Biomedical subjects

Ali C M Johnson

Publications and source records attributed to Ali C M Johnson.

16 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↗

PubMed2005

Ischemic proximal tubular injury primes mice to endotoxin-induced TNF-alpha generation and systemic release.

Endotoxemia (LPS) can exacerbate ischemic tubular injury and acute renal failure (ARF). The present study tested the following hypothesis: that acute ischemic damage sensitizes the kidney to LPS-mediated TNF-alpha generation, a process that can worsen inflammation and cytotoxicity. CD-1 mice underwent 15 min of unilateral renal ischemia. LPS (10 mg/kg iv), or its vehicle, was injected either 45 min before, or 18 h after, the ischemic event. TNF-alpha responses were gauged 2 h post-LPS injection by measuring plasma/renal cortical TNF-alpha and renal cortical TNF-alpha mRNA. Values were contrasted to those obtained in sham-operated mice or in contralateral, nonischemic kidneys. TNF-alpha generation by isolated mouse proximal tubules (PTs), and by cultured proximal tubule (HK-2) cells, in response to hypoxia-reoxygenation (H/R), oxidant stress, antimycin A (AA), or LPS was also assessed. Ischemia-reperfusion (I/R), by itself, did not raise plasma or renal cortical TNF-alpha or its mRNA. However, this same ischemic insult dramatically sensitized mice to LPS-mediated TNF-alpha increases in both plasma and kidney (approximately 2-fold). During late reperfusion, increased TNF-alpha mRNA levels also resulted. PTs generated TNF-alpha in response to injury. Neither AA nor LPS alone induced an HK-2 cell TNF-alpha response. However, when present together, AA+LPS induced approximately two- to fivefold increases in TNF-alpha/TNF-alpha mRNA. We conclude that modest I/R injury, and in vitro HK-2 cell mitochondrial inhibition (AA), can dramatically sensitize the kidney/PTs to LPS-mediated TNF-alpha generation and increases in TNF-alpha mRNA. That ischemia can "prime" tubules to LPS response(s) could have potentially important implications for sepsis syndrome, concomitant renal ischemia, and for the induction of ARF.

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↗

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

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↗

PubMed2003

Sepsis syndrome stimulates proximal tubule cholesterol synthesis and suppresses the SR-B1 cholesterol transporter.

BACKGROUND: Previous studies demonstrate that renal cortical/proximal tubule cholesterol accumulation is part of the renal "stress response." The present study was performed to help define underlying mechanisms, using experimental sepsis as a test model. METHODS: Male CD-1 mice and female low-density lipoprotein receptor (LDLR) knockout mice were injected with a heat-killed Escherichia coli suspension. Renal cortex and serum were obtained from these and control mice either 4, 6, or 18 hours later. Tissues samples were assayed for free cholesterol (FC), cholesteryl esters (CE), HMG CoA reductase (HMGCR) mRNA, and SR-B1 [the high-density lipoprotein (HDL) receptor/cholesterol transporter]. Statin effects on renal cortical HMGCR mRNA and FC/CE levels also were assessed. Finally, the impact of serum from septic versus normal mice on cultured proximal tubule (HK-2) cell cholesterol levels was assessed. RESULTS: Sepsis induced approximately 30% and 300 to 500% increases in renal FC and CE content, respectively. Cholesterol accumulation was not blunted in LDLR-/- mice versus their controls. Statin therapy also did not alter sepsis-induced renal FC/CE accumulation. However, statin treatment exerted no discernible intra-renal activity (for example, no rise in renal HMGCR mRNA), despite significant extra-renal activity (25% reduction in serum cholesterol; 400% increase in hepatic HMGCR mRNA). HK-2 cells exposed to septic serum sustained a 40% cholesterol increase, compared to cells exposed to control serum. This response was completely statin inhibited, proving that de novo synthesis was involved. Sepsis markedly suppressed renal levels of SR-B1 (an FC efflux protein). Renal HMGCR mRNA did not fall despite sepsis triggered cholesterol loading, indicating a failure of negative feedback activity. CONCLUSIONS: Sepsis-induced renal cholesterol accumulation is not simply an intrinsic renal response, since it can be enhanced by circulating "stress factors" that drive HMGCR activity. Sepsis also down-regulates SR-B1. Thus, decreased cell FC efflux, coupled with increased synthesis, may synergistically induce the post-sepsis cholesterol overload state.

Animals↗

PubMed2002

The mevalonate pathway during acute tubular injury: selected determinants and consequences.

Renal injury evokes tubular cholesterol accumulation, mediated in part by increased HMG CoA reductase (HMGCR) levels. The present study was undertaken to define potential molecular determinants of these changes and to ascertain the relative importance of increased cholesterol production versus mevalonate pathway-driven protein prenylation, on the emergence of the so-called postrenal injury "cytoresistant state." Cultured proximal tubule (HK-2) cells were subjected to Fe or ATP depletion injury, followed 1 to 24 hours later by assessments of: 1) sterol transcription factor expression (SREBP)-1 and -2); 2) HMGCR mRNA levels; and 3) Ras/Rho prenylation. HMGCR mRNA and Ras/Rho prenylation were also assessed after in vivo ischemic and Fe-mediated renal damage. Using specific inhibitors, the relative importance of protein prenylation versus terminal cholesterol synthesis on HK-2 cell susceptibility to injury was also assessed. Acute injury induced HK-2 cell SREBP disruption and reductions in HMGCR mRNA. Renal cortical HMGCR mRNA also fell in response to either in vivo ischemic or Fe-mediated oxidant damage. At 24 hours after in vitro/in vivo injury, a time of cholesterol buildup, no increase in Ras/Rho prenylation was observed. Prenylation inhibitors did not sensitize HK-2 cells to injury. Conversely, squalene synthase (terminal cholesterol synthesis) blockade sensitized HK-2 cells to both Fe and ATP depletion attack. We concluded that: 1) acute tubular cell injury can destroy SREBPs and lower HMGCR mRNA. This suggests that posttranscriptional/translational events are responsible for HMGCR enzyme and cholesterol accumulation after renal damage. 2) Injury-induced cholesterol accumulation appears dissociated from increased protein prenylation. 3) Cholesterol accumulation, per se, seems to be the dominant mechanism by which the mevalonate pathway contributes to the postrenal injury cytoresistant state.

Adenosine Triphosphate↗

PubMed2002

Parenteral iron formulations: a comparative toxicologic analysis and mechanisms of cell injury.

BACKGROUND: Multiple parenteral iron (Fe) formulations exist for administration to patients with end-stage renal disease. Although there are concerns regarding their potential toxicities, no direct in vitro comparisons of these agents exist. Thus, the present study contrasted pro-oxidant and cytotoxic potentials of four available Fe preparations: Fe dextran (Fe dext), Fe sucrose (Fe sucr), Fe gluconate (Fe gluc), and Fe oligosaccharide (Fe OS). METHODS: Differing dosages (0.06 to 1 mg/mL) of each compound were added to either (1) isolated mouse proximal tubule segments, (2) renal cortical homogenates, or (3) cultured human proximal tubule (HK-2) cells (0.5- to 72-hour incubations). Oxidant injury (malondialdehyde generation) and lethal cell injury (percentage of lactate dehydrogenase release; tetrazolium dye uptake) were assessed. Effects of selected antioxidants (glutathione [GSH], catalase, dimethylthiourea (DMTU), and sodium benzoate also were assessed. RESULTS: Each test agent induced massive and similar degrees of lipid peroxidation. Nevertheless, marked differences in cell death resulted (Fe sucr >> Fe gluc > Fe dext approximately Fe OS). This relative toxicity profile also was observed in cultured aortic endothelial cells. Catalase, DMTU, and sodium benzoate conferred no protection. However, GSH and its constituent amino acid glycine blocked Fe sucr-mediated cell death. The latter was mediated by mitochondrial blockade, causing free radical generation and a severe adenosine triphosphate depletion state. CONCLUSIONS: (1) parenteral Fes are highly potent pro-oxidants and capable of inducing tubular and endothelial cell death, (2) markedly different toxicity profiles exist among these agents, and (3) GSH can exert protective effects. However, the latter stems from GSH's glycine content, rather than from a direct antioxidant effect.

Animals↗

PubMed2002

Dephosphorylation of cell cycle-regulated proteins correlates with anoxia-induced suspended animation in Caenorhabditis elegans.

Some metazoans have evolved the capacity to survive severe oxygen deprivation. The nematode, Caenorhabditis elegans, exposed to anoxia (0 kPa, 0% O(2)) enters into a recoverable state of suspended animation during all stages of the life cycle. That is, all microscopically observable movement ceases including cell division, developmental progression, feeding, and motility. To understand suspended animation, we compared oxygen-deprived embryos to nontreated embryos in both wild-type and hif-1 mutants. We found that hif-1 mutants survive anoxia, suggesting that the mechanisms for anoxia survival are different from those required for hypoxia. Examination of wild-type embryos exposed to anoxia show that blastomeres arrest in interphase, prophase, metaphase, and telophase but not anaphase. Analysis of the energetic state of anoxic embryos indicated a reversible depression in the ATP to ADP ratio. Given that a decrease in ATP concentrations likely affects a variety of cellular processes, including signal transduction, we compared the phosphorylation state of several proteins in anoxic embryos and normoxic embryos. We found that the phosphorylation state of histone H3 and cell cycle-regulated proteins recognized by the MPM-2 antibody were not detectable in anoxic embryos. Thus, dephosphorylation of specific proteins correlate with the establishment and/or maintenance of a state of anoxia-induced suspended animation.

Adaptation, Physiological↗