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Membranous nephropathy associated with anti-PLA

Multifrequency bioimpedance spectroscopy (BIS) is an established method for assessing fluid status in chronic kidney disease (CKD). However, the technique is lacking in predictive value and accuracy. BIS algorithms assume constant tissue resistivity, which may vary with changing tissue ionic sodium concentration (Na+). This may introduce significant inaccuracies to BIS outputs. To investigate this, we used 23Na magnetic resonance imaging (MRI) to measure Na+ in muscle and subcutaneous tissues of 10 healthy controls (HC) and 20 patients with CKD 5 (not on dialysis). The extracellular (Re) and intracellular (Ri) resistance, tissue capacitance, extracellular (ECW) and total body water (TBW) were measured using BIS. Tissue water content was assessed using proton density-weighted MRI with fat suppression. BIS-derived volume indices were comparable in the two groups (OH: HC − 0.4 ± 0.9 L vs. CKD 0.5 ± 1.9 L, p = 0.13). However, CKD patients had higher Na+ (HC 21.2 ± 3.0, CKD 25.3 ± 7.4 mmol/L; p = 0.04) and significantly lower Re (HC 693 ± 93.6, CKD 609 ± 74.3 Ohms; p = 0.01); Ri and capacitance did not vary. Na+ showed a significant inverse linear relationship to Re (rs = − 0.598, p

Background: Membranous nephropathy is among the most common causes of nephrotic syndrome worldwide, with a high healthcare burden. Treatment using the modified Ponticelli regimen (mPR) has remained the standard of care for decades, but newer therapies such as rituximab offer promising results with reduced side effects. The cost of this treatment, however, is perceived as a barrier to widespread use, especially in resource limited healthcare systems.Methods: We developed a decision-analytic model to estimate the cost-effectiveness of rituximab versus the mPR from the perspective of the National Health Service in the UK over a 1 year, 5 year and lifetime horizon. Primary outcome is the cost-effectiveness of rituximab versus mPR at 5 years post-treatment. Secondary outcomes are cost-effectiveness at 1 and 10 years post-treatment and over a lifetime.Results: At 1-year post-treatment, rituximab therapy dominates mPR. At 5 years post-treatment, rituximab therapy is cheaper than the Ponticelli regimen but at a loss of 0.014 quality-adjusted life years (QALYs) with an incremental cost-effectiveness ratio (ICER) of £95 494.13. Over a lifetime, rituximab remains the cheaper option with an incremental cost of -£5251.03 but with a reduced quality of life (incremental QALY of -0.512) giving an ICER of £10 246.09.Conclusions: Our analysis indicates that rituximab has the potential to be a cost-effective treatment in the short and medium terms despite the high single-dose cost. This evaluation suggests that further research is warranted and highlights the need for a high-quality clinical trial to confirm the efficacy and cost-effectiveness of rituximab versus the current standard of care.

Background: Progressive chronic kidney disease (CKD) inevitably leads to salt and water retention and disturbances in the macro-and microcirculation. Objectives: We hypothesize that salt and water dysregulation in advanced CKD may be linked to inflammation and microvascular injury pathways. Methods: We studied 23 CKD stage 5 patients and 11 healthy controls (HC). Tissue sodium concentration was assessed using 23Sodium magnetic resonance (MR) imaging. Hydration status was evaluated using bioimpedance spectroscopy. A panel of inflammatory and endothelial biomarkers was also measured. Results: CKD patients had fluid overload (FO) when compared to HC (overhydration index: CKD = 0.5 ± 1.9 L vs. HC = –0.5 ± 1.0 L; p = 0.03). MR-derived tissue sodium concentrations were predominantly higher in the subcutaneous (SC) compartment (median [interquartile range] CKD = 22.4 mmol/L [19.4–31.3] vs. HC = 18.4 mmol/L [16.6–21.3]; p = 0.03), but not the muscle (CKD = 24.9 ± 5.5 mmol/L vs. HC = 22.8 ± 2.5 mmol/L; p = 0.26). Tissue sodium in both compartments correlated to FO (muscle: r = 0.63, p < 0.01; SC: rs = 0.63, p < 0.01). CKD subjects had elevated levels of vascular cell adhesion molecule (p < 0.05), tumor necrosis factor-alpha (p < 0.01), and interleukin (IL)-6 (p = 0.01) and lower levels of vascular endothelial growth factor-C (p = 0.04). FO in CKD was linked to higher IL-8 (r = 0.51, p < 0.05) and inversely associated to E-selectin (r = –0.52, p = 0.01). Higher SC sodium was linked to higher intracellular adhesion molecule (ICAM; rs = 0.54, p = 0.02). Conclusion: Salt and water accumulation in CKD appears to be linked with inflammation and endothelial activation pathways. Specifically IL-8, E-Selectin (in FO), and ICAM (in salt accumulation) may be implicated in the pathophysiology of FO and merit further investigation.

Background: Achieving euvolaemia using ultrafiltration (UF) during haemodialysis (HD) without inducing haemodynamic instability presents a major clinical challenge. Transcapillary refill is a key factor in sustaining the circulating blood volume (BV) during UF, which is in turn predicted by the rate of refilling. However, absolute plasma refilling rate (PRR), its determinants and variability with UF rate (UFR), have not been reported in the literature. Method: We studied paired HD sessions (n = 48) in 24 patients over 2 consecutive mid-week HD treatments. Plasma refilling was measured using real-time, minute-by-minute relative BV changes obtained from the integrated BV monitoring device during UF. A fixed bolus dilution approach at the start of HD was used to calculate absolute BV. The first control HD session was undertaken with a standard UFR required to achieve the prescribed target weight, while during the second study session, a fixed (high) UFR (1 L/h) was applied, either in the first (n = 12 patients) or in the final hour (n = 12 patients) of the HD session. Participants’ had their hydration status measured pre- and post-HD using multifrequency bioimpedance (BIS). Blood pressure was measured at 15-min intervals and blood samples were collected at 7 intervals during HD sessions. Results: The mean PRR during a standard 4-hr HD session was 4.3 ± 2.0 mL/kg/h and varied between 2 and 6 mL/kg/h. There was a mean time delay of 22 min (range 13.3–35.0 min) for onset of plasma refilling after the application of UF irrespective of standard or high UFRs. The maximum refilling occurred during the second hour of HD (mean max PRR 6.8 mL/kg/h). UFR (beta = 0.60, p < 0.01) and BIS derived pre-HD overhydration index (beta = 0.44, p = 0.01) were consistent, independent predictors of the mean PRR (R2 = 0.49) in all HD sessions. At high UFRs, PRR exceeded 10 mL/kg/h. The total overall plasma refill contribution to UF volume was not significantly different between standard and high UF. During interventions no significant haemodynamic instability was observed in the study. Conclusion:We describe absolute transcapillary refilling rate and its profile during HD with UF. The findings provide the basis for the development of UF strategies to match varying PRRs during HD. An approach to fluid removal, which is tailored to patients’ refilling rates and capacity, provides an opportunity for more precision in the practice of UF.

BACKGROUND Primary membranous nephropathy is associated with variable clinical course ranging from spontaneous remission to slow progression to end stage renal failure. Achieving remission confers better renal survival in primary membranous nephropathy (PMN). Longer term outcomes such as patient survival and relapse of active disease remain poorly understood. METHODS We performed a retrospective study of 128 consecutive adult patients diagnosed with biopsy proven PMN at a single UK centre between 1980 and 2010. These patients were followed prospectively over a median of 128 months. We assessed impact of persistent disease and relapse on Stage 5 chronic kidney disease (CKD-5) and patient survival and present longer term cumulative incidences of different end points. RESULTS One hundred patients achieved partial remission (PartRem) and 28 patients did not achieve remission (NoRem). Nine per cent of patients achieving first remission developed CKD-5 and 75% of those with NoRem developed CKD-5 [hazard ratio (HR) 0.07, 95% confidence interval 0.03-0.19). Relapse following PartRem occurred in 31 patients (31%) during follow-up and was significantly associated with progression to CKD-5. Progression to CKD-5 was strongly associated with death (47 versus 6%, HR 23.4; P < 0.01). Cumulative incidence at 15 years following first presentation included: death, 14%; CKD-5, 28%; and relapse 40% (in patients who achieved first remission). CONCLUSIONS Our data strongly suggest that mortality in PMN is seen in patients with disease progression to CKD-5. Achieving remission is strongly associated with improved renal survival after first presentation and following relapse. We suggest that patients who achieve remission should be followed up in longer term, and better strategies to help improve outcomes are needed in clinical practice.

Background Hyperphosphataemia in dialysis subjects is associated with increased mortality. However cause and effect has not been proven, and the ideal phosphate target range is unknown despite KDOQI’s call for studies over 12 years ago. The design and conduct of a randomized controlled trial is challenging because maintaining two groups within differing target ranges of serum phosphate has not been achieved over a long follow-up of 1 year, in a trial setting, before. The SPIRiT study examines the subject acceptance, recruitment and retention rates for such a study in which subjects were randomised to two distinct serum phosphate concentrations, then titrated and maintained over 12 months. Methods A two center trial of 104 hemodialysis subjects randomized to lower range LRG 0.8–1.4 mmol/L or 2.5–4.3 mg/dL) and higher range (HRG 1.8–2.4 mmol/L or 5.6–7.4 mg/dL) serum phosphate groups. Two months’ titration and ten months’ maintenance phase. Interventions were non-calcium phosphate binders, self-help questionnaires, with blood tests at specified time intervals. Results Thirteen percent of the eligible dialysis population were successfully recruited. A mean separation by serum phosphate of 1.1 mg/dL was achieved and maintained between the groups over 10 months. Drop-out rate was 27% with mortality 10%. Nine subjects in the HRG (17.6%) and two subjects in the LRG (3.8%) died during the study, however the study was not powered to detect significant differences in outcomes. Conclusion Randomizing dialysis subjects to separate treatment targets for serum phosphate can achieve a clinically significant sustained separation over 12 months. A large scale longer term study is required to examine outcomes including mortality. Trial registration The trial registration number is ISRCTN24741445 – Date of registration 16th January, retrospectively registered.

Rituximab induces nephrotic syndrome (NS) remission in two-thirds of patients with primary membranous nephropathy (MN), even after other treatments have failed. To assess the relationships among treatment effect, circulating nephritogenic anti-phospholipase A2 receptor (anti-PLA2R) autoantibodies and genetic polymorphisms predisposing to antibody production we serially monitored 24-hour proteinuria and antibody titer in patients with primary MN and long-lasting NS consenting to rituximab (375 mg/m2) therapy and genetic analyses. Over a median (range) follow-up of 30.8 (6.0–145.4) months, 84 of 132 rituximab-treated patients achieved complete or partial NS remission (primary end point), and 25 relapsed after remission. Outcomes of patients with or without detectable anti-PLA2R antibodies at baseline were similar. Among the 81 patients with antibodies, lower anti-PLA2R antibody titer at baseline (P=0.001) and full antibody depletion 6 months post-rituximab (hazard ratio [HR], 7.90; 95% confidence interval [95% CI], 2.54 to 24.60; P

Objective Arteriovenous fistula (AVF) maturation failure remains a significant problem with reported early failure rates around 50%. Suboptimal hemodynamics, variable surgical skills, and technique dependency are widely believed to contribute to AVF nonmaturation. The Optiflow (Bioconnect Systems, Ambler, Pa) is a novel anastomotic device placed in situ that has potential for improving hemodynamics and standardizing AVF placement. We report results from a prospective nonrandomized controlled pilot study designed to investigate the safety and performance of the Optiflow. Methods Forty-one participants underwent AVF formation using either a 3-mm or 4-mm Optiflow and 39 matched control participants underwent AVF formation using the standard technique at two sites. Patients were observed for 90 days after AVF placement. The primary end point was unassisted maturation, which was defined as an outflow vein with a diameter ≥5 mm and blood flow ≥500 mL/min measured by Doppler ultrasound. The secondary performance end point was unassisted patency, and the primary safety end point was freedom from device-related serious adverse events. Results Unassisted maturation rates at 14, 42, and 90 days were 76%, 72%, and 68%, respectively, for the Optiflow group and 67%, 68%, and 76%, respectively, in the control group ( P = .38, .69, and .47 at 14, 42, and 90 days). There was a trend to earlier maturation (assessed at 14 days) in the 4-mm Optiflow group compared with the control group ( P = .059). There were no device-related serious adverse events. Conclusions Maturation results for both the Optiflow and control groups were highly favorable compared with historical assisted maturation rates of approximately 50%. The Optiflow appears to be safe and effective in the placement of AVFs, with high maturation rates.

Aims: This study reports long-term outcomes after endovascular salvage (EVS) for acute dialysis fistula/graft dysfunction. Methods: All patients presenting with acute fistula or graft dysfunction, excluding primary failures, referred for endovascular salvage were included in this single-centre prospective study. Results: Altogether, 410 procedures were carried out in 232 patients. Overall, the incidence of thrombosis/occlusion (per patient-year) was 0.12 for fistulae and 0.9 for grafts. The anatomical success rate for EVS was 94% for fistulae and 92% for grafts. Primary patency rates for fistulae at 1, 6, 12, 24 and 36 months were 82, 64, 44, 34 and 26%, respectively, whereas secondary patency rates were 88, 84, 74, 69 and 61%, respectively. Primary patency rates for grafts at 1, 6 and 12 months were 50, 14 and 8%. The overall rate of complications was 6% with no incidence of symptomatic pulmonary embolism. In a Cox regression model, upper-arm location of fistula (HR 1.9, p = 0.04, n = 144) was associated with lower primary patency, whereas the presence of thrombosis was associated lower primary (HR 1.9, p = 0.004, n = 144) and secondary patency (HR 3.7, p < 0.001, n = 144). Aspirin therapy was associated with longer primary patency (HR 0.6, p = 0.02, n = 144) and secondary patency (HR 0.58, p = 0.08, n = 144). Conclusion: EVS is effective but longer-term outcomes are poor. Presence of thrombosis portends poor fistula survival and strategies for prevention need attention. Balloon maceration, our preferred declotting technique, is safe and the most cost-effective method. Aspirin therapy for patients presenting with failure of fistulae deserves further investigation.

Background: Extended haemodialysis (EHD) has been associated with better outcomes compared to conventional (CHD) regimes. The cardiovascular (CV) profile of these patients has not been assessed in detail. Methods: We report baseline demographic and CV phenotype of 36 CHD and 36 EHD participants to a longitudinal multicentre study. We measured pulse wave velocity (PWV), 24-h ambulatory blood pressure, sublingual dark-field capillaroscopy and vascular biomarkers. Results: EHD patients were younger (p < 0.01), with less CV comorbidity (p = 0.04) and higher dialysis vintage (p < 0.01). Higher PWV in CHD (p = 0.02) was not independent of demographic differences in the 2 groups. Biomarker profiles were similar in EHD and CHD but abnormal compared to healthy controls. Conclusion: Although CV profiles in these 2 cohorts were similar, EHD patients were distinct from the CHD population in terms of age and dialysis vintage and appear to comprise a unique group. Direct comparison of outcomes in these groups is challenging due to clinical bias.

Antibodies to the phospholipase A2 receptor 1 (PLA2R1) have been reported in 70% of cases of idiopathic membranous nephropathy (IMN). The genetic susceptibility of IMN has been accounted for by HLA DQA1 and PLA2R1 genes. Here we retrospectively quantified PLA2R antibodies by ELISA, and genotyped DQ alleles and PLA2R1 single-nucleotide polymorphisms for association with clinical criteria for disease activity at the time of first sample and with outcome over a median total follow-up of 90 months. In 90 prevalent patients with biopsy-proven IMN, anti-PLA2R antibodies were present in 75% of patients with IMN with active disease and were significantly higher than in patients in partial or complete remission at the time of antibody measurement. There was a differential IgG subclass response (4>2>3>1) at an early stage, i.e., within 6 months of biopsy. Levels of PLA2R antibodies were significantly linked to DQA1*05:01 and DQB1*02:01. Survival analysis of patients with IMN showed that PLA2R antibodies are significantly linked with outcome. Thus, high levels of PLA2R antibodies are linked with active disease and a higher risk of declining renal function during follow-up. Future therapeutic trials in IMN should monitor anti-PLA2R, as patients with a high antibody burden may benefit from earlier therapeutic intervention.

International audience; The M-type receptor for phospholipase A2 (PLA2R1) is the major target antigen in idiopathic membranous nephropathy (iMN). Our recent genome-wide association study showed that genetic variants in an HLA-DQA1 and phospholipase A2 receptor (PLA2R1) allele associate most significantly with biopsy-proven iMN, suggesting that rare genetic variants within the coding region of the PLA2R1 gene may contribute to antibody formation. Here, we sequenced PLA2R1 in a cohort of 95 white patients with biopsy-proven iMN and assessed all 30 exons of PLA2R1, including canonical (GT-AG) splice sites, by Sanger sequencing. Sixty patients had anti-PLA2R1 in serum or detectable PLA2R1 antigen in kidney tissue. We identified 18 sequence variants, comprising 2 not previously described, 7 reported as rare variants (

AIM: Primary membranous nephropathy (PMN) is associated with progression to end stage renal disease in some patients. Standard immunosuppressive therapy with cyclical cyclophosphamide and corticosteroids can be associated with significant adverse effects. We aimed to assess immunological and clinical response with intravenous pulse cyclophosphamide and oral steroids in patients with severe nephrotic syndrome - in a prospective observational cohort study at our centre.METHODS: 17 consecutive patients (9 New-incident and 8 relapses) with severe nephrotic syndrome received intravenous pulse cyclophosphamide and daily oral steroids after failure to achieve remission with supportive therapy alone. Immunosuppressive therapy was discontinued at 6 months or earlier if proteinuria regressed to RESULTS: Dose of cyclophosphamide received was 5.4gm in New-incident patients and 4.2gm in patients with relapses. All 17 patients achieved partial remission within 6 months: proteinuria improved from 656 to 102mg/mmol at 6-months and 55mg/mmol at 12-months (p CONCLUSION: Monthly intravenous pulse cyclophosphamide with oral steroids induced immunological and clinical partial remission at significantly reduced cyclophosphamide and steroid doses in PMN.

Encapsulating peritoneal sclerosis (EPS) is a potentially devastating complication of peritoneal dialysis\ud (PD). Diagnosis is often delayed due to the lack of effective and accurate diagnostic tools. We\ud therefore examined peritoneal effluent for potential biomarkers that could predict or confirm the\ud diagnosis of EPS and would be valuable in stratifying at-risk patients, and driving appropriate\ud interventions. Using prospectively collected samples from the Global Fluid Study and a cohort of\ud Greek PD patients, we utilized 2D SDSPAGE/ MS and iTRAQ to identify changes in the peritoneal\ud effluent proteome from patients diagnosed with EPS and controls matched for treatment exposure. We\ud employed a combinatorial peptide ligand library to compress the dynamic range of protein\ud concentrations, to aid identification of low-abundance proteins. In patients with stable membrane\ud function, fibrinogen γ-chain and heparan sulphate proteoglycan core protein progressively\ud increased over time on PD. In patients who developed EPS, collagen-α1(I), γ-actin and Complement\ud factors B and I were elevated up to five years prior to diagnosis. Orosomucoid-1 and a2-HSglycoprotein chain-B were elevated about one year before diagnosis, while apolipoprotein A-IV and\ud α1-antitrypsin were decreased compared to controls. Dynamic range compression resulted in an\ud increased number of proteins detected with improved resolution of protein spots, compared to the full\ud fluid proteome. Intelectin-1, dermatopontin, gelsolin and retinol binding protein-4 were elevated in\ud proteome-mined samples from patients with EPS compared to patients that had just commenced\ud peritoneal dialysis. Thus, prospective analysis of peritoneal effluent uncovered proteins indicative of\ud inflammatory and pro-fibrotic injury worthy of further evaluation as diagnostic/prognostic markers.

Background: In the United Kingdom, socioeconomic disadvantage has been associated with lower use of home dialysis, mostly peritoneal dialysis. In this study, we explore the role of a patient's sociodemographic, socioeconomic differences and the centre's influence on home haemodialysis (HD) prevalence. Methods: Data is derived from the cross-sectional arm of the UK multi-centre study investigating barriers and enablers of home HD (BASIC-HHD study). Centres were classified as low- (8%). Sociodemographic and socioeconomic status data were ascertained. Patients were enrolled in hospital HD (n = 213), home HD (n = 93) and predialysis groups (n = 222). Results: The treating renal centre to which the patient belonged was significantly associated with a patient's modality in prevalent HD groups and modality-choice in the “predialysis” group, in confounder-adjusted multivariable analyses. Non-white ethnicity was associated with lower odds of self-care dialysis modality choice (OR 0.21, 95% CI 0.07-0.62) and lower odds of home HD uptake in the prevalent HD group (OR 0.24, 95% CI 0.07-0.80). Other significant associations of home HD uptake in the HD cohort included lower age (OR 0.59, 95% CI 0.39-0.89), higher education (OR 2.99, 95% CI 1.25-7.16), home ownership (OR 0.26, 95% CI 0.09-0.70), childcare responsibility (OR 0.22, 95% CI 0.08-0.66) and unrestricted mobility (OR 0.31, 95% CI 0.11-0.91). Conclusion: “Centre” effect accounts for variation in home HD prevalence between renal units after accounting for sociodemographic parameters and co-morbidities. Unit practices and attitudes to home HD are likely to have a dominating impact on home HD prevalence rates and these aspects need to be explored systematically at the organisational level.

BACKGROUNDAntineutrophil cytoplasmic antibody (ANCA)-associated vasculitis is a severe condition encompassing two major syndromes: granulomatosis with polyangiitis (formerly known as Wegener's granulomatosis) and microscopic polyangiitis. Its cause is unknown, and there is debate about whether it is a single disease entity and what role ANCA plays in its pathogenesis. We investigated its genetic basis.METHODSA genomewide association study was performed in a discovery cohort of 1233 U. K. patients with ANCA-associated vasculitis and 5884 controls and was replicated in 1454 Northern European case patients and 1666 controls. Quality control, population stratification, and statistical analyses were performed according to standard criteria.RESULTSWe found both major-histocompatibility-complex (MHC) and non-MHC associations with ANCA-associated vasculitis and also that granulomatosis with polyangiitis and microscopic polyangiitis were genetically distinct. The strongest genetic associations were with the antigenic specificity of ANCA, not with the clinical syndrome. Anti-proteinase 3 ANCA was associated with HLA-DP and the genes encoding alpha(1)-antitrypsin (SERPINA1) and proteinase 3 (PRTN3) (P = 6.2x10(-89), P = 5.6x10(-12), and P = 2.6x10(-7), respectively). Anti-myeloperoxidase ANCA was associated with HLA-DQ (P = 2.1x10(-8)).CONCLUSIONSThis study confirms that the pathogenesis of ANCA-associated vasculitis has a genetic component, shows genetic distinctions between granulomatosis with polyangiitis and microscopic polyangiitis that are associated with ANCA specificity, and suggests that the response against the autoantigen proteinase 3 is a central pathogenic feature of proteinase 3 ANCA-associated vasculitis. These data provide preliminary support for the concept that proteinase 3 ANCA-associated vasculitis and myeloperoxidase ANCA-associated vasculitis are distinct autoimmune syndromes. (Funded by the British Heart Foundation and others.)

Background Patients with chronic kidney disease (CKD) need regular monitoring, usually by blood urea and creatinine measurements, needing venepuncture, frequent attendances and a healthcare professional, with significant inconvenience. Noninvasive monitoring will potentially simplify and improve monitoring. We tested the potential of transdermal reverse iontophoresis of urea in patients with CKD and healthy controls. Methods Using a MIC 2® Iontophoresis Controller, reverse iontophoresis was applied on the forearm of five healthy subjects (controls) and 18 patients with CKD for 3–5 h. Urea extracted at the cathode was measured and compared with plasma urea. Results Reverse iontophoresis at 250 μA was entirely safe for the duration. Cathodal buffer urea linearly correlated with plasma urea after 2 h (r = 0·82, P < 0·0001), to 3·5 h current application (r = 0·89, P = 0·007). The linear equations y = 0·24x + 1 and y = 0·21x + 4·63 predicted plasma urea (y) from cathodal urea after 2 and 3 h, respectively. Cathodal urea concentration in controls was significantly lower than in patients with CKD after a minimum current application of 2 h (P < 0·0001), with the separation between the two groups becoming more apparent with longer application (P = 0·003). A cathodal urea cut-off of 30 μM gave a sensitivity of 83·3% and positive predictive value of 87% CKD. During haemodialysis, the fall in cathodal urea was able to track that of blood urea. Conclusion Reverse iontophoresis is safe, can potentially discriminate patients with CKD and healthy subjects and is able to track blood urea changes on dialysis. Further development of the technology for routine use can lead to an exciting opportunity for its use in diagnostics and monitoring.

Background: Uremic toxins are typically measured in plasma and little is known of their interstitial concentrations. We undertook experiments to validate a microdialysis technique for simultaneous recovery of small and large uremic toxins in the subcutaneous interstitial fluid (ISF). Methods: Microdialysis catheters were inserted into the subcutaneous interstitium of 8 subjects (controls and uremic patients) and perfused using two different solutions at incremental flow rates to determine analyte recovery and ISF concentrations of urea and protein. Results: 10% dextran-40 perfusate allowed the determination of interstitial concentrations of urea and protein reliably, by virtue of the exponential decay of their concentrations in the microdialysate with incremental flow rates (R2 = 0.63–0.99). Interstitial and plasma urea correlated well (r = 0.95), as did interstitial urea from distant anatomical sites (r = 0.96). Conclusion: Cutaneous microdialysis with dextran-40 allows measurement of small and large molecule concentrations in ISF, creating an opportunity to characterize ISF in uremia.

BACKGROUND: Transplanted metanephroi vascularize and develop features of mature kidney. One group reported the intriguing finding that metanephric allografts and congenic, major histocompatibility complex-mismatched grafts developed without rejection in the absence of immunosuppression. Our experiments aim to investigate the hypothesis that metanephroi lack immunogenicity and identify immunosuppressives that do not inhibit development. METHODS: We transplanted syngeneic metanephric grafts, allografts, and class II mismatched transplants to adult rats along with control grafts to nude recipients. glomerular filtration rates (GFRs) were measured where possible and transplants assessed by histology, immunohistochemistry, electron microscopy, and polymerase chain reaction. RESULTS: Allografts underwent reliable growth and vascularization followed by vigorous rejection (n>200). Rejection was conserved across a class II-mismatched strain and when the earliest dissectable metanephric structures were transplanted. Immunosuppressive drugs other than cyclosporine demonstrated no in vivo toxicity to transplants and treatment with FTY720 and tacrolimus could ablate histologic evidence of allograft rejection. Syngeneic transplants exhibited function of up to 8% of a normal GFR. Renal mass reduction and growth factor treatment was associated with higher GFR than controls. The anatomical site of implantation was also linked strongly with achieved function. CONCLUSIONS: Fetal kidney rudiments can provide a source of functioning renal tissue. These results suggest that such structures are no less immunogenic than mature organs, but the observed rejection is controllable.

Heparanase (HSPE-1) and vascular endothelial growth factor (VEGF), proangiogenic growth factors, play important roles in the metastatic biology of ovarian cancer. The aim of the present study was to test for association between single nucleotide polymorphisms (SNPs) in HSPE-1 and VEGF and outcome in ovarian cancer. A mutational analysis was performed on the coding sequence of the HSPE-1 gene to define high-frequency SNPs. HSPE-1 polymorphisms, together with two SNPs in the VEGF gene, were studied in 136 patients with ovarian cancer. Patients were categorized into two groups, those with FIGO stages 1 and 2 (group 1) and those with stages 3 and 4 (group 2). We identified 10 polymorphisms in the HSPE-1 gene, those in introns 2, 3 and 5b, and exons 8, 13a and 13b occurring at a minor allele frequency of >/=10%. There was an increase in frequency of those individuals with a genotype that carried at least one copy of the intron 2 (C), exon 8 (G), exon 13a (C) haplotype (CGC) in group 2. Specifically there were 24% with this haplotype in group 2 versus 5% in group 1 (P = 0.0184, odds ratio 5.986, 95% confidence interval 1.340-26.752). Most of this association was captured by the intron 2 genotype, where carriage of the C allele was associated with stage (P = 0.0148, odds ratio 6.524, 95% confidence interval 1.401-27.921). There was no association between VEGF SNPs and stage of disease. The CGC HSPE-1 haplotype associates with stage in ovarian cancer. This haplotype may affect splicing of the HSPE-1 gene, as in silico it alters the presence of a splicing motif.

Prolonged recovery time (RT) affects patient's quality-of-life adversely. Shorter dialysis RT in home haemodialysis (HHD) noted in observational studies has been attributed to intensive dialysis regimens. Home patients adopt different haemodialysis (HD) schedules. There is insufficient literature on RT in HHD patients receiving different HD schedules.Data was prospectively collected for BASIC-HHD study, a multicentre study of home and hospital HD across five tertiary centres in the UK. Two hundred and eighty-eight patients answered the question, 'how long does it take for you to recover from a haemodialysis session?' Ninety-one patients (31.8%) of study population performed HHD. Participants completed neuropsychometric tests and depression and anxiety screening inventories.Recovery time is longest amongst 'in-centre' HD recipients (Mean 193 min; SD 295.37) and significantly higher than the mean RT of home HD recipients (Mean 67.3 min; SD 86.8). Within the home setting, RT was not significantly different between the intensive HD and conventional HD (67.8 vs 66.5 min; P 0.05) groups but higher residual urine volumes in the standard home group had significantly shorter RT. Location of HD, not intensity, remained significant (P = 0.001) in the unadjusted and adjusted multivariable analysis. Longer RT was associated with female gender, unemployed or retired 'work' status, 'non-white' ethnicity, lower predialysis systolic blood pressure and greater depression screening score.Home-based HD and higher residual urine volumes are significantly associated with shorter RT. This home advantage for RT may be sustained by preserving residual renal function (in early stages) and increasing HD intensity (in later stages) of dialysis therapy.

Background Extended criteria donor (ECD) and donation after circulatory death (DCD) kidneys are at increased risk of delayed graft function (DGF). Experimental evidence suggests that erythropoietin (EPO) attenuates renal damage in acute kidney injury. This study piloted the administration of high dose recombinant human EPO-beta at implantation of ECD and DCD kidneys, and evaluated biomarkers of kidney injury post-transplant. Methods Forty patients were randomly assigned to receive either rhEPO-b (100,000 iu) (n = 19 in the intervention group, as 1 patient was un-transplantable post randomisation), or placebo (n = 20) in this, double blind, placebo-controlled trial at Manchester Royal Infirmary from August 2007 to June 2009. Participants received either an ECD (n = 17) or DCD (n = 22) kidney. Adverse events, renal function, haematopoietic markers, and rejections were recorded out to 90 days post-transplant. Biomarkers of kidney injury (neutrophil gelatinase-associated lipocalin, Kidney Injury Molecule-1 and IL-18) were measured in blood and urine during the first post-operative week. Results The incidence of DGF (53% vs 55%) (RR = 1.0; CI = 0.5-1.6; p = 0.93) and slow graft function (SGF) (32% vs 25%) (RR = 1.1; CI = 0.5-1.9; p = 0.73) respectively, serum creatinine, eGFR, haemoglobin and haematocrit, blood pressure, and acute rejection were similar in the 2 study arms. High dose rhEPO-b had little effect on the temporal profiles of the biomarkers. Conclusions High dose rhEPO-b appears to be safe and well tolerated in the early post- transplant period in this study, but has little effect on delayed or slow graft function in recipients of kidneys from DCD and ECD donors. Comparing the profiles of biomarkers of kidney injury (NGAL, IL-18 and KIM-1) showed little difference between the rhEPO-b treated and placebo groups. A meta-analysis of five trials yielded an overall estimate of the RR for DGF of 0.89 (CI = 0.73; 1.07), a modest effect favouring EPO but not a significant difference. A definitive trial based on this estimate would require 1000-2500 patients per arm for populations with base DGF rates of 50-30% and 90% power. Such a trial is clearly unfeasible. Trial registration EudraCT Number 2006-005373-22 ISRCTN ISRCTN85447324 registered 19/08/09.

Vascular endothelial growth factor (VEGF) promotes angiogenesis, and elevated levels are found in plaques of psoriasis. Two VEGF polymorphisms, +405 and -460, are associated with early-onset psoriasis and are close to the functional activator protein-1 site (+419) through which retinoids, an established systemic therapy for psoriasis, can block production of VEGF. We report that peripheral blood mononuclear cells (PBMCs) and epidermal keratinocytes (KC) from patients with psoriasis demonstrate differential, genotype-dependent, regulation of VEGF. For PBMCs, VEGF genotype distinguishes two groups of patients with psoriasis - "high and low VEGF producers" (P < 0.001). In contrast, KC production of VEGF is not genotype dependent. However, the effects of all-trans retinoic acid (RA) on cellular expression of VEGF are determined by both cell type and genotype. RA inhibits KC production of VEGF in a genotype-dependent manner (P < 0.005) whereas RA stimulates PBMCs production irrespective of VEGF genotype (P < 0.001). We also report that the -460 VEGF polymorphism appears to have a clinical pharmacogenetic role in predicting response or non-response of psoriasis to acitretin (P = 0.01). In future, determination of VEGF gene polymorphisms and thus individual patient VEGF "signatures" may be used as a prognostic factor for psoriasis susceptibility/severity and as a means for optimizing treatment response.

Single-center experience of encapsulating peritoneal sclerosis inpatients on peritoneal dialysis for end-stage renal failure. Background Encapsulating peritoneal sclerosis (EPS) is a rare but serious complication in patients undergoing continuous ambulatory peritoneal dialysis (CAPD) or automated peritoneal dialysis (APD). It is characterized by a progressive, intra-abdominal, inflammatory process resulting in sheets of fibrous tissue that cover, bind, and constrict the viscera, thereby compromising the motility and function of the bowel. Although recent therapeutic approaches have been reported with variable success, the ability to detect reliably at an early stage patients at risk for EPS would be beneficial and allow treatment standardization. The aim of this study was to evaluate the clinical features of EPS and identify possible risk factors for its development in CAPD and APD patients. Methods This was a review of all cases of EPS in a single center over the last 5 years. Results There were 810 CAPD and APD patients, managed in our program over this period. We identified 27 cases of EPS, giving an overall of 3.3% in this population. The mean duration of CAPD before diagnosis of EPS was 72.6 ± 39.7 months (range 16-172). Sixteen cases required surgical treatment and were classified as severe; others were treated conservatively (mild to moderate group). Ten patients received tamoxifen treatment with apparent benefit. The overall mortality rate was 29.6%. Eight patients from the severe group and the entire moderate group survived on hemodialysis or transplantation at 48.71 and 27.63 months follow-up, respectively. Peritonitis rates were not different between the 2 groups and peritoneal history was unremarkable compared to overall peritonitis rates in the unit. Data on small solute transport were not available in all patients in this retrospective analysis. Conclusion EPS is a serious, life-threatening complication of CAPD. Most cases had PD duration of more than 4 years. Careful monitoring by CT scans of the peritoneal membrane in patients beyond 5 years, and early catheter removal in patients with peritoneal thickening should be considered for long-term CAPD patients. Treatment with tamoxifen may be of benefit in these patients.

Plasmodium falciparum malaria remains a major public health hazard in sub-Saharan African children. While the factors that determine the variations in clinical outcome of a malaria have not been completely defined, both host and parasite factors, as well as the complex molecular interactions between them have been implicated. The cyto-adherent properties of the P. falciparum -infected red blood cells are considered as key properties in the pathogenesis of malaria and the polymorphisms of the host adhesion molecules could contribute to the severity of malaria. Clinical information and blood samples were collected from 223 children from Ibadan (south-west Nigeria), median age of 34.5 months, presenting with different clinical manifestations of malaria—clinically asymptomatic parasitism (ACP), acute uncomplicated malaria (UM) and severe malaria (SM)—as defined by WHO criteria. The polymorphisms of genes coding for four human adhesion molecules at six different loci (ICAM-1 exons 2, 4 and 6, E-selectin exon 2, CD36 exon 10, and PECAM exon 3) were studied. DNA samples were prepared for further genotyping of the six exons mentioned above by PCR-RFLPs using the appropriate restriction digests for each loci. The ICAM-1 exon 4 locus was monomorphic. All the other loci were at Hardy-Weinberg equilibrium (HWE).The E-selectin locus had very low heterozygosity (∼0.06) in contrast to the other loci under study (0.23–0.44). Once the data was further processed for covariates (age and parasite density) and taking as the reference category the ACP group, results show that in the presence of the G allele at the ICAM-1 exon 6 there is an increased risk (3.6 times) of severe malaria. As far as the T allele in the E-selectin exon is concerned, the number of sampled DNAs with the T allele within both the UM and SM categories is too low for drawing any relevant conclusion at this stage. In conclusion, these results suggest that genetic polymorphisms at host adhesion molecules loci are an important variable in the susceptibility to severe malaria. Further studies of host loci are needed to further delineate which polymorphisms are associated with severe malaria and increase our knowledge of the biology of host-parasite interactions.

Background. Changes in renal vasculature, with vascular and interstitial fibrosis, are hallmarks of progression to chronic kidney disease (CKD) stage 5. Vascular endothelial growth factor (VEGF) is a potent angiogenic and vascular permeability factor. Transforming growth factor-b1 (TGF-b1) plays a critical role in promoting extracellular matrix (ECM) deposition and fibrosis. This study investigates whether genetic polymorphisms of VEGF or TGF-b1 are associated with (i) progressive decline in renal function in patients with glomerular disorders (cohort 1) and (ii) predisposition to CKD stage 5 in a separate group of renal transplant recipients with various primary diseases (cohort 2). Methods. Two patient groups were studied. Cohort 1 comprised 91 patients with biopsy-proven glomerular disease who were followed-up for 5 years before categorization as either non-progressors (with stable serum creatinine or � 30% increase over 5 years, n ¼ 39) or progressors (requiring dialysis, transplantation or whose serum creatinine increased by >30% over 5 years, n ¼ 52). Cohort 2 comprised 107 patients with various primary renal diseases, who had reached CKD stage 5 and undergone renal transplantation at the time of study. All patients were genotyped for the VEGF polymorphisms at positions � 460 (C/T) and þ405 (G/C). Linkage disequilibrium (LD) was established using EHplus. SNPHAP was used to estimate haplotype frequency and to infer haplotypes to all patients. Cohort 1 patients were genotyped for the TGF-b1 polymorphisms at positions � 800, � 509, codons 10 and 25. Genotyping was performed by polymerase chain reaction-restriction length polymorphism (PCR-RFLP). Results. In cohort 1, there was a significant increase in frequency of the � 460 VEGF CC genotype 30.8 vs 5.1%, P ¼ 0.008; odds ratio (OR), CC vs TT 10.67, 95% confidence interval (CI), 1.94–58.72 and C allele 56.7 vs 37.2%, P ¼ 0.009; OR 2.22, 95% CI, 1.21–4.04, in the progressor patients when compared with the non-progressors. In cohort 2, there was a significant increase in the VEGF � 460 CC genotype when compared with healthy volunteers 37 vs 20.8%, P ¼ 0.011; OR CC vs TT 1.59, 95% CI, 0.72–3.51. The � 460 and þ405 polymorphisms were in LD P

BackgroundHaemodialysis (HD) patients are predisposed to dysregulated fluid balance leading to extracellular water (ECW) expansion. Fluid overload has been closely linked with outcome in these patients. This has mainly been attributed to cardiac volume overload, but the relation between abnormalities in fluid status with micro-and macrovascular dysfunction has not been studied in detail. We studied the interaction of macro-and microvascular factors in states of normal and over-hydration in HD-dependent CKD.MethodsFluid compartments [total body water (TBW) and ECW] and overhydration index (OH) were measured with Multifrequency bio-impedance (BCM). Overhydration was defined as OH/ECW>7%. Overhydration was also assessed using the ECW/TBW ratio. Macrocirculation was assessed by pulse-wave velocity (PWV) and mean arterial pressure (MAP) measurements while microcirculation through sublingual capillaroscopy assessment of the Perfused Boundary Region of the endothelial glycocalyx (PBR 5-25mcg). A panel of pro-inflammatory and vascular serum biomarkers and growth factors was analysed.ResultsOf 72 HD participants, 30 were in normohydration (N) range and 42 overhydrated according to the OH/ECW ratio. Average ECW/TBW was 0.48 +/- 0.03. Overhydrated patients had higher MAP (122.9 +/- 22.5 v 111.7 +/- 22.2mmHg, p = 0.04) and comorbidities (median Davies score 1.5 v 1.0, p = 0.03). PWV (p = 0.25) and PBR 5-25mcg (p = 0.97) did not differ between the 2 groups. However, Vascular Adhesion Molecule (VCAM)-1, Interleukin-6 and Thrombomodulin, and reduced Leptin were observed in the overhydrated group. Elevation in VCAM-1 levels (OR 1.03; 95% CI 1.01-1.06; p = 0.02) showed a strong independent association with OH/ECW>7% in an adjusted logistic regression analysis and exhibited a strong linear relationship with ECW/TBW (Bata = 0.210, p = 0.03) in an also adjusted model.ConclusionExtracellular fluid overload is significantly linked to microinflammation and markers of endothelial dysfunction. The study provides novel insight in the cardiovascular risk profile associated with overhydration in uraemia.

Dysregulated vascular endothelial growth factor (VEGF) expression has been implicated as a major contributor to the development of a number of common disease pathologies. The aim of this study was to establish the extent of genetic variability within the VEGF gene and to determine whether this genetic variation influenced levels of VEGF protein expression. The promoter region and exon 1 of the VEGF gene were screened for polymorphisms using single-stranded conformation (SSCP) polymorphism analysis and direct PCR-sequencing. We identified 15 novel sequence polymorphisms most of which were rare. Eleven of these polymorphisms were single base substitutions, three were single base insertions and one was a two base deletion. Thirteen of the polymorphisms were located within the promoter and two in the 5′ untranslated region (5′UTR) of the gene. We established PCR-RFLP typing systems for ten of the polymorphisms. For the two common polymorphisms at −460 and +405, we developed a combined sequence specific priming (SSP) PCR typing system to determine the cis / trans orientation of each allele and hence, ascertain haplotypes. A significant correlation was observed between lipopolysaccharide (LPS) stimulated peripheral blood mononuclear cell (PBMC) VEGF protein production and genotype for the +405 polymorphism.

Circulating vascular endothelial growth factor is not increased during relapses of steroid-sensitive nephrotic syndrome. Background An uncharacterized circulating factor that increases vascular permeability has previously been described in childhood steroid-sensitive nephrotic syndrome (SSNS). The aim of this study was to determine whether this factor is vascular endothelial growth factor (VEGF), the recently described endothelial cell mitogen and enhancer of vascular permeability. Methods Plasma and urine VEGF levels were measured in children with SSNS in both relapse and remission and in normal age- and sex-matched controls. Semiquantitative reverse transcriptase-polymerase chain reaction studies investigating VEGF mRNA expression were performed on peripheral blood mononuclear cells isolated from children with SSNS in relapse and controls. In two experimental models (one-hour and three-day follow-up postinfusion), Sprague-Dawley rats were intravenously administered 50 μg rVEGF to determine whether this induced either proteinuria or glomerular histologic change. Results Plasma VEGF levels and urine VEGF/creatinine ratios were not elevated in SSNS relapse compared with remission and control samples. Peripheral blood mononuclear cell VEGF mRNA expression was no different in SSNS patients compared with controls. The administration of VEGF to rats induced an acute reversible fall in systemic blood pressure but did not result in the development of either proteinuria or glomerular histologic change. Conclusion Increased circulating VEGF levels are not responsible for the proteinuria observed during relapses of SSNS. Further studies are warranted to investigate intrarenal VEGF expression.

The kidneys and the interstitial compartment play a vital role in body fluid regulation. The latter may be significantly altered in renal dysfunction, but experimental studies are lacking. To help define this we measured the subcutaneous interstitial pressure, bioimpedance volumes, and edema characteristics in 10 healthy subjects and 21 patients with obvious edema and chronic kidney disease (CKD). Interstitial edema was quantified by the time taken for a medial malleolar thumb pit to refill and termed the edema refill time. Interstitial pressure was significantly raised in CKD compared to healthy subjects. Total body water (TBW), extracellular fluid volume (ECFV), interstitial fluid volume, the ratio of the ECFV to the TBW, and segmental extracellular fluid volume were raised in CKD. The ratio of the ECFV to the TBW and the interstitial fluid volume were the best predictors of interstitial pressure. Significantly higher interstitial pressures were noted in edema of 2 weeks or less duration. A significant nonlinear relationship defined interstitial pressure and interstitial fluid volume. Edema refill time was significantly inversely related to interstitial pressure, interstitial compartment volumes, and edema vintage. Elevated interstitial pressure in CKD with obvious edema is a combined function of accumulated interstitial compartment fluid volumes, edema vintage, and tissue mechanical properties. The edema refill time may represent an important parameter in the clinical assessment of edema, providing additional information about interstitial pathophysiology in patients with CKD and fluid retention.

Contains fulltext : 97321.pdf (Publisher’s version ) (Open Access) BACKGROUND: Idiopathic membranous nephropathy is a major cause of the nephrotic syndrome in adults, but its etiologic basis is not fully understood. We investigated the genetic basis of biopsy-proven cases of idiopathic membranous nephropathy in a white population. METHODS: We performed independent genomewide association studies of single-nucleotide polymorphisms (SNPs) in patients with idiopathic membranous nephropathy from three populations of white ancestry (75 French, 146 Dutch, and 335 British patients). The patients were compared with racially matched control subjects; population stratification and quality controls were carried out according to standard criteria. Associations were calculated by means of a chi-square basic allele test; the threshold for significance was adjusted for multiple comparisons (with the Bonferroni method). RESULTS: In a joint analysis of data from the 556 patients studied (398 men), we identified significant alleles at two genomic loci associated with idiopathic membranous nephropathy. Chromosome 2q24 contains the gene encoding M-type phospholipase A(2) receptor (PLA(2)R1) (SNP rs4664308, P=8.6x10(-29)), previously shown to be the target of an autoimmune response. Chromosome 6p21 contains the gene encoding HLA complex class II HLA-DQ alpha chain 1 (HLA-DQA1) (SNP rs2187668, P=8.0x10(-93)). The association with HLA-DQA1 was significant in all three populations (P=1.8x10(-9), P=5.6x10(-27), and P=5.2x10(-36) in the French, Dutch, and British groups, respectively). The odds ratio for idiopathic membranous nephropathy with homozygosity for both risk alleles was 78.5 (95% confidence interval, 34.6 to 178.2). CONCLUSIONS: An HLA-DQA1 allele on chromosome 6p21 is most closely associated with idiopathic membranous nephropathy in persons of white ancestry. This allele may facilitate an autoimmune response against targets such as variants of PLA2R1. Our findings suggest a basis for understanding this disease and illuminate how adaptive immunity is regulated by HLA.

To determine whether single nucleotide polymorphisms (SNPs) in the vascular endothelial growth factor (VEGF) gene are associated with severity of diabetic retinopathy.A case-control study was conducted in which 45 individuals with type 1 or 2 diabetes with proliferative diabetic retinopathy (PDR) and 61 individuals with type 1 or 2 diabetes without retinopathy (DWR) were genotyped for 14 SNPs in the VEGF promoter and gene.Three of the promoter SNP genotypes, -160C, -152A (rs13207351), and -116A (rs1570360), showed significant independent associations with PDR, as well as the minihaplotype CAA (P = 0.00017). Two promoter haplotypes were associated with severity of retinopathy: -460C, -417T, -172C, -165C, -160C, -152A, -141A, -116A, +405C was associated with PDR (OR [95% CI] = 29.92 [3.91, 228.78], P = 1.62 x 10(-5)) and -460C, -2417T, -172C, -165C, -160C, -152A, -141A, -116G, +405G was associated with DWR (OR = 0.05 [0.01, 0.35], P = 0.000373). Furthermore, two haplotype-tagged (ht) SNPs, +4618 (rs735286) and +5092 (rs2146323), and five htSNP haplotypes were associated with severity of retinopathy. When the nine promoter/5' untranslated region [UTR] and five htSNP genotypes were combined into a 14-SNP haplotype, a single haplotype, -460C, -417T, -172C, -165C, -160C, -152A, -141A, -116A, +405C, +674T, +4618C, +5092A, +9162C, +9512C was found to be significantly associated with the PDR group (OR = 18.45 [2.35, 144.67], P = 0.00622).A clear association was demonstrated between VEGF SNPs and severity of diabetic retinopathy. Furthermore, two of the htSNP haplotypes appear to be more generalized markers for angiogenesis, in that these have been found in prior work to be associated with neovascular age-related macular degeneration.

To invade the decidua and myometrium, extravillous trophoblast must degrade an assortment of extracellular matrix (ECM) components. The uterine wall is rich in heparan sulphate proteoglycans (HSPG), which interact with collagen, laminin and fibronectin, and bind a variety of growth factors. HSPG are catabolised by heparanase, an enzyme that is highly expressed in the placenta. The aim of this study was to investigate the role of heparanase in first trimester trophoblast invasion. First trimester cytotrophoblasts (CTB) were isolated by trypsin digestion followed by centrifugation on a Percoll gradient. Cells were cultured on Matrigel to promote an extravillous phenotype. Heparanase expression was studied by immunohistochemistry and confocal microscopy. Trophoblast invasion was assessed using an in vitro transwell assay. A high level of heparanase was observed in isolated first trimester trophoblast; however, a function-blocking antibody did not inhibit invasion of primary CTB or the extravillous trophoblast cell line SGHPL-4 at 21% oxygen. In contrast to cancer cells, heparanase expression was not increased following culture at 3% oxygen, and trophoblast invasion was not retarded by the blocking antibody under these conditions. Heparanase expression was observed in stromal cells and vascular endothelium in first trimester parietal decidua. Expression was evident on the cell surface and in the nucleus of trophoblast and decidual cells. In conclusion, trophoblast heparanase is not required for invasion in vitro. Its abundant expression suggests another role during pregnancy, perhaps in controlling the availability of ECM-bound growth factors or acting as a transcription factor. © 2008 Elsevier Ltd. All rights reserved.

BACKGROUND: Transplantation of embryonic kidneys (metanephroi) offers a potential solution to the problem of kidney donor shortage. The aim of this study was to characterise the haemodynamic capacity of transplanted rat metanephroi and to determine the number and maturity of the tubules. METHODS: Metanephroi from E15 Lewis rat embryos were transplanted adjacent to the abdominal aorta of uninephrectomised adult female syngeneic Lewis rats. Twenty-one days later, a single metanephros ureter was anastomosed to the host's urinary system. Three months later animals were prepared for standard clearance measurements. RESULTS: Effective renal blood flow (149 +/- 33 microl min(-1) per g kidney weight) and glomerular filtration rate (17 +/- 9 microl min(-1) per g kidney weight), standardised to kidney weight, were significantly lower in transplanted metanephroi compared with control adult kidneys (P < 0.001); renal vascular resistance (934 +/- 209 mmHg ml min(-1) per g kidney weight) was significantly higher (P < 0.001). Nephron number in transplanted metanephroi was significantly greater than that of E21 kidneys (P < 0.01) but lower than that of postnatal day (PND) 1 kidneys (P < 0.001). Angiotensin II type 2 receptor mRNA expression, a marker of nephrogenesis, was markedly reduced in metanephroi. Aquaporins 1 and 2, epithelial Na channel and Na-K-2Cl cotransporter type 2 mRNA and protein were expressed in transplanted metanephroi; the urea transporters-A1, 2 and 3 were absent. Vascular markers (alpha-smooth muscle actin and CD31) were identified in metanephroi but their expression did not differ from that of E21 and PND 1 kidneys. CONCLUSIONS: This study shows that metanephroi continue to develop post-transplantation but only reach a stage of development equivalent to that of a normal rat kidney at birth.

Renal parenchymal scarring (RPS) following urinary tract infection (UTI) is an important cause of renal morbidity in children. Studies have shown that the intensity of the inflammatory response following infection is related to the risk of RPS. However, genetic variability in this response has not been studied. Adhesion molecules play a crucial role in leucocyte recruitment following infection, and polymorphisms have been reported in the genes for key cell adhesion molecules. We have investigated the possibility that children who develop RPS following UTI may exhibit altered genotype or allele frequencies for polymorphisms of the intercellular adhesion molecule-1 (ICAM-1) (exons 4 and 6), E-selectin (exons 2 and 4), platelet endothelial cell adhesion molecule-1 (PECAM-1) (exon 3) and CD11b (3'UTR) genes, which may predict outcome of UTI. DNA was isolated from 99 children shown to have developed RPS, 43 children with no evidence of scarring (NS) following UTI and 170 healthy controls. Genotyping was performed by restriction fragment length polymorphism (RFLP) analysis. When the RPS group was compared with the NS group, there was a significant reduction in the frequency of the ICAM-1 exon 4 A allele (10.6 vs. 21.3%, respectively, chi2 = 6.01, P = 0.014). There was no significant difference in either allele or genotype frequency for any of the other polymorphisms studied. These data suggest that the A allele of the ICAM-1 exon 4 polymorphism may protect against the risk of RPS following UTI and may participate in the regulation of the inflammatory response following UTI.

A novel approach to circumventing the shortage in transplantable donor organs is the use of embryonic primordia that develop inside the host. Previously published work has shown that transplantation of rat fetal kidney primordia (metanephroi) onto the omentum of adult rat hosts results in growth and development of the metanephroi into functioning kidney units capable of providing a measurable renal function. However, for anatomical and physiological reasons the omentum may not provide the ideal site for transplantation and may limit the maximum renal function that the transplants can achieve. We postulate that it may be possible to increase the renal function of the transplants by transplantation to sites with increased blood flow. To test this we transplanted rat embryonic day 15 metanephroi into the retroperitoneal fat adjacent to major blood vessels in the peritoneum of unilaterally nephrectomized rats; 21 days later the transplants were examined and suitable transplants connected to the host urinary system. Approximately 130 days later the glomerular filtration rate of the connected transplants was analyzed. Our results show that transplantation of metanephroi to the regions highlighted in this study results in an increased presence of urinary cysts, suggesting increased early renal function in the transplants compared to metanephroi transplanted onto the omentum, but most importantly we show that we can increase the renal function of the transplants to a level comparable with other renal therapies such as dialysis. This work suggests life-sustaining renal function could be achieved through transplantation of renal primordia.

Heparanase activity is dysregulated in children with steroid-sensitive nephrotic syndrome.BackgroundImmune cells express heparanase, an endoglycosidase, able to degrade heparan sulfate glycosaminoglycan (HSGAG) in the glomerular capillary wall (GCW) and potentially induce proteinuria. The aim of this study was to determine whether dysregulated heparanase expression is associated with the heavy proteinuria of childhood steroid-sensitive nephrotic syndrome (SSNS).MethodsPlasma and urinary heparanase activity and peripheral blood mononuclear cell (PBMC) mRNA heparanase levels [real-time polymerase chain reaction (PCR)] were measured in children with SSNS in relapse and remission. Plasma and urinary heparanase activity was determined in adult patients with nephrotic syndrome and in age- and gender-matched controls.ResultsPlasma heparanase activity was reduced in SSNS with relapse (811.2 units) compared to remission (1147.96 units) (P = 0.003) and control subjects (1390.51 units) (P < 0.001). In adult nephrotic syndrome, plasma heparanase activity was significantly lower in patients compared to controls. However, there was no difference between remission and relapse states. In children, urinary heparanase activity/urinary creatinine ratio was highest in SSNS relapse (14.26 units/mg) compared with remission (7.43 units/mg) (P = 0.016) and controls (2.29) (P < 0.001). However, PBMC heparanase mRNA expression was not different between these three groups. In adult nephrotic syndrome, urinary heparanase activity/urinary creatinine levels were lower in both remission and relapse compared to controls and there was no difference between remission and relapse states.ConclusionIn childhood SSNS, there is a qualitative and quantitative difference in urinary heparanase activity expression that is not paralleled in adult nephrotic syndrom. These data suggest that dysregulated heparanase expression may play a significant role in the pathogenesis of SSNS, possibly through an abnormality in post-translational control of latent heparanase activation.

Diabetic retinopathy and nephropathy cause significant morbidity in patients with diabetes. Vascular endothelial growth factor (VEGF) is a potent angiogenic and vascular permeability factor and is implicated in both of these diabetes complications. We previously reported transfection studies showing the VEGF −460 and VEGF +405 polymorphisms to increase basal VEGF promoter activity by 71% compared with the wild-type sequence. Therefore, we investigated the association of these VEGF polymorphisms with proliferative diabetic retinopathy and diabetic nephropathy. DNA was isolated from 267 U.K. Caucasians with diabetes, comprising 69 patients with proliferative retinopathy and 198 patients with other grades of retinopathy. The distribution of VEGF −460 genotype was significantly different between the proliferative retinopathy and nonproliferative retinopathy groups (P = 0.027); specifically, carriage of the VEGF −460C allele was associated with proliferative diabetic retinopathy (odds ratio 2.5 [95% CI 1.20–5.23]). The VEGF −460 genotype was predictive of retinopathy, even after controlling for blood pressure, glycemic control, duration of diabetes, and obesity (P = 0.02). The VEGF +405 genotype did not associate with proliferative retinopathy, and neither polymorphism was associated with diabetic nephropathy. The VEGF −460C polymorphism is a positive independent predictive factor for the development of proliferative diabetic retinopathy. Increased VEGF production from high-expressing haplotypes, including −460C, may promote neovascularization.

BACKGROUND Evidence from animal models supports the hypothesis that dysregulated transforming growth factor beta(1) (TGF beta(1)) expression plays a role in chronic allograft rejection, the progression of diabetic nephropathy and fibrotic glomerulopathies. However, more evidence is required to support this hypothesis in man, and the current literature concerning blood TGF beta(1) levels in clinical studies is highly confused. We have investigated: (i) the hypothesis that the widespread practice of activating clinical samples prior to measurement of TGF beta(1) is detecting the platelet-released pool of TGF beta(1), artefactually generated on venepuncture and unrepresentative of the real circulating in vivo TGF beta(1) pool; and (ii) the effect of different immunosuppressive drugs on apparent TGF beta(1) plasma levels. METHODS The effect of two different venepuncture procedures on plasma TGF beta(1) was compared in 10 healthy volunteers, one procedure designed to minimize platelet activation and the other representing standard venepuncture practice in a clinic situation. Blood samples from 52 renal transplant recipients on either cyclosporine or tacrolimus immunosuppression were taken by standard venepuncture to investigate the effect of immunosuppressive drugs on plasma TGF beta(1). Plasma TGF beta(1) and beta thromboglobulin were measured by ELISA. RESULTS Among 10 healthy volunteers who underwent two different methods of venepuncture, eight of 10 had undetectable levels of TGF beta(1) (

It is accepted that antithymocyte globulin (ATG) preparations vary in their bioactivity and side effects. However, this is poorly documented in the literature. We compared the clinical course and cytokine response of heart transplant patients who had received either Merieux or Stanford ATG preparations. The serum cytokine response (interleukin [IL]-6, tumor necrosis factor [TNF]-alpha, IL-4, and IL-10) of 28 consecutive heart transplant recipients was measured for 14 days after surgery using ELISAs. The effect of various ATG preparations on cytokine stimulation of whole blood in vitro was also evaluated. There was a much greater in vivo IL-6 and TNF-alpha response to Merieux than to Stanford ATG (P < 0.0005). There was little IL-4 or IL-10 response with either preparation. No side effects could be attributed to either treatment. No significant difference was seen in the frequency of rejection at 30, 90, or 365 days. More infection episodes occurred in the group treated with Stanford ATG at 30 days (0.5 compared with 0.2 episodes/patient; P = 0.097), 90 days (1.2 compared with 0.5 episodes/patient; P = 0.17), and 365 days (2.8 compared with 1.8; P = 0.59), although none of these differences were statistically significant. When tested in vitro for cytokine stimulation, the in vivo pattern was confirmed, with Merieux ATG producing greater levels of TNF-alpha and IL-6 than Stanford ATG. The differences in cytokine stimulation may be reflected in different immunosuppressive activities. Further research to elucidate the important components of immunosuppressive activity while excluding potentially detrimental effects is important.

Urinary C3dg and C5b-9 indicate active immune disease in human membranous nephropathy. We have measured complement activation markers, C3dg and C5b-9 in plasma and urine from patients with idiopathic membranous nephropathy and IgA nephropathy. There was no significant difference in levels of plasma C5b-9 between the patient groups. However, high plasma concentrations of C3dg were associated significantly with IgA nephropathy with 45% of patients having levels over 25 U/ml (P < 0.001). High concentrations of urinary C3dg and C5b-9 were associated significantly with membranous nephropathy (43% and 43% of the patient group, respectively) compared to patients with IgA nephropathy (10% and 0%, respectively, P < 0.001). In a retrospective analysis of 31 patients with membranous nephropathy, 66% of patients with high initial urinary C5b-9 showed an unstable clinical course compared to 18% of patients with initially absent or low C5b-9 (P < 0.001). We suggest that high urinary C5b-9 identifies those patients with a membranous lesion which retains an active immunological component contributing to the pathology of progressive glomerular damage.

Sequential study of the IgA system in relapsing IgA nephropathy.Cellular and immunochemical parameters of the IgA system were studied in 15 subjects with IgA nephropathy (IgAN) and 15 age–matched controls. In IgAN remission no abnormalities of the IgA system were detectable by the methods used. In IgAN relapse, [macroscopic hematuria associated with upper respiratory tract infection (URTI) (N = 6)] there were rises in IgA-bearing B-lymphocytes (three of six), T helper/suppressor cell ratio (six of six) and pokeweed mitogen–induced IgA production (four of six). Total serum and salivary IgA were unchanged. Serum IgA profile (HPLC-ELISA) showed increases in polymer IgA (three of six). No such changes were found during URTI in controls. These findings support the view that an exaggerated IgA response to mucosal antigen challenge initiates glomerular damage and hematuria in IgAN.

Role of TGF-β1 in renal parenchymal scarring following childhood urinary tract infection. Background Significant variability exists in the outcome of renal parenchymal inflammation following urinary tract infection (UTI) in childhood as some children experience renal parenchymal scarring (RPS) while others do not scar. Since TGF-β1 is pro-fibrotic, we examined the role of this cytokine in RPS following UTI. Methods Five polymorphisms of the TGF-β1 gene were investigated as well as the relationship between these polymorphisms and TGF-β1 production by peripheral blood mononuclear cells (PBMC) in vitro. DNA was isolated from 91 children shown to have developed RPS, 43 children with no evidence of scarring (NS) following UTI, and 171 healthy controls. Genotyping was performed by restriction fragment length polymorphism (RFLP). PBMC were isolated from a subgroup of 24 patients from the total population. Cells were stimulated with LPS + PMA + PHA and then TGF-β1 production was determined by ELISA. Results Comparing the NS with the RPS group, there was an increase in the -800GA genotypes (18.6 vs. 7.4%, P = 0.05; χ 2 ) and the Leu 10 →Pro CT (62.8 vs. 41.5%, P = 0.021), and a decrease in the -509 TT genotype (0.0 vs. 8.5%, P = 0.049). PBMC TGF-β1 production was higher in those patients with the -800 GG compared to those with a GA genotype stimulation index [stimulated/unstimulated TGF-β1 levels were 1.54 interquartile range (IQR) 1.42 to 1.75 vs. 1.19, IQR 0.94 to 1.51, P = 0.031]. Conclusions There is an association between the TGF-β1 -800GA, -509 TT and Leu 10 →Pro CT genotypes and the presence or absence of RPS. The low TGF-β1 producer status of the -800GA genotype may protect against the development of a pro-fibrotic pathology.

A group of 108 male workers previously exposed to bladder carcinogens and a group of 26 patients with carcinoma of the bladder differed significantly from a control group comprising 63 healthy male donors with respect to their levels of tissue polypeptide antigen (TPA). The risk group exhibited 4-6 times more frequent elevations of TPA than did the group of healthy donors.

The IgG response to tetanus toxoid (TT) immunization was quantitated by by radioimmunoassay in patients with membranous nephropathy (MN) and healthy controls. Variation in subclass (ELISA) and electrical charge (isoelectric focussing, immunofixation & autoradiography) of the IgG response were also assessed. Total IgG and igG subclass responses were impaired in MN compared to controls, although this was only significant for IgG-3 (P less than 0 X 05). Non responders to TT were more common in MN, and response was independent of disease activity. No distinctive pattern of IgG subclass response or IgG spectrotype was seen in MN. Impaired response to TT was associated with HLA-DR3 among controls, and in MN (88 X 8% of whom were DR3) markedly depressed responses occurred in apparent DR3 homozygotes.

Background Encapsulating peritoneal sclerosis (EPS) is a rare but serious complication of peritoneal dialysis (PD). Gastrointestinal (GI) symptoms affect appetite and dietary intake. Adequate nutrition is especially important if surgical interventions are required. Aim To investigate the nutritional management of 23 EPS patients that underwent surgical intervention between 1999 and 2005 at Manchester Royal Infirmary, United Kingdom. Methods EPS was recognized by GI symptoms and diagnostically confirmed by laparotomy, computed tomographic scanning, or biopsy. Results Mean time on PD was 74 months (interquartile range 42 – 89 months). During the 12 months pre-diagnosis, 65% of the group showed significant weight loss ( p = 0.0001), with 8 patients losing >10% of body weight; 74% of patients experienced significant albumin decrease ( p = 0.001); and 56% of patients experienced GI symptoms during the 6 months pre-diagnosis. Nasogastric (NG) feeding was recommended for 8 patients but continued in only 1. 15 patients (mean albumin 27 g/L) commenced parenteral nutrition (PN); 9 patients recovered, with albumin increasing over the 6-month follow-up. Mean hospital time was 62 days for the group receiving neither NG nor PN, compared with 124.3 for the PN/NG group ( p = 0.04). In patients that died of EPS, albumin continued to fall at 3 months post-diagnosis. Conclusion There is currently little guidance for nutritional management of EPS. From this study we recommend ( 1 ) a high level of clinical suspicion for EPS, especially if PD patients have weight loss; ( 2 ) PN may be better than NG feeding but further studies into dual enteral nutrition and PN are needed; ( 3 ) aggressive nutritional supplementation pre- and postoperatively; and ( 4 ) dietitians need to recognize the high risk of refeeding syndrome.