Tubulointerstitial inflammation and progressive fibrosis are common pathways that lead to kidney failure in proteinuric nephropathies. Activation of the complement system has been implicated in the development of tubulointerstitial injury in clinical and animal studies, but the mechanism by which complement induces kidney injury is not fully understood. Here, we studied the effect of complement on the phenotype of tubular epithelial cells. Tubular epithelial cells exposed to serum proteins adopted phenotypic and functional characteristics of mesenchymal cells. Expression of E-cadherin protein decreased and expression of both ␣-smooth muscle actin protein and collagen I mRNA increased. Exposure of the cells to the complement anaphylotoxin C3a induced similar features. Treating with a C3a receptor (C3aR) antagonist prevented both C3a-and serum-induced epithelial-to-mesenchymal transition. In the adriamycin-induced proteinuria model, C3aR-deficient mice demonstrated less injury, preserved renal function, and improved survival compared with wild-type mice. Furthermore, the kidneys of C3aR-deficient mice had significantly less interstitial collagen I and ␣-smooth muscle actin. In summary, the complement anaphylotoxin C3a is an important mediator of glomerular and tubulointerstitial injury and can induce tubular epithelial-to-mesenchymal transition.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT• A number of pharmacokinetic studies have focused on S-warfarin. These have shown that demographic factors, such as bodyweight, genetic factors, such as CYP2C9 genotype, and interacting medicines, particularly amiodarone, contribute to the interindividual estimates of clearance. WHAT THIS STUDY ADDS• This study not only reinforces what has previously been learned about S-warfarin, but also provides an insight into the pharmacokinetics of R-warfarin. The study also focuses on individuals who are on long-term warfarin therapy, which is more reflective of clinical practice. BACKGROUNDWarfarin is a drug with a narrow therapeutic index and large interindividual variability in daily dosing requirements. Patients commencing warfarin treatment are at risk of bleeding due to excessive anticoagulation caused by overdosing. The interindividual variability in dose requirements is influenced by a number of factors, including polymorphisms in genes mediating warfarin pharmacology, co-medication, age, sex, body size and diet. AIMSTo develop population pharmacokinetic models of both R-and S-warfarin using clinical and genetic factors and to identify the covariates which influence the interindividual variability in the pharmacokinetic parameters of clearance and volume of distribution in patients on long-term warfarin therapy. METHODSPatients commencing warfarin therapy were followed up for 26 weeks. Plasma warfarin enantiomer concentrations were determined in 306 patients for S-warfarin and in 309 patients for R-warfarin at 1, 8 and 26 weeks. Patients were also genotyped for CYP2C9 variants (CYP2C9*1,*2 and *3), two single-nucleotide polymorphisms (SNPs) in CYP1A2, one SNP in CYP3A4 and six SNPs in CYP2C19. A base pharmacokinetic model was developed using NONMEM software to determine the warfarin clearance and volume of distribution. The model was extended to include covariates that influenced the between-subject variability. RESULTSBodyweight, age, sex and CYP2C9 genotype significantly influenced S-warfarin clearance. The S-warfarin clearance was estimated to be 0.144 l h -1 (95% confidence interval 0.131, 0.157) in a 70 kg woman aged 69.8 years with the wild-type CYP2C9 genotype, and the volume of distribution was 16.6 l (95% confidence interval 13.5, 19.7). Bodyweight and age, along with the SNPs rs3814637 (in CYP2C19) and rs2242480 (in CYP3A4), significantly influenced R-warfarin clearance. The R-warfarin clearance was estimated to be 0.125 l h -1 (95% confidence interval 0.115, 0.135) in a 70 kg individual aged 69.8 years with the wild-type CYP2C19 and CYP3A4 genotypes, and the volume of distribution was 10.9 l (95% confidence interval 8.63, 13.2). CONCLUSIONSOur analysis, based on exposure rather than dose, provides quantitative estimates of the clinical and genetic factors impacting on the clearance of both the S-and R-enantiomers of warfarin, which can be used in developing improved dosing algorithms.

Since their introduction to biological imaging, quantum dots (QDs) have progressed from a little known, but attractive technology to one that has gained broad application in many areas of biology. The versatile properties of these fluorescent nanoparticles have allowed investigators to conduct biological studies with extended spatiotemporal capabilities that were previously not possible. In this review, we focus on QD applications that provide enhanced quantitative information on protein dynamics and localization, including single particle tracking (SPT) and immunohistochemistry (IHC), and finish by examining prospects of upcoming applications, such as correlative light and electron microscopy (CLEM) and super-resolution. Advances in single molecule imaging, including multi-color and 3D QD tracking, have provided new insights into the mechanisms of cell signaling and protein trafficking. New forms of QD tracking in vivo have allowed for observation of biological processes with molecular level resolution in the physiological context of the whole animal. Further methodological development of multiplexed QD-based immunohistochemistry assays are allowing more quantitative analysis of key proteins in tissue samples. These advances highlight the unique quantitative data sets that QDs can provide to further our understanding of biological and disease processes.

Key Points• PD-L1 is variably expressed in MCs from patients with SM and CM.• PD-1 is expressed in MCs in a subset of patients with CM, but not SM.

To cite this article: Hatch E, Wynne H, Avery P, Wadelius M, Kamali F. Application of a pharmacogenetic-based warfarin dosing algorithm derived from British patients to predict dose in Swedish patients. J Thromb Haemost 2008; 6: 1038-40.The anticoagulation response to warfarin is influenced by a number of genetic and environmental factors. Two major genes affect warfarin dose requirements. The gene that encodes CYP2C9, the main enzyme responsible for S-warfarin metabolism, is highly polymorphic; two common allelic variants, CYP2C9 * 2 and CYP2C9 * 3, have been associated with low dose warfarin requirements and increased risk of bleeding [1]. Vitamin K epoxide reductase (VKOR) is the pharmacological target for warfarin. Polymorphisms in the VKOR complex subunit 1 (VKORC1) gene have been demonstrated to contribute to inter-individual differences in warfarin dose requirement, with (-1639) GG genotype patients requiring a significantly higher daily dose of warfarin than GA and AA genotypes to achieve the same target International Normalized Ratio (INR) [2].We recently demonstrated that the factors of patient age, height, and CYP2C9 and VKORC1 genotypes account for over half the variability in warfarin dose requirement in a cohort of British Caucasian patients [2]. A dosing algorithm was developed based on the regression model containing these factors. In the search for a wider applicability of the dosing algorithm, we set out to test its accuracy in predicting warfarin dose requirements in a cohort of Swedish Caucasian patients. We also examined the influence of concomitant use of drugs known to influence warfarin dose requirement on the accuracy of the predicted dose.Data were obtained on CYP2C9 and VKORC1 polymorphisms, and demographic information relating to an anonymized cohort of Swedish Caucasian patients, with a target INR of 2-3 and with stable maintenance warfarin dose, attending the anticoagulation clinic at Uppsala University Hospital, Uppsala, Sweden. The genotype information on this group of patients was available as part of an earlier study [3,4]. A patient was deemed as stable when his/her daily dose requirement had remained unchanged for at least the previous three visits to the clinic. Warfarin dose requirement for each patient was estimated using the published pharmacogenetic-based dosing algorithm [2].Statistical analysis of data was performed using Minitab 14 (Coventry, UK). Regression analysis between actual and estimated warfarin dose was assessed for the cohort as a whole and for those patients taking no interacting drugs. Concurrent drug use was input in the multiple regression analysis. A P value of <0.05 was considered as statistically significant. Data were analyzed from 88 patients (65 males) on stable warfarin doses aged 48-86 years, with a mean ± SD height of 175.7 ± 77 cm and weight of 81.9 ± 13.8 kg. Eight of the patients were smokers. The primary indication for anticoagulation was atrial fibrillation in 68 patients, pulmonary embolism/deep vein thrombosis in five patients and other t...

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