Amphiregulin stimulates liver regeneration after small-for-size mouse liver transplantation.

This study investigated whether amphiregulin (AR), a ligand of the epidermal growth factor receptor (EGFR), improves liver regeneration after small-for-size liver transplantation. Livers of male C57BL/6 mice were reduced to ~50% and ~30% of original sizes and transplanted. After transplantation, AR and AR mRNA increased in 50% but not in 30% grafts. 5-Bromodeoxyuridine (BrdU) labeling, proliferating cell nuclear antigen (PCNA) expression and mitotic index increased substantially in 50% but not 30% grafts. Hyperbilirubinemia and hypoalbuminemia occurred and survival decreased after transplantation of 30% but not 50% grafts. AR neutralizing antibody blunted regeneration in 50% grafts whereas AR injection (5 µg/mouse, iv) stimulated liver regeneration, improved liver function and increased survival after transplantation of 30% grafts. Phosphorylation of EGFR and its downstream signaling molecules Akt, mTOR, p70S6K, ERK and JNK increased markedly in 50% but not 30% grafts. AR stimulated EGFR phosphorylation and its downstream signaling pathways. EGFR inhibitor PD153035 suppressed regeneration of 50% grafts and largely abrogated stimulation of regeneration of 30% grafts by AR. AR also increased cyclin D1 and cyclin E expression in 30% grafts. Together, liver regeneration is suppressed in small-for-size grafts, as least in part, due to decreased AR formation. AR supplementation could be a promising therapy to stimulate regeneration of partial liver grafts.

Vitamin E succinate enhances steatotic liver energy status and prevents oxidative damage following ischemia/reperfusion.

We have previously shown that treatment of steatotic livers with vitamin E succinate decreases liver injury and increases survival after ischemia/reperfusion (I/R). It is now understood that compromised energy status is associated with increased injury following liver ischemia in the setting of hepatic steatosis at least partially as a result of increased reactive oxygen species (ROS) and induction of mitochondrial uncoupling protein-2 (UCP2). Given the association between ROS, mitochondrial function, and UCP2, it was our goal to determine whether the protective effects of vitamin E succinate were associated with decreased ROS injury, down-regulation of UCP2, or improvement of ATP levels following I/R. To test this, leptin deficient (ob/ob) mice with steatotic livers that had received other 50 IU of vitamin E succinate supplement per day or control chow for 7 days were subjected to total hepatic ischemia (15 minutes) followed by reperfusion. We measured liver expressions of ATP, glutathione (GSH), and UCP2 as well as mitochondrial DNA damage. Vitamin E treatment decreased hepatic UCP2 expression and increased ATP and GSH levels prior to I/R. These levels were maintained at 1 hour after I/R. At 24 hours, while hepatic UCP2 expression, ATP, and GSH levels were similar to those of mice not receiving vitamin E, mitochondrial DNA damage was blocked. These results revealed that vitamin E succinate decreased hepatic UCP2 expression, reduced oxidative stress, and improved mitochondrial function in mice with steatotic livers before and after I/R, identifying mechanisms of protection in this setting.

Vitamin E succinate reduces ischemia/reperfusion injury in steatotic livers.

Steatotic livers represent a growing proportion of marginal organs available for transplantation. These livers are highly prone to primary nonfunction following transplantation and are therefore routinely turned down for surgery. Given the elevated levels and sensitivity for reactive oxygen species (ROS) in these livers, we evaluated whether pretreatment with a targeted ROS scavenger, vitamin E succinate, increased survival and decreased injury after ischemia/reperfusion (I/R). For this study, ob/ob mice received 50 IU/d vitamin E succinate in supplemented vs control chow for 7 days, and were subjected to 15 minutes of total hepatic ischemia and 24 hours of reperfusion. Treatment resulted in a 5-fold decrease in serum alanine aminotransferase (ALT) levels after reperfusion, mirrored by significant decreases in hepatocellular necrosis. These results suggested that targeted antioxidants such as vitamin E succinate may prove to be highly applicable for the pretreatment of steatotic donor livers, increasing their tolerance for I/R and the transplantation process.

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.

Interactions between collagen IV and collagen-binding integrins in renal cell repair after sublethal injury.

Recent studies demonstrate that collagen IV selectively promotes the repair of physiological processes in sublethally injured renal proximal tubular cells (RPTC). We sought to further define the mechanisms of cell repair by measuring the effects of toxicant injury and stimulation of repair by L-ascorbic acid-2-phosphate (AscP), exogenous collagen IV, or function-stimulating integrin antibodies on the expression and subcellular localization of collagen-binding integrins (CBI) in RPTC. Expression of CBI subunits alpha1, alpha2, and beta1 in RPTC was not altered on day 1 after sublethal injury by S-(1,2-dichlorovinyl)-L-cysteine (DCVC). On day 6, expression of alpha1 and beta1 subunits remained unchanged, whereas a 2.2-fold increase in alpha2 expression was evident in injured RPTC. CBI localization in control RPTC was limited exclusively to the basal membrane. On day 1 after injury, RPTC exhibited a marked inhibition of active Na(+) transport and a loss of cell polarity characterized by a decrease in basal CBI localization and the appearance of CBI on the apical membrane. On day 6 after injury, RPTC still exhibited marked inhibition of active Na(+) transport and localization of CBI to the apical membrane. However, DCVC-injured RPTC cultured in pharmacological concentrations of AscP (500 microM) or exogenous collagen IV (50 microg/ml) exhibited an increase in active Na(+) transport, relocalization of CBI to the basal membrane, and the disappearance of CBI from the apical membrane on day 6. Function-stimulating antibodies to CBI beta1 did not promote basal relocalization of CBI despite stimulating the repair of Na(+)/K(+)-ATPase activity on day 6 after injury. These data demonstrate that DCVC disrupts integrin localization and that physiological repair stimulated by AscP or collagen IV is associated with the basal relocalization of CBI in DCVC-injured RPTC. These data also suggest that CBI-mediated repair of physiological functions may occur independently of integrin relocalization.

Calpains mediate acute renal cell death: role of autolysis and translocation.

The goals of this study were to determine 1) the expression of calpain isoforms in rabbit renal proximal tubules (RPT); 2) calpain autolysis and translocation, and calpastatin levels during RPT injury; and 3) the effect of a calpain inhibitor (PD-150606) on calpain levels, mitochondrial function, and ion transport during RPT injury. RT-PCR, immunoblot analysis, and FITC-casein zymography demonstrated the presence of only mu- and m-calpains in rabbit RPT. The mitochondrial inhibitor antimycin A decreased RPT mu- and m-calpain and calpastatin levels in conjunction with cell death and increased plasma membrane permeability. No increases in either mu- or m-calpain were observed in the membrane nor were increases observed in autolytic forms of either mu- or m-calpain in antimycin A-exposed RPT. PD-150606 blocked antimycin A-induced cell death, preserved calpain levels in antimycin A-exposed RPT, and promoted the recovery of mitochondrial function and active Na+ transport in RPT after hypoxia and reoxygenation. The present study suggests that calpains mediate RPT injury without undergoing autolysis or translocation, and ultimately they leak from cells subsequent to RPT injury/death. Furthermore, PD-150606 allows functional recovery after injury.

Collagen IV promotes repair of renal cell physiological functions after toxicant injury.

Collagen IV is found in the renal proximal tubular cell (RPTC) basement membrane and is a mediator of renal development and function. Pharmacological concentrations of L-ascorbic acid phosphate (AscP) promote the repair of physiological functions in RPTC sublethally injured by S-(1,2-dichlorovinyl)-L-cysteine (DCVC). We hypothesized that AscP promotes RPTC repair by stimulating collagen IV synthesis and/or deposition. RPTC exhibited increased synthesis but decreased deposition of collagen IV after DCVC exposure. In contrast, RPTC cultured in pharmacological concentrations of AscP maintained collagen IV deposition. The activity of prolyl hydroxylase was decreased in RPTC after DCVC injury, an effect that was partially attenuated in injured RPTC cultured in pharmacological concentrations of AscP. The addition of exogenous collagen IV to the culture media of DCVC-injured RPTC promoted the repair of mitochondrial function and Na(+)-K(+)-ATPase activity. However, neither collagen I, laminin, nor fibronectin promoted cell repair. These data demonstrate an association between AscP-stimulated deposition of collagen IV and exogenous collagen IV and repair of physiological functions, suggesting that collagen IV plays a specific role in RPTC repair after sublethal injury.

Progressive disruption of the plasma membrane during renal proximal tubule cellular injury.

The goal of this study was to examine the progression of plasma membrane disruption during cell injury using rabbit renal proximal tubules (RPT). The results demonstrated that the plasma membrane became permeable to larger and larger molecules as anoxia proceeded. At least three distinctive phases of membrane disruption were differentiated during anoxia. In phases 1, 2, and 3, plasma membranes became permeable to propidium iodide (PI, molecular weight = 668), 3 kDa dextrans, and 70 kDa dextrans or lactate dehydrogenase (LDH, molecular weight = 140 kDa), respectively. Phase 1 was reversible by reoxygenation but not prevented by the glycine. Phase 2 was inhibited by glycine. Phase 3 was inhibited by several membrane-permeable homobifunctional crosslinkers, dimethyl-pimelimidate (DMP), ethylene-glycolbis(succinimidylsuccinate), and dithiobis(succinimidylpropionate), but not by the membrane-impermeable crosslinker dithiobis(sulfosuccinimidylpropionate). In addition, DMP decreased RPT LDH release produced by mitochondrial inhibition (antimycin A), an oxidant (t-butylhydroperoxide) and a nephrotoxicant that is metabolized to an electrophile (tetrafluoroethyl-l-cysteine). These results identify (1) different phases of plasma membrane damage with increasing permeability during cell injury, (2) the reversibility of phase 1, (3) the relative site of action of the cytoprotectant glycine (prevents phase 2), and (4) the protective effects of chemical crosslinkers in RPT cell death produced by different toxicants.

Analysis of the cytotoxic properties of linoleic acid metabolites produced by renal and hepatic P450s.

Cytochrome P450 epoxidation of linoleic acid produces biologically active metabolites which have been associated with many pathological conditions that often lead to acute renal failure. In the present study, we evaluated the ability of specific cytochrome P450s to produce linoleic acid monoepoxides. We then tested the cytotoxic properties of linoleic acid, linoleic acid monoepoxides, and corresponding diols in a rabbit renal proximal tubule model. CYP1A2, CYP2E1, CYP2J2, CYP2J3, CYP2J5, and CYP2J9 metabolized linoleic acid at rates comparable to arachidonic acid and produced linoleic acid monoepoxides as major products. Cytotoxicity studies showed that linoleic acid, linoleic acid monoepoxides, and corresponding diols are toxic at pathologically relevant concentrations (100-500 microM). Concentration-dependent studies showed that linoleic acid and linoleic acid monoepoxides are the most toxic and induce mitochondrial dysfunction prior to cell death. Cytoprotectants known to block cell death associated with mitochondrial dysfunction and oxidative stress did not prevent cell death induced by linoleic acid and linoleic acid monoepoxides. This study shows that P450s in the CYP1 and CYP2 gene families metabolize linoleic acid to linoleic acid monoepoxides and that the monoepoxides, as well as linoleic acid, disrupt mitochondrial function without causing oxidative stress.

Phospholipase A(2)s in cell injury and death.

Phospholipase A(2)s (PLA(2)s) represent a family of esterases that hydrolyze the sn-2 ester bond in phospholipids, releasing free fatty acids and lysophospholipids. PLA(2)s are important in the signaling of several cellular processes and are known to play a significant role in inflammation. Studies also show that PLA(2)s are modulators of drug-, chemical-, and ischemia/reperfusion-induced cellular injury. The role of PLA(2)s in apoptosis and oncosis depends upon the PLA(2) isoform, the cell type, and the stimulus of injury. The purpose of this review is to discuss the functions of iPLA(2), cPLA(2) and sPLA(2) isoforms in oncosis and apoptosis, including oxidant-induced and receptor-mediated cell death. In addition, the measurement and modulation of PLA(2) is discussed.

Efficacy of novel calpain inhibitors in preventing renal cell death.

Inhibitors of calpains, calcium-activated neutral proteases, protect against cell death produced by anoxia and a variety of toxicants both in vitro and in vivo. The problems with known calpain inhibitors are a lack of specificity, low membrane permeability, and/or low potency. The goal of this study was to determine the effects of seven novel dipeptide and tripeptide calpain inhibitors on calpain activity and antimycin A-induced cell death in rabbit renal proximal tubule (RPT) suspensions. We chose the compounds based on their inhibitory constants for mu- versus m-calpain, specificity of the inhibitors for calpain, and membrane permeability. Only three of the compounds inhibited calpain in RPT and were cytoprotective (Z-Leu-Phe-COOH, Z-Leu-Abu-CONH-CH(2)-CH(OH)-Ph, and Z-Leu-Phe-CONH-Et). Interestingly, Z-Leu-Phe-COOEt, Z-Leu-Abu-CONH-CH(2)-CH(OH)-C(6)F(5), and Z-Leu-Abu-CONH-CH(2)-2-quinolinyl were greater than 60% cytoprotective but did not inhibit calpain in RPT. Z-Leu-Abu-CONH(CH(2))(3)-morpholine was neither cytoprotective nor inhibited calpain. Although these results suggest that six of the seven peptide calpain inhibitors are cell permeable, only three of them inhibited calpain activity in RPT and were cytoprotective. Their ability to inhibit calpain or produce cytoprotection did not correlate with their ability to selectively inhibit purified mu- or m-calpain. Thus it remains to be determined whether they inhibit mu-calpain, m-calpain, or both in RPT. These results also suggest that inhibition of other protease(s) in addition to calpains may be responsible for the cytoprotective actions of some compounds.

Ascorbic acid promotes recovery of cellular functions following toxicant-induced injury.

We have shown that renal proximal tubular cells (RPTC) recover cellular functions following sublethal injury induced by the oxidant t-butylhydroperoxide but not by the nephrotoxic cysteine conjugate dichlorovinyl-L-cysteine (DCVC). This study investigated whether L-ascorbic acid phosphate (AscP) promotes recovery of RPTC functions following DCVC-induced injury. DCVC exposure (200 microM; 100 min) resulted in 60% RPTC death and loss from the monolayer at 24 h independent of physiological (50 microM) or pharmacological (500 microM) AscP concentrations. Likewise, the DCVC-induced decrease in mitochondrial function (54%), active Na(+) transport (66%), and Na(+)-K(+)-ATPase activity (77%) was independent of the AscP concentration. Analysis of Na(+)-K(+)-ATPase protein expression and distribution in the plasma membrane using immunocytochemistry and confocal laser scanning microscopy revealed the loss of Na(+)-K(+)-ATPase protein from the basolateral membrane of RPTC treated with DCVC. DCVC-injured RPTC cultured in the presence of 50 microM AscP did not proliferate nor recover their physiological functions over time. In contrast, RPTC cultured in the presence of 500 microM AscP proliferated, recovered all examined physiological functions, and the basolateral membrane expression of Na(+)-K(+)-ATPase by day 4 following DCVC injury. These results demonstrate that pharmacological concentrations of AscP do not prevent toxicant-induced cell injury and death but promote complete recovery of mitochondrial function, active Na(+) transport, and proliferation following toxicant-induced injury.

Contribution of direct solvent injury to the dose-dependent kinetics of trichloroethylene: portal vein administration to rats.

Presystemic elimination of trichloroethylene (TCE), a common contaminant of drinking water, has been shown by Lee et al. (Toxicol. Appl. Pharmacol. 139, 262-271, 1996) to be inversely related to dose. When relatively high doses were administered to rats via the portal vein (PV), first-pass hepatic extraction became negligible. This phenomenon could result not only from metabolic saturation, but from suicidal destruction of cytochromes P450 and hepatocellular injury as well. The objectives of the current investigation were to: (a) clarify the relative roles of P450 depletion and hepatocellular toxicity in the apparent cessation of hepatic elimination of TCE in animals given relatively high doses of TCE via the PV; and (b) investigate mechanism(s) of hepatocellular injury under such exposure conditions. TCE (16 and 64 mg/kg body weight (bw) was incorporated into a 5% aqueous Alkamuls emulsion and injected via an indwelling jugular vein (JV) or PV cannula into male Sprague-Dawley rats. Some animals received 73.5 micromol/kg of p-nitrophenol (PNP), a competitive metabolic inhibitor of TCE, through the PV cannula 3 min before TCE. Administration of TCE via the PV resulted in deposition of relatively high levels of TCE in the liver. PV dosing resulted in lower total hepatic P450 levels than did JV dosing. PV dosing produced marked elevations of cytoplasmic enzymes in serum, but JV dosing did not. Decreases in hepatic P450 were not selective for cytochrome P4502E1. Histological examination of the liver of PV-dosed rats revealed periportal rather than centrilobular necrosis. PNP pretreatment failed to prevent the increase in serum enzymes, decrease in hepatic P450 content, and hepatic necrosis following PV TCE. It is concluded that PV injection of bolus doses of TCE >/= 16 mg/kg causes liver injury within minutes in rats, primarily through direct solvent action on hepatocellular membranes rather than by P450-mediated effects. This liver damage likely plays a modest role in reducing the liver's capacity to metabolize high PV doses of TCE.

Expression and localization of the neuronal glycine receptor beta-subunit in human, rabbit and rat kidneys.

The glycine receptor (GlyR) is a ligand-gated Cl- channel composed of two transmembrane subunits, alpha and beta, and gephyrin. The goal of this study was to determine whether the alpha- and/or beta-subunits of the GlyR are expressed in human, rabbit and/or rat kidneys. Screening of human and rat kidney cortex cDNA libraries identified polymerase chain reaction products that were identical to the neuronal GlyR beta-subunit. Sequencing revealed that rat kidney cortex and neuronal GlyR beta-subunits were identical. RNA isolated from the S2 segment of rabbit renal proximal tubules (RPT) and rat and rabbit kidney cortex was amplified following reverse transcription and gave similar results to that of human and rat kidney cDNA libraries. Degenerate primers against all GlyR alpha-subunits did not yield a product from rat and rabbit kidney cortex RNA, or from human and rat kidney cortex cDNA libraries. Immunofluorescence studies localized the beta-subunit and gephyrin to the basolateral membrane of rabbit RPT. These results provide compelling evidence for the GlyR beta-subunit, but not the alpha-subunit, in human, rabbit and rat kidney cortex.

Differential effects of EGF on repair of cellular functions after dichlorovinyl-L-cysteine-induced injury.

This study examined the repair of renal proximal tubule cellular (RPTC) functions following sublethal injury induced by the nephrotoxicant S-(1,2-dichlorovinyl)-L-cysteine (DCVC). DCVC exposure resulted in 31% cell death and loss 24 h following the treatment. Monolayer confluence recovered through migration/spreading but not proliferation after 6 days. Basal, uncoupled, and ouabain-sensitive oxygen consumption (QO2) decreased 47, 76, and 62%, respectively, 24 h after DCVC exposure. Na+-K+-ATPase activity and Na+-dependent glucose uptake were inhibited 80 and 68%, respectively, 24 h after DCVC exposure. None of these functions recovered over time. Addition of epidermal growth factor (EGF) following DCVC exposure did not prevent decreases in basal, uncoupled, and ouabain-sensitive QO2 values and Na+-K+-ATPase activity but promoted their recovery over 4-6 days. In contrast, no recovery of Na+-dependent glucose uptake occurred in the presence of EGF. These data show that: 1) DCVC exposure decreases mitochondrial function, Na+-K+-ATPase activity, active Na+ transport, and Na+-dependent glucose uptake in sublethally injured RPTC; 2) DCVC-treated RPTC do not proliferate nor regain their physiological functions in this model; and 3) EGF promotes recovery of mitochondrial function and active Na+ transport but not Na+-dependent glucose uptake. These results suggest that cysteine conjugates may cause renal dysfunction, in part, by decreasing RPTC functions and inhibiting their repair.

Proteases in renal cell death: calpains mediate cell death produced by diverse toxicants.

The role of proteases in renal cell death has received limited investigation. Calpains are non-lysosomal cysteine proteases that are Ca+2 activated. Calpain inhibitors that block the active site of calpains (calpain inhibitor 1 and 2) or the Ca+2 binding domain of calpains (PD150606) decreased calpain activity in rabbit renal proximal tubule (RPT) suspensions. The inhibition of calpain activity decreased cell death produced by the diverse toxicants antimycin A (mitochondrial inhibitor), tetrafluroethyl-L-cysteine (nephrotoxic halocarbon), bromohydroquinone (nephro-toxic quinone), t-butylhydroperoxide (model oxidant) and ionomycin (Ca+2 ionophore). In summary, calpains appear to play a common and critical role in cell injury produced by diverse toxicants with different mechanisms of action. The general cysteine protease inhibitor trans-epoxysuccinyl-L-leucylamido (4-guanidino)-butane (E-64) decreased antimycin A- and tetrafluoroethyl-L-cysteine-induced cell death but had no effect on bromohydroquinone- or t-butylhydroperoxide-induced cell death. Serine/cysteine protease inhibitors (antipain, leupeptin) were not cytoprotective to RPT exposed to any of the toxicants. The cytoprotection associated with E-64 correlated with inhibition of lysosomal cathepsins and E-64 was only cytoprotective after some cell death had occurred. Since some cell death occurred prior to the E-64 cytoprotective effect, lysosomal cathepsins may be released from dying cells and subsequently target the remaining viable cells.

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.

Analgesic nephropathy in rodents.

While it is clear that humans suffer from "classic" analgesic nephropathy, the causative agents and mechanisms are still not known. A review of the literature revealed that chronic acetaminophen exposure does not produce renal papillary necrosis in rodents or humans. In contrast, while chronic aspirin exposure to rodents results in renal papillary necrosis with renal morphological and functional changes similar to that described in humans, epidemiological studies do not implicate aspirin alone in human analgesic nephropathy. The difference in the effects of aspirin in humans and rats may be due to the inability of epidemiological studies to detect aspirin-induced analgesic nephropathy or more likely to the fact that species differences exist, with the rat being more sensitive than humans. With respect to combinations of aspirin and acetaminophen, with or without caffeine, there are minimal tightly controlled studies. In addition, there is little evidence of enhanced renal papillary necrosis in rodents treated with aspirin and acetaminophen combinations. In summary, it remains to be determined what chemical entities cause "classic" analgesic nephropathy in humans and the mechanisms of this toxicity such that preventative measures can be instituted. Elucidation of the mechanisms of analgesic nephropathy has been hampered due to the lack of animal models that closely mimic the human disease. Rodents do not appear to be an appropriate model.

Examination of the mechanisms of action of diverse cytoprotectants in renal cell death.

Glycine, strychnine, muscimol, allopregnanolone, and pregnenolone sulfate act in the late phase of renal cell injury, block Cl- influx and cell lysis induced by the mitochondrial inhibitor antimycin A, and promote the recovery of respiration and ion transport following hypoxia/reoxygenation. However, the mechanism of action of these compounds has not been completely elucidated. Recently, we have shown that calpains are critical mediators of renal cell death produced by diverse toxicants and that antimycin A exposure results in calpain translocation from the cytosol to the membrane fraction that is temporally associated with Cl- influx and precedes cell death/lysis. The current study examined the effects of a group of diverse cytoprotectants on calpain activity and determined if calpain inhibition plays a role in the cytoprotection produced by these compounds. The cytoprotection produced by glycine, strychnine, muscimol, allopregnanolone, and pregnenolone sulfate in rabbit renal proximal tubules exposed to antimycin A was associated with the inhibition of antimycin A-induced calpain translocation. None of the cytoprotectants had a direct effect on calpain activity. All of the cytoprotectants decreased calcium-ionophore-induced cell death. Glycine, strychnine, and muscimol also blocked antimycin A mediated extracellular Ca2+ influx. These data suggest that the cytoprotective mechanism of action of glycine, strychnine, and muscimol involves the inhibition of antimycin A mediated extracellular Ca2+ influx as well as calpain translocation and associated Cl- influx. In contrast, the mechanism of action of the neurosteroids results only from the blockade of calpain translocation and associated Cl- influx.

Stress response in a leporine renal cell model.

It is well established that renal proximal tubule (RPT) cells grown under standard in vitro conditions attenuate many of their in vivo properties and functions. Thus, the study of renal stress response mechanisms requires an appropriate cell culture model. In the present study, we compared the heat stress (10 min, 45 degrees C) response of freshly isolated RPT cells with that of RPT cells grown in vitro for 6 days under two different culture conditions: (1) SHAKE conditions, where oxygen levels and physiological functions are maintained via continuous media motion [Nowak G, Schnellmann RG: Am J Physiol 1996;271:C2072-2080] and (2) STILL conditions, involving standard cell culture which leads to partial hypoxia and a marked reduction in physiological functions. The freshly isolated RPT cells progressively synthesized heat shock proteins (HSPs) and stress glycoproteins (SGs) during a 3-hour culture period in vitro. Under these conditions, heat stress did not further increase HSP and SG synthesis. In RPT cells grown under SHAKE conditions, HSP70 synthesis was detected 1 h after heat stress and decreased below detection by 3 h. In contrast, the uptake of radiolabeled mannose into (glycoprotein) GP62 (Mr 62,000), GP50, and GP38 was observed in control SHAKE cultures and was not further increased after heat stress. These results are consistent with immunohistochemistry studies, where similar changes in HSP70 and GP50 expression were noted. RPT cells grown under STILL conditions showed both increased synthesis of HSP70 and increased glycosylation of GP62, GP50, and GP38 as early as 1 h after heat stress, but in contrast to SHAKE conditions, this heat-induced stress response further intensified at 3 h after heat stress. By 7 h after heating, HSP synthesis returned to control levels, while glycosylation of GP62 and GP50 remained elevated. Based on our results, we conclude that freshly isolated RPT cells exhibit a stress response that may be caused by acute cell isolation/culture stress. While this stress response unfolds, freshly isolated RPT cells appear unable to respond to additional heat stress. RPT cells grown under SHAKE and STILL conditions exhibit high rates of SG glycosylation, especially that of GP62, possibly reflecting a 'stress' condition associated with growth on plastic substrate. Concurrently, RPT cells from STILL cultures show a higher capacity for responding to acute heat stress than SHAKE cultures, evidenced by the transiently increased HSP synthetic rates. The interpretation of the renal stress response capacity, therefore, must be linked to a specific culture condition.