Dunja Rogić

Circulating Bone Morphogenetic Protein 1–3 Isoform Increases Renal Fibrosis

Bone morphogenetic proteins (BMPs) participate in organ regeneration through autocrine and paracrine actions, but the existence and effects of these proteins in the systemic circulation is unknown. Using liquid chromatography-mass spectrometry, we identified BMP6, GDF15, and the BMP1-3 isoform of the Bmp1 gene in plasma samples from healthy volunteers and patients with CKD. We isolated the endogenous BMP1-3 protein and demonstrated that it circulates as an active enzyme, evidenced by its ability to cleave dentin matrix protein-1 in vitro. In rats with CKD, administration of recombinant BMP1-3 increased renal fibrosis and reduced survival. In contrast, administration of a BMP1-3-neutralizing antibody reduced renal fibrosis, preserved renal function, and increased survival. In addition, treating with the neutralizing antibody was associated with low plasma levels of TGFβ1 and connective tissue growth factor. In HEK293 cells and remnant kidneys, BMP1-3 increased the transcription of collagen type I, TGFβ1, β-catenin, and BMP7 via a BMP- and Wnt-independent mechanism that involved signaling through an integrin β1 subunit. The profibrotic effect of BMP1-3 may, in part, be a result of the accompanied decrease in decorin (DCN) expression. Taken together, inhibition of circulating BMP1-3 reduces renal fibrosis, suggesting that this pathway may be a therapeutic target for CKD.

Do Guidelines for the Diagnosis and Monitoring of Diabetes Mellitus Fulfill the Criteria of Evidence-Based Guideline Development?

BACKGROUND Although the methodological quality of therapeutic guidelines (GLs) has been criticized, little is known regarding the quality of GLs that make diagnostic recommendations. Therefore, we assessed the methodological quality of GLs providing diagnostic recommendations for managing diabetes mellitus (DM) and explored several reasons for differences in quality across these GLs. METHODS After systematic searches of published and electronic resources dated between 1999 and 2007, 26 DM GLs, published in English, were selected and scored for methodological quality using the AGREE Instrument. Subgroup analyses were performed based on the source, scope, length, origin, and date and type of publication of GLs. Using a checklist, we collected laboratory-specific items within GLs thought to be important for interpretation of test results. RESULTS The 26 diagnostic GLs had significant shortcomings in methodological quality according to the AGREE criteria. GLs from agencies that had clear procedures for GL development, were longer than 50 pages, or were published in electronic databases were of higher quality. Diagnostic GLs contained more preanalytical or analytical information than combined (i.e., diagnostic and therapeutic) recommendations, but the overall quality was not significantly different. The quality of GLs did not show much improvement over the time period investigated. CONCLUSIONS The methodological shortcomings of diagnostic GLs in DM raise questions regarding the validity of recommendations in these documents that may affect their implementation in practice. Our results suggest the need for standardization of GL terminology and for higher-quality, systematically developed recommendations based on explicit guideline development and reporting standards in laboratory medicine.

The EC4 European Syllabus for Post-Graduate Training in Clinical Chemistry and Laboratory Medicine: version 4 - 2012

Abstract Laboratory medicine’s practitioners across the European community include medical, scientific and pharmacy trained specialists whose contributions to health and healthcare is in the application of diagnostic tests for screening and early detection of disease, differential diagnosis, monitoring, management and treatment of patients, and their prognostic assessment. In submitting a revised common syllabus for post-graduate education and training across the 27 member states an expectation is set for harmonised, high quality, safe practice. In this regard an extended ‘Core knowledge, skills and competencies’ division embracing all laboratory medicine disciplines is described. For the first time the syllabus identifies the competencies required to meet clinical leadership demands for defining, directing and assuring the efficiency and effectiveness of laboratory services as well as expectations in translating knowledge and skills into ability to practice. In a ‘Specialist knowledge’ division, the expectations from the individual disciplines of Clinical Chemistry/Immunology, Haematology/Blood Transfusion, Microbiology/ Virology, Genetics and In Vitro Fertilisation are described. Beyond providing a common platform of knowledge, skills and competency, the syllabus supports the aims of the European Commission in providing safeguards to increasing professional mobility across European borders at a time when demand for highly qualified professionals is increasing and the labour force is declining. It continues to act as a guide for the formulation of national programmes supplemented by the needs of individual country priorities.

Interpretation and management of INR results: A case history based survey in 13 countries

INTRODUCTION Standardisation of treatment with vitamin K antagonists (VKAs) is still an issue after 60 years of use. The study aimed to explore aspects of VKA monitoring in primary and secondary care. METHODS Two case histories were distributed to physicians in 13 countries. Case history A focused on a patient with atrial fibrillation on stable anticoagulation (latest INR 2.3). Physicians were asked about frequency of INR measurement, when to change the VKA dose, and the patients annual risk of ischemic stroke and bleeding. Case history B focused on a patient with an unexpected INR of 4.8, asking for the patients 48-hour bleeding risk, the immediate dose reduction and time until a repeat INR. RESULTS Altogether, 3016 physicians responded (response rate 8 - 38%), of which 82% were from primary care and 18% from secondary care. Answers varied substantially within and between countries regardless of level of care and VKA used. Median number of weeks between INR measurements was 4 - 6 weeks. Median threshold INR for increasing or decreasing the VKA dose was 1.9 and 3.1, respectively. Risk of ischemic stroke and bleeding were overestimated 2 - 3 times. In case history B, the median dose reduction the two first days was 75% for GPs and 55% for specialists, irrespective of estimates of bleeding risk; with one week to a repeat INR. CONCLUSION Variation in VKA monitoring is substantial implying clinical consequences. Guidelines seem either unknown or may be considered impracticable. Further efforts towards standardisation of VKA management are needed.

Carbendazim impends hepatic necrosis when combined with imazalil or cypermethrin.

Imazalil, cypermethrin and carbendazim are detected in plants for human nutrition. To explore whether their combinations, applied orally in low doses, would induce changes in metabolic patterns and hepatotoxicity, a subchronic in vivo experiment was conducted. Doses of 10mg/kg of imazalil (im) and cypermethrin (cy) and 20 mg/kg of carbendazim (car) and their combinations (im, 10 mg/kg+cy, 10mg/kg; im, 10mg/kg+car, 20mg/kg; car, 20 mg/kg + im, 10 mg/kg) were given to Swiss mice daily over 28 days. After 24 hr from the last dose, the relationships of cytotoxicity biomarkers were analysed: serum lactate dehydrogenase, aspartate transaminase, alanine transferase, amylase, alkaline phosphatase, creatine kinase, creatinine and total proteins. Individual pesticides showed different toxic potential (cy>im car) generally characterized by increase in enzyme activities. Histological analysis showed that cypermethrin, but not imazalil or carbendazim, alone can cause mild necrosis. Combinations generally caused decrease in the activity of enzymes, indicating liver damage. Low doses of carbendazim in combination with low doses of imazalil or cypermethrin caused very pronounced hepatic necrosis, more than any of the three individually applied pesticides or combination of imazalil and cypermethrin. In fruits and vegetables for human consumption, residues of these three pesticides and prolonged combined intake of low doses, which by themselves acutely would not cause any effect, may have similar hepatotoxic effects.

Constitutively Elevated Blood Serotonin Is Associated with Bone Loss and Type 2 Diabetes in Rats

Reduced peripheral serotonin (5HT) in mice lacking tryptophan hydroxylase (TPH1), the rate limiting enzyme for 5HT synthesis, was reported to be anabolic to the skeleton. However, in other studies TPH1 deletion either had no bone effect or an age dependent inhibition of osteoclastic bone resorption. The role of 5HT in bone therefore remains poorly understood. To address this issue, we used selective breeding to create rat sublines with constitutively high (high-5HT) and low (low-5HT) platelet 5HT level (PSL) and platelet 5HT uptake (PSU). High-5HT rats had decreased bone volume due to increased bone turnover characterized by increased bone formation and mineral apposition rate, increased osteoclast number and serum C-telopeptide level. Daily oral administration of the TPH1 inhibitor (LX1032) for 6 weeks reduced PSL and increased the trabecular bone volume and trabecular number of the spine and femur in high-5HT rats. High-5HT animals also developed a type 2 diabetes (T2D) phenotype with increased: plasma insulin, glucose, hemoglobin A1c, body weight, visceral fat, β-cell pancreatic islets size, serum cholesterol, and decreased muscle strength. Serum calcium accretion mediated by parathyroid hormone slightly increased, whereas treatment with 1,25(OH)2D3 decreased PSL. Insulin reduction was paralleled by a drop in PSL in high-5HT rats. In vitro, insulin and 5HT synergistically up-regulated osteoblast differentiation isolated from high-5HT rats, whereas TPH1 inhibition decreased the number of bone marrow-derived osteoclasts. These results suggest that constitutively elevated PSL is associated with bone loss and T2D via a homeostatic interplay between the peripheral 5HT, bone and insulin.

An international study of how laboratories handle and evaluate patient samples after detecting an unexpected APTT prolongation

Abstract Background: An unexpectedly detected prolonged activated partial thromboplastin time (APTT) can be a harmless laboratory finding, but can also reflect a thrombotic tendency or a bleeding disorder. The assistance of laboratory professionals in the interpretation of an unexpectedly detected prolonged APTT (uAPTT) is often required. The way in which uAPTTs are evaluated in laboratories was assessed in this international study with the aim of determining whether laboratory professionals are able to fulfill this need. Methods: Postanalytical practices after uAPTT were investigated and the mixing study methodology (if used) was studied by circulating a case report with a questionnaire to staff in the invited laboratories. In addition, the interpretations of those staff regarding the presence or absence of inhibitors in three APTT mixing study scenarios were examined. Results: Large within- and between-country variations were detected in both postanalytical practices and mixing study methodologies among the 990 responding laboratories, 90% of which were in 13 countries. Shortcomings regarding the investigation of uAPTTs leading to potentially incorrect or delayed clinical diagnoses were found in 88% of the laboratories. Of the laboratories to which the interpretative questions were sent, 49% interpreted all mixing study scenarios correctly. uAPTTs were investigated appropriately and all mixing study scenarios interpreted correctly in parallel in only 9.6% of the participating laboratories. Conclusions: The clinical requirement for the assistance of laboratory professionals in the interpretation of uAPTTs cannot be met at most of the participating laboratories. Laboratory professionals should be trained in the evaluation of ordinary laboratory tests, such as that for uAPTTs.

Evaluation of the Konelab 20XT clinical chemistry analyzer.

Abstract The Konelab 20XT (Thermo Electron Oy, Finland) is a clinical chemistry analyzer for colorimetric, immunoturbidimetric and ion-selective electrode measurements. The aim of our work was to evaluate the analytical performances of the Konelab 20XT according to the European Clinical Chemistry Laboratory Standards Guidelines. A total of 30 analytes including substrates, enzymes, electrolytes and specific proteins were tested. Investigation results showed low imprecision (within-run coefficient of variation was below 3.5% and between-day coefficient of variation was less than 2.5% for most analytes at all three levels studied) and acceptable accuracy of the analyzer. No significant sample- or reagent-related carry-over was found. It was demonstrated that the analytical system operates within the claimed linearity ranges. The results compared well with those obtained by instruments routinely used in our laboratory (Olympus AU2700, Behring Nephelometer II). In general, the data on interference by hemoglobin, hyperbilirubinemia and turbidity are in accordance with known facts. However, slight hemolysis was found to interfere with the alkaline phosphatase (ALP) assay and mild lipemia affected the glucose assay. The Konelab 20XT is an easy-to-use analyzer that is suitable for routine and emergency analyses in small laboratories.

The role of bone morphogenetic proteins in kidney development and repair

Members of TGF-β superfamily are secreted glycoproteins and have been shown to regulate biological processes as diverse as migration, proliferation and differentiation of pluripotent progenitor cells involved in the development of several organ systems during embryogenesis and in adult tissue repair [1, 2]. The kidney has been identified as a major site of bone morphogenetic protein-7 (BMP-7) synthesis during embryonal and post-natal development [1, 3, 4]. Gene knock-out [5, 6] and in vitro experiments [4, 7] demonstrated the importance of BMP-7 in kidney development. Many developmental features are recapitulated during renal injury, and BMPs may be important in both preservation of function and resistance to injury [8, 9]. BMP-7 has a cytoprotective and anti-inflammatory effect in models of acute and chronic renal failure [8, 9].

Reliability of CKD-EPI predictive equation in estimating chronic kidney disease prevalence in the Croatian endemic nephropathy area.

Introduction Chronic kidney disease (CKD) is a significant public health problem and it is not possible to precisely predict its progression to terminal renal failure. According to current guidelines, CKD stages are classified based on the estimated glomerular filtration rate (eGFR) and albuminuria. Aims of this study were to determine the reliability of predictive equation in estimation of CKD prevalence in Croatian areas with endemic nephropathy (EN), compare the results with non-endemic areas, and to determine if the prevalence of CKD stages 3-5 was increased in subjects with EN. Materials and methods A total of 1573 inhabitants of the Croatian Posavina rural area from 6 endemic and 3 non-endemic villages were enrolled. Participants were classified according to the modified criteria of the World Health Organization for EN. Estimated GFR was calculated using Chronic Kidney Disease Epidemiology Collaboration equation (CKD-EPI). Results The results showed a very high CKD prevalence in the Croatian rural area (19%). CKD prevalence was significantly higher in EN then in non EN villages with the lowest eGFR value in diseased subgroup. Conclusions eGFR correlated significantly with the diagnosis of EN. Kidney function assessment using CKD-EPI predictive equation proved to be a good marker in differentiating the study subgroups, remained as one of the diagnostic criteria for EN.