Vascular calcification and mineral bone disorder in chronic kidney disease

Chronic Kidney Disease (CKD), osteoporosis and mild hyponatremia are all prevalent chronic conditions that may coexist and are often under-recognized. Mineral-Bone Disorder begins early in the natural history of CKD and results in complex abnormalities of bone which ultimately confers a well-established increased risk of fragility fractures in End Stage Kidney Disease. Hyponatremia is a novel, usually renal mediated metabolic perturbation, that most commonly occurs independently of the stage of renal dysfunction but which may also predispose to increased fracture risk. The extent -if any- to which either early stages of renal dysfunction or the presence of hyponatremia contribute to fracture occurrence in the general population, independently of osteoporosis, is unclear. Renal transplantation is the treatment of choice for ESKD and although it restores endogenous renal function it typically fails to normalize either the long term cardiovascular or fracture risk. One potential mechanism contributing to these elevated long-term risks and to diminished Health Related Quality of Life is persistent, post-transplant hyperparathyroidism. In this study we retrospectively examine the association of renal function and serum sodium with Bone Mineral Density and fracture occurrence in a retrospective cohort of 1930 female members of the general population who underwent routine DXA scan. We then prospectively recruited a cohort of 90 renal transplant recipients in order to examine the association of post transplant parathyroid hormone (PTH) level with measures of CKD Mineral Bone Disorder, including, DXA Bone Mineral Density, Vascular Calcification (assessed using both abdominal radiography and CT techniques, as well as indirectly by carotid-femoral Pulse Wave Velocity) and Quality of Life (using the Short Form-12 and a PTH specific symptom score). In the retrospective DXA cohort, moderate CKD (eGFR 30-59ml/min/1.73m2) and hyponatremia (<135mmol/L) were associated with fracture occurrence, independently of BMD, with an adjusted Odds Ratio (95% Confidence Interval), of 1.37 (1.0, 1.89) and 2.25 (1.24, 4.09) respectively. In the renal transplant study, PTH was independently associated with the presence of osteoporosis, adjusted Odds Ratio (95% Confidence Interval), 1.15 (per 10ng/ml increment), (1.04, 1.26). The presence of osteoporosis but not PTH was independently associated with measures of vascular calcification, adjusted ß (95% Confidence Interval), 12.45, (1.16, 23.75). Of the eight quality-of-life domains examined, post-transplant PTH (per 10ng/ml increment), was only significantly and independently associated with reduced Physical Functioning, (95% Confidence Interval), 1.12 (1.01, 1.23). CKD and hyponatremia are both common health problems that may contribute to fracture occurrence in the general population, a major on-going public health concern. PTH and decreased Bone Mineral Density may signal sub-optimal long-term outcomes post renal transplantation, influencing bone and vascular health and to a limited extent long term Health Related Quality of Lif

The Role of Bid as a positive regulator of mutant SOD1 - induced TLR4 - NF-ҡB signalling in microglia

Amyotrophic Lateral Sclerosis is a debilitating, progressive and fatal neurodegenerative disorder resulting from the degeneration and death of motoneurons in the spinal cord and motor cortex which leads to muscle paralysis and death. Neuroinflammation is prominent in ALS pathogenesis, and other neurodegenerative disorders, and is believed to contribute to disease progression. Mutations in the superoxide dismutase 1 gene (SOD1) account for approximately 12 – 20 % of familial ALS cases. Microglia, the resident immune cells of the CNS, are activated concurrent with neuronal injury and mediate mutant SOD1 – induced neurotoxicity thus exacerbating disease progression. Additionally, mutant SOD1 has been shown to bind to the innate immune receptors Toll – like receptors -2 and -4 (TLR2, TLR4), which are highly expressed on microglia. The transcription factor nuclear factor ҡB (NF-ҡB) elicits the transcription of multiple pro-inflammatory genes, and its activation is upregulated in ALS pathology. The Bcl-2 family member Bid is demonstrated to promote NF-ҡB activation in astrocytes in response to TLR4 activation, and here it is also identified that Bid promotes TLR4 - NF-ҡB signalling in microglia, and that bid – deficiency attenuates the TLR4 – and mutant SOD1 - induced pro-inflammatory response. Detailed TLR4 - NF-ҡB pathway analysis identified an interaction between Bid and tumour necrosis factor α receptor adaptor protein 6 (TRAF6) in microglia, with Bid demonstrated to promote TRAF6 polyubiquitination. Further experiments demonstrated that Bid does not affect the activation of the Interleukin 1 receptor - associated kinase (IRAK) complex upon TLR4 activation; however reduced Peli1 protein levels are evident in the absence of Bid in TLR4 – activated microglia. Collectively the results demonstrate that Bid regulates TLR4 signalling by promoting TRAF6 ubiquitin ligase activity and therefore promotes downstream signalling to NF-ҡB. Depletion of microglial Bid offers a potential therapeutic target for the attenuation of the SOD1 – induced microglial mediated toxicity evident in ALS pathogenesis

Bid Promotes K63-Linked Polyubiquitination of Tumor Necrosis Factor Receptor Associated Factor 6 (TRAF6) and Sensitizes to Mutant SOD1-Induced Proinflammatory Signaling in Microglia.

Mutations in the superoxide dismutase 1 (SOD1) gene contribute to motoneuron degeneration and are evident in 20% of familial amyotrophic lateral sclerosis cases. Mutant SOD1 induces microglial activation through a stimulation of Toll-like receptors 2 and 4 (TLR2 and TLR4). In the present study, we identified the proapoptotic Bcl-2 family protein Bid as a positive regulator of mutant SOD1-induced TLR-nuclear factor-κB (NF-κB) signaling in microglia. bid-deficient primary mouse microglia showed reduced NF-κB signaling in response to TLR4 activation or exposure to conditioned medium derived from SOD1 (G93A) expressing NSC-34 cells. Attenuation of NF-κB signaling in bid-deficient microglia was associated with lower levels of phosphorylated IKKα/β and p65, with a delayed degradation of IκBα and enhanced degradation of Peli1. Upstream of IKK, we found that Bid interacted with, and promoted, the K63-linked polyubiquitination of the E3 ubiquitin ligase tumor necrosis factor receptor associated factor 6 (TRAF6) in microglia. Our study suggests a key role for Bid in the regulation of TLR4-NF-κB proinflammatory signaling during mutant SOD1-induced disease pathology. Bid promotes TLR4-NF-κB signaling by interacting with TRAF6 and promoting TRAF6 K63-linked polyubiquitination in microglia

Athletic groin pain (part 1): A prospective anatomical diagnosis of 382 patients - Clinical findings, MRI findings and patient-reported outcome measures at baseline

Background: Athletic groin pain remains a common field-based team sports time-loss injury. There are few reports of non-surgically managed cohorts with athletic groin pain. Aim: To describe clinical presentation/examination, MRI findings and patient-reported outcome (PRO) scores for an athletic groin pain cohort. Methods: All patients had a history including demographics, injury duration, sport played and standardised clinical examination. All patients underwent MRI and PRO score to assess recovery. A clinical diagnosis of the injured anatomical structure was made based on these findings. Statistical assessment of the reliability of accepted standard investigations undertaken in making an anatomical diagnosis was performed. Result: 382 consecutive athletic groin pain patients, all male, enrolled. Median time in pain at presentation was (IQR) 36 (16–75) weeks. Most (91%) played field-based ball-sports. Injury to the pubic aponeurosis (PA) 240 (62.8%) was the most common diagnosis. This was followed by injuries to the hip in 81 (21.2%) and adductors in 56 (14.7%) cases. The adductor squeeze test (90° hip flexion) was sensitive (85.4%) but not specific for the pubic aponeurosis and adductor pathology (negative likelihood ratio 1.95). Analysed in series, positive MRI findings and tenderness of the pubic aponeurosis had a 92.8% post-test probability. Conclusions: In this largest cohort of patients with athletic groin pain combining clinical and MRI diagnostics there was a 63% prevalence of PA injury. The adductor squeeze test was sensitive for athletic groin pain, but not specific individual pathologies. MRI improved diagnostic post-test probability. No hernia or incipient hernia was diagnosed. Clinical trial registration numberl: NCT02437942

The BCL-2 family protein Bid is critical for pro-inflammatory signaling in astrocytes.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motoneurons in the spinal cord, brainstem and motor cortex. Mutations in the superoxide dismutase 1 (SOD1) gene represent a frequent genetic determinant and recapitulate a disease phenotype similar to ALS when expressed in mice. Previous studies using SOD1(G93A) transgenic mice have suggested a paracrine mechanism of neuronal loss, in which cytokines and other toxic factors released from astroglia or microglia trigger motoneuron degeneration. Several pro-inflammatory cytokines activate death receptors and may downstream from this activate the Bcl-2 family protein, Bid. We here sought to investigate the role of Bid in astrocyte activation and non-cell autonomous motoneuron degeneration. We found that spinal cord Bid protein levels increased significantly during disease progression in SOD1(G93A) mice. Subsequent experiments in vitro indicated that Bid was expressed at relatively low levels in motoneurons, but was enriched in astrocytes and microglia. Bid was strongly induced in astrocytes in response to pro-inflammatory cytokines or exposure to lipopolysaccharide. Experiments in bid-deficient astrocytes or astrocytes treated with a small molecule Bid inhibitor demonstrated that Bid was required for the efficient activation of transcription factor nuclear factor-κB in response to these pro-inflammatory stimuli. Finally, we found that conditioned medium from wild-type astrocytes, but not from bid-deficient astrocytes, was toxic when applied to primary motoneuron cultures. Collectively, our data demonstrate a new role for the Bcl-2 family protein Bid as a mediator of astrocyte activation during neuroinflammation, and suggest that Bid activation may contribute to non-cell autonomous motoneuron degeneration in ALS

NF-κB regulates neuronal ankyrin-G via a negative feedback loop.

The axon initial segment (AIS) is a neuronal compartment defined by ankyrin-G expression. We here demonstrate that the IKK-complex co-localizes and interacts with the cytoskeletal anchor protein ankyrin-G in immunoprecipitation and proximity-ligation experiments in cortical neurons. Overexpression of the 270 kDa variant of ankyrin-G suppressed, while gene-silencing of ankyrin-G expression increased nuclear factor-κB (NF-κB) activity in primary neurons, suggesting that ankyrin-G sequesters the transcription factor in the AIS. We also found that p65 bound to the ank3 (ankyrin-G) promoter sequence in chromatin immunoprecipitation analyses thereby increasing ank3 expression and ankyrin-G levels at the AIS. Gene-silencing of p65 or ankyrin-G overexpression suppressed ank3 reporter activity. Collectively these data demonstrate that p65/NF-κB controls ankyrin-G levels via a negative feedback loop, thereby linking NF-κB signaling with neuronal polarity and axonal plasticity

Do social influences, awareness, or experience matter? Toward a better understanding of Farm-related Injury Risk Perception among agricultural science college students in Ireland

IntroductionFormal farm safety education/training should be tailored, in terms of the approach, content and delivery, to students undertaking agriculture education and training to enhance Farm-related Injury Risk Perception (FIRP). To this end, this paper assesses factor(s) explaining or predicting levels of FIRP amongst students studying for a degree in agriculture science.MethodsA cross-sectional online survey was conducted with a nationally representative sample of Bachelor of Agriculture Science (BAgrSc) students (N = 417) (aged 18–20) in Ireland. Descriptive [frequency and cross-tabulations) and inferential (Ordinal Logistic Regression (OLR)] analyses were applied to evaluate the effects of social influences, experience (of farming, of a near-miss or injury), and awareness (of others who were injured or killed on the farm) on FIRP.ResultsThe study found that social influences negatively affected FIRP (P &lt; 0.05). A relatively small number of students reported experiencing an injury (n = 56, 13.4%) that resulted in them being unable to participate in educational or social activities. A quarter of the respondents did, however, record experiencing a near-miss/close call (n = 106, 25.4%). A notable proportion (n = 144, 34.5%) of students had personal connections to someone who died as a consequence of a farm-related incident and 56.4% (n = 235) knew someone who was moderately or severely injured. OLR findings established that experiencing a severe injury, having a near-miss or close call, and awareness of a farm-related death or injury positively affects FIRP (P &lt; 0.05).ConclusionsPerception of farm risks amongst students in Ireland is low. Students who recorded higher levels of risk perception were, however, more likely to report experiencing a near-miss, close call or severe injury, or knowing someone who experienced a farm-related injury or fatality. Farmers, family or friends were found to negatively impact the FIRP and this reflects previous research findings. Our findings highlight the need for education and training programs to enhance opportunities for student peer-to-peer learning through sharing of experiences and/or knowledge of farm injuries and/or fatalities. Such activities will enhance awareness and understanding amongst the general population of students leading to increased FIRP and contribute to a reduction in risk-taking

Increased A20-E3 ubiquitin ligase interactions in bid-deficient glia attenuate TLR3- and TLR4-induced inflammation

Abstract Background Chronic pro-inflammatory signaling propagates damage to neural tissue and affects the rate of disease progression. Increased activation of Toll-like receptors (TLRs), master regulators of the innate immune response, is implicated in the etiology of several neuropathologies including amyotrophic lateral sclerosis, Alzheimer’s disease, and Parkinson’s disease. Previously, we identified that the Bcl-2 family protein BH3-interacting domain death agonist (Bid) potentiates the TLR4-NF-κB pro-inflammatory response in glia, and specifically characterized an interaction between Bid and TNF receptor associated factor 6 (TRAF6) in microglia in response to TLR4 activation. Methods We assessed the activation of mitogen-activated protein kinase (MAPK) and interferon regulatory factor 3 (IRF3) inflammatory pathways in response to TLR3 and TLR4 agonists in wild-type (wt) and bid-deficient microglia and macrophages, using Western blot and qPCR, focusing on the response of the E3 ubiquitin ligases Pellino 1 (Peli1) and TRAF3 in the absence of microglial and astrocytic Bid. Additionally, by Western blot, we investigated the Bid-dependent turnover of Peli1 and TRAF3 in wt and bid −/− microglia using the proteasome inhibitor Bortezomib. Interactions between the de-ubiquitinating Smad6-A20 and the E3 ubiquitin ligases, TRAF3 and TRAF6, were determined by FLAG pull-down in TRAF6-FLAG or Smad6-FLAG overexpressing wt and bid-deficient mixed glia. Results We elucidated a positive role of Bid in both TIR-domain-containing adapter-inducing interferon-β (TRIF)- and myeloid differentiation primary response 88 (MyD88)-dependent pathways downstream of TLR4, concurrently implicating TLR3-induced inflammation. We identified that Peli1 mRNA levels were significantly reduced in PolyI:C- and lipopolysaccharide (LPS)-stimulated bid-deficient microglia, suggesting disturbed IRF3 activation. Differential regulation of TRAF3 and Peli1, both essential E3 ubiquitin ligases facilitating TRIF-dependent signaling, was observed between wt and bid −/− microglia and astrocytes. bid deficiency resulted in increased A20-E3 ubiquitin ligase protein interactions in glia, specifically A20-TRAF6 and A20-TRAF3, implicating enhanced de-ubiquitination as the mechanism of action by which E3 ligase activity is perturbed. Furthermore, Smad6-facilitated recruitment of the de-ubiquitinase A20 to E3-ligases occurred in a bid-dependent manner. Conclusions This study demonstrates that Bid promotes E3 ubiquitin ligase-mediated signaling downstream of TLR3 and TLR4 and provides further evidence for the potential of Bid inhibition as a therapeutic for the attenuation of the robust pro-inflammatory response culminating in TLR activation

Additional file 1: of Increased A20-E3 ubiquitin ligase interactions in bid-deficient glia attenuate TLR3- and TLR4-induced inflammation

Figure S1. Increased A20-TRAF3, A20-Peli1, and TRAF6-Peli1 interactions in TLR3- and TLR4-stimulated bid-deficient glia and macrophages, compared with wt. (A) wt and bid −/− mixed glia were transfected with Ubiquitin-HA and stimulated for 1 h with LPS (100 ng/ml) 20 h following transfection. Co-immunoprecipitation of anti-TRAF6 was carried out for each sample, and TRAF6-A20 interactions were determined by Western blot using an anti-A20 antibody. TRAF6-A20 interactions were quantified using optical density (n = 1 experiment). (B) wt and bid-deficient mixed glia were transfected with TRAF6-FLAG and stimulated for 1 h with LPS (100 ng/ml) 20 h post transfection. The cells were lysed in RIPA buffer, and anti-FLAG was immunoprecipitated from each of the samples. Peli1 was detected by Western blot, indicating the interaction between TRAF6-FLAG and Peli1 (n = 1 experiment). (C) wt and bid-deficient macrophages were stimulated with PolyI:C (100 ng/ml) or LPS (100 ng/ml) for 1 h and lysed in RIPA buffer. Anti-Peli1 was co-immunoprecipitated from each sample, and Peli1-A20 interactions were determined by Western blot using an anti-A20 antibody (n = 1 experiment). (TIFF 1770 kb