'Endotoxin tolerance': TNF-alpha hyper-reactivity and tubular cytoresistance in a renal cholesterol loading state.

The term 'endotoxin tolerance' defines a state in which prior endotoxin (lipopolysaccharide (LPS)) exposure induces resistance to subsequent LPS attack. However, its characteristics within kidney have not been well defined. Hence, this study tested the impact of LPS 'preconditioning' (LPS-PC; 18 or 72 h earlier) on: (i) selected renal inflammatory mediators (tumor necrosis factor (TNF)-alpha, interleukin-10 (IL-10), monocyte chemotactic protein-1 (MCP-1), inducible nitric oxide synthase (iNOS), Toll-like receptor 4 (TLR4); protein or mRNA); (ii) cholesterol homeostasis (a stress reactant); and (iii) isolated proximal tubule (PT) vulnerability to hypoxia or membrane cholesterol (cholesterol oxidase/esterase) attack. Two hours post LPS injection, LPS-PC mice manifested reduced plasma TNF-alpha levels, consistent with systemic LPS tolerance. However, in kidney, paradoxical TNF-alpha hyper-reactivity (protein/mRNA) to LPS existed, despite normal TLR4 protein levels. PT TNF-alpha levels paralleled renal cortical results, implying that PTs were involved. LPS-PC also induced: (i) renal cortical iNOS, IL-10 (but not MCP-1) mRNA hyper-reactivity; (ii), PT cholesterol loading, and (iii) cytoresistance to hypoxia and plasma membrane cholesterol attack. A link between cholesterol homeostasis and cell LPS responsiveness was suggested by observations that cholesterol reductions in HK-2 cells (methylcyclodextrin), or reductions in HK-2 membrane fluidity (A2C), blunted LPS-mediated TNF-alpha/MCP-1 mRNA increases. In sum: (i) systemic LPS tolerance can be associated with renal hyper-responsiveness of selected components within the LPS signaling cascade (e.g., TNF-alpha, iNOS, IL-10); (ii) PT cytoresistance against hypoxic/membrane injury coexists; and (iii) LPS-induced renal/PT cholesterol accumulation may mechanistically contribute to each of these results.

Acute nephrotoxic and obstructive injury primes the kidney to endotoxin-driven cytokine/chemokine production.

Gram-negative sepsis is a frequent complication in patients with acute renal failure. This study tested whether acute tubular injury, for example, induced by cisplatin (CP) or urinary tract obstruction, enhances renal cytokine responses to endotoxin (lipopolysaccharide (LPS)), potentially contributing to tissue damage. CD-1 mice were subjected to CP or vehicle injection. After 24 or 72 h, LPS or its vehicle was given. At 2 h post LPS or vehicle administration, plasma/renal cortical tumor necrosis factor (TNF)-alpha, monocyte chemoattractant protein-1 (MCP-1), and interleukin-10, and their corresponding renal cortical mRNAs were assessed (representing pro-anti-inflammatory cytokines, and a chemokine, respectively). Comparable studies were conducted in mice 24 h post unilateral ureteral obstruction (UUO). Cultured human proximal tubular (HK-2) cell TNF-alpha responses to CP+/-LPS were also assessed. CP alone caused either minimal or no increases in cytokine levels. However, CP dramatically augmented cytokine responses to LPS (up to 5-10 x vs LPS alone). The cytokine increases were paralleled by changes in their mRNAs. UUO also sensitized to LPS. CP alone did not alter HK-2 cell TNF-alpha/mRNA. However, CP 'primed' the cells to LPS (approximately 50-100% greater TNF-alpha/mRNA increases vs LPS alone). CP+LPS also caused synergistic cell death (lactate dehydrogenase release). We conclude that (1) diverse forms of tubular injury can sensitize the kidney to LPS, increasing cytokine production; (2) proximal tubules are involved; (3) LPS 'priming' has broad-based consequences, impacting diverse pro- and anti-inflammatory pathways; and (4) increased transcriptional events may be at least partially involved.

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.

Mevastatin can increase toxicity in primary AMLs exposed to standard therapeutic agents, but statin efficacy is not simply associated with ras hotspot mutations or overexpression.

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is a rate-limiting enzyme in the mevalonate biochemical pathway and HMG-CoA reductase inhibitors (statins) show toxicity for certain tumors, including acute myeloid leukemia (AML). This toxicity has been attributed to statin inhibition of Ras isoprenylation in tumors like AML where oncogenic ras mutations and/or overexpression are common. We show that mevastatin kills certain AML cell lines and is more toxic to a majority of primary AML cell samples than to myeloid cells in bone marrow (BM) samples from normal donors, and that mevastatin can produce more than additive kill with standard chemotherapeutics. Mevastatin reduces Ras membrane localization, but statin sensitivity in primary AML cells is not consistently associated with ras mutations nor with Ras overexpression, suggesting that another mevalonate pathway by-product(s) is the statin target in at least some AMLs.

Renal cortical cholesterol accumulation is an integral component of the systemic stress response.

BACKGROUND: Direct tubular injury (such as ischemia or myohemoglobinuria) increases renal cortical cholesterol content. This study explored whether systemic forms of stress (such as heat shock or sepsis) can trigger renal cholesterol accumulation, and if so, whether increased 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGCR) expression might be involved. METHODS: Male CD-1 mice were subjected to glycerol-induced myohemoglobinuria (MH), systemic heat shock (HS), or E. coli sepsis. Free cholesterol (FC), cholesteryl esters (CE), and HMGCR (Western blot) levels were assessed 18 hours later. Statin effects on renal cholesterol levels and on the severity of MH-acute renal failure (ARF) were also determined. RESULTS: Sepsis and HS each induced dramatic FC and CE increments, comparable to those observed with myohemoglobinuria, and without inducing acute tubular necrosis (ATN). Part of the cholesterol increments was localized within plasma membrane (detergent resistant) microdomains (for example, rafts/caveolae). HS and MH each increased renal HMGCR, as well as HS protein (HSP-72) expression. Oxidant stress (Fe) imposed on cultured proximal tubule (HK-2) cells also enhanced HMGCR content. Conversely, sepsis did not raise renal HMGCR or HSP-72 levels. Statin therapy decreased the severity of MH-ARF and renal cholesterol content. However, this appeared to arise from a statin-mediated decrease in glycerol-induced extrarenal tissue damage (myolysis/LDH release). CONCLUSIONS: Cholesterol appears to be a renal 'acute phase reactant' with tissue levels increasing with either systemic stress (such as, heat shock, sepsis), or direct tissue damage (such as ATN). Increased HMGCR expression can contribute to this result. Mechanisms other than HMGCR induction also can mediate stress-induced cholesterol increments (for example, in the case of sepsis), and statins can mitigate MH-ARF. However, systemic anti-inflammatory effects, rather than a primary renal action, appear more likely to be involved.

P glycoprotein-mediated cholesterol cycling determines proximal tubular cell viability.

BACKGROUND: MDR P glycoproteins may help transport plasma membrane free cholesterol (FC) to the endoplasmic reticulum (ER), where it undergoes acylation, forming cholesterol esters (CE). This study assessed whether P glycoprotein inhibitors alter renal tubular FC/CE expression, thereby altering cell integrity. METHODS: Mouse proximal tubule segments (PTS) were exposed to chemically dissimilar P glycoprotein inhibitors [progesterone (prog), trifluoperazine (TFP), or cyclosporine A (CsA)]. Their effects on FC/CE and adenosine 5'-triphosphate (ATP) levels, phospholipid expression, lipid peroxidation, and cell viability (lactate dehydrogenase release; LDH) were assessed. P glycoprotein inhibitor effects on cultured proximal tubular (HK-2) cell viability and susceptibility to Fe-induced oxidant stress were also addressed. RESULTS: When applied to PTS, prog, TFP, and, to lesser extent, CsA induced dose-dependent ATP reductions (< or =90%), CE decrements (approximately 40%), and LDH release (< or =60%). No concomitant changes in lipid peroxidation or phospholipid profiles were observed. Ouabain did not preserve tubular ATP, suggesting that decreased ATP production, rather than increased consumption, was operative. Mechanisms leading to cell lysis were not identical, as glycine and arachidonic acid blocked prog- but not TFP-mediated cell death. When prog-driven CE reductions were attenuated in PTS with a procycling agent (cholesterol oxidase), decreased cell death resulted. P glycoprotein inhibitors also caused dose-dependent HK-2 cell death. Blocking Fe-mediated CE formation ( approximately x10) with sublethal CsA doses led to a marked increase in Fe-mediated cell death. CONCLUSIONS: P glycoproteins may be critical to tubule cholesterol transport. If blocked with pharmacologic agents, decreased ATP production, overt cell lysis, and/or a marked propensity to superimposed tubular cell injury can result.

Renal cholesterol accumulation: a durable response after acute and subacute renal insults.

UNLABELLED: Proximal tubular cholesterol levels rise within 18 hours of diverse forms of acute renal tubular injury (eg, myoglobinuria, ischemia/reperfusion, urinary tract obstruction). These increments serve to protect against further bouts of tubular attack (so-called "acquired cytoresistance"). Whether these cholesterol increments are merely transitory, or persist into the maintenance phase of acute renal failure (ARF), has not been previously defined. Furthermore, whether subacute/insidious tubular injury [eg, cyclosporine A (CSA), tacrolimus toxicity], nontubular injury (eg, acute glomerulonephritis), or physiological stress (eg, mild dehydration) impact renal cholesterol homeostasis have not been addressed. This study sought to resolve these issues. Male CD-1 mice were subjected to glycerol-induced ARF. Renal cortical-free cholesterol (FC) and cholesterol ester (CE) levels were determined 3, 5, 7, or 14 days later, and the values contrasted to prevailing blood-urea nitrogen concentrations. The impact of 40 minutes of unilateral renal ischemia plus reflow (3 to 6 days) on mouse cortical FC/CE content was also assessed. Additionally, FC/CE levels were measured in rat renal cortex either 10 days after CSA or tacrolimus therapy, or 48 hours after induction of nephrotoxic serum nephritis. Finally, the impact of overnight dehydration on mouse renal cortical/medullary FC/CE profiles was determined. Compared to sham-treated animals, glycerol, CSA, tacrolimus, ischemia-reperfusion, and nephrotoxic serum each induced dramatic CE +/- FC elevations, rising as much as 10x control values. In the glycerol model, striking correlations (r

Ethylene glycol-mediated tubular injury: identification of critical metabolites and injury pathways.

Ethylene glycol (EG) intoxication produces multisystem organ injury, including acute renal failure. Although EG must be metabolized to toxic intermediates to induce organ damage, the specific metabolite(s) responsible and the underlying pathogenic mechanisms remain poorly defined. To explore these issues, isolated mouse proximal tubular segments (PTSs) were incubated with either varying doses of EG or its prime metabolites (glycolate, glycoaldehyde, glyoxylate, or oxalate for 15 to 60 minutes). Injury was assessed by the percentage of lactate dehydrogenase (LDH) release, LDH destruction, adenosine triphosphate (ATP) depletion, or membrane phospholipid degradation. Toxicities were also assessed in cultured HK-2 cells over 18 hours (by MTT assay). EG, glycolate, and oxalate did not induce overt PTS injury. Conversely, glyoxylate and glycoaldehyde were highly toxic, causing profound ATP depletion and LDH release. Glycoaldehyde also caused enzyme (LDH) and selected phospholipid degradation (phosphatidylethanolamine, phosphatidylserine). These changes were not seen with glyoxylate treatment. Acidosis (pH 6.8) and glycine (2 mmol/L) each blocked glyoxylate, but not glycoaldehyde toxicity, indicating differing injury pathways. Only glycoaldehyde and glyoxylate induced marked HK-2 cell death. We conclude that glycoaldehyde and glyoxylate are the principal metabolites responsible for EG nephrotoxicity and do so by causing ATP depletion and phospholipid and enzyme destruction. Glycine and acidosis, by-products of EG metabolism, can attenuate glyoxylate-mediated injury. This suggests that naturally occurring but incomplete protective pathways may be operative during the evolution of EG cytotoxicity.

Cholesterol ester accumulation: an immediate consequence of acute in vivo ischemic renal injury.

BACKGROUND: Cholesterol is a major constituent of plasma membranes, and recent evidence indicates that it is up-regulated during the maintenance phase of acute renal failure (ARF). However, cholesterol's fate and that of the cholesterol ester (CE) cycle [shuttling between free cholesterol (FC) and CEs] during the induction phase of ARF have not been well defined. The present studies sought to provide initial insights into these issues. METHODS: FC and CE were measured in mouse renal cortex after in vivo ischemia (15 and 45 minutes)/reperfusion (0 to 120 minutes) and glycerol-induced myoglobinuria (1 to 2 hours). FC/CE were also measured in (1) cultured human proximal tubule (HK-2) cells three hours after ATP depletion and in (2) isolated mouse proximal tubule segments (PTSs) subjected to plasma membrane damage (with cholesterol oxidase, sphingomyelinase, phospholipase A2, or cytoskeletal disruption with cytochalasin B). The impact of cholesterol synthesis inhibition (with mevastatin) and FC traffic blockade (with progesterone) on injury-evoked FC/CE changes was also assessed. RESULTS: In vivo ischemia caused approximately threefold to fourfold CE elevations, but not FC elevations, that persisted for at least two hours of reperfusion. Conversely, myoglobinuria had no effect. Isolated CE increments were observed in ATP-depleted HK-2 cells. Neither mevastatin nor progesterone blocked this CE accumulation. Plasma membrane injury induced with sphingomyelinase or cholesterol oxidase, but not with phospholipase A(2) or cytochalasin B, increased tubule CE content. High CE levels, induced with cholesterol oxidase, partially blocked hypoxic PTS attack. CONCLUSIONS: In vivo ischemia/reperfusion acutely increases renal cortical CE, but not FC, content, indicating perturbed CE/FC cycling. The available data suggest that this could stem from specific types of plasma membrane damage, which then increase FC flux via aberrant pathways to the endoplasmic reticulum, where CE formation occurs. That CE levels are known to inversely correlate with both renal and nonrenal cell injury suggests the potential relevance of these observations to the induction phase of ischemic ARF.

Hypocalcemia: a pervasive metabolic abnormality in the critically ill.

Hypocalcemia has been reported in critically ill patients, most commonly in association with sepsis syndrome. However, the severity and incidence of hypocalcemia in nonseptic but critically ill patients has not been well defined. Therefore, the goal of this study was to identify and compare the frequency and degree of hypocalcemia in critically ill patients with differing underlying illnesses (those admitted to medical, surgical, trauma, neurosurgical, burn, respiratory, and coronary intensive care units [ICUs]; group A; n = 99). Results were compared with the frequency and degree of hypocalcemia in non-critically ill ICU patients (initially admitted to an ICU but discharged within 48 hours; group B; n = 50) or hospitalized non-ICU patients (group C; n = 50). Incidences of hypocalcemia (ionized calcium [Ca] < 1.16 mmol/L [less than normal]) were 88%, 66%, and 26% for groups A, B, and C, respectively (P: < 0.001). In group A, the frequency of hypocalcemia did not depend on the ICU setting or presence of sepsis. However, the occurrence of hypocalcemia correlated with both Acute Physiology and Chronic Health Evaluation II score (r = -0.39; P: < 0.001) and patient mortality (eg, hazard ratio for death, 1.65 for Ca decrements of 0.1 mmol/L; P: < 0.002). Hypomagnesemia, number of blood transfusions, and presence of acute renal failure were each associated with depressed Ca levels. A weak association (r = -0.12; P: = 0.09) was noted between serum Ca level and QT interval. Clinical concern stemming from hypocalcemia was underscored by the substantial use of intravenous (IV) Ca therapy ( approximately 2 to 3 g IV). We conclude that hypocalcemia is extremely common in hospitalized patients (up to 88%) and correlates with severity of illness, but not with a specific illness per se. Whether it directly impacts patient survival remains unknown. Resolution of this issue appears to be critical because of the frequency with which it leads to high-dose IV Ca therapy.

Changes in free and esterified cholesterol: hallmarks of acute renal tubular injury and acquired cytoresistance.

Acute tubular cell injury is accompanied by plasma membrane phospholipid breakdown. Although cholesterol is a dominant membrane lipid which interdigitates with, and impacts, phospholipid homeostasis, its fate during the induction and recovery phases of acute renal failure (ARF) has remained ill defined. The present study was performed to ascertain whether altered cholesterol expression is a hallmark of evolving tubular damage. Using gas chromatographic analysis, free cholesterol (FC) and esterified cholesterol (CE) were quantified in: 1) isolated mouse proximal tubule segments (PTS) after 30 minutes of hypoxic or oxidant (ferrous ammonium sulfate) injury; 2) cultured proximal tubule (HK-2) cells after 4 or 18 hours of either ATP depletion/Ca(2+) ionophore- or ferrous ammonium sulfate-mediated injury; and 3) in renal cortex 18 hours after induction of glycerol-induced myoglobinuric ARF, a time corresponding to the so-called "acquired cytoresistance" state (ie, resistance to further renal damage). Hypoxic and oxidant injury each induced approximately 33% decrements in CE (but not FC) levels in PTS, corresponding with lethal cell injury ( approximately 50 to 60% LDH release). When comparable CE declines were induced in normal PTS by exogenous cholesterol esterase treatment, proportionate lethal cell injury resulted. During models of slowly evolving HK-2 cell injury, progressive CE increments occurred: these were first noted at 4 hours, and reached approximately 600% by 18 hours. In vivo myoglobinuric ARF produced comparable renal cortical CE (and to a lesser extent FC) increments. Renal CE accumulation strikingly correlated with the severity of ARF (eg, blood urea nitrogen versus CE; r, 0.84). Mevastatin blocked cholesterol accumulation in injured HK-2 cells, indicating de novo synthesis was responsible. Acute tubule injury first lowers, then raises, tubule cholesterol content. Based on previous observations that cholesterol has cytoprotectant properties, the present findings have potential relevance for both the induction and maintenance phases of ARF.

Polyene antibiotics: relative degrees of in vitro cytotoxicity and potential effects on tubule phospholipid and ceramide content.

Polyene antibiotic administration is limited by dose-dependent nephrotoxicity. The latter is believed to be mediated by polyene anchoring to plasma membrane cholesterol, resulting in pore formation, abnormal ion/solute flux, adenosine triphosphate (ATP) declines, and, ultimately, a loss of tubule viability. The relative nephrotoxicity of these agents and their liposomal preparations has remained poorly defined. Thus, freshly isolated mouse proximal tubules or cultured human proximal tubule (HK-2) cells were exposed to either nystatin, amphotericin B, or three different polyene liposomal preparations (Nyotran, AmBisome, or Abelcet; 4 to 64 microg/mL). The impact of these agents on (1) plasma membrane injury (sodium-driven ATP consumption, assessed by ATP-adenosine diphosphate [ADP] ratios); (2) cellular susceptibility to superimposed injury (chemical hypoxia or ferrous ammonium sulfate-mediated oxidative stress; assessed by lactate dehydrogenase release); and (3) membrane cholesterol, phospholipid, and ceramide expression was assessed. Amphotericin B was more cytotoxic than nystatin (approximately 25% to 50% greater ATP-ADP ratio declines). Most of this toxicity could be eliminated by polyene liposomal formulation. Nevertheless, the liposomal polyenes still fully sensitized tubule cells to superimposed chemical hypoxic (antimycin/deoxyglucose), but not oxidant, attack. Nystatin and amphotericin B caused acute increments in tubule sphingomyelin-phosphatidylcholine ratios and ceramide content (indicating an impact on the plasma membrane extending beyond the classic view of pore formation with ion flux). In conclusion, (1) nystatin is seemingly less cytotoxic than amphotericin B (in contrast to the prevailing clinical view); (2) liposomal formulation markedly decreases this cytotoxicity; (3) despite this reduced toxicity, liposomal polyenes are still able to render tubule cells more vulnerable to selected forms of superimposed injury; and (4) acute alterations in plasma membrane phospholipid and ceramide expression are previously unrecognized consequences and potential mediators of polyene-mediated tubular cell attack.

Plasma membrane cholesterol: a critical determinant of cellular energetics and tubular resistance to attack.

BACKGROUND: Cholesterol is a major component of plasma membranes, forming membrane microdomains ("rafts" or "caveolae") via hydrophobic interactions with sphingolipids. We have recently demonstrated that tubule cholesterol levels rise by 18 hours following diverse forms of injury, and this change helps to protect kidneys from further damage (so-called acquired cytoresistance). The present study was undertaken to better define the effects of membrane cholesterol/microdomains on tubule homeostasis and cell susceptibility to superimposed attack. METHODS: Plasma membrane cholesterol was perturbed in normal mouse proximal tubular segments with either cholesterol esterase (CE) or cholesterol oxidase (CO). Alternatively, cholesterol-sphingomyelin complexes were altered by sphingomyelinase (SMase) treatment. Changes in cell energetics (ATP/ADP ratios + ouabain), viability [lactate dehydrogenase (LDH) release], phospholipid profiles, and susceptibility to injury (Fe-induced oxidant stress, PLA2, Ca2+ ionophore) were determined. The impacts of selected cytoprotectants were also assessed. RESULTS: Within 15 minutes, CE and CO each induced approximately 90% ATP/ADP ratio suppressions. These were seen prior to lethal cell injury (LDH release), and it was ouabain resistant (suggesting decreased ATP production, not increased consumption). SMase also depressed ATP without inducing cell death. After 45 minutes, CE and CO each caused marked cytotoxicity (up to 70% LDH release). However, different injury mechanisms were operative since (1) CE, but not CO, toxicity significantly altered cell phospholipid profiles, and (2) 2 mmol/L glycine completely blocked CE- but not CO-mediated cell death. Antioxidants also failed to attenuate CO cytotoxicity. Disturbing cholesterol/microdomains with a sublytic CE dose dramatically increased tubule susceptibility to Fe-mediated oxidative stress and Ca2+ overload, but not PLA2-mediated damage. CONCLUSION: Intact plasma membrane cholesterol/microdomains are critical for maintaining cell viability both under basal conditions and during superimposed attack. When perturbed, complex injury pathways can be impacted, with potential implications for both the induction of acute tubular damage and the emergence of the postinjury cytoresistance state.

Increased proximal tubular cholesterol content: implications for cell injury and "acquired cytoresistance".

BACKGROUND: Acute renal failure (ARF) leads to secondary adaptive changes that serve to protect proximal tubules from subsequent ischemic or toxic damage [so-called "acquired cytoresistance" (CR)]. A characteristic of CR is increased plasma membrane resistance to attack. Therefore, this study sought to identify potential changes in plasma membrane lipid composition in CR tubules/renal cortex and, if present, to test whether they might mechanistically contribute to the CR state. METHODS: Renal cortices/isolated tubules were obtained from CR mouse kidneys (18-hr postinduction of ischemia reperfusion, myoglobinuria, or ureteral obstruction). Their plasma membrane phospholipid/cholesterol profiles were compared with those observed in either control tissues or tissues obtained one to two hours post-renal damage (that is, prior to emergence of CR). RESULTS: Either no changes or inconsistent changes in phospholipid profiles were observed in CR tissues. Conversely, CR (vs. control) tissues demonstrated a consistent 25 to 50% increase in membrane cholesterol content. To ascertain whether cholesterol impacts tubule susceptibility to injury, its levels were reduced in proximal tubule (HK-2) cells with either (a) mevastatin, (b) a cholesterol "stripping" agent, (c) cholesterol oxidase, or (d) cholesterol esterase. Then cell susceptibility to injury [adenosine 5'-triphosphate (ATP) depletion; Fe-mediated oxidant stress] was assessed. In each instance, cholesterol reductions dramatically sensitized to superimposed injury (for example, a 2 to 3 times increase in the % of lactate dehydrogenase release). When cholesterol levels were restored to normal in CR tubules (with a "stripping" agent), an increased tubule susceptibility to injury resulted. Because cholesterol decreases membrane fluidity, the impact of a membrane-fluidizing agent (A2C) on cell injury was assessed. A2C dramatically sensitized HK-2 cells to superimposed attack. CONCLUSIONS: ARF leads to an up-regulation of proximal tubule cholesterol content. The latter may then contribute to acquired CR, possibly by stabilizing the plasma membrane via its antifluidizing effect.

Acute renal failure after cardiopulmonary bypass in related to decreased serum ferritin levels.

Acute renal failure (ARF) requiring dialysis occurs in up to 4% of patients after cardiopulmonary bypass (CPB). CPB leads to the generation of intravascular free hemoglobin, resulting in increased endothelial and renal tubular cell free iron, which is associated with renal injury. Conversely, renoprotection is conferred by processes that upregulate heme and iron sequestration pathways, such as ferritin. This study evaluates the influence of free hemoglobin generation during CPB and the capacity to sequester free iron on the occurrence of post-CPB renal insufficiency. Thirty consecutive patients undergoing CPB were enrolled in the study. Serum creatinine, free hemoglobin, and ferritin were measured preoperatively, at the end of bypass, and 24 and 48 h after surgery. Renal injury, as determined by an increase in the serum creatinine of > or =25% (ARF) by 48 h after surgery, occurred in 40% (12 of 30) of patients, and dialysis was necessary in 6.6% (2 of 30). Free hemoglobin levels increased in all patients but did not correlate with postoperative ARF. However, patients with preoperative serum ferritin levels < or =130 microg/L, the median value for the group, had a sixfold greater likelihood of developing ARF compared to patients with levels above this value (P = 0.03). Lower serum ferritin levels appear to be associated with the development of ARF. Serum ferritin levels may signify intravascular as well as endothelial and renal epithelial cell ability to bind free iron generated during CPB-induced hemolysis, and thus may help provide information regarding the risk for ARF.

Renal cortical ceramide patterns during ischemic and toxic injury: assessments by HPLC-mass spectrometry.

Ceramides are a class of signaling molecules that can acutely accumulate in tissues as part of a "stress response." They are classically measured by the diacylglycerol kinase assay, which, in general, measures total ceramide rather than individual moieties within the diverse ceramide family. The present study was undertaken to 1) adapt current HPLC-mass spectrometry technology for measuring individual renal ceramides, and 2) use this technique to more fully characterize the nature of the renal ceramide "stress" reaction. Renal cortical tissues were obtained from CD-1 mice under control conditions and 2 or 18 h after renal injury (ischemia-reperfusion and glycerol-mediated myohemoglobinuria). C24, C22, and C16 ceramides were identified in normal renal cortex, constituting 70, 10, and 20% of the total ceramide pool, respectively. Within each of these families, heterogeneity was apparent because of differing degrees of unsaturation (0-3 double bonds) in the constituent fatty acid of ceramide. Renal injury dramatically changed ceramide profiles: 1) total ceramide increased by approximately 300%; 2) although all ceramides participated in this reaction, they did so to differing degrees; 3) this caused pronounced changes in ceramide distribution patterns; 4) injury induced a striking shift toward unsaturated (vs. saturated) fatty acids within the C22 and C24 (but not the C16) ceramide pools; and 5) the extent of these qualitative changes differed according to the etiology of the initiating renal damage. Thus we conclude that ceramide stress response involves major qualitative (and not simply quantitative) changes in ceramide expression that are partially disease dependent. These findings underscore the fact that simply measuring total renal ceramide content (e.g., by diacylglycerol kinase assay) substantially oversimplifies the nature and, hence, the potential implications of the ceramide stress reaction.

Plasma membrane phospholipid integrity and orientation during hypoxic and toxic proximal tubular attack.

BACKGROUND: Acute cell injury can activate intracellular phospholipase A2 (PLA2) and can inhibit plasma membrane aminophospholipid translocase(s). The latter maintains inner/outer plasma membrane phospholipid (PL) asymmetry. The mechanistic importance of PLA2-mediated PL breakdown and possible PL redistribution ("flip flop") to lethal tubule injury has not been well defined. This study was performed to help clarify these issues. METHODS: Proximal tubule segments (PTS) from normal CD-1 mice were subjected to either 30 minutes of hypoxia, Ca2+ ionophore (50 microM A23187), or oxidant attack (50 microM Fe). Lethal cell injury [the percentage of lactate dehydrogenase (LDH) release], plasma membrane PL expression [two-dimensional thin layer chromatography (TLC)], and free fatty acid (FFA) levels were then assessed. "Flip flop" was gauged by preferential decrements in phosphatidylserine (PS) versus phosphatidylcholine (PC; PS/PC ratios) in response to extracellular (Naja) PLA2 exposure. RESULTS: Hypoxia induced approximately 60% LDH release, but no PL losses were observed. FFA increments suggested, at most 3% or less PL hydrolysis. Naja PLA2 reduced PLs in hypoxic tubules, but paradoxically, mild cytoprotection resulted. In contrast to hypoxia, Ca2+ ionophore and Fe each induced significant PL losses (6 to 15%) despite minimal FFA accumulation or cell death (26 to 27% LDH release). Arachidonic acid markedly inhibited PLA2 activity, potentially explaining an inverse correlation (r = -0.91) between tubule FFA accumulation and PL decrements. No evidence for plasma membrane "flip flop" was observed. In vivo ischemia reperfusion and oxidant injury (myohemoglobinuria) induced 0 and 24% cortical PL depletion, respectively, validating these in vitro data. CONCLUSIONS: (a) Plasma membrane PLs are well preserved during acute hypoxic/ischemic injury, possibly because FFA accumulation (caused by mitochondrial inhibition) creates a negative feedback loop, inhibiting intracellular PLA2. (b) Exogenous PLA2 induces PL losses during hypoxia, but decreased cell injury can result. Together these findings suggest that PL loss may not be essential to hypoxic cell death. (c) Oxidant/Ca2+ overload injury induces early PL losses, perhaps facilitated by ongoing mitochondrial FFA metabolism, and (d) membrane "flip flop" does not appear to be an immediate mediator of acute necrotic tubular cell death.

Calcitriol directly sensitizes renal tubular cells to ATP-depletion- and iron-mediated attack.

Vitamin Ds have been reported to have diverse effects on cell homeostasis, leading to suggestions that they have therapeutic applications extending beyond their traditional actions on the Ca2+/parathyroid/bone axis. As some of these potential indications carry an inherent risk of acute renal failure (ARF; eg, cancer chemotherapy and organ transplantation), the goal of this study was to assess whether vitamin Ds directly affect renal tubule injury responses. Cultured human proximal tubular (HK-2) cells were exposed to physiological or pharmacological doses of either calcitriol (D3) or a synthetic vitamin D2 analogue (19-nor) for 3 to 48 hours. Their impact on cell integrity (percent lactate dehydrogenase (LDH) release and tetrazolium dye MTT uptake) under basal conditions and during superimposed injuries (ATP depletion/Ca2+ ionophore or iron-mediated oxidant stress) were determined. As vitamin Ds can be anti-proliferative, cell outgrowth ([3H]thymidine uptake and crystal violet staining) was also tested. Finally, the action of D3 on in vivo ARF (glycerol-induced myoglobinuria) and isolated proximal tubule injury responses were assessed. D3 induced a rapid, dose-dependent increase in HK-2 susceptibility to both ATP-depletion/Ca2+-ionophore- and Fe-mediated attack without independently affecting cell integrity or proliferative responses. In contrast, D2 negatively affected only Fe toxicity and only after relatively prolonged exposure (48 hours). D3 dramatically potentiated in vivo ARF (two- to threefold increase in azotemia), suggesting potential in vivo relevance of the above HK-2 cell results. Proximal tubules, isolated from these glycerol-exposed mice, suggested that D3 can worsen tubule injury despite a parodoxic suppression of H2O2 production. In contrast, D3 had a mild negative impact on cellular energetics (depressed ATP/ADP ratios), and it accentuated plasma membrane phospholipid breakdown. The latter was observed in both glycerol-treated and control tubules, suggesting a primary role in the injury- potentiation effect of D3. Vitamins D(s) may directly, and differentially, increase proximal tubule cell susceptibility to superimposed attack. This property should be considered as new uses for these agents are defined.

Expression and role of parathyroid hormone-related protein in human renal proximal tubule cells during recovery from ATP depletion.

Parathyroid hormone (PTH)-related protein (PTHrP) is widely expressed in normal fetal and adult tissues and regulates growth and differentiation in a number of organ systems. Although various renal cell types produce PTHrP, and PTHrP expression in rat proximal renal tubules is upregulated in response to ischemic injury in vivo, the role of PTHrP in the kidney is unknown. To study the effects of injury on PTHrP expression and its consequences in more detail, the immortalized human proximal tubule cell line HK-2 was used in an in vitro model of ATP depletion to mimic in vivo renal ischemic injury. These cells secrete PTHrP into conditioned medium and express the type I PTH/PTHrP receptor. Treatment of confluent HK-2 cells for 2 h with substrate-free, glucose-free medium containing the mitochondrial inhibitor antimycin A (1 microM) resulted in 75% depletion of cellular ATP. After an additional 2 h in glucose-containing medium, cellular ATP levels recovered to approximately 75% of baseline levels. PTHrP mRNA levels, as measured in RNase protection assays, peaked at 2 h into the recovery period (at four times baseline expression). The increase in PTHrP mRNA expression was correlated with an increase in PTHrP protein content in HK-2 cells at 2 to 6 h into the recovery period. Heat shock protein-70 mRNA expression was not detectable under baseline conditions but likewise peaked at 2 h into the recovery period. Treatment of HK-2 cells during the recovery period after injury with an anti-PTHrP(1-36) antibody (at a dilution of 1:250) resulted in significant reductions in cell number and uptake of [3H]thymidine, compared with nonimmune serum at the same titer. Similar results were observed in uninjured HK-2 cells. It is concluded that this in vitro model of ATP depletion in a human proximal tubule cell line reproduces the pattern of gene expression previously observed in vivo in rat kidney after ischemic injury and that PTHrP plays a mitogenic role in the proliferative response after energy depletion.

Isoflurane alters proximal tubular cell susceptibility to toxic and hypoxic forms of attack.

BACKGROUND: Fluorinated anesthetics can profoundly alter plasma membrane structure and function, potentially impacting cell injury responses. Because major surgery often precipitates acute renal failure, this study assessed whether the most commonly used fluorinated anesthetic, isoflurane, alters tubular cell responses to toxic and hypoxic attack. METHODS: Mouse proximal tubule segments were incubated under control conditions or with a clinically relevant isoflurane dose. Cell viability (lactate dehydrogenase release), deacylation (fatty acid, such as C20:4 levels), and adenosine triphosphate (ATP) concentrations were assessed under one or more of the following conditions: (a) exogenous phospholipase A2 (PLA2) or C20:4 addition, (b) Ca2+ overload (A23187 ionophore), (c) increased metabolic work (Na ionophore), and (d) hypoxia- or antimycin A-induced attack. Isoflurane's effect on NBD phosphatidylserine uptake (an index of plasma membrane aminophospholipid translocase activity) was also assessed. RESULTS: Isoflurane alone caused trivial deacylation and no lactate dehydrogenase release. However, it strikingly sensitized to both PLA2- and A23187-induced deacylation and cell death. Isoflurane also exacerbated C20:4's direct membrane lytic effect. Under conditions of mild ATP depletion (Na ionophore-induced increased ATP consumption; PLA2-induced mitochondrial suppression), isoflurane provoked moderate/severe ATP reductions and cell death. Conversely, under conditions of maximal ATP depletion (hypoxia, antimycin), isoflurane conferred a modest cytoprotective effect. Isoflurane blocked aminophospholipid translocase activity, which normally maintains plasma membrane lipid asymmetry (that is, preventing its "flip flop"). CONCLUSIONS: Isoflurane profoundly and differentially affects tubular cell responses to toxic and hypoxic attack. Direct drug-induced alterations in lipid trafficking/plasma membrane orientation and in cell energy production are likely involved. Although the in vivo relevance of these findings remains unknown, they have potential implications for intraoperative renal tubular cell structure/function and how cells may respond to superimposed attack.