Torsten Slowinski

Endothelin-1 transgenic mice develop glomerulosclerosis, interstitial fibrosis, and renal cysts but not hypertension.

The human endothelin-1 (ET-1) gene under the control of its natural promoter was transferred into the germline of mice. The transgene was expressed predominantly in the brain, lung, and kidney. Transgene expression was associated with a pathological phenotype manifested by signs such as age-dependent development of renal cysts, interstitial fibrosis of the kidneys, and glomerulosclerosis leading to a progressive decrease in glomerular filtration rate. This pathology developed in spite of only slightly elevated plasma and tissue ET-1 concentrations. Blood pressure was not affected even after the development of an impaired glomerular filtration rate. Therefore, these transgenic lines provide a new blood pressure-independent animal model of ET-1-induced renal pathology leading to renal fibrosis and fatal kidney disease.

Effective elimination of dabigatran by haemodialysis. A phase I single-centre study in patients with end-stage renal disease.

Dabigatran, a specific, reversible direct thrombin inhibitor, is used to prevent ischaemic and haemorrhagic strokes in patients with atrial fibrillation. As with every anticoagulant, there is a need to rapidly reverse its effects in emergency situations. In an open-label, single-centre phase I study with two fixed multiple dosing periods, we investigated the pharmacokinetics, pharmacodynamics and safety of dabigatran before, during and after 4 hour haemodialysis sessions with either 200 or 400 ml/min targeted blood flow in seven end-stage renal disease patients without atrial fibrillation. Dabigatran was administered over three days in a regimen designed to achieve peak plasma concentrations comparable to those observed in atrial fibrillation patients receiving 150 mg b.i.d. and to attain adequate distribution of dabigatran in the central and peripheral compartments. Plasma concentration-time profiles were similar in both periods on Day 3 (Cmax: 176 and 159 ng/ml). Four hours of haemodialysis removed 48.8% and 59.3% of total dabigatran from the central compartment with 200 and 400 ml/minute targeted blood flow, respectively. The anticoagulant activity of dabigatran was linearly related to its plasma levels. There was a minor redistribution of dabigatran (<16%) after the end of the haemodialysis session. In conclusion, a 4 hour haemodialysis session can rapidly eliminate a substantial amount of dabigatran from the central compartment with a concomitant marked reduction in its anticoagulant activity. There was a clinically negligible redistribution of dabigatran after haemodialysis. These results demonstrate that haemodialysis can be a suitable approach to eliminate dabigatran in emergency situations.

Involvement of functional autoantibodies against vascular receptors in systemic sclerosis

Background Systemic sclerosis (SSc) features autoimmunity, vasculopathy and tissue fibrosis. The renin-angiotensin and endothelin systems have been implicated in vasculopathy and fibrosis. A role for autoantibody-mediated receptor stimulation is hypothesised, linking three major pathophysiological features consistent with SSc. Methods Serum samples from 478 patients with SSc (298 in the study cohort and 180 from two further independent cohorts), 372 healthy subjects and 311 control-disease subjects were tested for antibodies against angiotensin II type 1 receptor (AT1R) and endothelin-1 type A receptor (ETAR) by solid phase assay. Binding specificities were tested by immunoprecipitation. The biological effects of autoantibodies in microvascular endothelial cells in vitro were also determined, as well as the quantitative differences in autoantibody levels on specific organ involvements and their predictive value for SSc-related mortality. Results Anti-AT1R and anti-ETAR autoantibodies were detected in most patients with SSc. Autoantibodies specifically bound to respective receptors on endothelial cells. Higher levels of both autoantibodies were associated with more severe disease manifestations and predicted SSc-related mortality. Both autoantibodies exert biological effects as they induced extracellular signal-regulated kinase 1/2 phosphorylation and increased transforming growth factor β gene expression in endothelial cells which could be blocked with specific receptor antagonists. Conclusions Functional autoimmunity directed at AT1R and ETAR is common in patients with SSc. AT1R and ETAR autoantibodies could contribute to disease pathogenesis and may serve as biomarkers for risk assessment of disease progression.

Significant Differentiation of Focal Breast Lesions: Calculation of Strain Ratio in Breast Sonoelastography

RATIONALE AND OBJECTIVES Initial data suggest that elastography can improve the specificity of ultrasound in differentiating benign and malignant breast lesions. The aim of this study was to compare elastography and B-mode ultrasound to determine whether the calculation of strain ratios (SRs) can further improve the differentiation of focal breast lesions. MATERIALS AND METHODS A total of 227 women with histologically proven focal breast lesions (113 benign, 114 malignant) were included at two German breast centers. The women underwent a standardized ultrasound procedure using a high-end ultrasound system with a 9-MHz broadband linear transducer. B-mode scans and sonoelastograms were analyzed by two experienced readers using the Breast Imaging Reporting and Data System criteria. SRs were calculated from a tumor-adjusted region of interest (mean color pixel density) and a comparable region of interest placed in the lateral fatty tissue. Sensitivity, specificity, and cutoff values were calculated for SRs (receiver-operating characteristic analysis). RESULTS The women had a mean age of 54 years (range, 19-87 years). The mean lesion diameter was 1.6 +/- 0.9 cm. Sensitivity and specificity were 96% and 56% for B-mode scanning, 81% and 89% for elastography, and 90% and 89% for SRs. An SR cutoff value of 2.45 (area under the curve, 0.949) allowed significant differentiation (P < .001) of malignant (mean, 5.1 +/- 4.2) and benign (mean, 1.6 +/- 1.0) lesions. The quantitative method of SR calculation was superior to subjective interpretation of sonoelastograms and B-mode scans, with a positive predictive value of 89% compared to 68% and 84% for the other two methods. CONCLUSIONS Calculation of SRs contributes to the standardization of sonoelastography with high sensitivity and allows significant differentiation of benign and malignant breast lesions with higher specificity compared to B-mode ultrasound but not elastography.

Variable expression of P-glycoprotein in the human placenta and its association with mutations of the multidrug resistance 1 gene (MDR1, ABCB1)

The MDR1 gene product P-glycoprotein in the human placenta is important for protecting the fetus from unintended, harmful drug exposure, but also for limiting the access of therapeutic drugs to the fetus after maternal drug intake. A polymorphism in exon 26 of the MDR1 gene (C3435T) has previously been shown to be associated with reduced P-glycoprotein expression in the small intestine, kidney and lymphocytes. In the present study, we examined systematically whether MDR1 polymorphisms also have an impact on P-glycoprotein expression in the human placenta. MDR1 mRNA and P-glycoprotein were analysed in 73 full-term human placentas of Caucasians, as well as respective MDR1 genotypes/haplotypes, for the C3435T and G2677T/A polymorphisms of mothers and infants. MDR1 mRNA levels were not different between these genotype groups. However, P-glycoprotein expression was significantly lower when both mother and infant were homozygous for the 3435T allele (TT/tt) compared to maternal and fetal homozygotes for the C-allele (0.40 +/- 0.18 a.u. for TT/tt versus 0.66 +/- 0.30 a.u. for CC/cc, P = 0.01). Moreover, placentas from mothers carrying both polymorphisms (3435T and 2677T; TT/TT) also had a significantly lower P-glycoprotein expression (0.31 +/- 0.12 a.u.) compared to placentas of wild-type individuals (CC/GG, 0.71 +/- 0.31 a.u., P = 0.02). Taken together, the MDR1 polymorphisms C3435T and G2677T are associated with altered P-glycoprotein expression in the human placenta, and may have clinical consequences due to genetically determined, variable drug exposure of the fetus.

A safe citrate anticoagulation protocol with variable treatment efficacy and excellent control of the acid-base status.

Objective:Citrate anticoagulation is an excellent alternative to heparin anticoagulation for critically ill patients requiring continuous renal replacement therapy. In this article, we provide a safe and an easy-to-handle citrate anticoagulation protocol with variable treatment doses and excellent control of the acid–base status. Design:Prospective observational study. Setting:University hospital. Patients:One hundred sixty-two patients with acute renal failure requiring renal replacement therapy were enrolled in the study. Intervention:A continuous venovenous hemodialysis-based citrate anticoagulation protocol using a 4% trisodium solution, a specially designed dialysate fluid, and a continuous calcium infusion were used. The study period was 6 days. Hemofilters were changed routinely after 72 hours of treatment. The patients were grouped according to body weight, with patients below 60 kg body weight in group 1, patients with at least 60 kg and up to 90 kg body weight in group 2, and patients with a body weight of above 90 kg in group 3. Dialysate flow was adapted according to body size and matched approximately 2 L/hr for a patient with average body size. Blood flow, citrate flow, and calcium flow were adjusted according to the dialysate flow used. Measurements and Main Results:Median filter run time was 61.5 hours (interquartile range: 34.5–81.1 hours). Only 5% of all hemofilters had to be changed because of clotting. The prescribed treatment dose was achieved in all patients. Acid–base and electrolyte control were excellent in all groups. In the rare cases of metabolic disarrangement during citrate anticoagulation, acid–base values were rapidly corrected by modifying either the dialysate flow or alternatively the blood flow rate. Eight patients (5%) developed signs of citrate accumulation indicated by an increase of the total calcium >3 mmol/L or a need for high calcium substitution. Conclusions:We provide a safe and an easy-to-handle citrate anticoagulation protocol that allows an excellent acid–base and electrolyte control in critically ill patients with acute renal failure. The protocol can be adapted to patients’ need, allowing a wide spectrum of treatment doses.

Multicenter study of ultrasound real-time tissue elastography in 779 cases for the assessment of breast lesions: improved diagnostic performance by combining the BI-RADS®-US classification system with sonoelastography.

PURPOSE Hitachi real-time tissue elastography (HI-RTE) is an ultrasound technique that facilitates the estimation of tissue elasticity. Our study evaluates whether sonoelastography improves the differentiation of benign and malignant breast lesions. MATERIALS AND METHODS In a multicenter approach sonoelastography of focal breast lesions was carried out in 779 patients with subsequent histological confirmation. We present data from 3 study centers (Berlin, Bielefeld, Homburg/Saar) focusing on the sensitivity (SE), specificity (SP) and the positive (PPV) and negative predictive value (NPV) of sonoelastography. In addition we performed an analysis of the diagnostic performance, expressed by the pretest and posttest probability of disease (POD), in BI-RADS®-US 3 or 4 lesions as these categories can imply both malignant and benign lesions and a more precise prediction would be a preferable aim. RESULTS Sonoelastography demonstrated an improved SP (89.5 %) and an excellent PPV (86.8 %) compared to B-mode ultrasound (76.1 % and 77.2 %). Especially in dense breasts ACR III-IV, the SP was even higher (92.8 %). In BI-RADS-US 3 lesions, a suspicious elastogram significantly modified the POD from 8.3 % to a posttest POD of 45.5 %. In BI-RADS-US 4 lesions, we found a pretest POD of 56.6 %. The posttest POD changed significantly to 24.2 % with a normal elastogram and to 81.5 % with a suspicious elastogram. CONCLUSION Our data demonstrates that the complementary use of sonoelastography definitely improves the performance in breast diagnostics. Finally we present a protocol of how sonoelastography can be integrated into our daily practice.

Association of maternal G protein β3 subunit 825T allele with low birthweight

Weight at birth has been associated with an increased risk for cardiovascular disease and type 2 diabetes in adult life. We found an association between the maternal G protein beta3 subunit 825T allele and low birthweight in babies born to women without other risks for reduced fetal growth.

Erythropoietin-induced excessive erythrocytosis activates the tissue endothelin system in mice.

The endothelium controls blood flow and pressure by releasing several vasoactive factors, among them the vasodilator nitric oxide (NO) and the potent vasoconstrictor endothelin‐1 (ET‐1). Although increased NO levels have been found in excessive erythrocytosis, little is known concerning ET‐1 expression in this condition. Thus, we examined the endothelin system in transgenic mice that due to constitutive overexpression of erythropoietin (Epo) reached hematocrit levels of ~80%. Surprisingly, despite generalized vasodilatation, polycythemic mice exhibited a two‐ to fivefold elevation in ET‐1 mRNA levels in aorta, liver, heart, and kidney. In line with this, increased expression of ET‐1 protein was detected in the pulmonary artery by immunohistochemical analysis. Compared with their wild‐type littermates, aortic rings of Epo transgenic animals exhibited a marked reduction in vascular reactivity to ET‐1 and big ET‐1, but this effect was abrogated upon preincubation with the NO synthase inhibitor N‐nitro‐l‐arginine methyl ester (L‐NAME). Pretreatment of polycythemic mice with the ETA receptor antagonist darusentan for 3 wk significantly prolonged their survival upon acute exposure to L‐NAME. Taken together, these results demonstrate for the first time that excessive erythrocytosis induces a marked activation of the tissue endothelin system that results in increased mortality upon blockade of NO‐mediated vasodilatation. Because ETA antagonism prolonged survival after acute blockade of NO synthesis, endothelin may be regarded as a contributor to the adverse cardiovascular effects of erythrocytosis and may thus represent a new target in the treatment of cardiovascular disease associated with erythrocytosis.

Loss of insulin-induced activation of TRPM6 magnesium channels results in impaired glucose tolerance during pregnancy

Hypomagnesemia affects insulin resistance and is a risk factor for diabetes mellitus type 2 (DM2) and gestational diabetes mellitus (GDM). Two single nucleotide polymorphisms (SNPs) in the epithelial magnesium channel TRPM6 (V1393I, K1584E) were predicted to confer susceptibility for DM2. Here, we show using patch clamp analysis and total internal reflection fluorescence microscopy, that insulin stimulates TRPM6 activity via a phosphoinositide 3-kinase and Rac1-mediated elevation of cell surface expression of TRPM6. Interestingly, insulin failed to activate the genetic variants TRPM6(V1393I) and TRPM6(K1584E), which is likely due to the inability of the insulin signaling pathway to phosphorylate TRPM6(T1391) and TRPM6(S1583). Moreover, by measuring total glycosylated hemoglobin (TGH) in 997 pregnant women as a measure of glucose control, we demonstrate that TRPM6(V1393I) and TRPM6(K1584E) are associated with higher TGH and confer a higher likelihood of developing GDM. The impaired response of TRPM6(V1393I) and TRPM6(K1584E) to insulin represents a unique molecular pathway leading to GDM where the defect is located in TRPM6.