutamyltranspeptidase (GGT) activity of the RPT homogenates was equivalent in both preparations and stable over time. The ratio of suspension GGT activity to homogenate GGT activity doubled (0.4 to 0.8) during the incubation period. MECH tubules retained their tubule structure during 24 h of incubation whereas the ENZ tubules had a striking loss of tubular morphology over time. These results show that ENZ- and MECH-isolated renal proximal tubule suspensions exhibit similar biochemical properties in long-term incubations but differ in ouabain-sensitive QO2 and the retention of tubular morphology. The loss of tubular morphology and the increase in the rate of Na+,K(+)-ATPase activity in ENZ tubules may be secondary to the disruption of the tubular basement membrane.

Primary cultures of rabbit renal proximal tubule cells: II. Selected phase I and phase II metabolic capacities.

Specific characteristics of cells vary as a function of time in culture. We have determined the stability of selected Phase I and Phase II biotransformation capacities in rabbit renal proximal tubule cells in primary culture. When grown in hormonally-defined medium, proximal tubule cells lost Phase I metabolic capacity. Cytochrome P-450 content and associated mixed-function oxidase activities present in kidney cortex microsomes were not detectable after 14 days in culture. Phase II glutathione-dependent metabolic functions were well retained in cultured cells compared with freshly isolated proximal tubules (FIPT). Cellular total glutathione content was 2.8 mug/mg protein in FIPT compared with approximately 10 mug/mg protein in stable confluent cultures. A higher total glutathione content of 20.6 mug/mg was noted in preconfluent cultures. The glutathione redox state was initially perturbed in FIPT with 37% of the total glutathione present found in its oxidized form. Tubule cells recovered to a normal ratio (6-13% of total glutathione in the oxidized form) while in culture. The glutathione S-transferase activity in 4-day-old cells in culture was reduced to 50% of the 4 U/mg protein level found in FIPT. No appreciable further decline in glutathione S-transferase activity was detected during 15 days in culture. The level of gamma-glutamyl-transpeptidase (a brush-border enzyme necessary for glutathione uptake into proximal tubule cells) declined from 1499 mU/mg protein in homogenates of FIPT to 636 mU/mg in homogenates of 8-day-old cultured cells. A further decline in activity occurred during the next 7 days in culture. In conclusion, although Phase I metabolic functions were diminished in primary cultured rabbit proximal tubule cells, Phase II metabolic functions were retained at levels comparable with FIPT and well above those found in several established kidney cell lines.

Primary cultures of rabbit renal proximal tubule cells: I. Growth and biochemical characteristics.

Before the usefulness of a new in vitro model can be ascertained, the model must be properly defined and characterized. This study presents the growth rate and biochemical characteristics of rabbit renal proximal tubule cells in primary culture over a 2-wk culture period. When grown in a hormonally defined, antibiotic-free medium these cells form confluent monolayer cultures within 7 d after plating. Multicellular dome formation, an indicator of transepithelial solute transport, was expressed after confluent cultures were formed. The activity of the cytosolic enzyme, lactate dehydrogenase, and the lysosomal enzyme, N-acetyl-glucosaminidase, increased 14- and 2-fold during the first 8 d of culture, respectively. In contrast, the activity of a brush border enzyme, alkaline phosphatase, decreased 85% within the first 8 d of culture. Release of these enzyme markers into the culture medium, which are routinely used to measure cytotoxicity, stabilized after 8 d in culture. The ratio of cellular protein to DNA changed according to the state of cellular growth. Values rose from 0.035 mg protein/micrograms DNA in preconfluent cultures to 0.059 mg protein/micrograms DNA in confluent cultures. These results document the characteristics of a primary proximal tubule cell culture system for future studies in in vitro toxicology.

Cross-tolerance between ethanol and chlordiazepoxide.

Drug-induced hypothermia was used to investigate drug tolerance and cross-tolerance. C57BL/6J mice, which were injected with a single dose of chlordiazepoxide (CDP; 30 mg/kg) one day before and reinjected with an equivalent dose of CDP the next day, did not develop tolerance to the drug. However, ethanol-pretreated (3.5 g/kg, 24 hr earlier) mice, when injected with CDP (30 mg/kg), showed cross-tolerance to CDP. The cross-tolerance was short-lived (less than 48 hr). On the other hand, CDP-pretreated mice (30 mg/kg, 24 hr earlier) did not show cross-tolerance to ethanol. The lack of a reciprocating effect of CDP-pretreatment was not likely to be due to the difference in initial dosage between ethanol and CDP. It may be due to different rates of tolerance development or different mechanisms of actions between CDP and ethanol. Mice chronically treated with ethanol also showed a similar degree of cross-tolerance to CDP compared to those exposed to an acute dose of ethanol.

Nonaqueous reverse phase liquid chromatographic analysis for cholesterol in milk chocolate.

A method using liquid chromatography was developed for the analysis of cholesterol in milk chocolate products. The method involves saponification of the sample with methanolic KOH followed by extraction with ether. Potentially interfering components are eliminated through the use of a silica Sep-Pak cleanup step before injection. The nonaqueous reverse phase LC system consists of a C18 column and an isopropanol-hexane mobile phase with direct detection at 205 nm. Recoveries of 1, 3, and 5 mg cholesterol added to 1 g sample of milk chocolate were 88.6, 102.8, and 110.1%, respectively. Studies conducted with [4-14C]-cholesterol were undertaken to further document the accuracy of the method.

High performance liquid chromatographic analysis of cholesterol in milk.

A method was developed for analysis of cholesterol in whole milk powder and commercial liquid milk products. High performance liquid chromatography employed a nonaqueous reversed phase chromatographic system. Sample preparation was by saponification with methanolic potassium hydroxide followed by extraction with ether. Potential interfering substances were removed prior to injection by a simple clean-up procedure using a silica gel Sep-pakTM. The chromatographic system included a carbon-18 column with a hexane/isopropanol mobile phase and detection at 205 nm. Method validation and recovery studies using carbon-14 labeled cholesterol indicated excellent performance.