Bruce A. Pussell

The inhibitory effect of rosmarinic acid on complement involves the C5 convertase

Rosmarinic acid (RA), a naturally occurring extract from Melissa officinalis, inhibits several complement-dependent inflammatory processes and may have potential as a therapeutic agent for the control of complement activation in disease. Rosmarinic acid has been reported to have effects on both the classical pathway C3-convertase and on the cobra venom factor-induced, alternative pathway convertase. In order to define the mechanism of inhibition, the effect of RA on classical and alternative pathway lysis, C1q binding, the classical pathway convertase, the alternative pathway convertase, membrane attack pathway lysis and the generation of fragments of C3 and C5 during activation, was tested in vitro. The results showed that RA inhibited lysis by the classical pathway more than by the alternative pathway. This effect was dose-dependent with maximum inhibition of classical pathway lysis observed at 2.6 mmoles of RA. There was little effect on C1q binding or on the classical and alternative pathway convertases. However, there was highly significant inhibition of lysis of pre-formed EA43b cells by dilutions of human or rabbit serum in the presence of RA (1 mM); this was accompanied by inhibition of C5a generation. We conclude that the inhibitory effect of RA involves the C5 convertase. Such inhibition could be advantageous to the host in disorders where the terminal attack sequence plays a role in pathogenesis.

A Systematic Review of Conversion From Calcineurin Inhibitor to Mammalian Target of Rapamycin Inhibitors for Maintenance Immunosuppression in Kidney Transplant Recipients

This was a systematic review of randomized controlled trials comparing delayed conversion of mammalian target of rapamycin inhibitors (mTORi) for calcineurin inhibitors (CNIs) versus CNI continuation in kidney transplantation. Databases (2000–2012) and conference abstracts (2009–2012) were searched giving a total of 29 trials. Outcomes analyzed included GFR, graft loss, rejection and adverse events and were expressed as weighted mean differences (WMDs) or as risk ratios (RRs). Patients converted to mTORi up to 1 year posttransplant in intention‐to‐treat analysis had higher GFR compared with those remaining on CNI (WMD 0.28 mL/min/1.73 m2, 95% confidence interval [CI] 0.21–0.36; I2 = 68%, p < 0.001). Stratifying trials by time posttransplant or type of mTORi did not change the overall heterogeneity. For on‐treatment population, mTORi was associated with higher GFR (14.21 mL/min/1.73 m2, 10.34–18.08; I2 = 0%, p = 0.970) 2–5 years posttransplant. The risk of rejection at 1 year was higher in mTORi trials (RR 1.72, 1.34–2.22; I2 = 12%, p = 0.330). Discontinuation secondary to adverse events was more common in patients on mTORi, whereas the incidence of skin cancers and cytomegalovirus infection was lower in patients on mTORi. Conversion from CNI to mTORi is associated with short‐term improvements in GFR in a number of studies but longer‐term follow‐up data of graft and patient survival are required.

The influence of oral lipid loads on acylation stimulating protein (ASP) in healthy volunteers.

OBJECTIVES: To examine the hypothesis that a sustained rise in plasma acylation stimulating protein (ASP, C3a desarg) accompanies the elevation in triacylglycerol that follows the ingestion of an oral fat load.DESIGN: Following an overnight fast, blood samples were obtained from healthy volunteers while fasting and 15 min, 1, 2, 4, 6 and 8 h following ingestion of: (i) a liquid meal, rich in dairy fat (eight subjects) and (ii) a semi-liquid meal, with higher total fat content and rich in polyunsaturated fat (six subjects).SUBJECTS AND METHODS: Four male and four female volunteers (age range: 22–51 y; body mass index (BMI): 17.9–26.9 kg/m2) received the first meal. Six subjects (age range: 32–60 y; BMI: 18.0–28.4 kg/m2), including three from the first study, received the second meal using the same protocol. ASP and C5a were measured by radioimmunoassay (RIA) and the complement proteins C3, factor B and C5 by radial immunodiffusion or nephelometry. Tumour necrosis factor (TNF)-α was measured by enhanced ELISA, and plasma cholesterol and triacylglycerol by an automated enzymatic method. The presence of chylomicrons was assessed in post-prandial plasma samples taken after the second meal.RESULTS: There was no significant change in mean ASP concentration in either group at any time point, following ingestion of either meal. However, there was a significant positive linear trend in ASP following the second fat challenge (ANOVA; P<0.05). There was also no change in complement proteins, plasma cholesterol or TNF-α. Plasma triacylglycerol rose significantly after the first and second meals (P<0.05 and P<0.001 at 2 h post-prandially); the mean maximum rise above the fasting level was 58±41% and 89±38% respectively (mean±s.d.). Chylomicrons were detected in samples taken from each subject after the second meal. Analysis of individual ASP data showed a sustained rise in one subject after the first meal and two subjects after the second meal. Substantial variation in ASP concentration was observed in samples taken in the first 2 h post-prandially.CONCLUSION: There was no significant change in ASP nor other complement proteins for either group of subjects following ingestion of the lipid loads. Individual data showed substantial variation in post-prandial ASP, but multiple plasma sampling did not define the basis for this variation.

Sensory nerve excitability and neuropathy in end stage kidney disease

Background: Peripheral neuropathy is present in 65% of patients with end stage kidney disease (ESKD) starting dialysis. Studies of membrane potential and axonal ion channel function may help explain the pathophysiology. Objectives: To follow changes in median sensory axon excitability in patients with ESKD treated with haemodialysis, and correlate them with clinical rating scales and serum levels of potential neurotoxins. Methods: Sensory nerve action potentials were recorded from the second digit following stimulation of the median nerve in 12 ESKD patients. Stimulus–response behaviour using two stimulus durations, threshold electrotonus to 100 ms polarising currents, a current–threshold relation, and recovery of excitability following supramaximal stimulation were recorded before, during, and after haemodialysis. Serum concentrations of potential neurotoxins were measured. Results: Before dialysis, there were changes in nerve excitability consistent with axonal depolarisation: refractoriness was increased; superexcitability and depolarising threshold electrotonus were reduced. Following dialysis there were improvements in all indices, with correlations between excitability abnormalities and serum potassium measurements. Neuropathic symptoms correlated with excitability changes. Conclusions: Nerves are depolarised before haemodialysis in ESKD patients. The correlation of excitability abnormalities with potassium indicates that the achievement of normokalaemia should be a priority in treating such patients.

An International Comparison of the Effect of Policy Shifts to Organ Donation following Cardiocirculatory Death (DCD) on Donation Rates after Brain Death (DBD) and Transplantation Rates

During the past decade an increasing number of countries have adopted policies that emphasize donation after cardiocirculatory death (DCD) in an attempt to address the widening gap between the demand for transplantable organs and the availability of organs from donation after brain death (DBD) donors. In order to examine how these policy shifts have affected overall deceased organ donor (DD) and DBD rates, we analyzed deceased donation rates from 82 countries from 2000–2010. On average, overall DD, DBD and DCD rates have increased over time, with the proportion of DCD increasing 0.3% per year (p = 0.01). Countries with higher DCD rates have, on average, lower DBD rates. For every one-per million population (pmp) increase in the DCD rate, the average DBD rate decreased by 1.02 pmp (95% CI: 0.73, 1.32; p<0.0001). We also found that the number of organs transplanted per donor was significantly lower in DCD when compared to DBD donors with 1.51 less transplants per DCD compared to DBD (95% CI: 1.23, 1.79; p<0.001). Whilst the results do not infer a causal relationship between increased DCD and decreased DBD rates, the significant correlation between higher DCD and lower DBD rates coupled with the reduced number of organs transplanted per DCD donor suggests that a national policy focus on DCD may lead to an overall reduction in the number of transplants performed.

Association Between Calcineurin Inhibitor Treatment and Peripheral Nerve Dysfunction in Renal Transplant Recipients

Neurotoxicity is a significant clinical side effect of immunosuppressive treatment used in prophylaxis for rejection in solid organ transplants. This study aimed to provide insights into the mechanisms underlying neurotoxicity in patients receiving immunosuppressive treatment following renal transplantation. Clinical and neurophysiological assessments were undertaken in 38 patients receiving immunosuppression following renal transplantation, 19 receiving calcineurin inhibitor (CNI) therapy and 19 receiving a calcineurin‐free (CNI‐free) regimen. Groups were matched for age, gender, time since transplant and renal function and compared to normal controls (n = 20). The CNI group demonstrated marked differences in nerve excitability parameters, suggestive of nerve membrane depolarization (p < 0.05). Importantly, there were no differences between the two CNIs (cyclosporine A or tacrolimus). In contrast, CNI‐free patients showed no differences to normal controls. The CNI‐treated patients had a higher prevalence of clinical neuropathy and higher neuropathy severity scores. Longitudinal studies were undertaken in a cohort of subjects within 12 months of transplantation (n = 10). These studies demonstrated persistence of abnormalities in patients maintained on CNI‐treatment and improvement noted in those who were switched to a CNI‐free regimen. The results of this study have significant implications for selection, or continuation, of immunosuppressive therapy in renal transplant recipients, especially those with pre‐existing neurological disability.

Post‐streptococcal glomerulonephritis: studies on the interaction betwen nephritis strain‐associated protein (NSAP), complement and the glomerulus

Nephritis strain‐associated protein (NSAP), a streptokinase produced by strains of streptococci isolated from patients with acute glomerulonephritis, is believed to be a specific antigen which participates in the production of glomerular injury. In order to investigate the mechanisms by which NSAP induces damage we have examined its potential to activate complement in vitro and to bind to isolated human glomeruli. NSAP, both alone and in combination with specific antibody, caused depletion of complement in normal human serum as measured by total haemolytic complement activity and generation of the complement breakdown products, C3a and C4a. Furthermore, Scatchard analysis showed that NSAP bound tightly to human glomeruli (Ka of 400±240 times 106 M) when compared to non‐nephritic streptokinase (Ka of 7.3±4.1 times 106 M) and fully cationized human serum albumin (Ka of 0.6±0.04 times 106 M). These findings are consistent with the hypothesis that the deposition of streptococcal antigens within the glomerulus may precede the fixation of complement and specific antibody.

The complement system in Type 1 (insulin-dependent) diabetes

SummaryThe complement proteins C1q, r, s, C2, C4, C3, factor B, C5, C6, and the inhibitors, C1 inhibitor, factors I and H were measured in 35 patients with recently diagnosed Type 1 (insulin-dependent) diabetes, 76 patients with longer-duration disease (30 with complications) and 43 first-degree healthy relatives. We found that C1q, C4 and C3 were reduced significantly in all groups of patients (p<0.001 for each protein in recent onset and uncomplicated patients; p<0.01, p<0.01 and p<0.05 respectively, for patients with complications) compared to 60 control subjects and that C4 was also reduced in healthy relatives (p<0.001). C4 allotypes were examined in 63 subjects (selected from the patient groups) in order to clarify the role of null alleles in the production of the C4 abnormality. These showed serum C4 to be reduced significantly in 50 patients without null alleles (patient mean 0.24 g/l; control subject mean 0.34 g/l) (p< 0.0001), although levels were lowest in the 13 patients with one or more null alleles (mean 0.19 g/l). Finally, to examine the metabolic basis for the low concentrations of C4 and C3, the turnover of highly-purified, radiolabelled C4 and C3 was measured in seven recently diagnosed patients; four of these had low levels of C4. The data showed that three out of four of these patients had reduced synthesis of C3 and C4 and normal values for fractional catabolic rate. Two patients showed features of C4 hypercatabolism. We conclude that several early complement proteins are reduced in Type 1 diabetes, irrespective of duration or complications. These abnormalities may result from hypercatabolism or, more commonly reduction in protein synthesis. Genetic factors may influence the low serum concentration of C4.

Factors influencing glomerular filtration rate in renal transplantation after cyclosporine withdrawal using sirolimus-based therapy: a multivariate analysis of results at five years.

Abstract:  Changes in calculated glomerular filtration rate (GFR) from baseline to five yr were analyzed in relation to risk factors among renal transplant recipients. At three months after transplantation (baseline), 430 eligible patients receiving sirolimus (SRL), cyclosporine (CsA), and steroids (ST) were randomly assigned (1:1) to continue SRL–CsA–ST or have CsA withdrawn and SRL trough levels increased (SRL–ST group). For each risk factor, changes from baseline were compared within each treatment using a t‐test and between treatments using ANCOVA. Univariate then multivariate robust linear regression analyses were also performed. In the SRL–ST group, changes from baseline were not significantly different for any risk factor. With the exception of cold ischemia time >24 h, GFR values declined significantly for all risk factors in SRL–CsA–ST patients. For all risk factors, except second transplant or cold ischemia time >24 h, renal function was significantly different between groups. By order of significance in the multivariate analysis, treatment (p < 0.001), donor age (p < 0.001), proteinuria (p < 0.001), and biopsy‐confirmed rejection (p = 0.010) were significant predictors of GFR change from baseline. In conclusion, patients with risk factors for reduced renal function benefit from SRL maintenance therapy without CsA vs. those remaining on CsA.

Neuromuscular disease in the dialysis patient: an update for the nephrologist.

Neuromuscular disease is an extremely common complication of end‐stage kidney disease (ESKD), manifesting in almost all dialysis patients, and leading to weakness, reduced exercise capacity, and disability. Recent studies have suggested that hyperkalemia may underlie the development of neuropathy. As such, maintenance of serum K+ within normal limits between periods of dialysis in ESKD patients manifesting early neuropathic symptoms may reduce neuropathy development and progression. For patients with more severe neuropathic syndromes, increased dialysis frequency or a switch to high‐flux dialysis may prevent further deterioration, while ultimately, renal transplantation is required to improve and restore nerve function. Exercise training programs are beneficial for ESKD patients with muscle weakness due to neuropathy or myopathy, and are capable of improving exercise tolerance and quality of life. Specific treatments have recently been evaluated for symptoms of autonomic neuropathy, including sildenafil for impotence and midodrine for intra‐dialytic hypotension, and have been shown to be effective and well tolerated. Other important management strategies for neuropathy include attention to foot care to prevent callus and ulceration, vitamin supplementation, and erythropoietin. Treatment with membrane‐stabilizing agents, such as amitryptiline and gabapentin, are highly effective in patients with painful neuropathy.