Glycerol Injection Research Articles

Galectin-3 protects distal convoluted tubules in rhabdomyolysis-induced kidney injury

Advanced glycation endproducts (AGEs) contribute to cellular damage of various pathologies, including kidney diseases. Acute kidney injury (AKI) represents a syndrome seldom characterized by a single, distinct pathophysiological cause. Rhabdomyolysis-induced acute kidney injury (RIAKI) constitutes roughly 15% of AKI cases, yet its underlying pathophysiology remains poorly understood. Using a murine model of RIAKI induced by muscular glycerol injection, we observed elevated levels of AGEs and the AGE receptor galectin-3 (LGALS3) in the kidney. Immunofluorescence localized LGALS3 to distal nephron segments. According to transcriptomic profiling via next-generation sequencing, RIAKI led to profound changes in kidney metabolism, oxidative stress, and inflammation. Cellular stress was evident in both proximal and distal tubules, as shown by kidney injury markers KIM-1 and NGAL. However, only proximal tubules exhibited overt damage and apoptosis, as detected by routine morphology, active Caspase-3, and TUNEL assay, respectively. In vitro, distal convoluted tubule (DCT) cells challenged with AGEs underwent apoptosis, which was markedly enhanced by Lgals3 siRNA treatment. Thus, in RIAKI, the upregulation of LGALS3 may protect the distal nephron from AGE-mediated damage, while proximal tubules lacking LGALS3 stay at risk. Thus, stimulating LGALS3 in the proximal nephron, if achievable, may attenuate RIAKI.

Effects of Hemorrhagic Shock and Rhabdomyolysis on Renal Microcirculation, Oxygenation, and Function in a Female Swine Model.

Hemorrhagic shock (HS) and rhabdomyolysis (RM) are two important risk factors for acute kidney injury after severe trauma; however, the effects of the combination of RM and HS on kidney function are unknown. The purpose of this study was to determine the impact of RM and HS on renal function, oxygenation, perfusion, and morphology in a pig model. Forty-seven female pigs were divided into five groups: sham, RM, HS, HS and moderate RM (RM4/HS), and HS and severe RM (RM8/HS). Rhabdomyolysis was induced by intramuscular injection of glycerol 50% with a moderate dose (4 ml/kg for the RM4/HS group) or a high dose (8 ml/kg for the RM and RM8/HS groups). Among animals with HS, after 90 min of hemorrhage, animals were resuscitated with fluid followed by transfusion of the withdrawn blood. Animals were followed for 48 h. Macro- and microcirculatory parameters measurements were performed. RM alone induced a decrease in creatinine clearance at 48 h (19 [0 to 41] vs. 102 [56 to 116] ml/min for RM and sham, respectively; P = 0.0006) without alteration in renal perfusion and oxygenation. Hemorrhagic shock alone impaired temporarily renal microcirculation, function, and oxygenation that were restored with fluid resuscitation. The RM4/HS and RM8/HS groups induced greater impairment of renal microcirculation and function than HS alone at the end of blood spoliation that was not improved by fluid resuscitation. Mortality was increased in the RM8/HS and RM4/HS groups in the first 48 h (73% vs. 56% vs. 9% for the RM8/HS, RM4/HS, and HS groups, respectively). The combination of HS and RM induced an early deleterious effect on renal microcirculation, function, and oxygenation with decreased response to resuscitation and transfusion compared with HS or RM alone.

Muscle regeneration is not impaired by ablation of UCP-1-expressing cells

INTRODUCTION: Recently, cells expressing uncoupling protein 1 (UCP-1) have been proposed to impact muscle physiology. First, transplant of brown adipose tissue (which has high expression of UCP-1) enhanced regeneration in the injured mouse rotator cuff. Second, isolated UCP-1+ muscle fibroadipogenic progenitors (FAPs) treated to enhance UCP-1 expression improved injured mouse rotator cuff muscle pathology. However, it remains unknown whether endogenous UCP-1-expressing cells contribute to muscle regeneration. Therefore, the objective of this study was to assess whether mice that lack UCP-1-expressing cells demonstrate impaired muscle regeneration following toxin administration. METHODS: Experiments were performed using tibialis anterior (TA) and 5th toe extensor digitorum longus (EDL) muscle of wild type C57BL/6J (WT; n=22), and uncoupling protein 1 diphtheria Toxin A (UCP1-DTA; n=18) mice, which lack all UCP-1 expressing cells. All procedures were performed in accordance with the National Institute of Health’s Guide for the Use and Care of Laboratory Animals and were approved by Washington University School of Medicine IACUC. Muscle injury was induced using intramuscular injection of 50%v/v solution of glycerol. Regeneration was assessed at “early” (7 days post-injury (dpi)), “intermediate” (14 dpi) and “late” (21 dpi) stages. Outcome measures were cell population dynamics by flow cytometry, ex-vivo physiology testing and histological scoring as appropriate for each timepoint. RESULTS: Our preliminary data in early regeneration show that UCP1-DTA mice have significantly greater percentage of FAPs compared to WT mice (29.84 ± 1.24% higher cell population, p<0.0134), however no differences in muscle satellite (stem) cells between genotypes. Physiological and histological outcomes at intermediate and late-stage regeneration find a baseline deficit in muscle mass (23.69 ± 2.3% smaller, p<0.0001), type 2B fiber cross-sectional area (32.56 ± 4.8% smaller, p<0.0001) and normalized muscle active force generation (16.2 ± 3.3% lower force, p<0.0154) in UCP1-DTA compared with WT. However, mass and contractile forces recover to a similar extent between the genotypes following glycerol injection suggesting the phenotypic drivers of the reduced mass and contractile function do not affect muscle regeneration. CONCLUSIONS: Taken together, the data from this study point to a baseline growth deficit in mice lacking UCP-1-expressing cells. However, in two separate timepoints of regeneration (14 and 21dpi), we see that UCP1-DTA mice have similar recovery of mass and normalized contractile force to baseline values suggesting regeneration can proceed normally without UCP-1 expressing cells. However, further data collection will be needed at early-stage regeneration to help discern whether mice lacking UCP-1-expressing cells have initial changes in cell population dynamics given the increased FAP population at 7dpi. ACKNOWLEDGMENTS: This work was supported by R01AR075773 and R21AR071582 to GAM. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Renoprotective effect of thymoquinone against rhabdomyolysis-induced acute kidney injury in the rat model.

Rhabdomyolysis leads to the release of myoglobin, sarcoplasmic proteins, and electrolytes into the blood circulation causing acute kidney injury (AKI). Thymoquinone, a natural compound found in Nigella sativa seeds, has antioxidant and anti-inflammatory effects. This investigation assessed the renoprotective effect of thymoquinone on rhabdomyolysis-induced AKI in rats. Male Wistar rats were categorized into six groups (n = 6): 1. Control: (normal saline), 2. Glycerol (50 ml/kg, single dose, IM), 3-5: Glycerol + thymoquinone (1, 2.5 and 5 mg/kg, 4 days, IP), 6. Thymoquinone (5 mg/kg). On day 5, serum and kidney tissue were isolated and the amounts of serum creatinine and blood urea nitrogen (BUN), renal malondialdehyde (MDA), glutathione (GSH.), tumor necrosis factor-alpha (TNF-α), neutrophil gelatinase-associated lipocalin (NGAL), and pathological changes were evaluated. Glycerol increased creatinine, BUN, MDA, TNF-α, and NGAL levels. It decreased GSH amounts and caused renal tubular necrosis, glomerular atrophy, and myoglobin cast in kidney tissue. Co-administration of glycerol and thymoquinone reduced creatinine, BUN, histopathological alterations, and MDA levels, and enhanced GSH amounts. Administration of glycerol and thymoquinone (5 mg/kg) had no significant effect on TNF-α amount but decreased NGAL protein levels. The administration of thymoquinone (5 mg/kg) alone did not display a significant difference from the control group. Rhabdomyolysis from glycerol injection in rats can cause kidney damage. Thymoquinone may attenuate renal dysfunction and oxidative stress. However, the TNF-α level was not significantly affected. Further studies are needed to explore the potential therapeutic effects of thymoquinone in managing AKI.

Renal protection by ellagic acid in a rat model of glycerol-induced acute kidney injury.

Studies conducted on animal models have shown that the administration of glycerol can lead to kidney tissue damage and impaired renal function. This is believed to be caused by oxidative stress and inflammation, which in turn can result in elevated levels of blood urea nitrogen (BUN) and creatinine. These metabolites are commonly used as indicators of renal function. The aim of the current experimental research was to investigate the protective efficacy of ellagic acid in a rat model of rhabdomyolysis induced by glycerol. Sixty healthy adult male Wistar rats weighing between 250 - 300 g were divided into five equal groups including control, rhabdomyolysis (administered 8.00 mL kg-1 of glycerol), and three rhabdomyolysis plus various doses of ellagic acid (25.00, 50.00 and 100 mg kg-1 per day; 72 hr after receiving glycerol for 14 days successively) groups. Serum levels of BUN, creatinine, lactate dehydrogenase, alkaline phosphatase, electrolytes and inflammatory cytokines were evaluated in all rats. Histopathological studies were also performed on kidney tissues from all groups. The administration of ellagic acid resulted in a significant increase in renal function biomarkers compared to the rats with acute kidney injury. This increase was consistent with notable reductions in tumor necrosis factor-α levels and increases in interleukin-10 levels observed in blood samples. Furthermore, the improvement in histopathological indices observed in rats received ellagic acid confirmed its nephroprotective role. The results of the current experimental study suggest that ellagic acid can improve kidney damage following glycerol injection, potentially by modulating the inflammatory process.

Protective role of alpha-lipoic acid against rhabdomyolysis-induced acute kidney injury in rats.

Rhabdomyolysis, a potentially life-threatening condition, occurs when myoglobin is released from damaged muscle cells, leading to acute kidney injury (AKI). Alpha lipoic acid (ALA), an organosulfur compound known for its anti-oxidant and anti-inflammatory properties, was examined in this study for its potential impact on rhabdomyolysis-induced AKI in rats. Six groups of rats were included in the study, with each group consisting of six rats (n=6): Control, rhabdomyolysis, rhabdomyolysis treated with different doses of ALA (5, 10, and 20 mg/kg), and ALA alone (20 mg/kg) groups. Rhabdomyolysis was induced by intramuscular injection of glycerol on the first day of the experiment, while ALA was administered intraperitoneally for four consecutive days. Renal function parameters, oxidative stress markers, and histological changes in the kidneys were evaluated. Western blot analysis was performed to measure the levels of neutrophil gelatinase-associated lipocalin (NGAL) and tumor necrosis factor-alpha (TNF-α) proteins. A significant increase in serum urea, creatinine, renal malondialdehyde, NGAl, and TNF-α protein levels was observed in glycerol-injected rats. In addition, a significant decrease in glutathione was recorded. Compared to the rhabdomyolysis group, treatment with ALA recovered kidney histological and biochemical abnormalities. Results suggest that rhabdomyolysis-induced AKI is associated with increased oxidative stress and inflammation. Treatment with ALA improved kidney histological abnormalities and reduced oxidative stress markers in rats. Therefore, ALA may have a potential protective effect against rhabdomyolysis-induced AKI.

Protective Effect of Silymarin on Attenuating of Rhabdomyolysis -induced Acute Kidney Injury in Animal Models

Acute kidney injury is one of the most serious complications of rhabdomyolysis characterized by an acute loss of renal function. The study aims to investigate the renoprotective effect of silymarin against rhabdomyolysis-induced acute kidney injury in rats. Twenty-four healthy rats were equally divided into three groups: control, induction, and treatment groups. Rats in the control and induction groups received distilled water, while rats in the treatment group received silymarin (50 mg /kg/day p.o) for three consecutive days. On the first day, all rats (except rats of control group) received a single intramuscular injection of glycerol (10 mL/kg, 50% v/v in sterile saline) divided equally into each hind leg for induction of acute kidney injury. On the fourth day of experiment, blood and kidney,s were collected from all rats for biochemical and histopathological assay. Silymarin efficiently attenuated acute kidney injury caused by rhabdomyolysis evidenced by a significant decrease in serum creatinine and blood urea nitrogen(BUN), along with a significant reduction in renal homogenate levels of TNF-α, IL-6, NF-κB, caspase-3, and MDA with a significant increase in GSH level, which was further confirmed by improving renal histological changes when compared to that of induction group. This study found that silymarin has a potential attenuating effect on acute kidney injury due to its antioxidant, anti-inflammatory and antiapoptotic actions.

Gastric inhibitory polypeptide receptor antagonism suppresses intramuscular adipose tissue accumulation and ameliorates sarcopenia.

Intramuscular adipose tissue (IMAT) formation derived from muscle fibro-adipogenic progenitors (FAPs) has been recognized as a pathological feature of sarcopenia. This study aimed to explore whether genetic and pharmacological gastric inhibitory polypeptide (GIP) receptor antagonism suppresses IMAT accumulation and ameliorates sarcopenia in mice. Whole body composition, grip strength, skeletal muscle weight, tibialis anterior (TA) muscle fibre cross-sectional area (CSA) and TA muscle IMAT area were measured in young and aged male C57BL/6 strain GIP receptor (Gipr)-knockout (Gipr-/- ) and wild-type (Gipr+/+ ) mice. FAPs isolated from lower limb muscles of 12-week-old Gipr+/+ mice were cultured with GIP, and their differentiation into mature adipocytes was examined. Furthermore, TA muscle IMAT area and fibre CSA were measured in untreated Gipr-/- mice and GIP receptor antagonist-treated Gipr+/+ mice after glycerol injection into the TA muscles. Body composition analysis revealed that 104-week-old Gipr-/- mice had a greater proportion of lean tissue mass (73.7±1.2% vs. 66.5±2.7%, P<0.05 vs. 104-week-old Gipr+/+ mice) and less adipose tissue mass (13.1±1.3% vs. 19.4±2.6%, P<0.05 vs. 104-week-old Gipr+/+ mice). Eighty-four-week-old Gipr-/- mice exhibited increases in grip strength (P<0.05), weights of TA (P<0.05), soleus (P<0.01), gastrocnemius (P<0.05) and quadriceps femoris (P<0.01) muscles, and average TA muscle fibre CSA (P<0.05) along with a reduction in TA muscle IMAT area assessed by the number of perilipin-positive cells (P<0.0001) compared with 84-week-old Gipr+/+ mice. Oil Red O staining analysis revealed 1.6- and 1.7-fold increased adipogenesis in muscle FAPs cultured with 10 and 100nM of GIP (P<0.01 and P<0.001 vs. 0nM of GIP, respectively). Furthermore, both untreated Gipr-/- mice and GIP receptor antagonist-treated Gipr+/+ mice for 14days after glycerol injection into the TA muscles at 12weeks of age showed reduced TA muscle IMAT area (1.39±0.38% and 2.65±0.36% vs. 6.54±1.30%, P<0.001 and P<0.01 vs. untreated Gipr+/+ mice, respectively) and increased average TA muscle fibre CSA (P<0.01 and P<0.05 vs. untreated Gipr+/+ mice, respectively). GIP promotes the differentiation of muscle FAPs into adipocytes and its receptor antagonism suppresses IMAT accumulation and promotes muscle regeneration. Pharmacological GIP receptor antagonism may serve as a novel therapeutic approach for sarcopenia.

Penehyclidine Hydrochloride Improves Rhabdomyolysis-Mediated Acute Kidney Injury by Inhibiting Ferroptosis through the HIF-1α/MT1G Axis

Background: Penehyclidine hydrochloride (PHC) has been shown to be effective in the treatment of rhabdomyolysis (RM)-induced acute kidney injury (AKI). Our research sought to investigate the pharmacological effects and mechanisms of PHC on RM-induced AKI. Methods: RM-induced AKI models were established by FeG treatment and glycerol injection. Cell viability was analyzed by cell counting kit-8 assay. Reactive oxygen species (ROS) levels were examined by flow cytometry. The LDH, Fe2+, MPO, MDA, and GSH levels were measured using the corresponding kits. The interaction between HIF-1α and MT1G was analyzed by dual-luciferase reporter gene and chromatin immunoprecipitation assays. The kidney pathological alterations were examined by hematoxylin-eosin staining. The levels of serum creatinine, uric acid, and blood urea nitrogen were examined using ELISA. Ferroptosis-related proteins (SLC7A11, GPX4, and ACSL4) were analyzed by Western blot. Results: PHC administration increased FeG-treated HK-2 cell viability, reduced ROS, LDH, Fe2+, MPO, MDA, and ACSL4 levels, and raised GSH, SLC7A11, and GPX4 levels in cells, suggesting that PHC improved FeG-induced HK-2 cell ferroptosis and injury. PHC protected against AKI primarily by suppressing ferroptosis. HIF-1α blocked the SLC7A11/GPX4 pathway by transcriptionally activating MT1G. PHC alleviated glycerol-induced kidney injury in rats by inhibiting ferroptosis. Conclusion: PHC improved RM-mediated AKI by inhibiting ferroptosis through the HIF-1α/MT1G/SLC7A11/GPX4 axis.

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.

Clival-Meckel's Cave Angle: A Predictor of Glycerol Displacement in Percutaneous Glycerol Rhizotomy for Trigeminal Neuralgia.

Percutaneous glycerol rhizotomy successfully treats trigeminal neuralgia although failure rates and durability of the procedure are variable. Some of this variability in clinical outcome might be due to egress of glycerol from Meckel's cave (MC) because of surgical positioning and individual patient anatomy. In this article, we quantitatively analyzed the anatomic variances that affect glycerol fluid dynamics to better predict patients more amenable for percutaneous glycerol injections. Computed tomography imaging of 11 cadaveric heads was used to calculate bilateral Clival-Meckel's cave (CMC) and sella-temporal (ST) angles. Twenty-two cadaveric percutaneous injections of dyed glycerol into the Meckel's cave were performed using Härtel's approach, and the fluid movement was documented at prespecified intervals over 1 hour. The relationship between the angles and glycerol migration was studied. Specimens with basal cistern involvement by 60 minutes had significantly greater CMC angles (median [IQR]: basal cistern involvement = 74.5° [59.5°-89.5°] vs no basal cistern involvement = 58.0° [49.0°-67.0°]), U = 6.0, P < .001. This model may predict which patients will experience glycerol migration away from the Gasserian ganglion (area under the curve: 0.950, SE: 0.046, CI: 0.859-1.041, P < .001). Increased ST angle was associated with lateral flow of glycerol (r s = 0.639, P = .001), and CMC angle was associated with total area of dispersion (r s = -0.474, P = .026). Anatomic variation in skull base angles affects glycerol migration. Specifically, a more obtuse CMC angle was associated with a higher risk of posterior migration away from the Gasserian ganglion. This may be a reason for differing rates of surgical success. These results suggest that anterior head flexion for 60 minutes may prevent percutaneous glycerol rhizotomy failures and some patients with large CMC angles are more likely to benefit from postinjection head positioning. However, this clinical effect needs validation in vivo.

N-Acetylcysteine Improves Renal Function and Reduces Tissue Malondialdehyde Levels in Glycerol-Induced Acute Kidney Injury

Introduction: The etiology of myoglobinuric acute kidney injury involves oxidative injury brought on by the Fenton reaction and myoglobin redox cycle. Renal tubules may be harmed, and lipid peroxidation compounds with vasoconstrictor characteristics may be produced. N-acetylcysteine (NAC) is an antioxidant shown to improve renal microcirculation and have protective effects in various models of renal damage. The aim of the study was to demonstrate the protective impact of NAC in glycerol-induced rats by measuring tissue malondialdehyde (MDA) level and renal function test (RFT), and to determine the correlation between the protective effect and NAC dose.Methods: This study measured tissue malondialdehyde (MDA) and renal function to examine any protective effect of NAC in a glycerol-induced rat model and to determine whether the effect was dose-related. Five groups of male Wistar rats were used: 1) saline control group, (2) glycerol (50%, 8mL/kg, i.m) plus saline i.v group, 3) glycerol plus NAC (100 mg/kg)-treated group, 4) glycerol plus NAC (200 mg/kg)-treated group, 5) glycerol plus NAC (400 mg/kg)-treated group. At 24 hrs, after glycerol injection, rats were sacrificed, cardiac blood was taken for renal function measurement, and renal tissues were removed for thiobarbituric acid MDA level assessment.Results: Our study revealed that glycerol administration significantly amplified renal tissue MDA, serum creatinine, and BUN (blood urea nitrogen) levels. However, NAC administration dampened the MDA increment and renal function deterioration (p&lt;0.05). Moreover, tissue MDA, BUN, and serum creatinine levels were significantly correlated to NAC dose (r=0.485; r=0.491; rs=0.544, respectively; all p&lt;0.05), indicating that NAC protection declines by dose increments.Conclusion: In this glycerol-induced acute kidney injury rat model, the administration of intravenous NAC 100 mg/kg reduced lipid peroxidation and improved renal function. Nevertheless, the protective effect was diminished in higher doses.

Protective effect of thymol on glycerol-induced acute kidney injury

Acute kidney injury (AKI) is a syndrome characterized by an accelerating decrease in renal function in a short time. Thymol is one of the main components of thyme species and has a variety of pharmacological effects. Here, we investigated whether thymol could ameliorate rhabdomyolysis (RM)-induced AKI and its related mechanism. Glycerol was used to induce RM-associated AKI in rats. Rats received thymol (20 mg/kg/day or 40 mg/kg/day) gavage 24 h before glycerol injection until 72 h after injection daily. Kidney injury was identified by measuring serum creatinine (Scr) and urea levels and by H&E and PAS staining and immunohistochemistry (the expression of proliferating cell nuclear antigen (PCNA)). Renal superoxide dismutase (SOD), malondialdehyde (MDA), and oxidative stress-related Nrf2/HO-1 signaling pathways were measured. The expression of the inflammatory markers TNF-α, IL-6, MCP-1, and NF-κB was assessed by ELISA and western blotting. Finally, the expression of the PI3K/Akt signaling pathway was detected by western blotting. Glycerol administration induced obvious renal histologic damage and increased Scr, urea, and PCNA expression. Notably, thymol treatment attenuated these structural and functional changes and prevented renal oxidative stress, inflammatory damage and PI3K/Akt pathway downregulation associated with glycerol-induced AKI. In conclusion, thymol might have potential applications in the amelioration of AKI via its antioxidant and anti-inflammatory effects and upregulation of the PI3K/Akt signaling pathway.

Protective effect of citronellol in rhabdomyolysis-induced acute kidney injury in mice.

Acute kidney injury (AKI) is a serious pathophysiological event consequent to rhabdomyolysis. Inflammatory mechanisms play a role in the development of rhabdomyolysis-induced AKI. Citronellol (CT) is a naturally occurring monoterpene in essential oils of aromatic plant species. In this study, we explored the protective effects of citronellol on AKI resulting from glycerol-induced rhabdomyolysis. Rhabdomyolysis was induced by a single intramuscular injection of glycerol 50% (10mg/kg) in the thigh caudal muscle. Four groups of mice were assigned, including a control group, a group administered with glycerol to induce AKI as a model, a group treated with glycerol plus 50mg/kg CT, and a group treated with glycerol plus 100mg/kg CT. The renal function of mice from all groups was evaluated using kidney histopathological changes and kidney injury molecule-1 (KIM-1). Myoglobin levels were measured to detect rhabdomyolysis. Apoptosis was evaluated by renal cleaved caspase-3 and BAX levels. Both doses of citronellol (50mg/kg and 100mg/kg) significantly reduced KIM-1 mRNA expression and myoglobin levels compared to the glycerol group. In addition, citronellol resulted in lower cleaved caspase-3 and BAX in the renal tissue, indicating that citronellol exerted an anti-apoptotic effect in AKI. Citronellol showed a reno-protective effect against rhabdomyolysis-induced AKI, which may be attributed to its anti-apoptotic effects.

Glycerol induced paraspinal muscle degeneration leads to hyper-kyphotic spinal deformity in wild-type mice

Degenerative spinal disorders, including kyphotic deformity, are associated with a range of degenerative characteristics of the paraspinal musculature. It has therefore been hypothesized that paraspinal muscular dysfunction is a causative factor for degenerative spinal deformity; however, experimental studies demonstrating causative relationships are lacking. Male and female mice received either glycerol or saline injections bilaterally along the length of the paraspinal muscles at four timepoints, each separated by 2 weeks. Immediately after sacrifice, micro-CT was performed to measure spinal deformity; paraspinal muscle biopsies were taken to measure active, passive and structural properties; and lumbar spines were fixed for analysis of intervertebral disc (IVD) degeneration. Glycerol-injected mice demonstrated clear signs of paraspinal muscle degeneration and dysfunction: significantly (p < 0.01) greater collagen content, lower density, lower absolute active force, greater passive stiffness compared to saline-injected mice. Further, glycerol-injected mice exhibited spinal deformity: significantly (p < 0.01) greater kyphotic angle than saline-injected mice. Glycerol-injected mice also demonstrated a significantly (p < 0.01) greater IVD degenerative score (although mild) at the upper-most lumbar level compared to saline-injected mice. These findings provide direct evidence that combined morphological (fibrosis) and functional (actively weaker and passively stiffer) alterations to the paraspinal muscles can lead to negative changes and deformity within the thoracolumbar spine.

Volume maximised glycerol rhizolysis for trigeminal neuralgia: a single centre analysis of outcomes

Objectives Glycerol rhizotomy is an established treatment for medically refractory trigeminal neuralgia in select cases where microvascular decompression is contraindicated or not preferred. The standard approach is to inject a fixed volume of glycerol using Hartel’s technique into Meckel’s cave. We discuss a ‘volume-maximised’ technique of measuring the volume of Meckel’s cave using intra-operative fluoroscopy and injecting an equivalent volume of glycerol such that every patient receives a tailored quantity of glycerol dependent on the volume of Meckel’s cave. The safety and efficacy of this approach is analysed. Methods A retrospective analysis of 53 procedures where volume-maximised glycerol rhizolysis was utilised over a 7-year period (2012 –2018) at a single centre by the senior author was carried out. The incidence and duration of pain freedom and complications incurred over a median follow-up period of 8 years were analysed Results 37 procedures were carried out for typical trigeminal neuralgia, 13 for secondary trigeminal neuralgia, and 3 for atypical trigeminal neuralgia. Overall, pain freedom was achieved in 85% cases, and 92% in patients with typical trigeminal neuralgia. Median duration of pain freedom in patients with typical trigeminal neuralgia was 63 months, versus 6 months in those with secondary trigeminal neuralgia (p < 0.00001). 14 procedures (26.4%) resulted in mild and temporary complications. 54.7% of cases experienced hypoaesthesia in a distribution similar to or less extensive than the distribution of trigeminal neuralgia. The presence of hypoaesthesia post-procedure was highly predictive of longer pain freedom (95 months versus 8 months median pain freedom (p = 0.00003)). Conclusions Volume-maximised glycerol injection is safe and effective when compared to literature-reported outcomes post-standard volume glycerol injections. The duration of pain freedom achieved exceeds most literature-reported studies, with hypoaesthesia outcomes being comparable with previous studies. Pain freedom outcomes are more favourable in those with post-procedure hypoaesthesia.

Pentoxifylline and thiamine ameliorate rhabdomyolysis-induced acute kidney injury in rats via suppressing TLR4/NF-κB and NLRP-3/caspase-1/gasdermin mediated-pyroptosis

Acute kidney injury (AKI) is a common complication of rhabdomyolysis (RM), a syndrome characterized by skeletal muscle damage resulting in renal tubular oxidative stress, inflammation, and activated toll like receptor-4 (TLR-4) and NOD-like receptor protein-3 (NLRP-3) inflammasome. Pyroptosis is a programmed cell death mediated by NLRP-3 leading to the activation of caspase-1 and gasdermin D (GSDMD), the hallmark of pyroptosis. This study aims to investigate the renoprotective effects of two antioxidants; pentoxifylline (PTX) and thiamine (TM) via targeting the aforementioned pathways. RM-AKI was induced in male Albino Wistar rats by intramuscular injection of glycerol (50% v/v, 10 ml/kg). PTX (100 mg/kg, oral) and TM (25 mg/kg, i.p) were administered for 12 days prior glycerol injection and continued for 3 days following induction of RM-AKI. Serum creatinine, blood urea nitrogen (BUN), creatin kinase, lipid peroxides, total antioxidant activity, inflammatory markers (tumor necrosis factor-α, interleukin-1β, and nuclear factor kappa B), TLR4, NLRP-3, caspase-1, GSDMD and c-myc (an apoptotic marker) were estimated. Compared to AKI model, co-administered drugs revealed a significant improvement in renal function and pathology as indicated by the reduction in serum creatinine, BUN and protein cast accumulation. The elevations of oxidative stress, and inflammatory markers as well as the over-expression of c-myc were alleviated. Protein levels of TLR4, NLRP3, cleaved caspase-1, and GSDMD were significantly elevated in RM-AKI model, and this elevation was attenuated by the tested drugs. In conclusion, PTX and TM could be a potential renoprotective approach for patients with RM through targeting TLR4/NF-κB and NLRP-3/caspase-1/gasdermin mediated-pyroptosis pathways.

Renoprotective effect of vinpocetine and cilostazol on glycerol induced renal injury in male rats

Acute kidney injury (AKI) is characterized by a sudden loss of kidney function that is established by increased serum creatinine levels and decreased urinary output. AKI is one of a group of functional kidney conditions&#x0D; known as acute kidney disease and disorders (AKD), which can vary in severity and self-limiting to severe and chronic. Adminstrations of glycerol generate significant elevation in serum urea and creatinine that’s mean occurance of functional abnormalities in the kidney. Vinpocetine drug has many pharmacological targets with multiple action, phosphodiesterase inhibiters-1(PDE-1) inhibitor, a voltage-gated sodium channel, and Inhibitory kinase B (IKK) are 3 main molecule targets of vinpocetine. PDE1 has been implicated in the regulation of vasoconstriction, vascular and cardiac structure remodeling, and neuro-transmission. Cilostazol, a phosphodiesterase (PDE) III inhibitors, that widely used for many cases such as reduces direct vascular injury via different mechanism, such as vasodilation and antiplatelet action, anti-inflammation and platelet-leukocyte interaction minimisation, and inhibition of vascular proliferation via up-regulation of hepatocyte growth factors. In present study, we looked at the effect and mechanism of the drugs vinpocetine and cilostazol in an animal model of glycerol-induced AKI. Experiment done during the 14-day trial, rats were divided into five groups: the control group received 2ml/kg normal saline; the induction group received 10ml/kg intramuscular glycerol injection; the vinpocetine group received 5mg/kg via gavage for 14 days and on day 7 given glycerol IM, the cilostazol group received 50mg/kg for 14 days and on day 7 given glycerol IM, and the combination group received half dose vinpocetine (2.5mg/kg) and cilostazol (25mg/kg). We discovered that the induction group had higher levels of urea and creatinine, as well as increased inflammation and oxidative stress, and that their renal tissue showed morphological changes typical of AKI, whereas the combination groups reduced glycerol induce acute renal damage. This revealed that vinpocetine and cilostazol can reinforce renal rat protection by reducing serum urea and creatinine and improving histopathological changes.

PS-B08-9: INHIBITION OF PERIOSTIN PROTECTED FROM EXACERBATION OF INFLAMMATION IN RHABDOMYOLYSIS-INDUCED ACUTE KIDNEY INJURY

Objective: Rhabdomyolysis is the breakdown of damaged skeletal muscle and the leakage of muscle-cell contents, including myoglobin, and other sarcoplasmic proteins into the circulation. These products may be filtered through the glomeruli, leading to acute kidney injury (AKI) via several mechanisms, such as inflammation and intratubular obstruction secondary to protein precipitation. New therapeutic strategy to lessen AKI are urgently needed. Thus far, several cellular pathways, nuclear factor kappa beta (NF-KB) as well as including pro-inflammatory effects on epithelial and tubular epithelial cells, have been identified as the major avenues for the initiation of the matrix-producing cells in AKI. Recently, it is suggested that periostin, extracellular matrix, is involved in the development of inflammation through the modulation of NF-KB pathway. However, periostin on the progression of the inflammation protection in AKI due to rhabdomyolysis is unclear. This study aimed to investigate the role of periostin in a rhabdomyolysis mice model of AKI. Design and method: In vivo, rhabdomyolysis-induced AKI was created by an intramuscular injection of 50% glycerol (5 mg/kg body weight). The expression level of periostin during the progression of acute renal failure after glycerol intramuscular injection for C57BL/6J wild type (WT) mice model was investigated. The mice were sacrificed at 72 hours after glycerol injection. We have developed periostin null mice to examine the role of periostin on acute renal failure. The role of periostin were further investigated through in vitro methods. In vitro, the development of renal inflammation is associated with NF-KB pathway. To investigate the function of periostin, we administrated Hemin(100uM) on NIH-3T3 fibroblast cells and the subsequent signaling pathways was examined. Results: The expression of periostin was highly increased, peaking at approximately 72 hours after gycerol injection. Inflammation-associated mRNA such as Monocyte Chemotactic Protein-1, tumor necrosis factor-alpha and IL6 expression and tubular injury score in H-E staining was more reduced in periostin null mice than WT mice at 72 hours after gycerol injection. Finally, an intraperitoneal injection of periostin antibody suppressed the mortality. These findings suggest a causal link between periostin and inflammation in AKI induced by rhabdomyolysis. In addition, an intraperitoneal injection of antibody for periostin antibody might be a new therapeutic strategy against the development of AKI induced by rhabdomyolysis. Conclusions: Periostin were highly expressed in kidney via rhabdomyolysis, and were a positive regulator of AKI. Targeting periostin might offer a new therapeutic strategy against the development of renal failure.

Growth arrest and DNA damage-inducible alpha regulates muscle repair and fat infiltration through ATP synthase F1 subunit alpha.

Skeletal muscle fat infiltration is a common feature during ageing, obesity and several myopathies associated with muscular dysfunction and sarcopenia. However, the regulatory mechanisms of intramuscular adipogenesis and strategies to reduce fat infiltration in muscle remain unclear. Here, we identified the growth arrest and DNA damage-inducible alpha (GADD45A), a stress-inducible histone folding protein, as a critical regulator of intramuscular fat (IMAT) infiltration. To explore the role of GADD45A on IMAT infiltration and muscle regeneration, the gain or loss function of GADD45A in intramuscular preadipocytes was performed. The adipocyte-specific GADD45A knock-in (KI) mice and high IMAT-infiltrated muscle model by glycerol injection (50μL of 50% v/v GLY) were generated. RNA-sequencing, histological changes, gene expression, lipid metabolism, mitochondrial function and the effect of dietary factor epigallocatechin-3-gallate (EGCG) treatment (100mg/kg) on IMAT infiltration were studied. The unbiased transcriptomics data analysis indicated that GADD45A expression positively correlates with IMAT infiltration and muscle metabolic disorders in humans (correlation: young vs. aged people, Gadd45a and Cebpa, r2 =0.20, P<0.05) and animals (correlation: wild-type [WT] vs. mdx mice, Gadd45a and Cebpa, r2 =0.38, P<0.05; NaCl vs. GLY mice, Gadd45a and Adipoq/Fabp4, r2 =0.80/0.71, both P<0.0001). In vitro, GADD45A overexpression promotes intramuscular preadipocyte adipogenesis, upregulating the expression of adipogenic genes (Ppara: +47%, Adipoq: +28%, P<0.001; Cebpa: +135%, Fabp4: +16%, P<0.01; Pparg: +66%, Leptin: +77%, P<0.05). GADD45A knockdown robustly decreased lipid accumulation (Pparg: -57%, Adipoq: -35%, P<0.001; Fabp4: -37%, P<0.01; Leptin: -28%, P<0.05). GADD45A KI mice exhibit inhibited skeletal muscle regeneration (myofibres: -40%, P<0.01) and enhanced IMAT infiltration (adipocytes: +20%, P<0.05). These KI mice have impaired exercise endurance and mitochondrial function. Mechanistically, GADD45A affects ATP synthase F1 subunit alpha (ATP5A1) ubiquitination degradation (ubiquitinated ATP5A1, P<0.001) by recruiting the E3 ubiquitin ligase TRIM25, which decreases ATP synthesis (ATP production: -23%, P<0.01) and inactivates the cAMP/PKA/LKB1 signalling pathway (cAMP: -36%, P<0.01; decreased phospho-PKA and phospho-LKB1 protein content, P<0.01). The dietary factor EGCG can protect against muscle fat infiltration (triglyceride: -64%, P<0.05) via downregulating GADD45A (decreased GADD45A protein content, P<0.001). Our findings reveal a crucial role of GADD45A in regulating muscle repair and fat infiltration and suggest that inhibition of GADD45A by EGCG might be a potential strategy to combat fat infiltration and its associated muscle dysfunction.