Modulation of the Acetylcholine Receptor Clustering Pathway Improves Neuromuscular Junction Structure and Muscle Strength in a Mouse Model of Congenital Myasthenic Syndrome.

Introduction: Congenital myasthenic syndromes (CMS) are a diverse group of inherited neuromuscular disorders characterized by a failure of synaptic transmission at the neuromuscular junction (NMJ). CMS often present early with fatigable weakness and can be fatal through respiratory complications. The AGRN gene is one of over 30 genes known to harbor mutations causative for CMS. In this study, we aimed to determine if a compound (NT1654), developed to stimulate the acetylcholine receptor (AChR) clustering pathway, would benefit a mouse model of CMS caused by a loss-of-function mutation in Agrn (Agrn nmf380 mouse). Methods: Agrn nmf380 mice received an injection of either NT1654 or vehicle compound daily, with wild-type litter mates used for comparison. Animals were weighed daily and underwent grip strength assessments. After 30 days of treatment animals were sacrificed, and muscles collected. Investigations into NMJ and muscle morphology were performed on collected tissue. Results: While minimal improvements in NMJ ultrastructure were observed with electron microscopy, gross NMJ structure analysis using fluorescent labelling and confocal microscopy revealed extensive postsynaptic improvements in Agrn nmf380 mice with NT1654 administration, with variables frequently returning to wild type levels. An improvement in muscle weight and myofiber characteristics helped increase forelimb grip strength and body weight. Conclusions: We conclude that NT1654 restores NMJ postsynaptic structure and improves muscle strength through normalization of muscle fiber composition and the prevention of atrophy. We hypothesize this occurs through the AChR clustering pathway in Agrn nmf380 mice. Future studies should investigate if this may represent a viable treatment option for patients with CMS, especially those with mutations in proteins of the AChR clustering pathway.

Modulation of Agrin and RhoA Pathways Ameliorates Movement Defects and Synapse Morphology in MYO9A-Depleted Zebrafish.

Congenital myasthenic syndromes (CMS) are a group of rare, inherited disorders characterised by impaired function of the neuromuscular junction (NMJ). This is due to defects in one of the many proteins associated with the NMJ. In three patients with CMS, missense mutations in a gene encoding an unconventional myosin protein, MYO9A, were identified as likely causing their disorder. Preliminary studies revealed a potential involvement of the RhoA/ROCK pathway and of a key NMJ protein, agrin, in the pathophysiology of MYO9A-depletion. In this study, a CRISPR/Cas9 approach was used to generate genetic mutants of MYO9A zebrafish orthologues, myo9aa/ab, to expand and refine the morphological analysis of the NMJ. Injection of NT1654, a synthetic agrin fragment compound, improved NMJ structure and zebrafish movement in the absence of Myo9aa/ab. In addition, treatment of zebrafish with fasudil, a ROCK inhibitor, also provided improvements to the morphology of NMJs in early development, as well as rescuing movement defects, but not to the same extent as NT1654 and not at later time points. Therefore, this study highlights a role for MYO9A at the NMJ, the first unconventional myosin motor protein associated with a neuromuscular disease, and provides a potential mechanism of action of MYO9A-pathophysiology.

Plasma C-terminal agrin fragment and rapid kidney function decline in chronic kidney disease patients.

C-terminal agrin fragment (tCAF) is a promising biomarker for glomerular filtration. Data regarding biomarkers that have the ability to predict rapid progression of chronic kidney disease (CKD) are sparse but necessary in order to identify patients at high risk for rapid progression. This study addresses the value of tCAF as a predictor of rapid kidney function decline in CKD patients.We measured plasma tCAF in a retrospective observational cohort study of 277 prevalent CKD patients stage I-V. Using multivariable Cox proportional hazards regression analysis, we evaluated the association of tCAF with end-stage-renal-disease (ESRD), ≥30%-decline of estimated glomerular filtration rate (eGFR) and the composite endpoint of both, adjusting for eGFR, age, systolic blood pressure, proteinuria and diabetes.The median age was 58 [interquartile range 47, 71] years, 36% were female. Median tCAF level was 822 [594, 1232] pM, eGFR was 32 [19, 48] ml/min/1.73 m. tCAF was correlated to eGFR and proteinuria (r = -0.76 and r = 0.49, P < .001 resp.). During a follow-up of 57.1 [42.9, 71.9] weeks, 36 (13%) patients developed ESRD and 13 (5%) had an eGFR decline of ≥30% (composite endpoint: 49 (18%)). In multivariable analysis, each 100 pM higher tCAF was independently associated with ESRD (hazard ratio (HR) 1.05 (95%-CI 1.02-1.08)), ≥30% eGFR decline (HR 1.10 (1.03-1.18)) and the composite endpoint (HR 1.07 (1.04-1.1)).Plasma tCAF may identify CKD patients at risk for rapid kidney function decline independent of eGFR and other risk factors for eGFR loss such as proteinuria.

Increasing Agrin Function Antagonizes Muscle Atrophy and Motor Impairment in Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is a pediatric genetic disease, characterized by motor neuron (MN) death, leading to progressive muscle weakness, respiratory failure, and, in the most severe cases, to death. Abnormalities at the neuromuscular junction (NMJ) have been reported in SMA, including neurofilament (NF) accumulation at presynaptic terminals, immature and smaller than normal endplates, reduced transmitter release, and, finally, muscle denervation. Here we have studied the role of agrin in SMAΔ7 mice, the experimental model of SMAII. We observed a 50% reduction in agrin expression levels in quadriceps of P10 SMA mice compared to age-matched WT controls. To counteract such condition, we treated SMA mice from birth onwards with therapeutic agrin biological NT-1654, an active splice variant of agrin retaining synaptogenic properties, which is also resistant to proteolytic cleavage by neurotrypsin. Mice were analyzed for behavior, muscle and NMJ histology, and survival. Motor behavior was significantly improved and survival was extended by treatment of SMA mice with NT-1654. At P10, H/E-stained sections of the quadriceps, a proximal muscle early involved in SMA, showed that NT-1654 treatment strongly prevented the size decrease of muscle fibers. Studies of NMJ morphology on whole-mount diaphragm preparations revealed that NT-1654-treated SMA mice had more mature NMJs and reduced NF accumulation, compared to vehicle-treated SMA mice. We conclude that increasing agrin function in SMA has beneficial outcomes on muscle fibers and NMJs as the agrin biological NT-1654 restores the crosstalk between muscle and MNs, delaying muscular atrophy, improving motor performance and extending survival.

MYO9A deficiency in motor neurons is associated with reduced neuromuscular agrin secretion.

Congenital myasthenic syndromes (CMS) are a group of rare, inherited disorders characterized by compromised function of the neuromuscular junction, manifesting with fatigable muscle weakness. Mutations in MYO9A were previously identified as causative for CMS but the precise pathomechanism remained to be characterized. On the basis of the role of MYO9A as an actin-based molecular motor and as a negative regulator of RhoA, we hypothesized that loss of MYO9A may affect the neuronal cytoskeleton, leading to impaired intracellular transport. To investigate this, we used MYO9A-depleted NSC-34 cells (mouse motor neuron-derived cells), revealing altered expression of a number of cytoskeletal proteins important for neuron structure and intracellular transport. On the basis of these findings, the effect on protein transport was determined using a vesicular recycling assay which revealed impaired recycling of a neuronal growth factor receptor. In addition, an unbiased approach utilizing proteomic profiling of the secretome revealed a key role for defective intracellular transport affecting proper protein secretion in the pathophysiology of MYO9A-related CMS. This also led to the identification of agrin as being affected by the defective transport. Zebrafish with reduced MYO9A orthologue expression were treated with an artificial agrin compound, ameliorating defects in neurite extension and improving motility. In summary, loss of MYO9A affects the neuronal cytoskeleton and leads to impaired transport of proteins, including agrin, which may provide a new and unexpected treatment option.

Failed reinnervation in aging skeletal muscle.

BACKGROUND: Skeletal muscle displays a marked accumulation of denervated myofibers at advanced age, which coincides with an acceleration of muscle atrophy. METHODS: In this study, we evaluated the hypothesis that the accumulation of denervated myofibers in advanced age is due to failed reinnervation by examining muscle from young adult (YA) and very old (VO) rats and from a murine model of sporadic denervation secondary to neurotrypsin over-expression (Sarco mouse). RESULTS: Both aging rat muscle and Sarco mouse muscle exhibited marked fiber-type grouping, consistent with repeating cycles of denervation and reinnervation, yet in VO muscle, rapsyn at the endplate increased and was associated with only a 10 % decline in acetylcholine receptor (AChR) intensity, whereas in Sarco mice, there was a decline in rapsyn and a 25 % decrease in AChR intensity. Transcripts of muscle-specific kinase (21-fold), acetylcholine receptor subunits α (68-fold), ε (threefold) and γ (47-fold), neural cell adhesion molecule (66-fold), and runt-related transcription factor 1 (33-fold) were upregulated in VO muscle of the rat, consistent with the marked persistent denervation evidenced by a large proportion of very small fibers (>20 %). In the Sarco mice, there were much smaller increases in denervation transcripts (0-3.5-fold) and accumulation of very small fibers (2-6 %) compared to the VO rat, suggesting a reduced capacity for reinnervation in aging muscle. Despite the marked persistent denervation in the VO rat muscle, transcripts of neurotrophins involved in promoting axonal sprouting following denervation exhibited no increase, and several miRNAs predicted to suppress neurotrophins were elevated in VO rat. CONCLUSIONS: Our results support the hypothesis that the accumulation of denervated fibers with aging is due to failed reinnervation and that this may be affected by a limited neurotrophin response that mediates axonal sprouting following denervation.

Denervation drives mitochondrial dysfunction in skeletal muscle of octogenarians.

KEY POINTS: Mitochondria are frequently implicated in the ageing of skeletal muscle, although the role of denervation in modulating mitochondrial function in ageing muscle is unknown. We show that increased sensitivity to apoptosis initiation occurs prior to evidence of persistent denervation and is thus a primary mitochondrial defect in ageing muscle worthy of therapeutic targeting. However, at more advanced age, mitochondrial function changes are markedly impacted by persistent sporadic myofibre denervation, suggesting the mitochondrion may be a less viable therapeutic target. ABSTRACT: Experimental denervation modulates mitochondrial function, where changes in both reactive oxygen species (ROS) and sensitivity to permeability transition are implicated in the resultant muscle atrophy. Notably, although denervation occurs sporadically in ageing muscle, its impact on ageing muscle mitochondria is unknown. Because this information has important therapeutic implications concerning targeting the mitochondrion in ageing muscle, we examined mitochondrial function in skeletal muscle from four groups of humans, comprising two active (mean ± SD age: 23.7 ± 2.7 years and 71.2 ± 4.9 years) and two inactive groups (64.8 ± 3.1 years and 82.5 ± 4.8 years), and compared this with a murine model of sporadic denervation. We tested the hypothesis that, although some alterations of mitochondrial function in aged muscle are attributable to a primary organelle defect, mitochondrial dysfunction would be impacted by persistent denervation in advanced age. Both ageing in humans and sporadic denervation in mice increased mitochondrial sensitivity to permeability transition (humans, P = 0.004; mice, P = 0.01). To determine the contribution of sporadic denervation to mitochondrial function, we pharmacologically inhibited the denervation-induced ROS response. This reduced ROS emission by 60% (P = 0.02) in sporadically denervated mouse muscle, which is similar to that seen in humans older than 75 years (-66%, P = 0.02) but not those younger than 75 years. We conclude that an increased sensitivity to permeability transition is a primary mitochondrial defect in ageing muscle. However, at more advanced age, when muscle atrophy becomes more clinically severe, mitochondrial function changes are markedly impacted by persistent sporadic denervation, making the mitochondrion a less viable therapeutic target.

The C-Terminal Fragment of Agrin (CAF), a Novel Marker of Renal Function, Is Filtered by the Kidney and Reabsorbed by the Proximal Tubule.

Agrin, a multidomain proteoglycan and neurotrypsin, a neuronal serine protease, are important for forming (neuromuscular) synapses. Proteolytical activity of neurotrypsin produces a C-terminal fragment of agrin, termed CAF, of approximately 22 kDA molecular size which also circulates in blood. The presence of CAF in urine suggests either glomerular filtration or secretion into urine. Blood levels of CAF have been identified as a potential novel marker of kidney function. Here we describe that several nephron segments in the mouse kidney express agrin and neutrotrypsin in addition to the localization of both protein in the glomerulum. Agrin mRNA and protein was detected in almost all nephron segments and mRNA abundance was highest in the inner medullary collecting duct. Neurotrypsin mRNA was mostly detected in the thick ascending limb of the loop of Henle, the distal convoluted tubule, and the inner medullary collecting duct. Moreover, we show that the proximal tubule absorbs injected recombinant CAF by a process shared with receptor-mediated and fluid phase endocytosis. Co-injection of CAF with recombinant human transferrin, a substrate of the receptor-mediated endocytic pathway as well as with FITC-labelled dextran (10 kDa), a marker of fluid phase endocytosis, showed partial colocalization of CAF with both markers. Further colocalization of CAF with the lysosomal marker cathepsin B suggested degradation of CAF by the lysosome in the proximal tubule. Thus, the murine kidney expresses agrin and neurotrypsin in nephron segments beyond the glomerulum. CAF is filtered by the glomerulum and is reabsorbed by endocytosis by the proximal tubule. Thus, impaired kidney function could impair glomerular clearance of CAF and thereby increase circulating CAF levels.

Evaluation of C-terminal Agrin Fragment as a marker of muscle wasting in patients after acute stroke during early rehabilitation.

BACKGROUND: C-terminal Agrin Fragment (CAF) has been proposed as a novel biomarker for sarcopenia originating from the degeneration of the neuromuscular junctions. In patients with stroke muscle wasting is a common observation that predicts functional outcome. We aimed to evaluate agrin sub-fragment CAF22 as a marker of decreased muscle mass and physical performance in the early phase after acute stroke. METHODS: Patients with acute ischaemic or haemorrhagic stroke (n = 123, mean age 70 ± 11 y, body mass index BMI 27.0 ± 4.9 kg/m(2)) admitted to inpatient rehabilitation were studied in comparison to 26 healthy controls of similar age and BMI. Functional assessments were performed at begin (23 ± 17 days post stroke) and at the end of the structured rehabilitation programme (49 ± 18 days post stroke) that included physical assessment, maximum hand grip strength, Rivermead motor assessment, and Barthel index. Body composition was assessed by bioelectrical impedance analysis (BIA). Serum levels of CAF22 were measured by ELISA. RESULTS: CAF22 levels were elevated in stroke patients at admission (134.3 ± 52.3 pM) and showed incomplete recovery until discharge (118.2 ± 42.7 pM) compared to healthy controls (95.7 ± 31.8 pM, p < 0.001). Simple regression analyses revealed an association between CAF22 levels and parameters of physical performance, hand grip strength, and phase angle, a BIA derived measure of the muscle cellular integrity. Improvement of the handgrip strength of the paretic arm during rehabilitation was independently related to the recovery of CAF22 serum levels only in those patients who showed increased lean mass during the rehabilitation. CONCLUSIONS: CAF22 serum profiles showed a dynamic elevation and recovery in the subacute phase after acute stroke. Further studies are needed to explore the potential of CAF22 as a serum marker to monitor the muscle status in patients after stroke.

Comparison of Peritoneal Low-Molecular-Weight-Protein-Removal in CCPD and CAPD Patients Based on C-Terminal Agrin Fragment Clearance.

BACKGROUND/AIMS: This study compares the peritoneal elimination of the low-molecular-weight-protein (LMWP) C-terminal agrin fragment (tCAF, size 22 kDa), a promising biomarker for kidney function, in continuous cycling peritoneal dialysis (CCPD) and continuous ambulatory peritoneal dialysis (CAPD). METHODS: 103 sets of serum, 24h-urine and dialysate samples were obtained in 15 CCPD (63 sets) and 11 CAPD (40 sets) patients. Total, renal and peritoneal substrate removals/clearances were measured/calculated for tCAF, creatinine, blood-urea-nitrogen (BUN), cystatin C and albumin and correlated with the peritoneal transport type. RESULTS: Serum und urine concentrations of all biomarkers did not differ between both groups, urinary substrate removal was higher in CAPD patients for all biomarkers due to better residual renal function. Peritoneal substrate removal of tCAF and albumin were significantly higher in CAPD (tCAF: 35.3 vs. 19.3 µg/d, p<0.001; albumin: 4.3 vs. 3.7 g/d, p=0.001), whereas cystatin C and creatinine did not differ between CAPD and CCPD (cystatin: 7.7 vs. 6.1 mg/d, p=0.08, creatinine: 423.9 vs. 456.7 mg/d, p=0.241). BUN was better removed by CCPD (4846.6 vs. 3393.4 mg/d, p<0.001). CAPD patients with high-transporter characteristics had a higher peritoneal tCAF removal compared to high-average-transporters (49.8 vs. 28.4 µg/d, p<0.001), no differences could be detected in CCPD patients between these groups. CAPD patients using icodextrin twice/day had higher peritoneal clearance of tCAF compared to once daily (4.4 vs. 2.8 l/wk/1.73 m2 body-surface-area, p<0.001). CONCLUSIONS: CAPD was superior to CCPD concerning peritoneal tCAF removal. This finding was pronounced in high-transporters and CAPD patients using icodextrin twice daily.

Plasma total C-terminal agrin fragment (tCAF) as a marker for kidney function in patients with chronic kidney disease.

BACKGROUND: Total C-terminal agrin fragment (tCAF) is a new biomarker that was previously correlated with kidney function. This article studies the validity of tCAF as a biomarker for kidney function in chronic kidney disease (CKD). METHODS: Plasma tCAF, serum creatinine (Cr), cystatin C (CyC), blood urea-nitrogen (BUN) concentrations and estimated glomerular filtration rate (eGFR CKD-EPIcrea-cystatin) were assessed in 426 individuals [71 without CKD (CKD 0°) and 355 CKD patients]. In addition to descriptive statistics, univariate correlation between tCAF and biomarkers/eGFR was calculated; multiple linear regression modeling was applied between logarithmic (log) tCAF and log eGFR and adjusted for demographic data. The same methods were used to analyze the association of demographic factors and the different biomarkers adjusted for eGFR. RESULTS: Mean tCAF levels were 1012.2±789.9 pM. tCAF correlated with all biomarkers/eGFR in univariate analysis (eGFR: r=-0.77, Cr: r=0.74, BUN: r=0.66, CyC: r=0.75). Linear regression modeling revealed an excellent coefficient estimate between log tCAF and log eGFR (CKD-EPIcrea-cystatin) (-0.91, p<0.001). tCAF was the parameter least associated with demographic parameters in both univariate and multivariate regression modeling (only with age, coefficient estimate r=-0.159, p=0.001 in multivariate regression). CONCLUSIONS: In conclusion, tCAF is a promising biomarker for the assessment of kidney function in CKD patients showing an excellent correlation with eGFR and being less influenced by demographic parameters compared to conventional biomarkers. These preliminary results encourage further evaluation of tCAF in larger CKD cohorts and other clinical settings such as acute renal failure.

Early postoperative C-terminal agrin fragment (CAF) serum levels predict graft loss and proteinuria in renal transplant recipients.

BACKGROUND: C-terminal agrin fragment (CAF), cleavage product of agrin, was previously correlated with kidney function in renal transplant patients. This article studies the predictive value of CAF for long-term outcomes in renal transplant recipients. METHODS: In this observational cohort study, serum CAF, creatinine and blood-urea-nitrogen (BUN) concentrations and eGFR (CKD-EPI) were assessed 1-3 months after transplantation in 105 patients undergoing kidney transplantation. Cox regression models were used to analyse the predictive value of all parameters concerning all-cause mortality (ACM), graft loss (GL), doubling of creatinine/proteinuria at the end of follow-up. RESULTS: Median follow-up time was 3.1 years. The mean concentrations were 191.9±152.4 pM for CAF, 176±96.8 µmol/L for creatinine, 12.6±6.2 mmol/L for BUN and 44.9±21.2 mL/min for CKD-EPI formula, respectively. In univariate analysis CAF and BUN concentrations predicted ACM (CAF: HR=1.003, 1.1-fold risk, p=0.043; BUN: HR=1.037, 1.3-fold risk, p=0.006). Concerning GL, CAF (HR=1.006, 3.1-fold risk, p<0.001), creatinine (HR=2.396, 2.6-fold risk, p<0.001), BUN (HR=1.048, 1.7-fold risk, p=0.001) and eGFR (CKD-EPI) (HR=0.941, 0.45-fold risk reduction, p=0.006) showed a statistically significant association. CAF was the only parameter significantly associated with doubling of proteinuria (HR=1.005, 1.7-fold risk, p<0.001). In multiple regression analysis (CAF only) the association remained significant for GL and doubling of proteinuria but not ACM. CONCLUSIONS: Early postoperative serum CAF appears to be a useful tool for the assessment of long-term outcomes in renal transplant recipients. Most importantly it represents a promising predictor for the development of proteinuria.

C-Terminal Fragment of Agrin (CAF): A Novel Marker for Progression of Kidney Disease in Type 2 Diabetics.

BACKGROUND: Diabetes is the leading cause of CKD in the developed world. C-terminal fragment of agrin (CAF) is a novel kidney function and injury biomarker. We investigated whether serum CAF predicts progression of kidney disease in type 2 diabetics. METHODS: Serum CAF levels were measured in 71 elderly patients with diabetic nephropathy using a newly developed commercial ELISA kit (Neurotune®). Estimated glomerular filtration rate (eGFR) and proteinuria in spot urine were assessed at baseline and after 12 months follow up. The presence of end stage renal disease (ESRD) was evaluated after 24 months follow-up. Correlation and logistic regression analyses were carried out to explore the associations of serum CAF levels with GFR, proteinuria, GFR loss and incident ESRD. Renal handling of CAF was tested in neurotrypsin-deficient mice injected with recombinant CAF. RESULTS: We found a strong association of serum CAF levels with eGFR and a direct association with proteinuria both at baseline (r = 0.698, p<0.001 and r = 0. 287, p = 0.02) as well as after 12 months follow-up (r = 0.677, p<0.001 and r = 0.449, p<0.001), respectively. Furthermore, in multivariate analysis, serum CAF levels predicted eGFR decline at 12 months follow-up after adjusting for known risk factors (eGFR, baseline proteinuria) [OR (95%CI) = 4.2 (1.2-14.5), p = 0.024]. In mice, injected CAF was detected in endocytic vesicles of the proximal tubule. CONCLUSION: Serum CAF levels reflect renal function and are highly associated with eGFR and proteinuria at several time points. Serum CAF was able to predict subsequent loss of renal function irrespective of baseline proteinuria in diabetic nephropathy. CAF is likely removed from circulation by glomerular filtration and subsequent endocytosis in the proximal tubule. These findings may open new possibilities for clinical trial design, since serum CAF levels may be used as a selection tool to monitor kidney function in high-risk patients with diabetic nephropathy.

Detection of muscle wasting in patients with chronic heart failure using C-terminal agrin fragment: results from the Studies Investigating Co-morbidities Aggravating Heart Failure (SICA-HF).

AIMS: Skeletal muscle wasting affects 20% of patients with chronic heart failure and has serious implications for their activities of daily living. Assessment of muscle wasting is technically challenging. C-terminal agrin-fragment (CAF), a breakdown product of the synaptically located protein agrin, has shown early promise as biomarker of muscle wasting. We sought to investigate the diagnostic properties of CAF in muscle wasting among patients with heart failure. METHODS AND RESULTS: We assessed serum CAF levels in 196 patients who participated in the Studies Investigating Co-morbidities Aggravating Heart Failure (SICA-HF). Muscle wasting was identified using dual-energy X-ray absorptiometry (DEXA) in 38 patients (19.4%). Patients with muscle wasting demonstrated higher CAF values than those without (125.1 ± 59.5 pmol/L vs. 103.8 ± 42.9 pmol/L, P = 0.01). Using receiver operating characteristics (ROC), we calculated the optimal CAF value to identify patients with muscle wasting as >87.5 pmol/L, which had a sensitivity of 78.9% and a specificity of 43.7%. The area under the ROC curve was 0.63 (95% confidence interval 0.56-0.70). Using simple regression, we found that serum CAF was associated with handgrip (R = - 0.17, P = 0.03) and quadriceps strength (R = - 0.31, P < 0.0001), peak oxygen consumption (R = - 0.5, P < 0.0001), 6-min walk distance (R = - 0.32, P < 0.0001), and gait speed (R = - 0.2, P = 0.001), as well as with parameters of kidney and liver function, iron metabolism and storage. CONCLUSION: CAF shows good sensitivity for the detection of skeletal muscle wasting in patients with heart failure. Its assessment may be useful to identify patients who should undergo additional testing, such as detailed body composition analysis. As no other biomarker is currently available, further investigation is warranted.

C-terminal agrin fragment (CAF) reflects renal function in patients suffering from severe sepsis or septic shock.

BACKGROUND: One of the main causes of acute kidney injury (AKI) in patients treated on an intensive care unit (ICU) is sepsis. The identification of new biomarkers indicating the early development and future course of AKI are of utmost medical interest. The C-terminal agrin fragment (CAF) is measurable in blood serum and might reflect kidney function. Therefore, this study evaluates CAF in patients presenting to an internal ICU with severe sepsis or septic shock. Serum levels of CAF are correlated with biomarkers of kidney function, markers of systemic inflammation, and the presence of AKI and renal replacement therapy (RRT). METHODS: 61 patients suffering from severe sepsis or septic shock were included during the first 24 hours of ICU treatment and blood samples for biomarker measurements, i.e., CAF, creatinine, cystatin C, procalcitonin (PCT), interleukin 6, C reactive protein (CRP), and white blood cells (WBC) were collected on the first day of intensive care treatment. The number of RRT days and the incidence of AKI were documented. RESULTS: 13% of the patients (8/61) suffered from SIRS/sepsis, 20% (12/61) from severe sepsis, and 67% (41/61) from septic shock. Serum levels of CAF significantly correlated with creatinine (r = 0.623, p < 0.001) and cystatin C (r = 0.578, p < 0.001). Multiple linear regression analyses adjusting CAF for inflammatory parameters (i.e., WBC, CRP, interleukin 6, PCT), age, and gender showed a strong correlation between CAF and creatinine (r = 0.643, p < 0.001). Serum levels of CAF were significantly associated with the need of RRT (area under the curve (AUC) = 0.772, 95% CI: 0.641-0.903, p = 0.002) and the incidence of AKI (AUC = 0.721, 95% CI: 591-0.850, p = 0.004) as indicated by ROC analysis. CONCLUSIONS: In patients suffering from severe sepsis and septic shock, serum levels of CAF were significantly associated with kidney function and RRT and were not influenced by severe septic conditions.

C-terminal agrin fragment (CAF) as a serum biomarker for residual renal function in peritoneal dialysis patients.

BACKGROUND: C-terminal agrin fragment (CAF, size 22 kDa) is a promising new biomarker for kidney function. This study evaluated the usefulness of CAF as a serum biomarker for residual renal function (RRF) in patients undergoing automated peritoneal dialysis (APD). PATIENTS AND METHODS: Serum, urine and dialysate samples were obtained in 12 end-stage renal disease patients undergoing APD. Total, renal and peritoneal clearances were calculated for CAF, creatinine, blood urea nitrogen (BUN) and cystatin c. kt/V was computed, and RRF (in ml/min) was calculated as the arithmetic mean of creatinine and BUN clearance. Correlations between the biomarkers' serum concentrations, clearances, kt/V and RRF were computed. RESULTS: Serum CAF concentrations were highly correlated with serum concentrations of creatinine (r = 0.806, p = 0.002), BUN (r = 0.727, p = 0.007), cystatin c (r = 0.839, p = 0.001) and inversely to 24-h urinary output (r = -0.669, p = 0.017). RRF was inversely correlated with serum concentrations of CAF, cystatin c and creatinine being highest for CAF (r = -0.734, p = 0.007) followed by cystatin c (r = -0.65, p = 0.022) and creatinine (r = -0.606, p = 0.037). Serum BUN was not significantly associated with RRF (r = -0.497, p = 0.101). Age, weight and gender did not significantly affect serum CAF concentrations. CONCLUSION: Serum CAF provides a robust serum biomarker for RRF in peritoneal dialysis patients undergoing APD, possibly outperforming the value of conventional biomarkers.

Influence of high-flux hemodialysis and hemodiafiltration on serum C-terminal agrin fragment levels in end-stage renal disease patients.

C-terminal agrin fragment (CAF, 22 kDa) has been shown to be a promising new rapid biomarker for kidney function. This study evaluated the influence of hemodialysis (HD) and hemodiafiltration (HDF) treatment on serum CAF concentrations in patients with end-stage renal disease (ESRD). A total of 36 patients with ESRD undergoing chronic HD/HDF treatment were enrolled (21 high-flux-HD/Fx60 membrane, 7 high-flux-HD/Elisio19H membrane, and 8 HDF/Elisio19H membrane). On a midweek session, blood samples were obtained before, at halftime, and post-treatment. Dialysate samples were obtained 4 times during treatment. Serum and dialysate CAF, cystatin C, urea, and creatinine concentrations were measured. Reduction ratios (RRs), total solute removal, overall dialytic clearance, and instantaneous dialytic clearance at halftime were calculated and compared. Although HD/Elisio19H and HDF/Elisio19H treatments significantly reduced CAF concentrations (RR 46.6 ± 9.1% and 57.6 ± 11.7%, respectively, P = 0.018 and P = 0.001), HD/Fx60 treatment did not remove CAF from serum (RR 2.4 ± 15.4%, P = 0.25), there was no relevant CAF detection in dialysate. In the HD/Fx60 group, the RR of CAF was significantly lower compared with cystatin C, urea, and creatinine, in which significant removal was detected (37.9 ± 14.8%, 65.0 ± 10.7%, and 56.0 ± 9.8%, respectively, P < 0.001). CAF is a new biomarker for kidney function whose serum concentration is not influenced by conventional high-flux HD using Fx60 membrane. It might therefore represent a promising dialysis-independent biomarker for evaluation of kidney function, for example, in acute kidney failure.

Motoneuron loss is associated with sarcopenia.

OBJECTIVES: Sarcopenia, age-related muscle wasting, is associated with increased morbidity and mortality in the affected individuals. The pathogenesis of sarcopenia is not yet fully understood. A multifactorial concept is currently favored. The reduced number of motor units as a potential mechanism of muscle mass loss is explored in the present study. DESIGN: This is a cross-sectional study. SETTING: The participants were community-dwelling older adults. PARTICIPANTS: The participants were sarcopenic (75) and nonsarcopenic (74) according to the criteria of the European Working Group on Sarcopenia in Older People aged 65 to 94 years. MEASUREMENTS: The motor unit number index (MUNIX) of the hypothenar muscle was used to assess the number and size [motor unit size index (MUSIX)] of motor units. RESULTS: The participants with pathologic MUNIX and MUSIX (n = 23) are significantly more frequently sarcopenic (n = 17, P = .029) than nonsarcopenic (n = 6). The participants with pathologic MUNIX and MUSIX (n = 23) had significantly less muscle mass than the nonsarcopenic controls (P < .001). After adjusting for age and sex, only gait speed has shown no difference between the 2 groups. Pearson's correlation coefficient between MUSIX and the reciprocal value of MUNIX is 0.87 (P < .001). CONCLUSIONS: Sarcopenia induced by a small number of motoneurons can be identified by applying the MUNIX method to the hypothenar muscle. An enlargement of motor units because of motoneuron loss seems to preserve physical performance.

Elevated troponin in septic patients in the emergency department: frequency, causes, and prognostic implications.

BACKGROUND: According to the "Third Universal Definition of Myocardial Infarction", cardiac troponin (cTn) is defined to be elevated, if the value is above the 99th percentile of a normal reference population. Especially in emergency medicine, this leads to pathological values more often than before this definition has been founded. Severe sepsis and septic shock frequently cause a rise of cTn, but there is only limited data about its role in septic patients in the emergency department (ED). Therefore, we investigated the frequency, main causes, and prognostic relevance of elevated high-sensitive troponin T (hsTnT) in septic patients in the ED. METHODS: Adults presenting at the ED with sepsis were included in the study. HsTnT was measured soon after admission. Main influencing factors were investigated, and the prognostic value was evaluated. RESULTS: 197 of the 313 analysed patients (62.9 %) showed an elevated hsTnT, with significantly higher rates in patients with severe sepsis and septic shock than in uncomplicated sepsis. APACHE II score, creatinine, and coronary heart disease were found to influence hsTnT independently. Nevertheless, patients with uncomplicated sepsis and without relevant renal insufficiency also showed notable rates of elevated hsTnT: 51.6 % (uncomplicated sepsis) and 34.5 % (no relevant renal failure), respectively. HsTnT showed a prognostic value with higher levels in non-survivors and an AUC of 0.72, p < 0.001. CONCLUSIONS: In the ED, sepsis is a relevant cause of elevated cTn, which underlines the role of sepsis as a differential diagnosis in non-ACS patients with positive cTn. A rise of cTn may be an indicator of poor outcome.

Injection of a soluble fragment of neural agrin (NT-1654) considerably improves the muscle pathology caused by the disassembly of the neuromuscular junction.

Treatment of neuromuscular diseases is still an unsolved problem. Evidence over the last years strongly indicates the involvement of malformation and dysfunction of neuromuscular junctions in the development of such medical conditions. Stabilization of NMJs thus seems to be a promising approach to attenuate the disease progression of muscle wasting diseases. An important pathway for the formation and maintenance of NMJs is the agrin/Lrp4/MuSK pathway. Here we demonstrate that the agrin biologic NT-1654 is capable of activating the agrin/Lrp4/MuSK system in vivo, leading to an almost full reversal of the sarcopenia-like phenotype in neurotrypsin-overexpressing (SARCO) mice. We also show that injection of NT-1654 accelerates muscle re-innervation after nerve crush. This report demonstrates that a systemically administered agrin fragment has the potential to counteract the symptoms of neuromuscular disorders.