Biomedical subjects

Richard A Zager

Publications and source records attributed to Richard A Zager.

At least 19 recordsLinked to original sources

PubMed2006

Toll-like receptor (TLR4) shedding and depletion: acute proximal tubular cell responses to hypoxic and toxic injury.

Acute renal failure (ARF) induces tubular hyperresponsiveness to TLR4 ligands, culminating in exaggerated renal cytokine/chemokine production. However, the fate of TLR4 protein during acute tubular injury remains unknown. The study sought new insights into this issue. Male CD-1 mice were subjected to 1) unilateral ischemia-reperfusion (I/R), 2) cisplatin (CP) nephrotoxicity, or 3) glycerol-induced myohemoglobinuric ARF. Renal cortical TLR4 protein (Western blotting, immunohistochemistry) and TLR4 mRNA levels (RT-PCR) were determined thereafter (90 min-4 days). Urinary TLR4 excretion post-I/R or CP injection was also assessed. To gain proximal tubule-specific results, TLR4 protein and mRNA were quantified in posthypoxic or oxidant (Fe)-challenged isolated mouse tubules. Finally, TLR4 mRNA was determined in antimycin A-injured cultured proximal tubular (HK-2) cells. Acute in vivo renal injury reduced proximal tubule TLR4 content. These changes corresponded with the appearance of TLR4 fragment(s) in urine and a persistent increase in renal cortical TLR4 mRNA. Isolated proximal tubules responded to injury with rapid TLR4 reductions, dramatic extracellular TLR4 release, and increases in TLR4 mRNA. Glycine blocked these processes, implying membrane pore formation was involved. HK-2 cell injury increased TLR4 mRNA, but not protein levels, suggesting intact transcriptional, but not translational, pathways. Diverse forms of acute tubular injury rapidly reduce proximal tubular TLR4 content. Plasma membrane TLR4 release through glycine-suppressible pores, possibly coupled with a translation block, appears to be involved. Rapid postinjury urinary TLR4 excretion suggests its potential utility as a "biomarker" of impending ARF.

Animals↗

PubMed2006

Acute renal failure: determinants and characteristics of the injury-induced hyperinflammatory response.

Acute renal failure (ARF) markedly sensitizes mice to endotoxin (LPS), as evidenced by exaggerated renal cytokine/chemokine production. This study sought to further characterize this state by testing the following: 1) does anti-inflammatory heme oxygenase-1 (HO-1) upregulation in selected ARF models prevent this response? 2) Is the ARF hyperresponsive state specifically triggered by LPS? 3) Does excess iNOS activity/protein nitrosylation participate in this phenomenon? and 4) are upregulated Toll receptors involved? Mice with either 1) rhabdomyolysis-induced ARF (massive HO-1 overexpression), 2) cisplatin nephrotoxicity, 3) or HO-1 inhibition (Sn protoporphyrin) were challenged with either LPS (a TLR4 ligand), lipoteichoic acid (LTA; a TLR2 ligand), or vehicle. Two hours later, renal and plasma TNF-alpha/mRNA, MCP-1/mRNA, renal nitrotyrosine/iNOS mRNA, and plasma cytokines were assessed. Renal TLR4 was gauged by mRNA and Western blot analysis. Both ARF models markedly hyperresponded to both LPS and LTA, culminating in exaggerated TNF-alpha, MCP-1, and iNOS/nitrotryosine increments. This was despite the fact that HO-1 exerted anti-inflammatory effects. TLR4 levels were either normal (cisplatin), or markedly depressed ( approximately 50%; rhabdomyolysis) in the ARF kidneys, despite the LPS hyperresponsive state. 1) The ARF kidney can hyperrespond to chemically dissimilar Toll ligands; 2) HO-1 does not prevent this response; 3) excess NO/protein nitrosylation can result; and 4) this hyperresponsiveness can be expressed with either normal or reduced renal TLR4 expression. This suggests that diverse signaling pathways may be involved.

Acute Kidney Injury↗

PubMed2006

Levosimendan protects against experimental endotoxemic acute renal failure.

Endotoxemia induces a hemodynamic form of acute renal failure (ARF; renal vasoconstriction +/- reduced glomerular ultrafiltration coefficient, K(f); minimal/no histological damage). We tested whether levosimendan (LS), an ATP-sensitive K+ (K(ATP)) channel opener with cardiac ionotropic and possible anti-inflammatory properties, might have utility in combating this form of ARF. CD-1 mice were injected with LPS +/- LS. LS effects on LPS-induced systemic inflammation (plasma TNF-alpha/MCP-1; cardiorenal mRNAs), plasma NO levels, and azotemia were assessed. Because K(ATP) channel opening has been reported to mediate hypoxic tubular injury, possible adverse LS effects on ischemic ARF and ATP depletion injury were sought. Effects of diazoxide (another K(ATP) channel agonist) and glibenclamide (a channel antagonist) on hypoxic tubular injury also were assessed. Finally, the ability of LS to alter rat mesangial cell (MC) contraction in response to ANG II (elevated in sepsis) was tested. LS conferred almost complete protection against LPS-induced ARF, without any apparent reduction in the LPS-induced inflammatory response. Neither LS nor diazoxide altered ATP depletion-mediated tubule injury (in vivo or in vitro). Conversely, glibenclamide induced a marked and direct cytotoxic effect. LS completely blocked ANG II-induced MC contraction, an action likely to increase K(f). We concluded that 1) LS can confer marked protection against LPS-induced ARF; 2) this likely stems from vasoactive properties, rather than reductions in LPS-induced inflammation; and 3) K(ATP) channel agonists (but not antagonists) appear to be devoid of toxic proximal tubular cell effects. This suggests that LS, and other K(ATP) channel agonists, have a margin of safety if employed in situations (sepsis syndrome, heart failure) in which severe renal vasoconstriction might lead to ischemic ARF.

Acute Kidney Injury↗

PubMed2005

IGF-1 induces rat glomerular mesangial cells to accumulate triglyceride.

Rat glomerular mesangial cells (MC) become lipid-laden foam cells when they are exposed to IGF-1. IGF-1 increased accumulation of triglyceride (TG) 2.5-fold in MC after 7 days. TG accumulation resulted from enhanced macropinocytosis and decreased efflux secondary to a 40-50% reduction in peroxisome proliferator-activated receptor (PPAR)-delta (PPARdelta). There was no evidence of primary or secondary changes in cholesterol or TG synthesis, increased uptake by LDL or scavenger receptors, or reduced efflux via ATP-binding cassette A-1. Although the lipid moiety taken up can be influenced by the concentration of cholesterol or TG in the medium, in standard medium MC preferentially accumulate TG. TG-rich MC foam cells fail to contract in response to angiotensin II (Berfield AK, Andress DL, and Abrass CK. Kidney Int 62: 1229-1237, 2002); however, their migratory response to IGF binding protein-5 is unaffected. This differs from cholesterol loading, which impairs both phagocytosis and migration. These findings have important implications for understanding the mechanisms that contribute to lipid accumulation in MC and the functional consequences of different forms of foam cells. These observations are relevant to understanding vascular disease and progressive renal diseases that are accelerated by abnormalities in lipid metabolism.

Animals↗

PubMed2005

Renal tubular triglyercide accumulation following endotoxic, toxic, and ischemic injury.

BACKGROUND: Cholesterol accumulates in renal cortical proximal tubules in response to diverse forms of injury or physiologic stress. However, the fate of triglycerides after acute renal insults is poorly defined. This study sought new insights into this issue. METHODS: CD-1 mice were subjected to three diverse models of renal stress: (1) endotoxemia [Escherichia coli lipopolysaccharide (LPS), injection]; (2) ischemia/reperfusion (I/R); or (3) glycerol-induced rhabdomyolysis. Renal cortical, or isolated proximal tubule, triglyceride levels were measured approximately 18 hours later. To gain mechanistic insights, triglyceride levels were determined in (1) proximal tubules following exogenous phospholipase A(2) (PLA(2)) treatment; (2) cultured HK-2 cells after mitochondrial blockade (antimycin A) +/- serum; or (3) HK-2 cells following "septic" (post-LPS) serum, or exogenous fatty acid (oleate) addition. RESULTS: Each form of in vivo injury evoked three-to fourfold triglyceride increases in renal cortex and/or proximal tubules. PLA(2) treatment of proximal tubules evoked acute, dose-dependent, triglyceride formation. HK-2 cell triglyceride levels rose with antimycin A. With serum present, antimycin A induced an exaggerated triglyceride loading state (vs. serum alone or antimycin A alone). "Septic" serum stimulated HK-2 triglyceride formation (compared to control serum). Oleate addition caused striking HK-2 cell triglyceride accumulation. Following oleate washout, HK-2 cells were sensitized to adenosine triphosphate (ATP) depletion or oxidant attack. CONCLUSION: Diverse forms of renal injury induce dramatic triglyceride loading in proximal tubules/renal cortex, suggesting that this is a component of a cell stress response. PLA(2) activity, increased triglyceride/triglyceride substrate (e.g., fatty acid) uptake, and possible systemic cytokine (e.g., from LPS) stimulation, may each contribute to this result. Finally, in addition to being a marker of prior cell injury, accumulation of triglyceride (or of its constituent fatty acids) may predispose tubules to superimposed ATP depletion or oxidant attack.

Acute Kidney Injury↗

PubMed2005

Triglyceride accumulation in injured renal tubular cells: alterations in both synthetic and catabolic pathways.

BACKGROUND: Triglycerides can accumulate in injured tissues, a process thought to represent flux of excess, cytotoxic, free fatty acids into nontoxic triglyceride storage pools. However, this view may be overly simplistic, given that multiple pathways may impact triglyceride levels. This study sought new insights into this issue. METHODS: Cultured human proximal tubule [human kidney-2 (HK-2)] cells or in vivo kidney were subjected to injuries known to increase triglyceride levels approximately three- to fourfold [HK-2 cells antimycin A-induced mitochondrial blockade; in vivo glycerol-induced rhabdomyolysis; endotoxemia). Six reverse transcription-polymerase chain reactions (RT-PCRs) were used to monitor mouse/human mRNAs for renal fatty acid transport protein (FATP2), or triglyceride-synthesizing enzymes (acyl-coenzyme A:diacylglycerol acyltransferases DGAT1 and DGAT2). Fatty acid synthase (FAS) and FATP2 were gauged by Western blot. FAS, FATP2, mitochondrial respiration, and phospholipase A2 (PLA(2)) effects on cell triglyceride accumulation were probed. Finally, tissue lipase activity was assessed. RESULTS: Antimycin A up-regulated multiple determinants of HK-2 cell triglyceride formation, including FATP2, FAS, DGAT1, and DGAT2 (proteins and/or mRNAs). However, neither FAS- nor FATP2-inhibition eliminated antimycin A-induced triglyceride loading, indicating the latter's multifactorial basis. PLA(2) activity increased FFA and triglyceride content. Rhabdomyolysis and endotoxemia altered multiple triglyceride homeostatic mechanisms. However, these changes were model-dependent and did not closely parallel those in HK-2 cells. Lipase activity signficantly fell (glycerol) or rose (endotoxemia) with different forms of tissue damage. CONCLUSION: Injury-induced triglyceride accumulation stems from multiple, and disease-specific, changes in triglyceride synthetic and degradative pathways. Simple flux of excess FFAs into triglyceride pools is an overly simplistic view of the post-injury-triglyceride loading state.

Acute Kidney Injury↗

PubMed2005

Parenteral iron treatment induces MCP-1 accumulation in plasma, normal kidneys, and in experimental nephropathy.

INTRODUCTION: Monocyte chemoattractant protein-1 (MCP-1) promotes renal inflammation, thereby contributing to acute and chronic nephropathies. Its production is stimulated by oxidative stress. Thus, this study tested whether pro-oxidant iron/carbohydrate complexes, used to treat iron deficiency, induce MCP-1 in renal/extrarenal tissues, in plasma, and in the setting of experimental nephropathy. METHODS: CD-1 mice received 2 mg of intravenous iron [complexed with dextran (iron dextran), sucrose (iron sucrose), or gluconate (iron gluconate)]. Renal MCP-1 and/or its mRNA were measured 3 hours to 7 days post-iron injection. Iron effects on liver, lung, spleen, and heart MCP-1 mRNA, and on peritoneal lavage fluid MCP-1 concentrations were assessed. Iron pretreatment effects on MCP-1 levels in unilaterally obstructed kidneys vs. contralateral kidneys were determined. Finally, iron gluconate's influence on proximal tubule [human kidney-2 (HK-2)] cell MCP-1 levels was assessed. RESULTS: Iron sucrose (the primary test agent) markedly increased plasma and renal MCP-1 levels. It also induced multiorgan MCP-1 mRNA increments (liver > spleen > kidney > lung > heart). Iron gluconate was more potent than iron sucrose; conversely, iron dextran had no discernible effect. The iron dextran and iron sucrose-induced renal MCP-1 mRNA increments ( approximately 4x) were persistent, lasting for at least 3 to 7 days. Iron gluconate raised MCP-1 levels in peritoneal lavage fluid. It also doubled MCP-1 in unilaterally obstructed kidneys (ureteral ligation) without altering contralateral (control kidney) MCP-1 content. Iron gluconate raised HK-2 cell MCP-1, implying a direct proximal tubule effect. CONCLUSION: Iron sucrose and iron gluconate (but not iron dextran) can induce MCP-1 generation in renal and extrarenal tissues, possibly via transcriptional events. This may dramatically impact renal disease-induced MCP-1 increments. Finally, iron can increase peritoneal lavage fluid MCP-1 levels. Whether the above changes have implications for renal disease progression, and/or for peritoneal inflammation/peritoneal dialysis efficiency, are issues which may need to be addressed.

Acute Disease↗

PubMed2004

Parenteral iron compounds sensitize mice to injury-initiated TNF-alpha mRNA production and TNF-alpha release.

Intravenous Fe is widely used to treat anemia in renal disease patients. However, concerns of potential Fe toxicity exist. To more fully define its spectrum, this study tested Fe's impact on systemic inflammation following either endotoxemia or the induction of direct tissue damage (glycerol-mediated rhabdomyolysis). The inflammatory response was gauged by tissue TNF-alpha message expression and plasma TNF-alpha levels. CD-1 mice received either intravenous Fe sucrose, -gluconate, or -dextran (FeS, FeG, or FeD, respectively; 2 mg), followed by either endotoxin (LPS) or glycerol injection 0-48 h later. Plasma TNF-alpha was assessed by ELISA 2-3 h after the LPS or glycerol challenge. TNF-alpha mRNA expression (RT-PCR) was measured in the kidney, heart, liver, lung, and spleen with Fe +/- LPS treatment. Finally, the relative impacts of intramuscular vs. intravenous Fe and of glutathione (GSH) on Fe/LPS- induced TNF-alpha generation were assessed. Each Fe preparation significantly enhanced LPS- or muscle injury-mediated TNF-alpha generation. This effect was observed for at least 48 h post-Fe injection, a time at which plasma iron levels were increased by levels insufficient to fully saturate transferrin. Fe did not independently increase plasma TNF-alpha or tissue mRNA. However, it potentiated postinjury-induced TNF-alpha mRNA increments and did so in an organ-specific fashion (kidney, heart, and lung; but not in liver or spleen). Intramuscular administration, but not GSH treatment, negated Fe's ability to synergize LPS-mediated TNF-alpha release. We conclude 1) intravenous Fe can enhance TNF-alpha generation during LPS- or glycerol-induced tissue damage; 2) increased TNF-alpha gene transcription in the kidney, heart, and lung may contribute to this result; and 3) intramuscular administration, but not GSH, might potentially mitigate some of Fe's systemic toxic effects.

Anemia↗

PubMed2004

Cholesterol synthesis and import contribute to protective cholesterol increments in acute myeloid leukemia cells.

Cholesterol levels are abnormally increased in many acute myeloid leukemia (AML) samples exposed in vitro to chemotherapy. Blocking these acute cholesterol responses selectively sensitizes AML cells to therapeutics. Thus, defining the molecular mechanisms by which AML cells accomplish these protective cholesterol increments might elucidate novel therapeutic targets. We now report that the levels of mRNAs encoding the cholesterol synthesis-regulating enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and the cholesterol-importing low-density lipoprotein (LDL) receptor were both increased by daunorubicin (DNR) or cytarabine (ARA-C) treatments in almost three fourths of cultured AML samples. However, less than one third of AML samples significantly increased LDL accumulation during drug treatments, suggesting that de novo synthesis is the primary mechanism by which most AML cells increase cholesterol levels during drug exposures. LDL increments were not correlated with cholesterol increments in ARA-C-treated AML samples. However, LDL and cholesterol increments did correlate in DNR-treated AML samples where they were measured, suggesting that a subset of AMLs may rely on increased LDL accumulation during treatment with particular drugs. Our data suggest that cholesterol synthesis inhibitors may improve the efficacy of standard antileukemia regimens, but that for maximum benefit, therapy may need to be tailored for individual patients with leukemia.

Biological Transport↗

PubMed2004

Adenovirus nephritis in hematopoietic stem-cell transplantation.

BACKGROUND: Although adenovirus (ADV) infections may involve many different organs, kidney infection is seldom reported in association with hematopoietic stem-cell transplantation (HSCT). METHODS: In the present study, the diagnosis of ADV nephritis was established by the culture isolation of adenovirus or the immunocytochemical (ICC) demonstration of the adenoviral hexon protein. The clinical description of ADV nephritis was derived from retrospective review of clinical records to identify signs, symptoms, outcomes, and associated complications. ADV nephritis was characterized as a pathologic entity by the histologic and ICC analysis of tissue from the kidney and all other major organs to establish the distribution of the virus and the associated gross and microscopic alterations. RESULTS: ADV nephritis was diagnosed in 21 HSCT patients, in 2 by biopsy and in 19 at autopsy. Focal signs of BK nephropathy were present in only one patient. Twenty had received allogeneic marrow and one had undergone autologous transplantation. Graft-versus-host disease was a risk factor. ADV nephritis was associated with acute renal failure in 90% of the infected patients. Prodromal symptoms included fever, hematuria, and flank pain. Adenoviruria was present in 78% of the patients. Kidney infection as determined by viral antigen ICC predominantly involved the tubular epithelial cells. ADV organ tropism was striking, with sero-types from subgenus B, cluster 2, primarily responsible for cases involving predominantly the urinary system. ADV infection was a major cause of death in 17 patients. CONCLUSIONS: ADV nephritis is a specific renal complication in HSCT patients that can be diagnosed by renal biopsy in patients with hematuria and adenoviruria.

Acute Kidney Injury↗

PubMed2004

Proximal tubular cytochrome c efflux: determinant, and potential marker, of mitochondrial injury.

BACKGROUND: Cytochrome c (cyt c) is released from mitochondria after tissue injury, but little is known of its subsequent fate. This study was undertaken to ascertain: (1) does cyt c readily gain access to the extracellular space; (2) if so, what are some determinants of this process; and (3) might cyt c release be a potentially useful marker of in vivo tissue damage. METHODS: Isolated mouse proximal tubules (PT) were subjected to site 1 (rotenone; Rot), site 2 (antimycin A, AA), or site 3 (hypoxic) respiratory chain blockade (+/- 2 mmol/L glycine, to prevent plasma membrane disruption/cell death). Alternatively, oxidant injury was imposed (Fe(2+) or cholesterol oxidase). Extra- and intracellular cyt c levels were quantified by Western blot. Plasma or urine cyt c levels were also determined after rhabdomyolysis or ischemic acute renal failure (ARF) (in mice), or clinical ARF. RESULTS: AA, Rot, and hypoxia caused variable degrees of PT cyt c release (AA >> rot approximately hypoxia), but at most, <20% of total cell content was involved. In contrast, Fe(2+) evoked approximately 65% cyt c efflux, and cholesterol oxidation caused approximately 100% cyt c release. Glycine did not block cyt c efflux, dissociating this process from plasma membrane disruption/necrotic cell death. After rhabdomyolysis, plasma cyt c levels rose and correlated with the severity of ARF (r, 0.93 vs. BUNs). Cyt c was detected in urine after both experimental and clinical ARF. CONCLUSION: Cell cyt c release is dependent on the site and the type of mitochondrial injury sustained. Oxidative injury, in general, and cholesterol oxidation, in particular, seem particularly relevant in this regard. After mitochondrial release, cyt c traverses plasma membranes, eventuating in the extracellular space. The data suggest that plasma and/or urine cyt c appearance might function as a clinically useful in vivo marker of mitochondrial stress and the tissue injury sustained.

Acute Kidney Injury↗

PubMed2004

Parenteral iron nephrotoxicity: potential mechanisms and consequences.

BACKGROUND: Parenteral iron administration is a mainstay of anemia management in renal disease patients. However, concerns of potential iron toxicity persist. Thus, this study was conducted to more fully gauge iron toxicologic profiles and potential determinants thereof. METHODS: Isolated mouse proximal tubule segments (PTS) or cultured proximal tubular [human kidney (HK-2)] cells were exposed to four representative iron preparations [iron sucrose (FeS), iron dextran (FeD), iron gluconate (FeG), or iron oligosaccharide (FeOS)] over a broad dosage range (0, 30 to 1000 microg iron/mL). Cell injury was assessed by lactate deyhdrogenase (LDH) release, adenosine triphosphate (ATP) reductions, cell cytochrome c efflux, and/or electron microscopy. In vivo toxicity (after 2 mg intravenous iron injections) was assessed by plasma/renal/cardiac lipid peroxidation [malondialdehyde (MDA)], renal ferritin (protein)/heme oxygenase-1 (HO-1) (mRNA) expression, electron microscopy, or postiron injection PTS susceptibility to attack. RESULTS: In each test, iron evoked in vitro toxicity, but up to 30x differences in severity (e.g., ATP declines) were observed (FeS > FeG > FeD = FeOS). The in vitro differences paralleled degrees of cell (HK-2) iron uptake. In vivo correlates of iron toxicity included variable increases in renal MDA, ferritin, and HO-1 mRNA levels. Again, these changes appeared to parallel in vivo (glomerular) iron uptake (seen with FeS and FeG, but not with FeD or FeOS). Iron also effected in vivo alterations in proximal tubule cell homeostasis, as reflected by the "downstream" emergence of tubule resistance to in vitro oxidant attack. CONCLUSION: Parenteral iron formulations have potent, but highly variable, cytotoxic potentials which appear to parallel degrees of cell iron uptake (FeS > FeG >> FeD or FeOS). That in vitro injury can be expressed at clinically relevant iron concentrations, and that in vivo glomerular iron deposition/injury may result, suggest caution is warranted if these agents are to be administered to patients with active renal disease.

Animals↗

PubMed2004

Parenteral iron therapy exacerbates experimental sepsis.

BACKGROUND: Catalytic iron can potentiate systemic inflammation via its pro-oxidant effects. This raises the possibility that parenteral iron administration might exacerbate a concomitant septic state. This study sought to experimentally test this hypothesis. METHODS: Male CD-1 mice were subjected to experimental sepsis via intraperitoneal injection of heat-killed Escherichia coli +/- concomitant intravenous iron sucrose (Venofer; 2 mg). Nonseptic mice +/- iron therapy served as controls. Plasma tumor necrosis factor-alpha (TNF-alpha) levels were assessed 2 hours postinjections (serving as an inflammatory marker). Oxidative stress was gauged in heart or kidney tissue (at either 4 or 24 hours) by heme oxygenase-1 (HO-1) mRNA or protein levels. Overall sepsis severity was assessed by morbidity/mortality rates (at 24 hours). RESULTS: Iron alone or sepsis alone each induced oxidant stress in heart and kidney (HO-1 mRNA/protein increases). When iron and E. coli were coadministered, additive or synergistic HO-1 mRNA/protein increments resulted. Iron injection alone only slightly raised TNF-alpha levels (from 0 to 2.3 pg/mL; P= 0.01). However, iron approximately doubled the TNF-alpha increments which arose from the septic state (1400 --> 2600 pg/mL). Neither sepsis alone, nor iron alone, induced any mortality and no mice became moribund (0/24 mice). However, when iron + sepsis were combined, approximately 60% of mice either died (5/12) or developed a moribund (2/12) state (P= 0.005). CONCLUSION: Parenteral iron administration can induce systemic oxidative stress and modest TNF-alpha release. However, when iron is given during experimental sepsis, profound increases in both processes, and approximately 60% mortality, result. Given that renal failure patients have decreased antioxidant defenses and intermittently develop bacteremia, the potential for parenteral iron therapy to exacerbate clinical sepsis needs to be addressed.

Animals↗

PubMed2003

Cytotoxicity of S-conjugates of the sevoflurane degradation product fluoromethyl-2,2-difluoro-1-(trifluoromethyl) vinyl ether (Compound A) in a human proximal tubular cell line.

Fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (FDVE) is a fluorinated alkene formed by degradation of the volatile anesthetic sevoflurane in anesthesia machines. FDVE is nephrotoxic in rats but not humans. Rat FDVE nephrotoxicity is attributed to FDVE glutathione conjugation and bioactivation of subsequent FDVE-cysteine S-conjugates, in part by renal beta-lyase. Although FDVE conjugation and metabolism occur in both rats and humans, the mechanism for selective toxicity in rats and lack of effect in humans is incompletely elucidated. This investigation measured FDVE S-conjugate cytotoxicity in cultured human proximal tubular HK-2 cells, and compared this with known cytotoxic S-conjugates. HK-2 cells were incubated with FDVE and its GSH, cysteine S-mercapturic acid, cysteine S-sulfoxide, and mercapturic acid sulfoxide conjugates (0.1-2.7 mM) for 24 h. Cytotoxicity was determined by lactate dehydrogenase (LDH) release, total LDH, and the ability of viable cells to reduce a tetrazolium-based compound (MTT). FDVE was cytotoxic only at concentrations >/=0.9 mM. No increase in LDH release was observed with either FDVE-GSH conjugate. The FDVE-cysteine conjugates S-(1,1-difluoro-2-fluoromethoxy-2-(trifluoromethyl) ethyl)-L-cysteine (DFEC) and (Z)-S-(1-fluoro-2-fluoromethoxy-2-(trifluoromethyl) vinyl)-L-cysteine ((Z)-FFVC) caused significant differences in LDH release and MTT reduction only at 2.7 mM; (Z)-FFVC was slightly more cytotoxic. Both S-(1,1-difluoro-2-fluoromethoxy-2-(trifluoromethyl) ethyl)-L-cysteine sulfoxide (DFEC-SO) and (Z)-N-acetyl-S-(1-fluoro-2-fluoromethoxy-2-(trifluoromethyl) vinyl)-L-cysteine sulfoxide ((Z)-N-Ac-FFVC-SO) caused slightly greater changes in LDH release or total LDH than the corresponding equimolar DFEC and (Z)-N-acetyl-S-(1-fluoro-2-fluoromethoxy-2-(trifluoromethyl) vinyl)-L-cysteine ((Z)-N-Ac-FFVC) conjugates. In contrast to FDVE S-conjugates, S-(1,2-dichlorovinyl)-L-cysteine was markedly cytotoxic, at concentrations as low as 0.1 mM. These results show that human proximal tubular cells are relatively resistant to FDVE and FDVE S-conjugate cytotoxicity. This may partially explain the lack of FDVE nephrotoxicity in humans.

Anesthetics, Inhalation↗

PubMed2003

Proximal tubular cholesterol loading after mitochondrial, but not glycolytic, blockade.

Diverse forms of injury cause proximal tubular cholesterol accumulation. However, underlying mechanisms in general, and those involved with ATP depletion injury in particular, remain poorly defined. To help elucidate this issue, cholesterol homeostasis and its determinants were assessed after partial ATP depletion states. Serum-exposed HK-2 cells were subjected to mild ATP depletion, induced by mitochondrial inhibition (antimycin A; AA) or glycolytic blockade (2-deoxyglucose; DG). Four or 18 h later, cell cholesterol levels, hydroxymethylglutaryl (HMG)-CoA reductase (HMGCR), the LDL receptor (LDL-R), and ABCA1/SR-B1 cholesterol transporters were assessed. AA and DG each induced mild, largely sublethal ATP depletion injury. Each also caused significant HMGCR increments and SR-B1 decrements and left ABCA1 intact. In contrast, only AA increased the LDL-R, and only AA evoked a cholesterol-loading state (approximately 25% up). One-half of this increase was statin inhibitable, and one-half could be blocked by serum deletion, implying that both synthetic and nonsynthetic (e.g., LDL-R transport) pathways were involved. The AA-induced HMGCR and LDL-R protein changes were paralleled by their mRNAs, suggesting the presence of altered transcriptional events. We conclude that 1) sublethal ATP depletion, whether induced by mitochondrial or glycolytic blockade, can upregulate HMGCR and decrease SR-B1, and these changes represent a previously unrecognized ATP depletion "phenotype"; 2) mitochondrial blockade can also upregulate the LDL-R and evoke a cholesterol-loading state; 3) the latter likely occurs via synthetic and transport pathways; and 4) the mitochondrion may be a critical, and previously unrecognized, determinant of postinjury cell cholesterol homeostasis, potentially by impacting the LDL-R.

ATP Binding Cassette Transporter 1↗

PubMed2003

Acute tubular injury causes dysregulation of cellular cholesterol transport proteins.

Acute renal injury causes accumulation of free and esterified cholesterol (FC, CE) in proximal tubules, mediated, at least in part, by increased cholesterol synthesis. Normally, this would trigger compensatory mechanisms such as increased efflux and decreased influx to limit or reverse the cholesterol overload state. This study sought to determine the integrity of these compensatory pathways following acute renal damage. Rhabdomyolysis-induced acute renal failure was induced in mice by glycerol injection. Normal mice served as controls. After 18 hours, BUN levels and renal cortical FC/CE content were determined. Expression of ABCA-1 and SR-B1 (cholesterol efflux proteins) were assessed by Western blot. Renal cortical LDL receptor (LDL-R; a cholesterol importer) regulation was gauged by quantifying its mRNA. To obtain proximal tubule cell-specific data, the impact of oxidant (Fe) stress on cultured HK-2 cell LDL-R, SR-B1, and ABCA-1 proteins and their mRNAs (versus controls) was assessed. Glycerol evoked marked azotemia and striking FC/CE increments (44%, 384%, respectively). Paradoxically, renal cortical SR-B1 and ABCA-1 protein reductions and LDL-R mRNA increments resulted. Fe-induced injury suppressed HK-2 cell SR-B1, ABCA-1, and their mRNAs. LDL-R protein rose with the in vitro Fe challenge. Renal tubular cell injury causes dysregulation of SR-B1, ABCA-1, and LDL-R protein expression, changes which should contribute to a cholesterol overload state. Reductions in HK-2 cell SR-B1 and ABCA-1 mRNAs and increases in renal cortical LDL-R mRNA imply that this dysregulation reflects, at least in part, altered genomic/transcriptional events.

ATP Binding Cassette Transporter 1↗

PubMed2003

Experimental glomerulopathy alters renal cortical cholesterol, SR-B1, ABCA1, and HMG CoA reductase expression.

Previous studies indicate that acute tubular injury causes free cholesterol (FC) and cholesteryl ester (CE) accumulation within renal cortex/proximal tubules. This study assessed whether similar changes occur with glomerulopathy/nephrotic syndrome, in which high-circulating/filtered lipoprotein levels increase renal cholesterol supply. Potential adaptive changes in cholesterol synthetic/transport proteins were also assessed. Nephrotoxic serum (NTS) or passive Heymann nephritis (PHN) was induced in Sprague-Dawley rats. Renal injury (blood urea nitrogen, proteinuria) was assessed 2 and 7 days (NTS), or 10 and 30 days (PHN) later. FC and CE levels in renal cortex, isolated glomeruli, and proximal tubule segments were determined. SR-B1 (a CE influx protein), ABCA1 (a FC exporter), and HMG CoA reductase protein/mRNA levels were also assessed. FC was minimally elevated in renal cortex (0 to 15%), the majority apparently localizing to proximal tubules. More dramatic CE elevations were found ( approximately 5 to 15x), correlating with the severity of proteinuria at any single time point (r >/= 0.85). Cholesterol increments were associated with decreased SR-B1, increased ABCA1, and increased HMG CoA reductase (HMGCR) protein and its mRNA. Tubule (HK-2) cell culture data indicated that SR-B1 and ABCA1 levels are responsive to cholesterol supply. Experimental nephropathy can increase renal FC, and particularly CE, levels, most notably in proximal tubules. These changes are associated with adaptations in SR-B1 and ABCA1 expression, which are physiologically appropriate changes for a cholesterol overload state. However, HMGCR protein/mRNA increments can also result. These seem to reflect a maladaptive response, potentially contributing to a cell cholesterol overload state.

ATP Binding Cassette Transporter 1↗

PubMed2003

Radiographic contrast media-induced tubular injury: evaluation of oxidant stress and plasma membrane integrity.

BACKGROUND: Experimental and clinical investigations suggest that oxidant stress is a critical determinant of radiocontrast nephropathy (RCN), and that N acetyl cysteine (NAC) can prevent this damage. This study addresses these issues directly at the tubular cell level. Potential alternative mechanisms for RCN have also been sought. METHODS: Isolated mouse proximal tubule segments (PTS), or cultured proximal tubule (HK-2) cells, were subjected to radiocontrast media (RCM) (Ioversol, Optiray 320) exposure, followed by assessments of cellular viability [% lactate dehydrogenase (LDH) release, tetrazolium dye (MTT), uptake] and lipid peroxidation. These experiments were conducted in the absence or presence of a variety of antioxidants [NAC, glutathione (GSH), superoxide dismutase, catalase] or pro-oxidant (GSH depletion, heme oxygenase inhibition) strategies. RCM effects on mitochondrial and plasma membrane integrity were also assessed. RESULTS: RCM exposure did not induce PTS lipid peroxidation. Neither antioxidant nor pro-oxidant interventions mitigated or exacerbated RCM-induced tubular cell injury, respectively. RCM impaired mitochondrial integrity, as assessed by ouabain-resistant ATP reductions, and by cytochrome c release (before cell death). RCM also induced plasma membrane damage, as indicated by loss of key resident proteins (NaK-ATPase, caveolin) and by increased susceptibility to phospholipase A2 (PLA2) attack (increase of >/=2 times in free fatty acid and NaK-ATPase release). Hyperosmolality could not account for RCM's toxic effects. CONCLUSION: RCM toxicity can be dissociated from tubular cell oxidant stress. Alternative mechanisms may include mitochondrial injury/cytochrome c release and plasma membrane damage. The latter results in critical protein loss, as well as a marked increase in plasma membrane susceptibility to exogenous/endogenous PLA2 attack.

Adenosine Triphosphate↗