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Lipin 1 is an intracellular protein acting as a phosphatidic acid phosphohydrolase enzyme controlling lipid metabolism. Human recessive mutations in LPIN1 cause recurrent, early-onset myoglobinuria, a condition normally associated with muscle pain and weakness. Whether and how lipin 1 deficiency in humans leads to peripheral neuropathy is yet unclear. Herein, two novel compound heterozygous mutations in LPIN1 with neurological disorders, but no myoglobinuria were identified in an adult-onset syndromic myasthenia family. The present study sought to explore the pathogenic mechanism of LPIN1 in muscular and neural development. Methods: The clinical diagnosis of the proband was compared to the known 48 cases of LPIN1 recessive homozygous mutations. Whole-exome sequencing was carried out on the syndromic myasthenia family to identify the causative gene. The pathogenesis of lipin 1 deficiency during somitogenesis and neurogenesis was investigated using the zebrafish model. Whole-mount in situ hybridization, immunohistochemistry, birefringence analysis, touch-evoke escape response and locomotion assays were performed to observe in vivo the changes in muscles and neurons. The conservatism of the molecular pathways regulated by lipin 1 was evaluated in human primary glioblastoma and mouse myoblast cells by siRNA knockdown, drug treatment, qRT-PCR and Western blotting analysis. Results: The patient exhibited adult-onset myasthenia accompanied by muscle fiber atrophy and nerve demyelination without myoglobinuria. Two novel heterozygous mutations, c.2047A>C (p.I683L) and c.2201G>A (p.R734Q) in LPIN1 , were identified in the family and predicted to alter the tertiary structure of LPIN1 protein. Lipin 1 deficiency in zebrafish embryos generated by lpin1 morpholino knockdown or human LPIN1 mutant mRNA injections reproduced the myotomes defects, a reduction both in primary motor neurons and secondary motor neurons projections, morphological changes of post-synaptic clusters of acetylcholine receptors, and myelination defects, which led to reduced touch-evoked response and abnormalities of swimming behaviors. Loss of lipin 1 function in zebrafish and mammalian cells also exhibited altered expression levels of muscle and neuron markers, as well as abnormally enhanced Notch signaling, which was partially rescued by the specific Notch pathway inhibitor DAPT. Conclusions: These findings pointed out that the compound heterozygous mutations in human LPIN1 caused adult-onset syndromic myasthenia with peripheral neuropathy. Moreover, zebrafish could be used to model the neuromuscular phenotypes due to the lipin 1 deficiency, where a novel pathological role of over-activated Notch signaling was discovered and further confirmed in mammalian cell lines., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Myoglobinuric acute renal failure (MARF) may develop after severe muscle injury. Heme oxygenase-1 (HO-1), a stress-response protein, has been implicated as a protective agent against MARF. We hypothesized that hyperbaric oxygen therapy (HBOT) may alleviate MARF by inducing renal HO-1 expression. Wistar-Albino rats were randomly assigned into three groups: Control (n = 4), MARF (n = 8), MARF + HBO (n = 8). MARF was induced by intramuscular glycerol (50%, 8 mL/kg) injection. Saline (8 mL/kg) was injected into the hind limb of the animals in the control group. Animals in the MARF + HBO group received two sessions of HBO therapy (90 min at 2.5 atm) 2 and 18 h after glycerol injection. Serum and tissue samples were taken at 24 h. Serum urea and creatinine levels increased in the MARF and MARF + HBO groups confirming the development of MARF. But, serum urea and creatinine levels were similar in MARF and MARF + HBO groups. Oxidative stress parameters were similar among all groups. Histological renal injury score was similar in MARF and MARF + HBO groups. HO-1 level, determined by immunohistochemistry, was significantly higher in MARF and MARF + HBO groups, compared to the control group. Although HO-1 level in MARF + HBO group was higher than MARF group, it was not statistically significant. We found that HBOT did not reduce renal injury in experimental MARF model. HBOT is used to reduce the muscle damage after crush injury, which may be accompanied by MARF. Therefore, more studies are needed to understand the effects of HBO treatment on renal functions after MARF.

One large group of hereditary myopathies characterized by recurrent myoglobinuria, almost invariably triggered by exercise, comprises metabolic disorders of two main fuels, glycogen and long-chain fatty acids, or mitochondrial diseases of the respiratory chain. Differential diagnosis is required to distinguish the three conditions, although all cause a crisis of muscle energy. Muscle biopsy may be useful when performed well after the episode of rhabdomyolysis. Molecular genetics is increasingly the diagnostic test of choice to discover the underlying genetic basis.

Rugby union is a sport governed by the impacts of high force and high frequency. Analysis of physiological markers following a game can provide an understanding of the physiological response of an individual and the time course changes in response to recovery. Urine and saliva were collected from 11 elite amateur rugby players 24 h before, immediately after, and at 17, 25, 38, 62 and 86 h post-game. Myoglobin, salivary immunoglobulin A and cortisol were analysed by ELISA, whereas neopterin and total neopterin were analysed by high-performance liquid chromatography. There was a significant post-game increase of all four markers. The increases were cortisol 4-fold, myoglobin 2.85-fold, neopterin 1.75-fold and total neopterin 2.3-fold when corrected with specific gravity. All significant changes occurred post-game only, with markers returning to and remaining at baseline within 17 h. The intensity of the game caused significant changes in key physiological markers of stress. They provide an understanding of the stress experienced during a single game of rugby and the time course changes associated with player recovery. Neopterin provides a new marker of detecting an acute inflammatory response in physical exercise, while specific gravity should be considered for urine volume correction post-exercise.

Rhabdomyolysis accounts for ∼10% of acute kidney injuries. In glycerol-induced myoglobinuric acute kidney injury, we found an increase in the nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear protein, a key redox-sensitive transcription factor, and Nrf2-regulated genes and proteins including upregulation of heme oxygenase-1. In in vitro studies, pretreatment of LLC-PK1 cells with an activator of Nrf2 before myoglobin exposure significantly decreased oxidant generation and cytotoxicity, whereas Nrf2 inhibition and gene silencing exacerbated the injury. Chlormethiazole, a specific CYP2E1 transcription inhibitor, prevented an increase in catalytic iron in the kidneys, decreased oxidative stress, blocked nuclear translocation of the Nrf2 protein, decreased heme oxygenase-1 upregulation, and provided functional and histological protection against acute kidney injury. CYP2E1 inhibitors and gene silencing in renal tubular epithelial cells significantly decreased reactive oxygen species generation and provided marked protection against myoglobin-induced cytotoxicity. Thus, during CYP2E1-induced oxidative stress, the transcription factor Nrf2 has a pivotal role in the early adaptive response. Inhibition of CYP2E1 coupled with the prior induction of Nrf2 may be a valuable tool to reduce CYP2E1-mediated rhabdomyolysis-induced acute kidney injury.

Myoglobinuric acute renal failure (ARF) is a uremic syndrome caused by traumatic or non-traumatic skeletal muscle breakdown and intracellular elements that are released into the bloodstream. We hypothesized that hyperbaric oxygen (HBO) therapy could be beneficial in the treatment of myoglobinuric ARF caused by rhabdomyolysis. A total of 32 rats were used in the study. The rats were divided into four groups: control, control+hyperbaric oxygen (control+HBO), ARF, and ARF+hyperbaric oxygen (ARF+HBO). Glycerol (8 ml/kg) was injected into the hind legs of each of the rats in ARF and ARF+HBO groups. 2.5 atmospheric absolute HBO was applied to the rats in the control+HBO and ARF+HBO groups for 90 min on two consecutive days. Plasma urea, creatinine, sodium, potassium, calcium, aspartate aminotransferase, alanine aminotransferase, lactic dehydrogenase, creatinine kinase and urine creatinine and sodium were examined. Creatinine clearance and fractional sodium excretion could then be calculated. Superoxide dismutase, catalase, glutathione and malondialdehyde (MDA) levels were assessed in renal tissue. Tissue samples were evaluated by Hematoxylin-eosin, PCNA and TUNEL staining histopathologically. MDA levels were found to be significantly decreased whereas SOD and CAT were twofold higher in the ARF+HBO group compared to the ARF group. Renal function tests were ameliorated by HBO therapy. Semiquantitative evaluation of histopathological findings indicated that necrosis and cast formation was decreased by HBO therapy and TUNEL staining showed that apoptosis was inhibited. PCNA staining showed that HBO therapy did not increase regeneration. Ultimately, we conclude that, in accordance with our hypothesis, HBO could be beneficial in the treatment of myoglobinuric ARF.

Exercise intolerance with myalgia, muscle stiffness, and recurrent rhabdomyolysis due to mutations in the DMD gene can mimic metabolic myopathies leading to delayed or inaccurate diagnoses. In this retrospective chart review, we report 3 unrelated boys with exertional myalgia, muscle stiffness, myoglobinuria, and normal neurological examination due to an identical point mutation in the DMD gene: a hemizygous T-to-C change in exon 15 (c.1724T>C) resulting in an amino acid substitution of leucine to proline at codon 575. Two of the 3 boys had normal dystrophin immunostaining and Western blot analysis in muscle. This missense mutation has been reported twice before, with at least 1 patient exhibiting rhabdomyolysis. Our report, however, is the first to describe in detail the clinical findings associated with this specific mutation. Further studies and clinical reports are needed to better understand the pathogenicity of the mutation.

A mitochondria-targeted chimeric compound consisting of a rhodamine derivative linked to a plastoquinone molecule (10-(6'-plastoquinonyl)decylrhodamine, SkQR1) was studied under conditions of acute brain or kidney damage. A protective effect of this compound was demonstrated in a model of focal brain ischemia, rat kidney ischemia/reperfusion, myoglobinuria (rhabdomyolysis, or crush syndrome), and pyelonephritis. We found that a single intraperitoneal injection of SkQR1 diminishes the size of the ischemic zone in the brain and improves performance of a test characterizing neurological deficit in ischemic animals. Control substance not containing plastoquinone appeared to be not neuroprotective. The data show that SkQR1 is a nephroprotectant and neuroprotectant, which can be due to the antioxidative action of this Skulachev cation.

Rhabdomyolysis or crush syndrome is a pathology caused by muscle injury resulting in acute renal failure. The latest data give strong evidence that this syndrome caused by accumulation of muscle breakdown products in the blood stream is associated with oxidative stress with primary role of mitochondria. In order to evaluate the significance of oxidative stress under rhabdomyolysis we explored the direct effect of myoglobin on renal tubules and isolated kidney mitochondria while measuring mitochondrial respiratory control, production of reactive oxygen and nitrogen species and lipid peroxidation. In parallel, we evaluated mitochondrial damage under myoglobinurea in vivo. An increase of lipid peroxidation products in kidney mitochondria and release of cytochrome c was detected on the first day of myoglobinuria. In mitochondria incubated with myoglobin we detected respiratory control drop, uncoupling of oxidative phosphorylation, an increase of lipid peroxidation products and stimulated NO synthesis. Mitochondrial pore inhibitor, cyclosporine A, mitochondria-targeted antioxidant (SkQ1) and deferoxamine (Fe-chelator and ferryl-myoglobin reducer) abrogated these events. Similar effects (oxidative stress and mitochondrial dysfunction) were revealed when myoglobin was added to isolated renal tubules. Thus, rhabdomyolysis can be considered as oxidative stress-mediated pathology with mitochondria to be the primary target and possibly the source of reactive oxygen and nitrogen species. We speculate that rhabdomyolysis-induced kidney damage involves direct interaction of myoglobin with mitochondria possibly resulting in iron ions release from myoglobin's heme, which promotes the peroxidation of mitochondrial membranes. Usage of mitochondrial permeability transition blockers, Fe-chelators or mitochondria-targeted antioxidants, may bring salvage from this pathology.

We report a case of rosuvastatin induced rhabdomyolysis in a low risk patient, who presented with five-day history of generalized muscle pain, weakness and easy fatigability associated with passing dark urine. Initial investigations showed creatinine 140micromol/L, creatine kinase (CK) 4566 U/L and serum myoglobin 2694 ng/ml with a significant increase in urine myoglobin. Although there were no obvious risk factors, the patient was diagnosed with rosuvastatin induced rhabdomyolysis. The drug was stopped on the first day of admission and the patient was initiated on intravenous fluid with cautious monitoring of serum electrolytes. On the following days the level of creatine kinase and serum myoglobin returned toward normal and consequently he was discharged without statins but on dietary therapy. On follow-up evaluation, the patient was symptom free his serum creatinine was 106micromol/L, whereas his LDL cholesterol was 2.1mmol/L. The rosuvastatin induced rhabdomyolysis is discussed and the danger of its use in low risk patients is emphasized.

1. Free radicals and nitric oxide (NO) play a crucial role in the pathogenesis of myoglobinuric acute renal failure (ARF). The aim of the present study was to investigate the effects of caffeic acid phenethyl ester (CAPE), an anti-oxidant, on the myoglobinuric ARF induced by intramuscular hypertonic glycerol injection. 2. Thirty rats were divided equally into three groups. Rats in group 1 were given saline and those in groups 2 and 3 were injected with glycerol (10 mL/kg, i.m.). Concomitant and 24 h after glycerol injection, CAPE (10 micromol/kg, i.p.) was administered to group 3 rats. Forty-eight hours after glycerol injection, blood samples and kidney tissues of rats were taken under anaesthesia. 3. Plasma concentrations of urea, creatinine, malondialdehyde (MDA) and NO were determined, as were superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities and MDA levels in kidney tissues. Kidney morphology was also investigated. 4. In the group receiving CAPE, although SOD enzyme activity was found to be increased, we failed to find any protective effect of CAPE on other parameters investigated. Moreover, although CAPE significantly decreased NO levels, it increased plasma concentrations of urea and MDA. 5. We suggest that the effect of CAPE in decreasing NO concentrations may further increase the renal ischaemia in this model. Thus, CAPE may have a worsening rather than beneficial effect under these conditions in this model of ARF.

A(3) adenosine receptor (AR) activation and inhibition worsen and improve, respectively, renal function after ischemia-reperfusion (I/R) injury in rats. We sought to further characterize the role of A(3) ARs in modulating renal function after either I/R or myoglobinuric renal injury. A(3) knockout mice had significantly lower plasma creatinines compared with C57 controls 24 h after I/R or myoglobinuric renal injury. C57 control mice pretreated with the A(3) AR antagonist [3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5 dicarboxylate] or agonist [0.125 mg/kg N(6)-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (IB-MECA)] demonstrated improved or worsened renal function, respectively, after I/R or myoglobinuric renal injury. Higher doses of IB-MECA were lethal in C57 mice subjected to renal ischemia. H(1) but not H(2) histamine receptor antagonist prevented death in mice pretreated with IB-MECA before renal ischemia. Improvement in renal function was associated with significantly improved renal histology. In conclusion, preischemic A(3) AR activation (0.125 mg/kg IB-MECA) exacerbated renal I/R injury in mice. Mice lacking A(3) ARs or blocking A(3) ARs in wild-type mice resulted in significant renal protection from ischemic or myoglobinuric 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.

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.

Myoglobin has a relatively high molecular weight of 17,000 Da and is poorly cleared by dialysis (diffusion). However, elimination of myoglobin might be enhanced by an epuration modality based on convection for solute clearances. We present a single case of myoglobin-induced renal failure (peak creatine kinase level: 313,500 IU/l) treated by continuous venovenous hemofiltration (CVVH). Our purpose was to evaluate the efficiency of such a modality using an ultrafiltration rate of 2 to 3 l/h for myoglobin removal and clearance. The hemofilter was a 0.9 m(2) polyacrylonitrile (AN69) membrane Multiflow-100 (Hospal-Gambro, St-Leonard, Canada) and the blood flow rate was maintained at 150 ml/min by an AK-10 pump (Hospal-Gambro, St-Leonard, Canada). The ultrafiltration bag was placed 60 cm below the hemofilter and was free of pump control or suction device. Serum myoglobin concentration was 92,000 microg/l at CVVH initiation and dropped to 28,600 microg/l after 18 h of the continuous modality. The mean sieving coefficient for myoglobin was 0.6 during the first 9 h of therapy and this decreased to 0.4 during the following 7 h. Mean clearance of myoglobin was 22 ml/min, decreasing to 14 ml/min during corresponding periods, while the mean ultrafiltration rates were relatively stable at 2,153 +/- 148 ml/h and 2,074 +/- 85 ml/h, respectively. In contrast to myoglobin, the sieving coefficeint for urea, creatinine, and phosphorus remained stable at 1.0 during the first 16 h of CVVH. More than 700 mg of myoglobin were removed by CVVH during the entire treatment. In conclusion, considerable amounts of myoglobin can be removed by an extracorporeal modality allowing important convective fluxes and middle molecule clearances, such as CVVH at a rate of 2 to 3 l/h using an AN69 hemofilter. If myoglobin clearance had been maintained at 22 ml/min, 32 l of serum would have been cleared per day. However, the sieving coefficient of myoglobin decreased over time, probably as a consequence of protein coating and/or blood clotting of the hemofilter. Whereas myoglobin can be removed by CVVH, it remains unknown at this point if such a modality, applied early, can alter or shorten the course of myoglobinuric acute renal failure.

A 21-year-old patient developed rhabdomyolysis during his nineteenth week of treatment with clozapine for drug-resistant schizophrenia. No risk factors for rhabdomyolysis were found, but the calcium-dependent potassium efflux, normally responsible for membrane hyperpolarization and muscle refractoriness, was severely decreased in the patient's red blood cells. Clozapine is speculated to cause rhabdomyolysis in patients with defective calcium-activated K+ channels.

Intramuscular injection of hypertonic glycerol solution to rats results in acute renal injury. In this model, the proximal tubules are characteristically damaged. After glycerol injection renal glutathione (GSH) levels drastically decreased. On the other hand, stress protein heme oxygenase-1 (HO-1) was induced. When N-acetyl cysteine was administered to rats before 1 h glycerol injection, renal function was obviously improved. In this condition, the renal GSH content were sustained in the normal levels and HO-1 protein levels were decreased compared with those of glycerol-treated rats. Induction of HO-1 was accompanied by reduced renal GSH content. In addition, to investigate whether the location of HO-1 protein induced by glycerol injection is restricted to injured region or not in the kidney, we determined the localization of HO-1 protein using immunohistochemical staining. HO-1 protein was identified in the epithelia of the distal tubules, Henle's loop and collecting ducts, but not in the injured proximal tubules.

The purpose of the present study was to analyse the course of patients hospitalised with electrical burn wounds in the past 25 years at a major children's hospital in the United States in order to devise safe and cost effective management strategies for these patients. The study was a retrospective chart review of patients with electrical injuries admitted to the hospital between 1971 and 1995. We identified 127 children who were included in the study. Injuries resulted from biting an electrical cord (oral injury) (n = 48), placing an object into an electrical socket (outlet injury) (n = 33), contacting a low voltage wire or appliance indoors (low voltage household injury) (n = 25), contacting a high voltage wire outdoors (high voltage wire injury) (n = 18), or being struck by lightning (n = 3). A retrospective review revealed that the great majority of patients with low voltage electrical injuries did not need admission to the hospital and could have been cared for on an outpatient basis. Almost every patient with high voltage injury had a justified admission due to the severity of the injury. On the basis of these results we conclude that we can safely reduce the number of admissions to the hospital for children with low voltage minor electrical injuries.

Exercise intolerance associated with myalgias, muscle cramps or myoglobinuria may be associated with a dystrophinopathy. A search for abnormal dystrophin expression (using immunohistochemistry, immunoblot and DNA analysis) was carried out in a series of 15 patients. They were selected because they presented exercise intolerance, negative biochemical tests (lipid, glycogen and mitochondrial metabolism) and abnormal immunohistochemistry with at least one anti-dystrophin antibody (anti-Dys 1, rod domain; anti-Dys 2, C terminus; anti-Dys 3, N terminus). Lack of anti-Dys 1 immunoreactivity was seen in three patients and abnormal immunoreactivity with all three anti-dystrophin antibodies in two. Immunoblot confirmed the dystrophinopathy in these five patients only, and multiplex polymerase chain reaction DNA analysis revealed a deletion in the dystrophin gene in two of these patients, affecting the proximal part of the rod domain in one and the distal part of this domain in the other. The clinical, biological and histopathological features of the five patients reported here, together with the previous cases reported in the literature, are described and reveal that exercise intolerance associated with dystrophinopathy displays characteristic clinical, biological and immunohistochemical features and defines a new dystrophinopathy phenotype. The absence of staining in the rod domain provides a secure diagnosis of this syndrome. Dystrophinopathy is one etiology of idiopathic myoglobinuria, requiring genetic counseling.

Myoglobin induces renal injury by mechanisms that remain incompletely defined. Acidosis has been suggested as an important factor in myoglobinuric renal failure, and urine alkalization is routinely recommended for its prevention. We tested this hypothesis by exploring the effects of acid-base balance upon myoglobin nephrotoxicity in vivo and in vitro. In isolated rat kidneys at normal pH, myoglobin at concentrations of 25-250 mg/dl minimally affected renal perfusion flow, glomerular filtration rate (GFR) and tubular sodium reabsorption (TRNa). By contrast, at pH 7.1 myoglobin induced vasoconstriction, reduced GFR and TRNa and increased hypoxic injury to medullary thick ascending limbs. These changes were largely reproduced by perfusing kidneys with hematin, suggesting its release from myoglobin in acidosis. Chronic alkalosis or acidosis was induced in rats by supplementing drinking water with 0.28 M NaHCO3 or NH4Cl, respectively. Acute renal failure, produced in control animals by myoglobin infusion (38 mg/100 g body weight), was comparably prevented by both chronic alkalosis and acidosis. Acute intravenous or oral acid load provided similar protection. Thus, although acidosis exacerbates myoglobin toxicity in isolated perfused kidneys, acute or chronic exogenous acid load prevents renal damage in vivo. This may underscore the protective properties of solute load, a consequence of preconditioning, and suggests that, in the crush syndrome, endogenous acidosis rather than being an independent risk factor is a marker of tissue hypoperfusion and organism susceptibility to myoglobin renal toxicity.

This article presents a brief description of what is known about the cause and pathogenesis of chronic intermittent rhabdomyolysis in horses. Clinically applicable diagnostic tests and published results in affected horses, prophylaxis, and treatment of the acute case are discussed.

Polymyositis rarely leads to myoglobinuric acute renal failure. One such case is reported here, and certain common features in reported cases are discussed. Given therapeutic advances, these patients should do well despite the dismal prognosis reported in the literature, but early diagnosis and prompt treatment are essential.

A case is described in which, after administration of diclofenac for 13 days for arthritis attributed to gout, the patient experienced erythema multiforme followed by muscle weakness, elevation of serum creatine phosphokinase (CPK) level from 101 to 83,770 U/L, 100% muscle isoenzyme, blood urea nitrogen (BUN) level from 15 to 87 mg/dL, creatinine level from 1.0 to 2.1 mg/dL and urine myoglobin level to 1,190 micrograms/dL (N < 1.2). The diagnosis was rhabdomyolysis due to diclofenac, with myoglobinuria resulting in mild renal failure. Treatment consisted of discontinuing diclofenac and administering sufficient fluids to prevent progression of myoglobinuric renal failure. Serum CPK level gradually returned to normal by day 50, BUN and creatinine levels by day 28, and muscle strength between day 90 and 180. Rhabdomyolysis due to diclofenac or to other nonsteroidal antiinflammatory drugs has not been reported.

Iron catalyzed free radical formation and lipid peroxidation are accepted mechanisms of heme protein-induced acute renal failure. However, the source(s) of those free radicals which trigger lipid peroxidation in proximal tubular cells remains unknown. This study tested the potential involvement of mitochondrial electron transport, xanthine oxidase activity, and arachidonic acid metabolism in the heme-induced peroxidative state. The impact of cytosolic Ca2+ loading also was assessed. Rhabdomyolysis was induced in mice by glycerol injection, and two hours later heme-laden proximal tubular segments (PTS) were isolated for study. PTS from normal mice served as controls. During 30 to 60 minute incubations, heme loaded PTS developed progressive cytotoxicity (LDH release) and iron-dependent lipid peroxidation (malondialdehyde, MDA, generation; inhibited by deferoxamine). Site 2 (antimycin A) or site 3 (cyanide, hypoxia) mitochondrial respiratory chain inhibition completely blocked lipid peroxidation, whereas site 1 inhibition (rotenone) doubled its extent (presumably by shunting NADH through NADH dehydrogenase, a free radical generating system). Conversely, these agents did not substantially alter MDA in normal PTS. Normal and heme loaded PTS developed comparable degrees of LDH release during respiratory blockade irrespective of increased or decreased MDA production (indicating that lipid peroxidation was not a critical determinant of cell death). Neither increasing free arachidonic acid (PLA2 treatment) nor adding cyclooxygenase/lipoxygenase/cytochrome p450 inhibitors conferred a consistent protective effect. Altering free Ca2+ status (chelators; ionophore addition) and xanthine oxidase inhibition had no discernible impacts. Despite mitochondrial free radical production, mitochondrial function, as assessed by the ATP/ADP ratio, seemingly remained intact. In conclusion, (1) the terminal mitochondrial respiratory chain is the dominant source of free radicals which trigger PTS lipid peroxidation; (2) iron is a required secondary factor; (3) although mitochondria fuel lipid peroxidation, they do not appear to be critical targets of the heme-induced oxidant attack.

We evaluated the feasibility of magnetic resonance imaging (MRI) for early detection of tubular injury by monitoring changes in the apparent diffusion coefficient (ADC) of renal water in a rat model of myohemoglobinuric glycerol-induced acute renal failure (ARF). Diffusion-weighted MRI was performed concurrently with measurements of serum creatinine and blood urea nitrogen (BUN), evaluation of renal perfusion with dynamic contrast-enhanced MRI, and renal morphological examination. ADC values in the cortex and outer medulla significantly declined within minutes after the glycerol administration (70-75% of control at 4 min and 50-60% of control at 15 min). Contrast-enhanced MRI demonstrated renal hypoperfusion at 20 min after the onset of injury. Light microscopy showed normal glomeruli and edematous tubular epithelial cells at 10 and 30 min, with more severe swelling and protein casts at 30 min. No changes in serum creatinine or BUN levels were detected. We hypothesize that decrease in renal ADC may be attributed to renal ischemia and to subsequent intracellular accumulation of diffusion-restricted water. Similar imaging evaluation in other experimental models of ARF, and in patients, will define the diagnostic value of renal ADC changes in early detection of acute tubular injury.

Rat experiments demonstrated that acute renal myoglobinuria was characterized by high renal lipid peroxidation rates. Prior administration of the antioxidant ionol reduced the activation of lipid peroxidation in renal tissue and diminished the severity of renal dysfunctions.

Muscle carnitine was measured in 3 severely handicapped children with acute myoglobinuria. Muscle total carnitine levels were 9.6 +/- 3.0 nmol/mg non collagen protein (m +/- SD, control 15.7 +/- 2.8, n = 19), and free 6.5 +/- 3.0 nmol/mg non collagen protein (m +/- SD, control 12.9 +/- 3.7, n = 19), muscle free carnitine was significantly low in them. Serum carnitine was measured in 22 severely handicapped children. Serum total carnitine was 48.9 +/- 12.3 nmol/ml (m +/- SD, control 60.3 +/- 11.7, n = 14), and free 37.7 +/- 10.0 nmol/ml (m +/- SD, control 46.0 +/- 12.5, n = 14), serum carnitine was also significantly decreased. Secondary muscle and serum carnitine depletion might be one of the potential cause of myoglobinuria in severely handicapped children.

Mannitol (M) and deferoxamine (DFO) can each protect against myohemoglobinuric acute renal failure (MH-ARF). This study assessed M-DFO interactions during MH-ARF to help discern mechanisms of renal injury, and to define whether M + DFO exerts additive or synergistic antioxidant/cytoprotective effects. Rats subjected to the glycerol model of MH-ARF were treated with (a) M; (b) DFO; (c) M + DFO; or (d) no protective agents. Relative degrees of protection (24-h plasma urea/creatinine concentrations) were M + DFO greater than M greater than DFO greater than or equal to no therapy. To assess whether catalytic Fe is generated during MH-ARF, the bleomycin assay was applied to plasma/urine samples obtained 0-2 h post-glycerol injection. Although striking plasma and urinary increments were noted, excess renal hydroxyl radical (.OH) production was not apparent (gauged by the salicylate trap method). M increased catalytic Fe excretion (four times), whereas DFO eliminated its urinary (but not plasma) activity. To determine direct M/DFO effects on proximal tubular cell oxidant injury, isolated rat proximal tubular segments (PTS) were incubated with toxic dosages of FeSO4 or H2O2. Despite inducing cell injury (lactic dehydrogenase release), Fe caused no .OH production. DFO conferred dose-dependent cytoprotection, correlating with increased, not decreased, .OH generation. Although M scavenged this .OH excess, it had no additive or independent, protective effect. H2O2 cytotoxicity correlated with increased catalytic Fe (but not .OH) generation. The fact that DFO (but not .OH scavengers [M and dimethylthiourea]) blocked H2O2 toxicity implied Fe-dependent, .OH-independent cell killing. In conclusion, (a) striking catalytic Fe generation occurs during MH-ARF, but augmented intrarenal .OH production may not develop; (b) DFO can block Fe toxicity despite a prooxidant effect; (c) H2O2 PTS toxicity is Fe, but possibly not .OH, dependent; and (d) M does not mitigate oxidant PTS injury, either in the presence or absence of DFO, suggesting that its additive benefit with DFO in vivo occurs via a diuretic, not antioxidant effect.

Myocardial damage was studied in the dog heart with experimentally induced myonephropathic metabolic syndrome (MNMS). The animals underwent a ligation of the infrarenal arteries with a re-establishment of arterial flow 5 hours after the operation (group 1) and all showed the typical phenomena of MNMS and revealed basophilic changes in the myocardial cells fixed with 4 per cent formalin in 2 per cent calcium acetate. These degenerated cells were distributed in either the left or right ventricle, or even in both as several foci composed of a considerable number of the myocardial cells. Most of these cells showed a significantly enhanced expression of immunoreactive copper-zinc superoxide dismutase in their sarcoplasm. With luxol fast blue staining, the basophilic myocardial cells appeared to be deep blue in color which indicated an accumulation of phospholipid. Such basophilic cells in the animals undergoing a sham operation (group 2) were only sporadically observed in the myocardium fixed with the same fixative. The present study including morphological procedures indicates that dog MNMS causes severe myocardial damage with superoxidation due to an excessive production of free radicals after the re-establishment of arterial flow.

A competitive enzyme immunoassay (C-EIA) was developed for the measurement of serum and urinary levels of human heart fatty acid-binding protein (hh-FABP), and the appearance and time-course changes of hh-FABP levels were evaluated in patients with acute myocardial infarction (AMI). Control serum and urinary hh-FABP levels, which were determined in 86 serum and 42 urine samples from 86 patients without AMI, were found to range between 0 and 2.8 ng/mL. Serial determinations performed on 11 patients with AMI demonstrated that hh-FABP levels were significantly elevated in the first serum and urine samples obtained within 14 h of the onset of clinical symptoms. Two serum and 2 urine samples obtained only 1.5 h after the onset of symptoms already showed elevated hh-FABP levels, while in the same serum samples the activity of the myocardial-specific isoenzyme of creatine kinase (CK-MB) was still normal. Maximal serum and urinary hh-FABP levels appeared between 5 and 10 h after symptoms developed, and fell sharply towards normal thereafter. The hh-FABP levels in serum and urine both peaked earlier than the elevation of CK-MB activity in serum. The presence of hh-FABP in serum and/or urine seems to be a marker for myocardial damage and could be used as a useful tool for the early diagnosis of AMI.

The frequency of myoglobinuric renal failure is estimated between 8 and 20%. Despite early onset of therapy often the use of renal substitution by hemodialysis or hemofiltration is required. This study of the clinical course of nine patients with myoglobinuric acute renal failure reveals continuous arterio-venous hemofiltration (CAVH) to have an effective clearance for myoglobin. Thus, the time until recovery of renal function as well as the frequency of secondary complications in rhabdomyolysis induced acute renal failure can be distinctly reduced.

The case of an adolescent with idiopathic recurrent myoglobinuria is reported. The following features are emphasized: (1) persistence of limb weakness and elevated CK levels between the attacks, (2) a constant myopathic pattern in EMG, and (3) chronic morphologic abnormalities of myopathy in two muscle biopsies. All known causes of myoglobinuria were investigated and ruled out.

Part I highlights the mechanisms of glomerular filtration and tubular reabsorption of plasma proteins, selected characteristics of urinary proteins based upon electrophoretic properties and recent advances in clinical laboratory analysis of proteinuria. Both structural characteristic of the glomerular capillary wall and molecular properties of plasma proteins are important determinants of glomerular filtration. Proteins filtered by the glomerulus subsequently appear in urine only after escaping the efficient mechanisms of tubular reabsorption. Albumin is one such protein and constitutes the major protein in normal urine although trace amounts of alpha, beta, and gamma globulins are also detectable. Several techniques of protein analysis have thus been developed to specifically measure albumin as well as other plasma proteins. Other methods have been adapted to measure total urinary protein content enabling the clinician to readily monitor renal function in health and disease. The second part of this review will consider conditions associated with proteinuria in both asymptomatic individuals and patients with renal disease. Asymptomatic proteinuria encompasses states of excess protein excretion during conditions of orthostasis, exercise, travel to high altitude of fever. Proteinuria during renal disease has received considerable interest as a means to monitor kidney function. It is therefore classified according to the type of damage incurred: (1) glomerular-type where large molecular weight proteins are excreted (2) tubular-type where small molecular weight proteins are excreted and (3) mixed-type characterized by both large and small molecular weight proteinuria.

Two adult brothers became ill within 48 hours of each other, and both had severe myoglobinuria. One brother died of oliguric renal failure. The other did not have renal failure and survived. Acute influenza A infection was documented serologically and from throat washings in the surviving brother, and by isolation of the influenza A virus from throat cultures and lung tissue of the brother who died. It is not certain whether a genetic myopathy made these brothers susceptible to viral-induced myoglobinuria, but a normal response of venous lactate to ischemic work excluded lack of phosphorylase or phosphofructokinase as a cause of the myoglobinuria in the surviving brother. Neither brother had a history of recurrent episodes of myoglobinuria precipitated by exercise, cold, or fasting, thus making carnitine palmityl transferase deficiency unlikely.

A 14-year-old boy had myoglobinuria and renal failure after intense exercise; a year earlier he had experienced a milder episode. There was no consanguinity and no family history of neuromuscular diseases or hemolytic anemia. Strength was normal. Forearm ischemic exercise caused prolonged contracture with no rise of venous lactate. Muscle morphology showed only a mild increase of lipid droplets. Glycogen concentration was normal. Muscle phosphoglycerate kinase (PGK) activity was 5% of the normal mean, and all other glycolytic enzymes were normal. The residual PGK activity of muscle was heat stable but showed slower than normal electrophoretic mobility and decreased Michaelis constants for 3-phosphoglycerate and adenosine triphosphate. The enzyme defect was also expressed in erythrocytes and in fibroblast and muscle cultures. PGK activity was decreased in tissues from the patient's mother but normal in the father. PGK deficiency is an X-linked recessive trait usually associated with hemolytic anemia, mental retardation, and seizures; myopathy had not been recognized previously. Muscle PGK deficiency is now added to two other newly recognized glycolytic defects, phosphoglycerate mutase and lactate dehydrogenase deficiencies, as a cause of recurrent myoglobinuria.

A 36-year-old man was admitted because of sopor and dark urine after intravenous amphetamine injection. He subsequently developed myoglobinuria and acute renal failure. Serum myoglobin value was remarkably elevated to 83,000 ng/ml, and urine myoglobin was 400,000 ng/ml. Renal biopsy revealed tubular degeneration and tubular obstruction with myoglobin casts by immunofluorescence examination. Amphetamine-induced rhabdomyolysis was suspected to cause myoglobinuric acute renal failure.

Muscle phosphoglycerate mutase (PGAM) activity was markedly decreased (6% of the normal mean) in a 17-year-old girl with recurrent myoglobinuria after intense exercise. Muscle biopsy showed increased PAS stain; glycogen concentration was twice normal. Studies of anaerobic glycolysis in vitro showed decreased lactate production with glycogen, and with all hexose phosphate glycolytic intermediates, which was corrected by addition of purified PGAM to the reaction mixtures. A defect of the M subunit of PGAM was documented by electrophoretic, heat lability, and mercury inhibition studies. Intermediate PGAM activities (39 and 50% of normal) were found in muscle biopsies from the patient's asymptomatic parents. These data confirm the clinical, morphologic, and biochemical features described in the first patient with PGAM deficiency and suggest autosomal-recessive transmission of the trait.

The use of an immunohistochemical technic offers a very sensitive method to detect myoglobin in formalin-fixed, paraffin-embedded tissues. Thus, the technic is suitable for retrospective studies. Myoglobin can be demonstrated at the brush border and apical cytoplasm of the tubular epithelium of proximal tubules. Granular substances and casts in the lumens also react positively with antimyoglobin. Our findings confirm the results of a previous animal study, showing that myoglobin is filtered through glomeruli and reabsorbed by the proximal tubules.

A patient with severe metabolic acidosis in association with rhabdomyolysis and nonoliguric acute renal failure is presented. Evaluation of his acidosis indicated a large excess of unmeasured anions as well as evidence of a renal acidifying defect. We conclude that the metabolic acidosis seen with myoglobinuric acute renal failure may represent a mixed acid-base disturbance.

The lower detection limit of a radioimmunoassay for human myoglobin was determined by two "precision from day to day" methods (90% and 3 s methods), and by two "precision in series" methods (95% confidence range and the method of Markowetz & Munz). According to Markowetz & Munz, the lower detection limit (as a measure of sensitivity) is the lowest myoglobin concentration which, in 15-fold assays, shows no binding value in common with that of the next highest concentration, and no activity value in common with that of the reference binding value. On theoretical grounds (precision in series) and from a practical standpoint (determination of the lower detection limit using one sample assay), this method is the most suitable for the determination of the lower detection limit as a measure of sensitivity. It remains to be seen whether this is a valid generalization for other methods.