Altered cholesterol localization and caveolin expression during the evolution of acute renal failure

BACKGROUND: Renal cortical/proximal tubule cholesterol accumulation, with preferential localization within plasma membrane "detergent resistant microdomains" (DRMs: rafts/caveolae), is a hallmark of the maintenance phase of acute renal failure (ARF). This study addressed two related issues: (1) Are maintenance-phase cholesterol increases accompanied by an up-regulation of caveolin, a DRM/caveolar-associated cholesterol binding protein? (2) Is DRM cholesterol/caveolin homeostasis acutely altered during the induction phase of ARF? METHODS: Mouse kidneys were subjected to ischemia +/- reperfusion (I/R) followed by assessment of cholesterol DRM partitioning. Acute cell injury effects on potential caveolin release from isolated proximal tubules or into urine also were assessed. Finally, renal cortical/isolated proximal tubule caveolin levels were determined 18 hours after I/R or myoglobinuric ARF. RESULTS: Acute ischemia causes a rapid shift of cholesterol into cortical DRMs (>22%). Cholesterol migration into DRMs also was observed in ATP-depleted cultured proximal tubule (HK-2) cells. Acute hypoxic or toxic tubule injury induced plasma membrane caveolin release (Western blot). By the maintenance phase of ARF, marked renal cortical/proximal tubule caveolin increases resulted. CONCLUSIONS: Acute proximal tubular injury damages caveolar/DRM structures, as determined by cholesterol maldistribution and caveolin release. Post-injury, there is a dramatic up-regulation of renal cortical/proximal tubule caveolin, suggesting an increased caveolar mass. These findings indicate, to our knowledge for the first time, that dysregulation of caveolae/raft microdomain expression is a correlate of, and potential participant in, the induction and maintenance phases of ischemic and toxic forms of experimental ARF.     

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.     

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.     

Porcine hepatic phospholipid efflux during reperfusion after cold ischemia

BACKGROUND: Cold preservation produces hepatic injury that is difficult to assess during early reperfusion. The value of reperfusion plasma choline phospholipid in predicting subsequent organ function is documented in these studies. MATERIALS AND METHODS: Livers of female Yorkshire pigs were prepared for transplantation. After 2 h of cold ischemia the reperfusion plasma was evaluated for choline phospholipid and cholesterol. These values were correlated with bile secretion, hepatic hemodynamics, oxygen uptake, and plasma sorbitol dehydrogenase levels. RESULTS: The isolated porcine liver demonstrates a rapid efflux of choline phospholipids into plasma during early reperfusion after cold preservation. After this initial efflux no subsequent plasma increment occurred. These choline-phospholipid increments were isolated in plasma higher density (d > 1.063) lipoproteins and were not accompanied by equivalent increases in cholesterol. Neither biliary reflux nor lecithin cholesterol acyl transferase abnormalities contributed appreciably to the phospholipid increments in reperfusion plasma. Livers with the largest efflux of choline phospholipids had the most impaired circulatory and bile secretory function at 4 h of reperfusion. CONCLUSION: The immediate increase of choline phospholipids, particularly lysophosphatidylcholine, in reperfusion plasma after cold ischemia provides an index of the injury occurring during this interval and correlates with early organ function.     

Rhabdomyolysis

Key points Rhabdomyolysis describes the destruction or disintegrationof striated muscle; it is an important cause of acute renalfailure. Creatinine kinase concentration is the most sensitiveand useful indicator of muscle injury in rhabdomyolysis. Themost important intervention is early aggressive crystalloidfluid resuscitation. Life-threatening hyperkalaemia is a commoncause of death and must be treated promptly. Myoglobin-inducedrenal failure has an excellent prognosis.   The term rhabdomyolysis describes the breakdown or disintegrationof striated muscle. Although a broad range of conditions canresult in rhabdomyolysis, the final common pathway of myocytenecrosis involves a rapid increase in intracytoplasmic calcium.This leads to the release of myocyte constituents into the circulation,which can produce life-threatening complications including acutehyperkalaemia and acute renal failure (ARF). Rhabdomyolysis is a common cause of ARF, especially in timesof conflict or after major disasters     

Proximal tubules from caspase-1-deficient mice are protected against hypoxia-induced membrane injury.

BACKGROUND: Caspase-1 is a proinflammatory caspase via activation of the cytokine IL-18. We have recently demonstrated that the caspase-1-mediated production of IL-18 plays a deleterious role in ischaemic acute renal failure (ARF) which is independent of neutrophils and CD4+ T cells. The role of caspase-1 in hypoxia-induced membrane injury of proximal tubules (PT) in vitro is unknown. METHODS: Freshly isolated mouse PT exposed to 25 min of hypoxia were used to study the role of caspases, caspase-1 and IL-18 in hypoxia-induced membrane injury. Lactate dehydrogenase (LDH) release into the PT medium was used as a biochemical parameter of cell membrane damage. IL-18 was determined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. RESULTS: PT pre-incubated with the novel pancaspase inhibitor IDN-8050 were protected; LDH release (%) was 35+/-3 in vehicle-treated hypoxic PT and 21+/-2 in IDN-8050-treated hypoxic PT (P<0.01, n=6). To investigate the mechanism of protection and examine the role of caspase-1 specifically, PT were isolated in parallel from wild-type and caspase-1- deficient (-/-) mice. PT from caspase-1-/-mice demonstrated less hypoxia-induced membrane injury. LDH release was 37+/-2 in wild-type hypoxic PT and 28+/-2 in caspase-1-/-hypoxic PT (P<0.01, n=12). IL-18 was detected in PT by immunoblotting and ELISA. PT pre-incubated with IL-18 binding protein, an inhibitor of IL-18, were not protected. CONCLUSIONS: These studies demonstrate a deleterious effect of the proinflammatory caspase, caspase-1, on PT in vitro in the absence of inflammatory cells and vascular effects.     

Clinical study of influenza-associated rhabdomyolysis with acute renal failure

AIMS: Influenza-associated rhabdomyolysis induces renal failure with a fatal outcome. The aim of this study is to evaluate the clinical features, diagnosis, and treatment efficacy of influenza-associated rhabdomyolysis patients with acute renal failure (ARF). MATERIALS AND METHODS: The subjects included 6 patients who had presented with rhabdomyolysis and ARF due to influenza infection on admission to our university hospital and its 2 affiliated hospitals between January 2002 and February 2004. We retrospectively examined the cases. RESULTS: All the patients (n = 6) were males, and none of them had received an influenza vaccine. The viruses were identified as influenza A (n = 5) and B (n = 1). Muscular weakness was observed in many cases (n = 5), whereas pain or tenderness was observed in only 1 case (n = 1). For anuric or oliguric patients (n = 4), blood purification therapy was performed, while for patients in whom the urine volume was normal (n = 2), conservative therapy was administered. CONCLUSION: Careful medical attention is necessary when patients have muscle pain and weakness. Early recognition of rhabdomyolysis allows prompt institution of an appropriate therapy that includes blood purification and may minimize the renal dysfunction associated with this disorder.     

Tempol, a membrane-permeable radical scavenger, reduces oxidant stress-mediated renal dysfunction and injury in the rat

BACKGROUND: The generation of reactive oxygen species (ROS) contributes to the pathogenesis of renal ischemia-reperfusion injury. The aim of this study was to investigate the effects of tempol in (1) an in vivo rat model of renal ischemia/reperfusion injury and on (2) cellular injury and death of rat renal proximal tubular (PT) cells exposed to oxidant stress in the form of hydrogen peroxide (H2O2). METHODS: Male Wistar rats underwent bilateral renal pedicle clamping for 45 minutes followed by reperfusion for six hours. Tempol (30 mg/kg/h), desferrioxamine (DEF; 40 mg/kg/h), or a combination of tempol (30 mg/kg/h) and DEF (40 mg/kg/h) were administered prior to and throughout reperfusion. Plasma concentrations of urea, creatinine, Na+, gamma-glutamyl transferase (gammaGT), aspartate aminotransferase (AST), and urinary Na+ and N-acetyl-beta-D-glucosaminidase (NAG) were measured for the assessment of renal function and reperfusion injury. Kidney myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured for assessment of polymorphonuclear (PMN) cell infiltration and lipid peroxidation, respectively. Renal sections were used for histologic grading of renal injury and for immunohistochemical localization of nitrotyrosine and poly(ADP-ribose) synthetase (PARS). Primary cultures of rat PT cells were incubated with H2O2 (1 mmol/L for 4 h) either in the absence or presence of increasing concentrations of tempol (0.03 to 10 mmol/L), DEF (0.03 to 10 mmol/L), or a combination of tempol (3 mmol/L) or DEF (3 mmol/L). PT cell injury and death were determined by evaluating mitochondrial respiration and lactate dehydrogenase (LDH) release, respectively. RESULTS: In vivo, tempol significantly reduced the increase in urea, creatinine, gammaGT, AST, NAG, and FENa produced by renal ischemia/reperfusion, suggesting an improvement in both renal function and injury. Tempol also significantly reduced kidney MPO activity and MDA levels, indicating a reduction in PMN infiltration and lipid peroxidation, respectively. Tempol reduced the histologic evidence of renal damage associated with ischemia/reperfusion and caused a substantial reduction in the staining for nitrotyrosine and PARS, suggesting reduced nitrosative and oxidative stress. In vitro, tempol significantly attenuated H2O2-mediated decrease in mitochondrial respiration and increase in LDH release from rat PT cells, indicating a reduction in cell injury and death. Both in vivo and in vitro, the beneficial actions of tempol were similar to those obtained using the Fe2+ chelator DEF. However, coadministration of DEF and tempol did not produce any additional beneficial actions against renal ischemia/reperfusion injury or against oxidative stress-mediated PT cell injury/death. CONCLUSION: Our results suggest that the membrane-permeable radical scavenger, tempol, reduces the renal dysfunction and injury associated with ischemia/reperfusion of the kidney.     

Inhibitors of poly (ADP-ribose) synthetase protect rat proximal tubular cells against oxidant stress.

BACKGROUND: The generation of reactive oxygen species (ROS) has been implicated in the pathogenesis of renal ischemia-reperfusion injury. ROS produce DNA strand breaks that lead to the activation of the DNA-repair enzyme poly (ADP-ribose) synthetase (PARS). Excessive PARS activation results in the depletion of its substrate, nicotinamide adenine dinucleotide (NAD) and subsequently of adenosine 5'-triphosphate (ATP), leading to cellular dysfunction and eventual cell death. The aim of this study was to investigate the effect of various PARS inhibitors on the cellular injury and death of rat renal proximal tubular (PT) cells exposed to hydrogen peroxide (H2O2). METHODS: Rat PT cell cultures were incubated with H2O2 (1 mM) either in the presence or absence of the PARS inhibitors 3-aminobenzamide (3-AB, 3 mM), 1,5-dihydroxyisoquinoline (0.3 mM) or nicotinamide (Nic, 3 mM), or increasing concentrations of desferrioxamine (0.03 to 3 mM) or catalase (0.03 to 3 U/ml). Cellular injury and death were determined using the MTT and lactate dehydrogenase (LDH) assays, respectively. H2O2-mediated PARS activation in rat PT cells and the effects of PARS inhibitors on PARS activity were determined by measurement of the incorporation of [3H]NAD into nuclear proteins. RESULTS: Incubation of rat PT cells with H2O2 significantly inhibited mitochondrial respiration and increased LDH release, respectively. Both desferrioxamine and catalase reduced H2O2-mediated cellular injury and death. All three PARS inhibitors significantly attenuated the H2O2-mediated decrease in mitochondrial respiration and the increase in LDH release. Incubation with H2O2 produced a significant increase in PARS activity that was significantly reduced by all PARS inhibitors. 3-Aminobenzoic acid (3 mM) and nicotinic acid (3 mM), structural analogs of 3-AB and Nic, respectively, which did not inhibit PARS activity, did not reduce the H2O2-mediated injury and necrosis in cultures of rat PT cells. CONCLUSION: We propose that PARS activation contributes to ROS-mediated injury of rat PT cells and, therefore, to the cellular injury and cell death associated with conditions of oxidant stress in the kidney.     

Prognostic value, kinetics and effect of CVVHDF on serum of the myoglobin and creatine kinase in critically ill patients with rhabdomyolysis

BACKGROUND: (I) To investigate the kinetics of the myoglobin and creatine kinase (CK) in rhabdomyolysis. Especially to describe those patients in whom an isolated increase in the myoglobin or the CK occurred at a later stage. (II) To evaluate the sensitivity of the myoglobin and the CK as prognostic tools for the development of Acute renal failure (ARF). (III) To investigate the effect of continuous venovenous haemodiafiltration (CVVHDF) on the myoglobin elimination in ARF. PATIENTS AND METHODS: Prospective and retrospective cohort study carried out in an ICU of a university hospital. A total of 47 critically ill patients with rhabdomyolysis and a plasma myoglobin > 5000 microg l(-1) were admitted between July 1998 and July 2003. RESULTS: (I) The myoglobin peaked 0.66 +/- 0.6 days before the CK. The elimination kinetics of the myoglobin was faster than for the CK. (II) Fifty percent developed ARF. Mortality in the ARF patients was 52% compared to 14% in the non-ARF patients. The sensitivity and specificity of developing ARF were higher with the myoglobin in comparison to the CK. (III) In non-ARF, t(1/2) CK was 25.5 h and t(1/2) myoglobin was 17 h (13-23). In those with ARF treated with CVVHDF, t(1/2) CK was 24.8 and t(1/2) myoglobin was 21 h (17-29). CONCLUSION: (I) The myoglobin peaked earlier than the CK. (II) The myoglobin was a better prognostic tool than the CK. However, the myoglobin also has a wide interindividual range. (III) Though the myoglobin is eliminated in ultrafiltration t(1/2) myoglobin, it was not faster in patients with ARF treated with CVVHDF compared to non-ARF patients.     

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.     

Utility of Serum Creatinine, Creatine Kinase and Urinary Myoglobin in Detecting Acute Renal Failure due to Rhabdomyolysis in Trauma and Electrical Burns Patients

Rhabdomyolysis due to trauma and burns is an important cause of acute renal failure (ARF) secondary to myoglobinuria. To prevent morbidity and mortality from ARF due to rhabdomyolysis, early detection of ARF by monitoring the biochemical parameters such as serum creatinine, serum creatine kinase (CK), and urinary myoglobin (UM) can be helpful. The aims of the study were (1) to detect ARF due to rhabdomyolysis using serum creatinine, serum CK, and UM in trauma and electrical burn patients (2) to compare utility of these parameters in early prediction of ARF in patients of rhabdomyolysis. A total of 50 patients with trauma and electrical burns were included in the study. Serum creatinine, serum CK, and UM measurements were done at the time of admission and after 48 h. Diagnosis of ARF was made in the patients by Rifle’s criteria. The presence of significant elevation of creatinine, serum CK, and UM at the time of admission and after 48 h was compared in patients developing ARF by Fisher’s exact test. Fifteen of the 50 patients developed ARF as per the defined criteria. Of these, 9 patients (60 %) had raised level of serum creatinine above 1.4 mg% at admission and 14 patients (93.33 %) had CK level >1250 U/L at admission, whereas UM was positive in 6 (40 %) patients. Serum creatinine was significantly raised in all of the 15 ARF patients (100 %) after 48 h of admission and serum CK was raised in 14 of the 15 ARF patients (93.33 %). UM was negative in all the patients after 48 h of admission. Statistical analysis showed that rise in serum CK on admission was significantly increased in patients developing ARF as compared with serum creatinine and UM (P < 0.0001). On admission, CK is a better predictor of ARF due to rhabdomyolysis than creatinine and UM. Initial creatinine is a better predictor of ARF due to rhabdomyolysis than UM. UM assay is not a good investigation for early prediction of ARF in rhabdomyolysis.     

Exogenous arachidonic acid promotes insulin release from intact or permeabilized rat islets by dual mechanisms. Putative activation of Ca2+ mobilization and protein kinase C

A number of indirect studies suggest a role for endogenous arachidonic acid (AA) in pancreatic islet function. To probe the effects of this fatty acid, AA and other polyunsaturated fatty acids were exogenously provided in Ca2+-free medium to avoid the formation of insoluble or impermeant Ca2+-arachidonate complexes. Concentrations of AA of greater than or equal to 3 microM induced potent and sustained but reversible 45Ca efflux from prelabeled intact (or digitonin-permeabilized) islets; AA also induced insulin release at somewhat higher concentrations. Other unsaturated fatty acids (erucic, oleic, linoleic, linolenic, dihomo-gamma-linolenic, eicosapentaenoic, docosahexaenoic acids) were generally less active than AA itself, indicating a structure-function relationship. The effects of AA were saturable, were inhibitable by cooling, and were not accompanied in parallel by 51Cr release or trypan blue retention, suggesting a nontoxic mechanism. At low concentrations (3.3-16 microM), at which AA does not stimulate insulin release, AA-induced 45Ca efflux was not reduced by pretreatment with ionomycin (to deplete membrane-bound Ca2+ stores), suggesting stimulation of Ca2+ extrusion through the plasma membrane. At higher concentrations (greater than or equal to 25 microM), at which AA promotes insulin release, further Ca2+ efflux was stimulated, which was blunted by pretreatment with ionomycin (as well as by trifluoperazine). Conversely, pretreatment with AA obliterated the effects of ionomycin (3 microM) on cellular Ca2+ mobilization. Thus, AA also mobilizes Ca2+ from intracellular organelles, leading to a rise in free cytosolic Ca2+ (as previously reported). AA-induced 45Ca efflux and insulin release were independent of the presence of extracellular Na+ and did not require the oxygenation of AA. Dose-response curves comparing 45Ca efflux and insulin secretion suggested that AA also stimulates hormone release by at least one other mechanism in addition to Ca2+ mobilization. This second stimulatory effect of AA could be seen in digitonin-permeabilized islets, where changes in cytosolic free Ca2+ concentration were vitiated by EGTA-containing buffers. Such secretion was also saturable and was inhibited by cooling or by spermine (which inhibits protein kinase C in the islet). Furthermore, AA-induced secretion from either intact or permeabilized islets was blunted by prolonged pretreatment of islets with a phorbol ester to deplete them of protein kinase C. Thus, exogenous arachidonic acid seems to be a complete secretagogue, having stimulatory effects both on Ca2+ mobilization and Ca2+-related secretory processes, putatively the activation of protein kinase C.     

'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.     

Rhabdomyolysis and acute renal failure in children

Acute renal failure (ARF) is an important complication of rhabdomyolysis. However, the contributing factors to the development of ARF in children with rhabdomyolysis remain obscure. The aim of this study was to clarify the factors contributing to the development of ARF in children with rhabdomyolysis. This is a retrospective review of the clinical characteristics, laboratory data, pediatric risk of mortality (PRISM) scores, the occurrence of systemic inflammatory response syndrome (SIRS) criteria, and the number of dysfunctional organs in 18 children with rhabdomyolysis seen in our hospital between 1991 and 2000. The patients were divided into an ARF group (n=9) and a non-ARF group (n=9). All patients with ARF had more than two dysfunctional organs. The incidence of dehydration, serum concentrations of myoglobin, creatinine kinase, aspartate aminotransferase, and lactate dehydrogenase, PRISM scores, and the numbers of SIRS criteria and dysfunctional organs were higher in the ARF group than the non-ARF group. The blood pH and base excess, and urinary pH were lower in the ARF group than in the non-ARF group. These results suggest that ARF is more likely to develop in the presence of dehydration, metabolic acidosis, or severe muscle damage, or with multiple organ failure in children with acute rhabdomyolysis. Received: 12 April 2001 / Revised: 20 August 2001 / Accepted: 21 August 2001     

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.     

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.     

Acute renal failure in rhabdomyolysis.

Fifteen to 30% of patients develop acute renal failure (ARF) following rhabdomyolysis and rhabdomyolysis accounts for 5 to 9% of all ARF. Experimental studies revealed two critical factors that predispose to myoglobinuric ARF: hypovolemia/dehydration and aciduria. At the nephron level, three basic mechanisms underlie heme protein toxicity: renal vasoconstriction with decreased renal blood flow, intraluminal cast formation and direct heme protein-induced cytotoxicity. During the early phase of myoglobinuric ARF, hemodynamic process are mainly involved in glomerumar filtration rate decrease while tubular mechanisms occur in the late phase. Critical factors which predispose to myoglobinuric ARF in animal models--i.e. hypovolemia/dehydration and aciduria--are also encountered in human epidemiological studies. Prevention of myoglobinuric ARF rely on rapid and adequate correction of fluid deficits with saline, bicarbonates and mannitol. The choice of hemodialysis technique in the case of constituted ARF strongly depends on the site of intervention, especially in the case of rescue operation. The care of myoglobinuric ARF in intensive care unit do not differ from this of ARF from other causes.     

过度训练致急性肾损伤的临床研究

目的:了解过度训练致急性肾损伤(OTIAKI)的发病情况及临床特点。方法:对我院9年间(2001年5月～2010年8月)因5km武装越野跑住院患者的临床病理特点和预后进行分析。结果:83例患者符合入选标准,单纯尿检异常32例,横纹肌溶解症24例,横纹肌溶解伴急性肾衰竭27例。重症OTIAKI的患者除肾脏损害外,还可表现为意识障碍、抽搐、肌痛、棕色尿,血清UREA、CR、CK、CK-MB、LDH、ALT、HBDH等指标明显异常。肾脏病理检查提示为肾小球和肾小管的轻微病变。83例患者死亡1例,病死率为1.2%。结论:过度训练致急性肾损伤已成为近年来急性肾衰竭的重要病因;血清酶升高的幅度越大,病情越重;血清酶水平恢复越慢,预后越差;早期应用肾剂量的多巴胺和山莨菪碱有助于肾功能的恢复,病情严重者及时行血液净化治疗。     

Ameliorative effect of hepatocyte growth factor on glycerol-induced acute renal failure with acute tubular necrosis

BACKGROUND/AIMS: Hepatocyte growth factor (HGF), a multi-potent growth factor, is known to promote regeneration of damaged renal epithelial cells. Glycerol injection into rats induces severe acute renal failure (ARF) with ischemia and tubular necrosis, a model which shares many features with human ARF or rhabdomyolysis. We investigated the efficacy of HGF in this glycerol-induced ARF rat model. METHODS: ARF was induced by intramuscular injection of glycerol into the hind limbs of male Wistar rats. HGF (0.25 mg/kg/shot) or vehicle was administered intravenously 1 h before and 1, 3, 5, 8, 24 and 36 h after glycerol injection. Biochemical parameters for serum and urine were measured and histological analyses of the kidneys were performed. We also analyzed endogenous HGF expression and phosphorylation of c-Met/HGF receptor in the kidneys of glycerol-induced ARF rats. RESULTS: Glycerol treatment caused severe ARF which invariably led to death of the rats. Repeated administration of HGF protected rats from death caused by severe ARF. Histological analyses revealed that HGF treatment reduced necrosis of tubular cells in the renal cortex. Serum/urine biochemical parameters also showed that renal dysfunction was improved by HGF administration. Intravenous administration of HGF enhanced phosphorylation of the c-Met/HGF receptor and mitogen-activated protein kinase in the kidney. In the vehicle-treated group the renal endogenous HGF concentration decreased and there was no change in c-Met/HGF receptor phosphorylation. CONCLUSION: These results indicate that HGF effectively accelerated the recovery of renal function and improved survival in glycerol-induced ARF rats.