Rhabdomyolysis aggravates renal iron accumulation and acute kidney injury in a humanized mouse model of sickle cell disease.

Individuals with sickle cell disease (SCD) are at greater risk of rhabdomyolysis, a potentially life-threatening condition resulting from the breakdown of skeletal muscle fibers. Acute kidney injury (AKI) is one of the most severe complications of rhabdomyolysis. Chronic kidney and cardiovascular disease, which account for SCD mortality, are long-term consequences of AKI. Although SCD elevates the risks of rhabdomyolysis-induced sudden death, the mechanisms that underlie rhabdomyolysis-induced AKI in SCD are unclear. In the present study, we show that, unlike their control non-sickling (AA) counterparts, transgenic homozygous SCD (SS; Townes model) mice exhibited 100% mortality 8-24 h after intramuscular glycerol injection. Five hours after glycerol injection, SS mice showed a more significant increase in myoglobinuria and plasma creatine kinase levels than AA mice. Basal plasma heme and kidney tissue iron levels were significantly higher in SS than in AA mice. In contrast to AA, glycerol-induced rhabdomyolysis aggravated these parameters in SS mice. Rhabdomyolysis also amplified oxidative stress in SS compared to AA mice. Glycerol-treated SS mice exhibited worse renal function, exemplified by a reduction in GFR with a corresponding increase in plasma and urinary biomarkers of early AKI and renal tubular damage. The free radical scavenger and Fenton chemistry inhibitor, TEMPOL, ameliorated rhabdomyolysis-induced AKI in the SS mice. These findings demonstrate that oxidative stress driven by renal iron accumulation amplifies rhabdomyolysis-induced AKI in SCD mice.

Abnormal myosin post-translational modifications and ATP turnover time associated with human congenital myopathy-related RYR1 mutations.

AIM: Conditions related to mutations in the gene encoding the skeletal muscle ryanodine receptor 1 (RYR1) are genetic muscle disorders and include congenital myopathies with permanent weakness, as well as episodic phenotypes such as rhabdomyolysis/myalgia. Although RYR1 dysfunction is the primary mechanism in RYR1-related disorders, other downstream pathogenic events are less well understood and may include a secondary remodeling of major contractile proteins. Hence, in the present study, we aimed to investigate whether congenital myopathy-related RYR1 mutations alter the regulation of the most abundant contractile protein, myosin. METHODS: We used skeletal muscle tissues from five patients with RYR1-related congenital myopathy and compared those with five controls and five patients with RYR1-related rhabdomyolysis/myalgia. We then defined post-translational modifications on myosin heavy chains (MyHCs) using LC/MS. In parallel, we determined myosin relaxed states using Mant-ATP chase experiments and performed molecular dynamics (MD) simulations. RESULTS: LC/MS revealed two additional phosphorylations (Thr1309-P and Ser1362-P) and one acetylation (Lys1410-Ac) on the ß/slow MyHC of patients with congenital myopathy. This method also identified six acetylations that were lacking on MyHC type IIa of these patients (Lys35-Ac, Lys663-Ac, Lys763-Ac, Lys1171-Ac, Lys1360-Ac, and Lys1733-Ac). MD simulations suggest that modifying myosin Ser1362 impacts the protein structure and dynamics. Finally, Mant-ATP chase experiments showed a faster ATP turnover time of myosin heads in the disordered-relaxed conformation. CONCLUSIONS: Altogether, our results suggest that RYR1 mutations have secondary negative consequences on myosin structure and function, likely contributing to the congenital myopathic phenotype.

Enriched Pathways of Calcium Regulation, Cellular/Oxidative Stress, Inflammation, and Cell Proliferation Characterize Gluteal Muscle of Standardbred Horses between Episodes of Recurrent Exertional Rhabdomyolysis.

Certain Standardbred racehorses develop recurrent exertional rhabdomyolysis (RER-STD) for unknown reasons. We compared gluteal muscle histopathology and gene/protein expression between Standardbreds with a history of, but not currently experiencing rhabdomyolysis (N = 9), and race-trained controls (N = 7). Eight RER-STD had a few mature fibers with small internalized myonuclei, one out of nine had histologic evidence of regeneration and zero out of nine degeneration. However, RER-STD versus controls had 791/13,531 differentially expressed genes (DEG). The top three gene ontology (GO) enriched pathways for upregulated DEG (N = 433) were inflammation/immune response (62 GO terms), cell proliferation (31 GO terms), and hypoxia/oxidative stress (31 GO terms). Calcium ion regulation (39 GO terms), purine nucleotide metabolism (32 GO terms), and electron transport (29 GO terms) were the top three enriched GO pathways for down-regulated DEG (N = 305). DEG regulated RYR1 and sarcoplasmic reticulum calcium stores. Differentially expressed proteins (DEP ↑N = 50, ↓N = 12) involved the sarcomere (24% of DEP), electron transport (23%), metabolism (20%), inflammation (6%), cell/oxidative stress (7%), and other (17%). DEP included ↑superoxide dismutase, ↑catalase, and DEP/DEG included several cysteine-based antioxidants. In conclusion, gluteal muscle of RER-susceptible Standardbreds is characterized by perturbation of pathways for calcium regulation, cellular/oxidative stress, inflammation, and cellular regeneration weeks after an episode of rhabdomyolysis that could represent therapeutic targets.

ACSL4 contributes to ferroptosis-mediated rhabdomyolysis in exertional heat stroke.

BACKGROUND: Rhabdomyolysis (RM) is a common complication of exertional heat stroke (EHS) and constitutes a direct cause of death. However, the mechanism underlying RM following EHS remains unclear. METHODS: The murine EHS model was prepared by our previous protocol. RNA sequencing is applied to identify the pathological pathways that contribute to RM following EHS. Inhibition of the acyl-CoA synthetase long-chain family member 4 (ACSL4) was achieved by RNA silencing in vitro prior to ionomycin plus heat stress exposure or pharmacological inhibitors in vivo prior to heat and exertion exposure. The histological changes, the iron accumulation, oxidized phosphatidylethanolamines species, as well as histological evaluation and levels of lipid metabolites in skeletal muscle tissues were measured. RESULTS: We demonstrated that ferroptosis contributes to RM development following EHS. Ferroptosis inhibitor ferrostatin-1 administration once EHS onset significantly ameliorated the survival rate of EHS mice from 35.357% to 52.288% within 24 h after EHS (P = 0.0028 compared with control) and markedly inhibited RM development induced by EHS. By comparing gene expression of between sham heat rest (SHR) (n = 3) and EHS (n = 3) mice in the gastrocnemius (Gas) muscle tissue, we identified that Acsl4 mRNA expression is elevated in Gas muscle tissue of EHS mice (P = 0.0038 compared with SHR), so as to its protein levels (P = 0.0001 compared with SHR). Followed by increase in creatine kinase (CK) and myoglobin (MB) levels, the labile iron accumulation, decrease in glutathione peroxidase 4 (GPX4) expression, and elevation of lipid peroxidation products. From in vivo and in vitro experiments, inhibition of Acsl4 significantly improves muscle cell death caused by EHS, thereby ameliorating RM development, followed by reduction in CK and MB levels by 30-40% (P < 0.0001; n = 8-10) and 40% (P < 0.0001; n = 8-10), restoration of GPX4 expression, and decrease in lipid peroxidation products. Mechanistically, ACSL4-mediated RM seems to be Yes-associated protein (YAP) dependent via TEA domain transcription factor1/TEA domain transcription factor4. CONCLUSIONS: These findings demonstrate an important role of ACSL4 in mediating ferroptosis activation in the development of RM following EHS and suggest that targeting ACSL4 may represent a novel therapeutic strategy to limit the skeletal muscle cell death and prevent RM after EHS.

COVID-19 and Myositis: What We Know So Far.

PURPOSE: Myositis as a rare manifestation of COVID-19 is only recently being reported. This review examines the current literature on COVID-19-induced myositis focusing on etiopathogenesis, clinical presentations, diagnostic practices, and therapeutic challenges with immunosuppression, and the difficulties experienced by rheumatologists in established myositis in the COVID-19 era. RECENT FINDINGS: COVID-19 is associated with a viral myositis attributable to direct myocyte invasion or induction of autoimmunity. COVID-19-induced myositis may be varied in presentation, from typical dermatomyositis to rhabdomyolysis, and a paraspinal affliction with back pain. It may or may not present with acute exponential elevations of enzyme markers such as creatine kinase (CK). Virus-mediated muscle inflammation is attributed to ACE2 (angiotensin-converting enzyme) receptor-mediated direct entry and affliction of muscle fibers, leading on to innate and adaptive immune activation. A greater recognition of the stark similarity between anti-MDA5-positive myositis with COVID-19 has thrown researchers into the alley of exploration - finding common etiopathogenic basis as well as therapeutic strategies. For patients with established myositis, chronic care was disrupted during the pandemic with several logistic challenges and treatment dilemmas leading to high flare rates. Teleconsultation bridged the gap while ushering in an era of patient-led care with the digital transition to tools of remote disease assessment. COVID-19 has brought along greater insight into unique manifestations of COVID-19-related myositis, ranging from direct virus-induced muscle disease to triggered autoimmunity and other etiopathogenic links to explore. A remarkable shift in the means of delivering chronic care has led patients and caregivers worldwide to embrace a virtual shift with teleconsultation and opened doorways to a new era of patient-led care.

Stevioside protects against rhabdomyolysis-induced acute kidney injury through PPAR-γ agonism in rats.

We explored the potential role of peroxisome proliferator activated receptor-γ (PPAR-γ) in stevioside-mediated renoprotection using rhabdomyolysis-induced acute kidney injury (AKI) model in rats. Rhabdomyolysis refers to intense skeletal muscle damage, which further causes AKI. Glycerol (50% w/v, 8 ml/kg) was injected intramuscularly in rats to induce rhabdomyolysis. After 24 hr, AKI was demonstrated by quantifying serum creatinine, urea, creatinine clearance, microproteinuria, and electrolytes in rats. Further, oxidative stress was measured by assaying thiobarbituric acid reactive substances, generation of superoxide anion, and reduced glutathione levels. Additionally, serum creatine kinase (CK) level was assayed to determine glycerol-induced muscle damage in rats. Pathological changes in rat kidneys were studied using hematoxylin-eosin and periodic acid Schiff staining. Moreover, the expression of apoptotic markers (Bcl-2, Bax) in rat kidneys was demonstrated by immunohistochemistry. Stevioside (10, 25, and 50 mg/kg) was administered to rats, prior to the induction of AKI. In a separate group, bisphenol A diglycidyl ether (BADGE, 30 mg/kg), a PPAR-γ receptor antagonist was given prior to stevioside administration, which was followed by rhabdomyolysis-induced AKI in rats. The significant alteration in biochemical and histological parameters in rats indicated AKI, which was attenuated by stevioside treatment. Pretreatment with BADGE abrogated stevioside-mediated renoprotection, which is suggestive of the involvement of PPAR-γ in its renoprotective effect. In conclusion, stevioside protects against rhabdomyolysis-induced AKI, which may be attributed to modulation of PPAR-γ expression.

Prediction of the pharmacokinetics and pharmacodynamics of topiroxostat in humans by integrating the physiologically based pharmacokinetic model with the drug-target residence time model.

Topiroxostat is a selective xanthine oxidoreductase (XOR) inhibitor for the management of hyperuricemia in patients with or without gout. In this work, we aim to employ the physiologically based pharmacokinetic (PBPK) model with the drug-target residence time model to predict and characterize both the pharmacokinetics (PK) and pharmacodynamics (PD) of topiroxostat in humans. The plasma concentration-time profile of topiroxostat was simulated based on drug properties and human physiology parameters. The predictive power of this PBPK model was then demonstrated by comparison of stimulated to observed pharmacokinetic parameters. The utility of the model was further demonstrated through predicting the oral absorption and disposition characteristics of topiroxostat in humans. Finally, by combining the PBPK model and the drug-target residence time model, we successfully predicted the target occupancy and built the relationship between PK and PD using in vitro, in vivo and in silico information. The results showed that topiroxostat exhibited significant in vivo pharmacological activity even after the complete clearance of this drug from the liver (target site), which may be due to the long residence time of the binary topiroxostat-XOR complex. This work may be helpful to guide future investigations of topiroxostat and also provides a novel strategy for PK/PD studies.

5-Aminolevulinic acid exerts renoprotective effect via Nrf2 activation in murine rhabdomyolysis-induced acute kidney injury.

AIM: Acute kidney injury (AKI) is associated with chronic kidney disease, as well as high mortality, but effective treatments for AKI are still lacking. A recent study reported the prevention of renal injury, such as ischemia-reperfusion injury, by 5-aminolevulinic acid (ALA), which induces an antioxidant effect. The current study aimed to investigate the effect of ALA in a rhabdomyolysis-induced mouse model of AKI created by intramuscular injection of 50% glycerol. METHODS: Rhabdomyolysis-induced AKI was induced by an intramuscular injection of glycerol (5 mL/kg body weight) into mice. Administration of ALA (30 mg/kg, by gavage) was started from 48 h before or 24 h after glycerol injection. The mice were sacrificed at 72 h after glycerol injection. The roles of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1), which is one of the Nrf2-related antioxidants, were further investigated through in vivo and in vitro methods. RESULTS: 5-aminolevulinic acid markedly reduced renal dysfunction and tubular damage in mice with rhabdomyolysis-induced AKI. ALA administration decreased oxidative stress, macrophage infiltration, and inflammatory cytokines and apoptosis. The expression of Nrf2 was upregulated by ALA administration. However, administration of Zinc protoporphyrin-9 (ZnPPIX) to inhibit HO-1 activity did not abolish these improvements by ALA. The expression of Nrf2-associated antioxidant factors other than HO-1 was also increased. CONCLUSION: These findings indicate that ALA exerts its antioxidant activity via Nrf2-associated antioxidant factors to provide a renoprotective effect against rhabdomyolysis-induced AKI.

N-(2-hydroxyphenyl)acetamide and its gold nanoparticle conjugation prevent glycerol-induced acute kidney injury by attenuating inflammation and oxidative injury in mice.

The protective activity of N-(2-hydroxyphenyl)acetamide (NA-2) and NA-2-coated gold nanoparticles (NA-2-AuNPs) in glycerol-treated model of acute kidney injury (AKI) in mice was investigated. NA-2 (50 mg/kg) and NA-2-AuNPs (30 mg/kg) were given to the animals for four days followed by 24-h water deprivation and injection of 50% glycerol (10 ml/kg im). The animals were sacrificed on the next day. Blood and kidneys were collected for biochemical investigations (urea and creatinine), histological studies (hematoxylin and eosin; and periodic acid-Schiff staining), immunohistochemistry (actin and cyclooxygenase-2, Cox-2), and real-time RT-PCR (inducible nitric oxide synthase, iNOS; nuclear factor-κB p50, NFκB; hemeoxygenase-1, HO-1; and kidney injury molecule-1, Kim-1). NA-2 protected renal tubular necrosis and inflammation, though the result of NA-2-AuNPs was better than compound alone and it also exhibited the activity at far less dose. The test compound and its gold nano-formulation decreased the levels of serum urea and creatinine level in the treated animals. Both NA-2 and NA-2-AuNPs also conserved actin cytoskeleton, and lowered COX-2 protein expression. Moreover, the mRNA expressions of iNOS and NFkB p50 were down-regulated, and HO-1 and Kim-1 genes were up-regulated. We conclude that NA-2 and NA-2-AuNPs ameliorates kidney inflammation and injury in glycerol-induced AKI animal model via anti-oxidant and anti-inflammatory mechanisms which make it a suitable candidate for further studies. We believe that these findings will contribute in the understanding of the mechanism of action of paracetamol-like drugs and can be considered for clinical research for the prevention of AKI.

Acquired perforating calcific collagenosis in a drug addict with rhabdomyolysis and transient hypercalcemia.

Acquired perforating calcific collagenosis (APCC), which is characterized by the calcification of dermal collagen fibers with subsequent transepidermal elimination and perforation, is an extremely rare entity. Thus far, it has only been reported in a patient with direct contact exposure to calcium chloride. Here, we report a unique case of APCC occurring in a drug addict admitted for rhabdomyolysis. The present case is a 20-year-old male patient hospitalized for drug-related rhabdomyolysis and multiple organ damage. During hospitalization, he gradually developed unusual skin rashes. There were multiple confluent umbilicated and keratotic erythematous to brownish papules and plaques with scratch-like linear plaques on his lower abdomen, inguinal areas and gluteal sulci. Also, multiple well-demarcated flesh-colored rough, hard and thin plaques with a "crepe paper"-like texture were found on the bilateral popliteal fossae, olecranon fossae and axillae. The histopathology of two biopsied lesions demonstrated acquired perforating calcific collagenosis. The lesions appeared during the rhabdomyolysis-related hypercalcemia phase and resolved spontaneously after the calcium level returned to normal. This is the first reported case of disseminated APCC occurring during transient hypercalcemia due to rhabdomyolysis.

The effect of oral N-acetylcystein on prevention of extensive tissue destruction in electrical burn injury.

BACKGROUND: Electric burn patients usually suffer permanent injury and sequelae. Salvage of the zone of stasis is an important topic in the treatment of burn patients. N-Acetylcysteine (NAC), as an antioxidant, has effect on the saving zone of stasis and extensive rhabdomyolisis. The aim of this study was therefore to evaluate the effect of oral NAC on tissue destruction indicators in an electric burn rat model. MATERIAL AND METHODS: An experimental study was conducted with thirty six male Wistar albino rats divided into 2 groups. Group A (n=18) and group B (n=18) were electrical burn injury groups without and with NAC therapy, respectively. The extent of burn wounds were evaluated by planimetry using a digital wound measuring device. Blood samples were obtained to analyze creatine kinase (CK) levels as a marker of extensive rhabdomiolysis on the first hour after electric injury (baseline) and on the 7th day to see the antioxidant effect of NAC. RESULTS: A significant decrease in tissue destruction was seen by the necrotic area on day 7 in the NAC therapy group compared to the control group (mean 2.26±1.05cm2 versus mean 7.12±3.30cm2 respectively; p=0.001), which was confirmed by the level of serum CK (day 7: group A, mean 140±51U/L versus Group B, mean 102±6U/L; p=0.007). CONCLUSION: A decrease in electric burn necrotic area and tissue damage in the group using NAC treatment was demonstrated. NAC might have a beneficial effect in the treatment of electrical burns. Further experimental and clinical studies with NAC treatment are necessary to confirm these results.

Primary carnitine deficiency in a 57-year-old patient with recurrent exertional rhabdomyolysis.

Rhabdomyolysis is an emergency requiring rapid diagnosis and suitable aetiological treatment. We describe the case of a 57-year-old man with recurrent exertional rhabdomyolysis who was diagnosed with systemic primary carnitine deficiency (SPCD). Clinical examination was normal, creatine kinase levels were elevated, plasma free carnitine concentration was mildly decreased, muscle biopsy demonstrated lipid accumulation, carnitine uptake in cultured fibroblasts was decreased and genetic analysis identified a homozygous pathologic c.1181_1183del in the SLC22A5 gene. Rhabdomyolysis did not recur after treatment with oral L-carnitine was introduced. SPCD is a rare autosomal recessive disorder of carnitine transportation usually manifesting as an infantile (hepatic) or a childhood myopathic (cardiac) condition and rarely affecting adults. Our case indicates that SPCD should be considered in the aetiological evaluation of adult patients with recurrent exertional rhabdomyolysis, even in the absence of myopathy and cardiomyopathy.

Electrical injury - a dual center analysis of patient characteristics, therapeutic specifics and outcome predictors.

BACKGROUND: Electrical injuries represent life-threatening emergencies. Evidence on differences between high (HVI) and low voltage injuries (LVI) regarding characteristics at presentation, rhabdomyolysis markers, surgical and intensive burn care and outcomes is scarce. METHODS: Consecutive patients admitted to two burn centers for electrical injuries over an 18-year period (1998-2015) were evaluated. Analysis included comparisons of HVI vs. LVI regarding demographic data, diagnostic and treatment specific variables, particularly serum creatinine kinase (CK) and myoglobin levels over the course of 4 post injury days (PID), and outcomes. RESULTS: Of 4075 patients, 162 patients (3.9%) with electrical injury were analyzed. A total of 82 patients (50.6%) were observed with HVI. These patients were younger, had considerably higher morbidity and mortality, and required more extensive burn surgery and more complex burn intensive care than patients with LVI. Admission CK and myoglobin levels correlated significantly with HVI, burn size, ventilator days, surgical interventions, amputation, flap surgery, renal replacement therapy, sepsis, and mortality. The highest serum levels were observed at PID 1 (myoglobin) and PID 2 (CK). In 23 patients (14.2%), cardiac arrhythmias were observed; only 4 of these arrhythmias occurred after hospital admission. The independent predictors of mortality were ventilator days (OR 1.27, 95% CI 1.06-1.51, p = 0.009), number of surgical interventions (OR 0.47, 95% CI 0.27-0.834, p = 0.010) and limb amputations (OR 14.26, 95% CI 1.26-162.1, p = 0.032). CONCLUSIONS: Patients with electrical injuries, HVI in particular, are at high risk for severe complications. Due to the need for highly specialized surgery and intensive care, treatment should be reserved to burn units. Serum myoglobin and CK levels reflect the severity of injury and may predict a more complex clinical course. Routine cardiac monitoring > 24 h post injury does not seem to be necessary.

A dual role of miR-22 in rhabdomyolysis-induced acute kidney injury.

AIM: In acute kidney injury (AKI), regions of the kidney are hypoxic. However, for reasons yet unknown, adaptation to hypoxia through hypoxia-inducible factor (HIF) is limited. Here, we studied miR-22, a potential HIF repressor, in normal kidneys, as well as in rhabdomyolysis-induced AKI, a condition where miR-22 is up-regulated. METHODS: AKI in mice was provoked by IM injection of glycerol. Tissue homogenates were processed to determine the levels of candidate RNAs and proteins, as well as global gene expression profiles. Reporter assays quantified in vitro miR-22 activity and its modulation by mimic or inhibitor molecules, under normoxia or hypoxia (1% O2 ) respectively. In vivo, anti-miR-22 molecules were applied to normal mice or prior to induction of AKI. Renal outcome was assessed by measuring plasma creatinine, plasma urea and the levels of the injury markers Kim-1 and Ngal. RESULTS: Renal miR-22 is inducible by hypoxia and represses hypoxia-inducible factor (HIF). Specific inhibition of miR-22 regulates 1913 gene transcripts in kidneys controls and 3386 in AKI, many of which are involved in development or carcinogenesis. Specific inhibition of miR-22 up-regulates tissue protective HIF target genes, yet renal function and injury markers are unchanged or worsened. CONCLUSIONS: miR-22 is a HIF repressor constitutively expressed in the adult kidney and up-regulated in AKI. Specific inhibition of miR-22 is efficient in vivo and profoundly affects renal gene expression in health and disease, including up-regulation of HIF. However, the net effect on rhabdomyolysis-induced AKI outcome is neutral or even negative.

Acute lumbar paraspinal compartment syndrome: a systematic review.

While still a rare entity, acute lumbar paraspinal compartment syndrome has an increasing incidence. Similar to other compartment syndromes, acute lumbar paraspinal compartment syndrome is defined by raised pressure within a closed fibro-osseous space, limiting tissue perfusion within that space. The resultant tissue ischaemia presents as acute pain, and if left untreated, it may result in permanent tissue damage. A literature search of 'paraspinal compartment syndrome' revealed 21 articles. The details from a case encountered by the authors are also included. A common data set was extracted, focusing on demographics, aetiology, clinical features, management and outcomes. There are 23 reported cases of acute compartment syndrome. These are typically caused by weight-lifting exercises, but may also result from other exercises, direct trauma or non-spinal surgery. Pain, tenderness and paraspinal paraesthesia are key clinical findings. Serum creatine kinase, magnetic resonance imaging and intracompartment pressure measurement confirm the diagnosis. Half of the reported cases have been managed with surgical fasciotomy, and these patients have all had good outcomes relative to those managed with conservative measures with or without hyperbaric oxygen therapy. These good outcomes were despite significant delays to operative intervention. The diagnostic uncertainty and subsequent delay to fasciotomy result from the rarity of this disease entity, and a high level of suspicion is recommended in the appropriate setting. This is particularly true in light of the current popularity of extreme weight lifting in non-professional athletes. Operative intervention is strongly recommended in all cases based on the available evidence.

Evaluation of Trace Elements in Augmentation of Statin-Induced Cytotoxicity in Uremic Serum-Exposed Human Rhabdomyosarcoma Cells.

Patients with end-stage kidney disease (ESKD) are at higher risk for rhabdomyolysis induced by statin than patients with normal kidney function. Previously, we showed that this increase in the severity of statin-induced rhabdomyolysis was partly due to uremic toxins. However, changes in the quantity of various trace elements in ESKD patients likely contribute as well. The purpose of this study is to determine the effect of trace elements on statin-induced toxicity in rhabdomyosarcoma cells exposed to uremic serum (US cells) for a long time. Cell viability, apoptosis, mRNA expression, and intracellular trace elements were assessed by viability assays, flow cytometry, real-time RT-PCR, and ICP-MS, respectively. US cells exhibited greater simvastatin-induced cytotoxicity than cells long-time exposed with normal serum (NS cells) (non-overlapping 95% confidence intervals). Intracellular levels of Mg, Mn, Cu, and Zn were significantly less in US cells compared to that in NS cells (p < 0.05 or 0.01). Pre-treatment with TPEN increased simvastatin-induced cytotoxicity and eliminated the distinction between both cells of simvastatin-induced cytotoxicity. These results suggest that Zn deficiencies may be involved in the increased risk for muscle complaints in ESKD patients. In conclusion, the increased severity of statin-induced rhabdomyolysis in ESKD patients may be partly due to trace elements deficiencies.

Pharmacological Inhibition of Macrophage Toll-like Receptor 4/Nuclear Factor-kappa B Alleviates Rhabdomyolysis-induced Acute Kidney Injury.

BACKGROUND:: Acute kidney injury (AKI) is the most common and life-threatening systemic complication of rhabdomyolysis. Inflammation plays an important role in the development of rhabdomyolysis-induced AKI. This study aimed to investigate the kidney model of AKI caused by rhabdomyolysis to verify the role of macrophage Toll-like receptor 4/nuclear factor-kappa B (TLR4/NF-κB) signaling pathway. METHODS:: C57BL/6 mice were injected with a 50% glycerin solution at bilateral back limbs to induce rhabdomyolysis, and CLI-095 or pyrrolidine dithiocarbamate (PDTC) was intraperitoneally injected at 0.5 h before molding. Serum creatinine levels, creatine kinase, the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6, and hematoxylin and eosin stainings of kidney tissues were tested. The infiltration of macrophage, mRNA levels, and protein expression of TLR4 and NF-κB were investigated by immunofluorescence double-staining techniques, reverse transcriptase-quantitative polymerase chain reaction, and Western blotting, respectively. In vitro, macrophage RAW264.7 was stimulated by ferrous myoglobin; the cytokines, TLR4 and NF-κB expressions were also detected. RESULTS:: In an in vivo study, using CLI-095 or PDTC to block TLR4/NF-κB, functional and histologic results showed that the inhibition of TLR4 or NF-κB alleviated glycerol-induced renal damages (P < 0.01). CLI-095 or PDTC administration suppressed proinflammatory cytokine (TNF-α, IL-6, and IL-1ß) production and macrophage infiltration into the kidney (P < 0.01). Moreover, in an in vitro study, CLI-095 or PDTC suppressed myoglobin-induced expression of TLR4, NF-κB, and proinflammatory cytokine levels in macrophage RAW264.7 cells (P < 0.01). CONCLUSION:: The pharmacological inhibition of TLR4/NF-κB exhibited protective effects on rhabdomyolysis-induced AKI by the regulation of proinflammatory cytokine production and macrophage infiltration.

Activation of autophagy is involved in the protective effect of 17ß-oestradiol on endotoxaemia-induced multiple organ dysfunction in ovariectomized rats.

Oestrogens have been reported to attenuate acute inflammation in sepsis. In this study, the effects of long-term oestrogen replacement with 17ß-oestradiol (E2 ) on endotoxaemia-induced circulatory dysfunction and multiple organ dysfunction syndrome were evaluated in ovariectomized (Ovx) rats. E2 (50 µg/kg, s.c., 3 times/week) was administered for 8 weeks, followed by the induction of endotoxaemia by intravenous infusion of lipopolysaccharides (LPS; 30 mg/kg/4 hrs). Oestrogen deficiency induced by ovariectomy for 9 weeks augmented the LPS-induced damage, including endotoxic shock, myocardial contractile dysfunction, renal dysfunction and rhabdomyolysis. Cardiac levels of NF-κB p65, iNOS and oxidized glutathione, free radical production in skeletal muscles, myoglobin deposition in renal tubules, and plasma levels of plasminogen activator inhibitor-1, TNF-α, and IL-6 were more pronounced in the Ovx + LPS group than in the Sham + LPS group. Long-term treatment of E2 prevented this amplified damage in Ovx rats. Six hours after LPS initiation, activation of the autophagic process, demonstrated by increases in Atg12 and LC3B-II/LC3B-I ratios, and induction of haem oxygenase (HO)-1 and heat-shock protein (HSP) 70 protein expression in myocardium were increased significantly in the Ovx + E2  + LPS group. These results suggest that activation of autophagy and induction of HO-1 and HSP70 contribute to the protective effect of long-term E2 replacement on multiple organ dysfunction syndrome in endotoxaemia.

Biological Membrane-Packed Mesenchymal Stem Cells Treat Acute Kidney Disease by Ameliorating Mitochondrial-Related Apoptosis.

The mortality of rhabdomyolysis-induced AKI remains high because no effective therapy exists. We investigated a new therapeutic method using MSCs. The aim of this study was to investigate the therapeutic potential and anti-apoptotic mechanisms of action of MSCs in the treatment of AKI induced by glycerol in vivo and in vitro. We used Duragen as a biological membrane to pack MSCs on the glycerol-injured renal tissue in vivo. The anti-apoptotic mechanism was investigated. In vitro, HK-2 cells were incubated with ferrous myoglobin and MSCs-conditioned medium, followed by cell proliferation and apoptosis assays. We founded that packing MSCs on the injured renal tissue preserved renal function, ameliorated renal tubular lesions, and reduced apoptosis in the mice with glycerol-induced AKI. The MSC-conditioned medium improved HK-2 cell viability and inhibited apoptosis. These effects were reversed by the PI3K inhibitor LY294002. Biological membrane packing of MSCs on the renal tissue has a therapeutic rescue function by inhibiting cell apoptosis in vivo. MSCs protect renal cells from apoptosis induced by myoglobin in vitro. We have thus demonstrated MSCs reduced rhabdomyolysis-associated renal injury and cell apoptosis by activating the PI3K/Akt pathway and inhibiting apoptosis.