Diglycolic acid inhibits succinate dehydrogenase activity, depletes mitochondrial membrane potential, and induces inflammation in an SH-SY5Y neuroblastoma model of neurotoxicity in vitro.

Diethylene glycol is a toxic industrial solvent resulting in a well-defined toxidrome. Diglycolic acid (DGA) has been identified as the metabolite responsible for the nephrotoxicity and hepatotoxicity. These studies assess the mechanism of DGA-induced neurotoxicity, specifically addressing the known ability of DGA to chelate calcium (Ca2+) in solution and inhibit mitochondrial complex II. SH-SY5Y cells were seeded into 96-well plates to assess intracellular Ca2+ chelation, complex II activity, mitochondrial membrane potential (ΔΨm), ATP production, and release of inflammatory cytokines TNF-α and IL-1ß with 2-, 4-, 6-, 24-, and 48-h DGA exposure. Peak Ca2+ chelation occurred at 4 h in cells treated with 6.25-50 mM DGA; however, effects were transient. Complex II activity was significantly decreased at all DGA concentrations tested, with 12.5 mM DGA causing 80% inhibition and 25 and 50 mM DGA causing 97 and 100% inhibition, respectively. Subsequently, 12.5-50 mM DGA concentrations significantly decreased ΔΨm at all time points. 50 mM DGA significantly increased release of TNF-α and IL-1ß after 24 and 48 h with significantly decreased ATP production observed at the same time points and concentration. These studies demonstrate that the DGA-induced mechanism of SH-SY5Y cell death involves complex II inhibition leading to mitochondrial depolarization, and subsequent ATP depletion with accompanying inflammatory cytokine release. These results indicate a direct mechanism of DGA-induced neurotoxicity in vitro, similarly observed in other DEG-affected target organs.

Risks and Risk Factors for Contralateral Amputation in Patients who have Undergone Amputation for Chronic Limb Threatening Ischaemia.

OBJECTIVE: The aim of this study was to determine the cumulative incidence of, and the risk factors associated with, contralateral amputation in patients with chronic limb threatening ischaemia (CLTI). METHODS: This was a retrospective cohort study of patients with incident unilateral transmetatarsal (TM), transtibial (TT), or transfemoral (TF) amputation secondary to CLTI, identified from the National Veterans Affairs Surgical Quality Improvement Program database (2004 - 2014). Thirteen potential pre-operative risk factors for contralateral amputation were considered. A competing risk analysis to estimate the cumulative incidence of contralateral amputation was performed using a Fine-Gray subdistribution hazard model. The effect of risk factors on contralateral amputation was estimated by computing subdistribution hazard ratios (sub-HR) with 95% confidence intervals (CI). RESULTS: From the database, 7 360 patients met the inclusion criteria. The contralateral amputation risk was 7.7% and was greatest in those who underwent a TF amputation (9.7%), followed by TT (7.4%) and TM amputation (6.6%) (p < .001). Among the 588 contralateral amputations, 50% were at the TF level, 34% at the TT level, and 16% at the TM level. The adjusted risk of contralateral amputation was greater in those who underwent an incident TF amputation or were Black or Hispanic. The factor that contributed to risk of contralateral amputation to the greatest extent was dialysis (sub-HR, 2.3; 95% CI 1.7 - 3.0; p < .001) while those who were obese (compared with underweight) were at lowest risk (0.67; 95% CI 0.46 - 0.97; p = .030). CONCLUSION: The one year risk of contralateral amputation in patients with CLTI is related to incident amputation level, medical comorbidities, correlates with race/ethnicity, and body mass index at the time of the incident amputation. The identified risk factors are largely not modifiable; however, they can be used to help identify populations at elevated risk.

Cloning, function, and localization of human, canine, and Drosophila ZIP10 (SLC39A10), a Zn2+ transporter.

Zinc (Zn2+) is the second most abundant trace element, but is considered a micronutrient, as it is a cofactor for many enzymes and transcription factors. Whereas Zn2+ deficiency can cause cognitive immune or metabolic dysfunction and infertility, excess Zn2+ is nephrotoxic. As for other ions and solutes, Zn2+ is moved into and out of cells by specific membrane transporters: ZnT, Zip, and NRAMP/DMT proteins. ZIP10 is reported to be localized at the apical membrane of renal proximal tubules in rats, where it is believed to play a role in Zn2+ import. Renal regulation of Zn2+ is of particular interest in light of growing evidence that Zn2+ may play a role in kidney stone formation. The objective of this study was to show that ZIP10 homologs transport Zn2+, as well as ZIP10, kidney localization across species. We cloned ZIP10 from dog, human, and Drosophila ( CG10006), tested clones for Zn2+ uptake in Xenopus oocytes and localized the protein in renal structures. CG10006, rather than foi (fear-of-intimacy, CG6817) is the primary ZIP10 homolog found in Drosophila Malpighian tubules. The ZIP10 antibody recognizes recombinant dog, human, and Drosophila ZIP10 proteins. Immunohistochemistry reveals that ZIP10 in higher mammals is found not only in the proximal tubule, but also in the collecting duct system. These ZIP10 proteins show Zn2+ transport. Together, these studies reveal ZIP10 kidney localization, a role in renal Zn2+ transport, and indicates that CG10006 is a Drosophila homolog of ZIP10.

The development and validation of the AMPREDICT model for predicting mobility outcome after dysvascular lower extremity amputation.

OBJECTIVE: The objective of this study was the development of AMPREDICT-Mobility, a tool to predict the probability of independence in either basic or advanced (iBASIC or iADVANCED) mobility 1 year after dysvascular major lower extremity amputation. METHODS: Two prospective cohort studies during consecutive 4-year periods (2005-2009 and 2010-2014) were conducted at seven medical centers. Multiple demographic and biopsychosocial predictors were collected in the periamputation period among individuals undergoing their first major amputation because of complications of peripheral arterial disease or diabetes. The primary outcomes were iBASIC and iADVANCED mobility, as measured by the Locomotor Capabilities Index. Combined data from both studies were used for model development and internal validation. Backwards stepwise logistic regression was used to develop the final prediction models. The discrimination and calibration of each model were assessed. Internal validity of each model was assessed with bootstrap sampling. RESULTS: Twelve-month follow-up was reached by 157 of 200 (79%) participants. Among these, 54 (34%) did not achieve iBASIC mobility, 103 (66%) achieved at least iBASIC mobility, and 51 (32%) also achieved iADVANCED mobility. Predictive factors associated with reduced odds of achieving iBASIC mobility were increasing age, chronic obstructive pulmonary disease, dialysis, diabetes, prior history of treatment for depression or anxiety, and very poor to fair self-rated health. Those who were white, were married, and had at least a high-school degree had a higher probability of achieving iBASIC mobility. The odds of achieving iBASIC mobility increased with increasing body mass index up to 30 kg/m2 and decreased with increasing body mass index thereafter. The prediction model of iADVANCED mobility included the same predictors with the exception of diabetes, chronic obstructive pulmonary disease, and education level. Both models showed strong discrimination with C statistics of 0.85 and 0.82, respectively. The mean difference in predicted probabilities for those who did and did not achieve iBASIC and iADVANCED mobility was 33% and 29%, respectively. Tests for calibration and observed vs predicted plots suggested good fit for both models; however, the precision of the estimates of the predicted probabilities was modest. Internal validation through bootstrapping demonstrated some overoptimism of the original model development, with the optimism-adjusted C statistic for iBASIC and iADVANCED mobility being 0.74 and 0.71, respectively, and the discrimination slope 19% and 16%, respectively. CONCLUSIONS: AMPREDICT-Mobility is a user-friendly prediction tool that can inform the patient undergoing a dysvascular amputation and the patient's provider about the probability of independence in either basic or advanced mobility at each major lower extremity amputation level.

Sulfate and thiosulfate inhibit oxalate transport via a dPrestin (Slc26a6)-dependent mechanism in an insect model of calcium oxalate nephrolithiasis.

Nephrolithiasis is one of the most common urinary tract disorders, with the majority of kidney stones composed of calcium oxalate (CaOx). Given its prevalence (US occurrence 10%), it is still poorly understood, lacking progress in identifying new therapies because of its complex etiology. Drosophila melanogaster (fruitfly) is a recently developed model of CaOx nephrolithiasis. Effects of sulfate and thiosulfate on crystal formation were investigated using the Drosophila model, as well as electrophysiological effects on both Drosophila (Slc26a5/6; dPrestin) and mouse (mSlc26a6) oxalate transporters utilizing the Xenopus laevis oocyte heterologous expression system. Results indicate that both transport thiosulfate with a much higher affinity than sulfate Additionally, both compounds were effective at decreasing CaOx crystallization when added to the diet. However, these results were not observed when compounds were applied to Malpighian tubules ex vivo. Neither compound affected CaOx crystallization in dPrestin knockdown animals, indicating a role for principal cell-specific dPrestin in luminal oxalate transport. Furthermore, thiosulfate has a higher affinity for dPrestin and mSlc26a6 compared with oxalate These data indicate that thiosulfate's ability to act as a competitive inhibitor of oxalate via dPrestin, can explain the decrease in CaOx crystallization seen in the presence of thiosulfate, but not sulfate. Overall, our findings predict that thiosulfate or oxalate-mimics may be effective as therapeutic competitive inhibitors of CaOx crystallization.

Reporting standards of the Society for Vascular Surgery for endovascular treatment of chronic lower extremity peripheral artery disease: Executive summary.

Recommended reporting standards for lower extremity ischemia were last published by the Society for Vascular Surgery in 1997. Since that time, there has been a proliferation of endovascular therapies for the treatment of chronic peripheral arterial disease. The purpose of this document is to clarify and update these standards, specifically for reports on endovascular treatment. The document is divided into sections: Claudication Reporting, Critical Limb Ischemia Reporting, Preintervention Assessment and Nonanatomic Treatment, Intervention, Outcome Measures - Procedural, Outcome Measures - Disease Specific, and Complications.

Reporting standards of the Society for Vascular Surgery for endovascular treatment of chronic lower extremity peripheral artery disease.

Peripheral arterial disease (PAD) represents a spectrum from asymptomatic stenosis to limb-threatening ischemia. The last decade has seen a tremendous increase in the variety of endovascular devices and techniques to treat occlusive disease. Like many evolving technologies, the literature surrounding therapy for endovascular arterial disease consists of mixed-quality manuscripts without clear standardization. Accordingly, critical evaluation of the reported results may be problematic. As such, providers and their patients make treatment decisions without the full benefit of a comparative effectiveness framework. The purpose of this document is to provide a summary for the reporting of endovascular revascularization techniques in the setting of chronic disease. Much of the work in this document is based on prior publications and standards proposed by the Society for Vascular Surgery. We have also made recommendations based on current literature and have attempted to acknowledge shortcomings and areas for future research. The various sections contain summaries of required reporting standards and should serve as a guide for the design of clinical trials and as reference for journal editors and reviewers when considering scientific work pertaining to endovascular therapy for chronic lower extremity arterial disease. An Appendix is provided with commonly used abbreviations in this document.

Diethylene glycol-induced toxicities show marked threshold dose response in rats.

Diethylene glycol (DEG) exposure poses risks to human health because of widespread industrial use and accidental exposures from contaminated products. To enhance the understanding of the mechanistic role of metabolites in DEG toxicity, this study used a dose response paradigm to determine a rat model that would best mimic DEG exposure in humans. Wistar and Fischer-344 (F-344) rats were treated by oral gavage with 0, 2, 5, or 10g/kg DEG and blood, kidney and liver tissues were collected at 48h. Both rat strains treated with 10g/kg DEG had equivalent degrees of metabolic acidosis, renal toxicity (increased BUN and creatinine and cortical necrosis) and liver toxicity (increased serum enzyme levels, centrilobular necrosis and severe glycogen depletion). There was no liver or kidney toxicity at the lower DEG doses (2 and 5g/kg) regardless of strain, demonstrating a steep threshold dose response. Kidney diglycolic acid (DGA), the presumed nephrotoxic metabolite of DEG, was markedly elevated in both rat strains administered 10g/kg DEG, but no DGA was present at 2 or 5g/kg, asserting its necessary role in DEG-induced toxicity. These results indicate that mechanistically in order to produce toxicity, metabolism to and significant target organ accumulation of DGA are required and that both strains would be useful for DEG risk assessments.

Tartaric acid induces toxicity in Madin-Darby canine kidney cells, but not human kidney-2 cells in vitro, and is prevented by organic anion transporter inhibition and human OAT-4 transfection.

OBJECTIVES: To evaluate the effect of tartaric acid (TTA) on Madin-Darby canine kidney (MDCK) cells compared to human kidney (HK)-2 cells. Secondarily, to evaluate the effects of probenecid, an organic anion transporter (OAT)-1 inhibitor, as well as human (h)OAT-4 transfection into MDCK cells to prevent TTA-induced cytotoxicity through decreasing accumulation via OAT-1 uptake inhibition or increasing OAT-4-mediated TTA efflux. DESIGN: Seventy-two-hour TTA concentration response and inhibitor studies in immortalized cell lines. SETTING: School of Pharmacy biomedical research laboratory and tissue culture facility. ANIMALS/SAMPLES: MDCK and HK-2 immortalized cell lines. INTERVENTIONS: Both cell lines were treated with increasing concentrations of TTA for 72 hours. Additionally, MDCK cells were co-incubated with TTA and increasing concentrations of probenecid or had been transfected with hOAT-4 and subsequently treated with TTA for 72 hours. MEASUREMENTS AND MAIN RESULTS: Media and samples were collected and lactate dehydrogenase (LDH) release was measured. LDH release was measured to assess TTA-induced cytotoxicity after 72 hours. LDH was not significantly increased in the HK-2 cells at any concentration but was significantly increased in the MDCK cells from 10 to 100 mM. LDH concentrations were significantly decreased (61%) in MDCK cells incubated with 50 mM TTA and probenecid when compared to TTA alone. hOAT-4 MDCK cell transfection also significantly reduced LDH release (57%) when comparing the transfected MDCK cells to the nontransfected MDCK cells treated with 50 mM TTA. CONCLUSIONS: TTA is a species-specific nephrotoxicant in dogs due to an interspecies difference in OAT-4 expression. Inhibiting TTA uptake in MDCK cells in vitro using the OAT-specific inhibitor, probenecid, prevents TTA-induced cytotoxicity.

Lack of efflux of diglycolic acid from proximal tubule cells leads to its accumulation and to toxicity of diethylene glycol.

Diethylene glycol (DEG) mass poisonings have resulted from ingestion of adulterated pharmaceuticals, leading to proximal tubular necrosis and acute kidney injury. Diglycolic acid (DGA), one of the primary metabolites, accumulates greatly in kidney tissue and its direct administration results in toxicity identical to that in DEG-treated rats. DGA is a dicarboxylic acid, similar in structure to Krebs cycle intermediates such as succinate. Previous studies have shown that DGA is taken into kidney cells via the succinate-related dicarboxylate transporters. These studies have assessed whether the DGA that is taken up by primary cultures of human proximal tubule (HPT) cells is effluxed. In addition, a possible mechanism for efflux, via organic anion transporters (OATs) that exchange external organic anions for dicarboxylates inside the cell, was assessed using transformed cell lines that actively express OAT activities. When HPT cells were cultured on membrane inserts, then loaded with DGA and treated with the OAT4/5 substrate estrone sulfate or the OAT1/3 substrate para-aminohippurate, no DGA efflux was seen. A repeat of this experiment utilizing RPTEC/TERT1 cells with overexpressed OAT1 and OAT3 had similar results. In these cells, but not in HPT cells, co-incubation with succinate increased the uptake of PAH, confirming the presence of OAT activity in the RPTEC/TERT1 cells. Thus, despite OATs stimulation in cells with OAT activity, there was little to no efflux of DGA from the cells. This study concluded that DGA is poorly transported out of cells and that stimulation of OAT transporters is not a viable target for reducing DGA accumulation in cells.

A hypoxia-driven occurrence of chronic kidney disease and osteoporosis in COPD individuals: New insights into environmental cadmium exposure.

Humans are exposed to cadmium via a variety of anthropogenic and natural pathways. Hypoxia, a key pathophysiological consequence of chronic obstructive pulmonary disease (COPD), as well as anemia, induce expression of many genes, including divalent metal transporter (DMT-1) , to induce cell adaptation to decreased pO2. DMT-1 then becomes increasingly expressed in a majority of organs, specifically the duodenum and the kidney. DMT-1 serves as an iron transporter; however, it can transport other physiologically important elements, including manganese (Mn2+) and zinc (Zn2+), as well as highly toxic divalent cations such as cadmium (Cd2+). Chronic obstructive pulmonary disease (COPD) is a highly prevalent, non-communicable disease in populations > 40 years of age, and is a leading cause of death worldwide. Occurrence of comorbidities accompanying COPD, such as chronic kidney disease (CKD) and osteoporosis increase the mortality rate and costs of treatment. As cadmium has been shown to be significantly osteo- and nephrotoxic, its hazardous effects could deteriorate bone microarchitecture and decrease kidney function positioning it as a likely environmental contributor to comorbidity development. In this review, we highlight the important contribution of hypoxia-induced DMT-1 expression mediating a cadmium (Cd2+) overload-induced CKD and osteoporosis axes. Furthermore, individuals who suffer from chronic lung disease with hypoxic respiratory failure, such as severe COPD appear to be significantly more sensitive to cadmium toxicity than healthy individuals.

Role of Plasma Membrane Dicarboxylate Transporters in the Uptake and Toxicity of Diglycolic Acid, a Metabolite of Diethylene Glycol, in Human Proximal Tubule Cells.

Diethylene glycol (DEG) mass poisonings have resulted from ingestion of pharmaceuticals mistakenly adulterated with DEG, typically leading to proximal tubular necrosis and acute kidney injury. The metabolite, diglycolic acid (DGA) accumulates greatly in kidney tissue and its direct administration results in toxicity identical to that in DEG-treated rats. DGA is a dicarboxylic acid, similar in structure to metabolites like succinate. These studies have assessed the mechanism for cellular accumulation of DGA, specifically whether DGA is taken into primary cultures of human proximal tubule (HPT) cells via sodium dicarboxylate transporters (NaDC-1 or NaDC-3) like those responsible for succinate uptake. When HPT cells were cultured on membrane inserts, sodium-dependent succinate uptake was observed from both apical and basolateral directions. Pretreatment with the NaDC-1 inhibitor N-(p-amylcinnamoyl)anthranilic acid (ACA) markedly reduced apical uptakes of both succinate and DGA. Basolateral uptake of both succinate and DGA were decreased similarly following combined treatment with ACA and the NaDC-3 inhibitor 2,3-dimethylsuccinate. When the cells were pretreated with siRNA to knockdown NaDC-1 function, apical uptake of succinate and toxicity of apically applied DGA were reduced, while the reduction in basolateral succinate uptake and basolateral DGA toxicity was marginal with NaDC-3 knockdown. DGA reduced apical uptake of succinate but not basolateral uptake. This study confirmed that primary HPT cells retain sodium dicarboxylate transport functionality and that DGA was taken up by these transporters. This study identified NaDC-1 as a likely and NaDC-3 as a possible molecular target to reduce uptake of this toxic metabolite by the kidney.

Effects of covert subject actions on percent body fat by air-displacement plethsymography.

Air-displacement plethysmography (ADP) is used for estimation of body composition, however, some individuals, such as athletes in weight classification sports, may use covert methods during ADP testing to alter their apparent percent body fat. The purpose of this study was to examine the effect of covert subject actions on percent body fat measured by ADP. Subjects underwent body composition analysis in the Bod Pod following the standard procedure using the manufacturer's guidelines. The subjects then underwent 8 more measurements while performing the following intentional manipulations: 4 breathing patterns altering lung volume, foot movement to disrupt air, hand cupping to trap air, and heat and cold exposure before entering the chamber. Increasing and decreasing lung volume during thoracic volume measurement and during body density measurement altered the percent body fat assessment (p < 0.001). High lung volume during thoracic gas measures overestimated fat by 3.7 ± 2.1 percentage points. Lowered lung volume during body volume measures overestimated body fat by an additional 2.2 ± 2.1 percentage points. The heat and cold exposure, tapping, and cupping treatments provided similar estimates of percent body fat when compared with the standard condition. These results demonstrate the subjects were able to covertly change their estimated ADP body composition value by altering breathing when compared with the standard condition. We recommend that sports conditioning coaches, athletic trainers, and technicians administering ADP should be aware of the potential effects of these covert actions. The individual responsible for administering ADP should remain vigilant during testing to detect deliberate altered breathing patterns by athletes in an effort to gain a competitive advantage by manipulating their body composition assessment.

Diethylene glycol and its metabolites induce cell death in SH-SY5Y neuronal cells in vitro.

Diethylene glycol (DEG) intoxication results in metabolic acidosis, renal and hepatic dysfunction, and late-stage neurotoxicity. Though the renal and hepatic toxicity of DEG and its metabolites 2-hydroxyethoxyacetic acid (2-HEAA) and diglycolic acid (DGA) have been well characterized, the resultant neurotoxicity has not. SH-SY5Y neuroblastoma cells were incubated with all 3 compounds at increasing concentrations for 24, 48, or 120 h. At all 3 time points, 50 mmol/L DGA and 100 mmol/L DEG showed significant Annexin V and propidium iodide (PI) staining with additional concentrations showing similar staining patterns at 24 h (100 mmol/L DGA) and 48 h (50 mmol/L DEG, 100 mmol/L DGA). Only the 200 mmol/L 2-HEAA concentration induced SH-SY5Y cell death. Interestingly at 24 and 48 h, 100 mmol/L DEG induced significant increases in apoptotic cell death markers, which progressed to necrosis at 120 h. Similar to DEG, 50 mmol/L DGA induced significant increases in SH-SY5Y cell apoptosis and necrosis markers at both 24 and 48 h. As expected, high DGA concentrations (100 mmol/L) at 120 h induced significant SH-SY5Y cell necrosis with no apoptosis detected. However, at 120 h lower DGA concentrations (20 mmol/L) significantly increased oligonucleosome formation alone and in combination with 2-HEAA or DEG. Taken together, these results indicate that DGA and DEG at threshold concentrations induce neurotoxicity in SH-SY5Y cells.

Lead (Pb2+)-induced calcium oxalate crystallization ex vivo is ameliorated via inositol 1,4,5-trisphosphate receptor (InsP3R) knockdown in a Drosophila melanogaster model of nephrolithiasis.

Nephrolithiasis causes severe pain and is a highly recurrent pathophysiological state. Calcium-containing stones, specifically calcium oxalate (CaOx), is the most common type accounting for approximately 75 % of stone composition. Genetic predisposition, gender, geographic region, diet, and low fluid intake all contribute to disease pathogenesis. However, exposure to environmental pollutants as a contribution to kidney stone formation remains insufficiently studied. Lead (Pb2+) is of particular interest as epidemiological data indicate that low-level exposure (BLL = 0.48-3.85 µM) confers a 35 % increased risk of developing CaOx nephrolithiasis. However, mechanisms underlying this association have yet to be elucidated. Drosophila melanogaster provide a useful genetic model where major molecular pathophysiological pathways can be efficiently studied. Malpighian tubules (MT) were isolated from either Wild-Type or InsP3R knockdown flies and treated with oxalate (5 mM) ± Pb2+ (2µM) for 1 h. Following exposure, MTs were imaged and crystals quantified. CaOx crystal number and total area were significantly increased (˜5-fold) in Pb2+(pre-treatment) + oxalate-exposed MTs when compared to oxalate alone controls. However, CaOx crystal number and total crystal area in Pb2+ + oxalate-exposed InsP3R knockdown MTs were significantly decreased (˜3-fold) indicating the role for principal cell-specific InsP3R-mediated Ca2+ mobilization as a mechanism for Pb2+-induced increases in CaOx crystallization inset model of nephrolithiasis.

Pro-apoptotic effects of 1'-acetoxychavicol acetate in human breast carcinoma cells.

The tropical ginger compound, 1'-acetoxychavicol acetate (ACA) possesses cancer chemopreventive properties in several models but its effects on breast cancer have not been fully evaluated. In this study, the effects of ACA on human breast carcinoma-derived MCF-7 and MDA-MB-231 cell viability were assessed using trypan blue exclusion analysis. ACA significantly decreased cell viability in a time- and dose-dependent manner, with effective concentrations 10-50 microM. Apoptosis was confirmed by morphological examination of cells through light microscopy, 4,6-diamidino-2-phenylindole dihydrochloride staining, and annexin V/Alexa Fluor 488 staining visualized using flow cytometry. ACA also increased protein expression of the activated form of caspase-3 in MDA-MB-231 cells. Addition of antioxidants N-acetylcysteine, ascorbic acid, or trolox prevented the loss of viability caused by ACA using trypan blue uptake as a marker. These results suggest ACA may have potential anticancer effects against breast carcinoma cells by inducing apoptosis.

Diglycolic acid, the toxic metabolite of diethylene glycol, chelates calcium and produces renal mitochondrial dysfunction in vitro.

CONTEXT: Diethylene glycol (DEG) has caused many cases of acute kidney injury and deaths worldwide. Diglycolic acid (DGA) is the metabolite responsible for the renal toxicity, but its toxic mechanism remains unclear. OBJECTIVE: To characterize the mitochondrial dysfunction produced from DGA by examining several mitochondrial processes potentially contributing to renal cell toxicity. MATERIALS AND METHODS: The effect of DGA on mitochondrial membrane potential was examined in normal human proximal tubule (HPT) cells. Isolated rat kidney mitochondria were used to assess the effects of DGA on mitochondrial function, including respiratory parameters (States 3 and 4), electron transport chain complex activities and calcium-induced opening of the mitochondrial permeability transition pore. DGA was compared with ethylene glycol tetraacetic acid (EGTA) to determine calcium chelating ability. DGA cytotoxicity was assessed using lactate dehydrogenase leakage from cultured proximal tubule cells. RESULTS: DGA decreased the mitochondrial membrane potential in HPT cells. In rat kidney mitochondria, DGA decreased State 3 respiration, but did not affect State 4 respiration or the ADP/O ratio. DGA reduced glutamate/malate respiration at lower DGA concentrations (0.5 mmol/L) than succinate respiration (100 mmol/L). DGA inhibited Complex II activity without altering Complex I, III or IV activities. DGA blocked calcium-induced mitochondrial swelling, indicating inhibition of the calcium-dependent mitochondrial permeability transition. DGA and EGTA reduced the free calcium concentration in solution in an equimolar manner. DGA toxicity and mitochondrial dysfunction occurred as similar concentrations. DISCUSSION: DGA inhibited mitochondrial respiration, but without uncoupling oxidative phosphorylation. The more potent effect of DGA on glutamate/malate respiration and the inhibition of mitochondrial swelling was likely due to its chelation of calcium. CONCLUSION: These results indicate that DGA produces mitochondrial dysfunction by chelating calcium to decrease the availability of substrates and of reducing equivalents to access Complex I and by inhibiting Complex II activity at higher concentrations.

Injury patterns in big ten conference football.

BACKGROUND: In 1998, the National Collegiate Athletic Association legislated a decrease in the number of scrimmages and full-contact practices allowed during the spring season. HYPOTHESIS: A significantly increased risk of injury faced by the same player during the spring versus fall practice sessions does exist, but the National Collegiate Athletic Association regulations will not have the intended effect of reducing injury rates to equal or below those of the fall practice sessions. STUDY DESIGN: Retrospectively analyzed, descriptive study of prospectively collected injury surveillance data followed by a prospective, controlled, repeated-measures study after the rule change. METHODS: Phase 1: the Big Ten Conference Sports Injury Surveillance System database was reviewed from spring 1992 through fall 1997 for all reportable injuries. Phase 2: a prospective investigation was instituted from spring 1998 through fall 2000. This protocol was modified to also document updated individual player position descriptions and string as well as exposures for the old fall and spring practice categories (scrimmages, full contact, and limited contact) and the new spring practice designations (full pads with and without tackling, helmets only, spring game, and other scrimmages). RESULTS: Phase 1: There were 3950 fall injuries and 1007 spring injuries, with 469 of the 1007 attributable to the "spring risk factor." There was a statistically significant increase in injury rate in the spring (19.8) versus fall (10.6). Scrimmages (incidence density ratio = 2.4) and limited-contact practices (incidence density ratio = 2.5) showed more than twice the fall injury rate. Phase 2: There were 1502 fall injuries and 648 spring injuries, with 192 attributable to the spring risk factor. There was a 3-fold increase in injury rate in the spring (incidence density ratio = 3.2). Although the noncontact, helmets-only practices produced the lowest injury rates, the nonspring game scrimmages and the limited-contact practices defined as "practice with pads but without tackling" displayed the highest injury rates. In both spring and fall, nonplayers exhibited the highest injury rates for all string groups. CONCLUSIONS: There was and still is a statistically significant increase in spring practice injury risk. The 1998 rule change resulted in an even greater increase in spring practice injury risk. If the goal is to minimize the number of spring practice injuries, it will be best accomplished by limiting the number of scrimmages and limited-contact sessions.