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BACKGROUND AND AIMS Anti-neutrophil-cytoplasmatic-antibody-associated vasculitis (AAV) is a life-threatening autoimmune disease. It is a necrotizing vasculitis of small- and medium-sized vessels. T-cells play a pivotal role in pathogenesis and mediating damage in the vessels. Exogen ATP (adenosine triphosphate) is normally in a low nanomolar range. Under inflammatory conditions, various cells can release ATP to the extracellular department. In this case, ATP is a DAMP (damage-associated molecular pattern), but its effect on disease mechanisms in AAV is not well understood. It is the aim of the study to determine the effect of exogenous ATP on T-cells in healthy controls and AAV-patients. METHOD A total of 22 AAV-patients in remission and 13 healthy controls were recruited. Peripheral blood mononuclear cells (PBMC) were isolated and then labeled with a tracking dye. The PBMC were cultured for 3 days with different concentrations of ATP in presence or absence of anti-CD3/CD28 stimulation. After 3 days, the cells were restimulated with PMA/Ionomycin and Brefeldin A for 4 hours. Cytokine production and cell proliferation were assessed by flow cytometry. Baseline was defined as T-cell proliferation in absence of exogenous ATP. The fold change was calculated by: $\frac{{{\rm{T}} - {\rm{cell\ proliferation\ with\ ATP}}}}{{{\rm{T}} - {\rm{cell\ proliferation\ without\ ATP}}}}$ . The P-value for paired values was calculated by the Wilcoxon matched-pairs signed rank test. The P-value for unpaired groups was calculated by the Mann Whitney U-test. RESULTS The mean T-cell proliferation of CD4+-T-cells for healthy controls was significantly different comparing 0 µM ATP and 500 µM ATP (mean proliferated fraction, 0 µM ATP versus 500 µM ATP: 40.48% versu 22.58%, P = .0002). In presence of ATP, a suppression of T-cell proliferation was found in patients and healthy controls (patients versus healthy controls, fold change T-cell proliferation from baseline with 50 µM ATP: 0.81 ± 0.03 versus 0.79 ± 0.03, P = .60; 125 µM ATP: 0.75 ± 0.03 versus 0.70 ± 0.04, P = .38; 250 µM ATP: 0.68 ± 0.03 versus 0.59 ± 0.05, P = .17; 500 µM ATP: 0.53 ± 0.03 versus 0.54 ± 0.05, P = .77); the higher the concentration of ATP, the stronger the suppression of proliferation. IFNγ+-CD4+-T-cells of healthy controls were also suppressed by exogenous ATP (0 µM ATP versus 500 µM ATP, CD4+ T-cells: %IFNγ+: 19.94% versus 10.59%, P = .0002). INFγ production by CD4+-T-cells was reduced after addition of ATP in both patients and healthy controls (patients versus healthy controls, fold change CD4+-IFNγ+ -T-cells from baseline with 50 µM ATP: 0.69 ± 0.04 versus 0.64 ± 0.04, P = 0.45; 125 µM ATP: 0.62 ± 0.04 versus 0.58 ± 0.04, P = .63; 250 µM ATP: 0.62 ± 0.04 versus 0.49 ± 0.04, P = .11; 500 µM ATP: 0.59 ± 0.05 versus 0.51 ± 0.05, P = .56). CONCLUSION Exogenous ATP induced suppression of T-cells in healthy controls and might be important for the maintenance of immunotolerance. DAMP-mediated inhibitory mechanisms were functional in AAV-patients in remission.

Background and Aims Arterial hypertension is one of the most common diseases of the cardiovascular system worldwide and is still the cause of most deaths in Germany. Data on interactions of the endothelin-system with the renin-angiotensin- and the sympathoadrenergic system in the regulation of systemic hemodynamics in humans are lacking. In our present investigation we study the effects of Endothelin A-, Alpha1- and Angiotensin II-type-1-receptor antagonization on the systemic pressor effects of intravenous Endothelin-1-application in young, healthy men. In addition, we analyzed the effects of the genetic variations of the GNB3 C825T-polymorphism on hemodynamic changes. GNB3 825CT/TT-allele-carriers are considered to have a higher risk for multiple diseases with structural, vascular degeneration, such as arterial hypertension, diabetes mellitus and obesity. Method 21 healthy male volunteers were included in this double- blind, randomized, placebo-controlled cross-over study and were studied on four days. Endothelin-1 (ET-1) (0.5, 1, 2.5, 5 ng/kg/min for 20 min each) was given intravenous 2.0 hours after oral application of either placebo or Doxazosin, 3.5 hours after oral application of Candesartan (Candesartan 8 mg) or in the presence of a continuous infusion of the ET-A-selective antagonist BQ123 (60 μg/min). Blood pressure (BP) and heart rate (HR) were recorded and total peripheral resistance (TPR) was measured using impedance cardiography. ET-1-dose-response curves were analyzed with ANOVA. Data are presented as mean ± SD. Since we suspected an effect of the GNB3 C825T-polymorphism we divided the overall collective into 2 sub-collectives according to the GNB3 C825T-genotypes (n = 21, GNB3 825CC: n = 10, GNB3 825CT/TT: n = 11). Our analyses considered the overall collective and compared the sub-collectives intraday and interday. Results ET-1 increased systolic blood pressure (SBD) (p ≤ 0,01), diastolic blood pressure (DBD) and mean arterial pressure (MBP) as well as total peripheral resistance (TPR) (each p ≤ 0,001) with decreasing heart rate (HR5) (p ≤ 0,05). Elevation of blood pressure existed in both sub-collectives (GNB3 825CC: SBD & MBD: p ≤ 0,01, DBP & TPR: p ≤ 0,05, GNB3 825CT/TT: DBD, MBD & TPR: p ≤ 0,01, SBP p ≤ 0,05). Antagonization of ETA-receptors reversed the effect in the overall collective as well as in the sub-collectives. Both, Doxazosin, as well as Candesartan led to a decrease in blood pressure, however, dose-response relationship was influenced more by doxazosin (DBD: p ≤ 0,001, MBD: p ≤ 0,01) than by candesartan (all values: p > 0,05). For both drugs, blood pressure and TPR remained elevated under maximum ET-1-application compared to baseline measurement. Blood pressure dependent heart rate changes were observed in the overall collective and in GNB3 825CC-allele-carriers under sole ET-1-therapy (p ≤ 0.05) (Fig. 1). Candesartan reversed the effect of ET-1 on the sub-collectives (p > 0.05). GNB3 825CT/TT-allele-carriers showed no reduction in heart rate under ET-1-application, but with accompanying candesartan therapy (p ≤ 0.01) (Fig. 2). The genotype collectives thus behaved oppositely to the drugs in this respect. Conclusion In summary, ET-1 increased systolic, diastolic and mean arterial blood pressure as well as systemic vascular resistance. Doxazosin, Candesartan and BQ123 led to a decrease in blood pressure. Blood pressure and TPR remained elevated under maximum ET-1 application plus Candesartan or Doxazosin. The heart rate changes of the genotype-separated sub-collectives were opposite when ET-1 was administered compared to ET-1 and Candesartan.

Background and Aims Anti-neutrophil-cytoplasmic-antibody (ANCA)-associated-vasculitis (AAV) is an autoimmune small-vessel-vasculitis. T-cells play a pivotal role in pathogenesis as drivers of autoantibody formation and vasculitic damage. However, the involvement of T-cells in renal vasculitis is understood poorly. IL-17C is secreted by epithelial parenchymal cells enhancing the pathogenicity of Th17 cells. Furthermore, IL-17C is a chemotactic factor for Th17 cells. The aim of this study was to assess the role of Th17 cells and IL-17C in the pathogenesis of ANCA-vasculitis in an animal model of AAV. Method Wistar-Kyoto-rats were immunized with myeloperoxidase (MPO) in Freunds Adjuvant to induce AAV. Control rats were immunized with Freunds Adjuvant only. Albuminuria was determined weekly and rats were culled after two, four and six weeks. At the time of harvest, renal T-cells were isolated and characterized by flow cytometry. Antigen-specificity was determined by ELiSPOT. Petechial bleedings on the lung surface reflecting pulmonary vasculitis were quantified after harvest. Gene expression in spleen, kidneys and lungs was studied by PCR and is expressed as fold change over controls. IL-17C levels were measured in sera by ELISA. Results MPO-rats developed detectable titres of anti-MPO by week two. By week six, all MPO-animals developed renal vasculitis with significant albuminuria and pulmonary vasculitis. Accordingly, MPO animals showed significant crescent formation as compared to the controls (% of affected glomeruli: 11.4 ±10.5% vs. 0.4 ±0.7%, p Conclusion Th17 and Th22 cells are drivers of renal inflammation in ANCA-vasculitis. The role of IL-17C in this cascade needs to be determined.

Background and Aims B cell activating factor (BAFF) is a cytokine which drives B cell survival and maturation. Several studies have shown that elevated BAFF levels in renal transplant patients are associated with increased risk for the development of donor specific antibodies and antibody mediated rejection. It was the aim of this study to investigate neutrophils as cellular source of BAFF in renal transplant patients. Method Neutrophils (NT) were freshly isolated from whole blood of healthy controls (HC) and renal transplant patients (RTX). After isolation, purity of neutrophils was usually above 98%. Neutrophils were stimulated with LPS or TNFα in presence of Granulocyte-colony stimulating factor (GCSF) or Granulocyte macrophage colony-stimulating factor (GMCSF). In selected conditions, FK506 or rapamycin was added. Supernatants were harvested after 20 hours of culture and BAFF levels were determined by ELISA. Results GCSF/TNFα and GCSF/LPS were the most potent stimuli leading to BAFF secretion of NT in HC (403 ±64 pg/ml and 421 ±69 pg/ml). NT derived RTX showed similar capacity to secrete BAFF as compared to HC (GCSF/TNFα: 515 ±31 pg/ml and GCSF/LPS: 539.4 ±36 pg/ml). Treatment of cultures with rapamycin reduced BAFF levels (515 ±100 pg/ml vs. 348 ±27 pg/ml, p Conclusion NT may enhance of B cell maturation and survival via BAFF. BAFF-secretion by NT can be suppressed with mTOR inhibitors. In renal transplantation, NT might promote formation of allo-antibodies and drive antibody mediated rejection.

Background The C5 complement inhibitor eculizumab is a first-line treatment in atypical haemolytic uraemic syndrome (aHUS). Therapy with eculizumab is associated with a highly increased risk for meningococcal infection. Therefore, vaccination is highly recommended before beginning treatment. Efficacy of quadrivalent meningococcal vaccines (MenACWY) in patients treated with the C5 complement inhibitor eculizumab in aHUS has not yet been determined. Methods Patients with aHUS received one dose of a MenACWY conjugate vaccine before eculizumab treatment commenced. Bactericidal titres against meningococcal serogroups A, C, W and Y were determined using baby rabbit complement in 25 patients. Results Full immune response to meningococcal vaccination was detected in five patients (20%), while seven patients (28%) showed no immune response in any of the tested serogroups. The remaining 13 patients showed incomplete immune response with proof of protective antibody titres for one to three serogroups without perceptible preference for any serogroup. Bactericidal titres after re-vaccination were available for 17 patients. Nine patients with incomplete immune response after first vaccinations showed protective antibody titres for all serogroups after re-vaccination. Kidney function had improved in >50% of patients at the time of re-vaccination compared with the time of first vaccination and immunosuppressive therapy was only applied to re-vaccinated patients following kidney transplantation. Conclusions Immunogenicity of first quadrivalent meninongococcal vaccination is insufficient in patients with aHUS. Booster response is promising, but incomplete. Therefore, establishing antibiotic prophylaxes seems pivotal.

Background: Serum creatinine (SCR) and blood urea nitrogen (BUN) determine the glomerular filtration rate (GFR) improperly in acute renal failure. Serum cystatin C (CYS) has the potential to be a more precise marker for GFR. The aim of this study was to compare the sensitivity of SCR, BUN and CYS with respect to the detection of acute renal failure in mice. Methods: In an ischaemia reperfusion (I/R) injury model, mice suffered 60-min left kidney ischaemia and right nephrectomy. In a nephrectomy model, mice were nephrectomized to a different extent: from unilateral (3/6Nx) to bilateral nephrectomy (BiNx). Blood samples were collected 2, 12 or 24 h post-op. Results:SCR,BUNandCYSincreasedsignificantlyinthe I/R-model in comparison to sham mice and 3/6Nx mice at 12 and 24 h post-op (SCR P = 0.009; BUN P < 0.001 and CYS P < 0.004). There were no significant differences in all three markers between 3/6Nx and sham-operated mice. In graded nephrectomy, BUN and CYS showed already significantly the loss of kidney in 4/6Nx mice 12 h postop [BUN (mg/dl): sham 26.4 ± 3.5, 4/6Nx 52.3 ± 13.4, P < 0.01; CYS (mg/l): sham 0.08 ± 0.03, 4/6Nx 0.15 ± 0.04, P < 0.01], whereas SCR was only significantly increasedin5/6NxandBiNxmice24hpost-op[SCR(mg/dl): sham 0.39 ± 0.05, 4/6Nx 0.52 ± 0.07, P = 0.13, 5/6Nx 1.00 ± 0.29, P < 0.01]. In the longitudinal experiment, CYS showed the renal damage significantly earlier and to a larger extent (2 h: SCR 57 ± 15%, BUN 40 ± 16%, CYS 295 ± 143%, P < 0.001). Conclusions: CYS can be used as a reliable and precise marker for renal function in mouse models. CYS is more sensitive than SCR, and it shows renal damage earlier than SCR and BUN.

BACKGROUND Proximal tubules subjected to hypoxia in vitro under conditions relevant to ischaemia in vivo develop an energetic deficit that is not corrected even after full reoxygenation. We have provided evidence that accumulation of nonesterified fatty acids (NEFA) is the primary reason for this energetic deficit. In this study, we have further investigated the mechanism for the NEFA-induced energetic deficit. METHODS Mitochondrial membrane potential (Deltapsi) was measured in digitonin-permeabilized, freshly isolated proximal tubules by safranin O uptake. Addition of the potassium/proton exchanger nigericin enables the determination of the mitochondrial proton motive force (Deltap) and the proton gradient (DeltapH). ATP was measured luminometrically and NEFA colorimetrically. RESULTS Tubule ATP content was depleted after hypoxia and recovered incompletely, even after full reoxygenation. Mitochondrial safranin O uptake was decreased in proximal tubules after hypoxia and reoxygenation (H/R). This decrease was attenuated by delipidated bovine serum albumin (dBSA) or citrate. Addition of nigericin increased safranin O uptake of mitochondria in normoxic proximal tubules, but not in proximal tubules after H/R. Addition of dBSA restored the effect of nigericin to increase mitochondrial safranin O uptake. Addition of the NEFA oleate had the same impact on mitochondrial safranin O uptake as subjecting proximal tubules to H/R. CONCLUSION The mechanism of the NEFA-induced energetic deficit in freshly isolated rat proximal tubules induced by H/R is characterized by impaired ATP production after full reoxygenation, impaired recovery of Deltapsi and Deltap, abrogation of DeltapH and sensitivity to citrate, consistent with involvement of the tricarboxylate carrier. The data support the concept that protonophoric uncoupling by NEFA movement on anion carriers plays a critical role in proximal tubule mitochochondrial dysfunction after H/R.

Background. An increased CD4⁺ CD25⁺ T-cell population is observed in Wegener's granulomatosis (WG). This T-cell population is not well characterized yet and their contribution to the disease pathogenesis remains obscure. Methods. Thirty patients with WG and 18 healthy controls (HC) were included in this study. The disease activity and extension were measured by the Birmingham Vasculitis Activity Score (BVAS) and the Disease Extent Index (DEI). Lymphocytes from peripheral blood were analysed by FACS for the expression of CD4, CD25, CD134 and GITR. Cytokine expression in these subsets was assessed too. Nasal, lung and renal tissues from WG patients were immunohistochemically stained for CD3 and CD134. Results. The percentage of CD134⁺ as well as GITR⁺ expressing CD4 ⁺CD25⁺ lymphocytes was increased in patients as compared to HC (37 ± 12% versus 27 ± 8%, P = 0.005; 18 ± 9% versus 11 ± 6%, P = 0.003). The expression of CD134 and GITR showed a significant correlation with disease activity (r = 0.5, P = 0.009; r = 0.55, P = 0.001). Most of these displayed the phenotype of effector memory T-cells (94 ± 4% and 91 ± 6%). CD134 T-cells were found in tissues affected by WG. Conclusions. CD4⁺CD25⁺ effector memory T-cells expressing CD134 and GITR seem to play a role in disease mechanisms, as suggested by their close association with disease activity and their participation in inflammatory process. © The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

Background. Acute myocardial infarction (AMI) has been reported and is associated with poor outcome in the course of thrombotic microangiopathies (TMA). However, data are very limited in regard to the clinical characteristics, risk factors and outcome of AMI during TMA. Furthermore, current AMI definitions based on troponins are more sensitive and specific to detect myocardial injury. Methods. We retrospectively analysed 74 consecutive patients with 78 TMA episodes. TMA was defined as platelets below 150 � 10 9 /l, haemolytic anaemia, elevated lactate dehydrogenase (LDH) and increased red cell fragmentation, and AMI as serum troponin I above 1 ng/ml with symptoms of myocardial ischaemia and/or appropriate electrocardiography (ECG) alterations. Results. AMI occurred in 14 TMA episodes (18%) (9 non- and 5 ST-segment elevation AMI). AMI occurred 5±3 days after TMA diagnosis, predominately in clinically suspected thrombotic thrombocytopenic purpura (TTP) as TMA subtype. Independent risk factors for subsequent AMI were TTP (RR 2.2; 95% CI 1.1–5.6), and serum LDH above 1000 U/l (RR 2.7; 95% CI 1.3–7.2) as well as serum troponin I above 0.20 ng/ml at TMA presentation (RR 13.5; 95% CI 2.6–86.8). LDH above 1000 U/l together with troponin I above 0.20 ng/ml had a sensitivity of 86% (95% CI 60–96%) and a specificity of 95% (95% CI 86–98%) to predict AMI in the later course of TMA. AMI contributed substantially to morbidity causing left ventricular dysfunction in three of eight survivors and potentially accounted for the death in five of six non-survivors. Conclusions. AMI is an early, frequent and severe complication during TMA. AMI occurs especially in TTP, and serum LDH above 1000 U/l in combination with serum troponin I above 0.20 ng/ml at TMA presentation are excellent predictors of subsequent AMI.

Patients were subdivided into two groups: those with good and those with impaired graft functions. Patients Background. Persistent hyperparathyroidism after with good graft function had stable serum creatinine renal transplantation (Rtx) has been reported in several levels (132 mmol/ lo r1.5 mg/dl ) well below the studies. However these studies evaluated biochemical mean serum creatinine concentration during the study bone parameters either only during a short time period period. The significant changes in AP, BAP, and OC (up to 6 months) or for a longer time period, but with occurred irrespective of renal function. However, long intervals in between. Therefore, we prospectively patients with impaired graft function (n=65) had evaluated biochemical bone parameters of kidneysignificantly higher PTH-levels (70 pg/ml higher) than transplant recipients at short intervals for 2 years patients with good graft function (n=64), P