HLA-DR8 Subtyping by Polymerase Chain Reaction (PCR) - DNA Conformation Polymorphism (DCP) Analysis : a simple and practical genotyping method

Two hundred Japanese panels were serologically typed for human leukocyte antigen (HLA) - DR to assign 65 HLA-DRS haplotypes, which were then subdivided into two genotypes, i.e., DRB1 *0802 and DRB1 *0803, by a polymerase chain reaction (PCR) - based, simple, and practical method. The panels possessing DR8 specificity were firstly subjected to PCR with a couple of primers specifically to amplify their DR52 associated group - DRB1 genes. PCR products were then denatured in the presence of formamide, electrophoresed in a non-denaturing polyacrylamide gel, and visualized by silver staining. The same DRB1 products of these samples were also mixed with the DRB1 *1302, and simultaneously analyzed by the same procedure. Electrophoretic mobilities of the samples were compared with those of the typing standards to genotype their DRS-DRB1 alleles by using the characteristic polymorphism in the single-stranded DNAs and the heteroduplexes. This method, designated PCR - DNA conformation polymorphism (DCP) analysis, allowed for genotyping of the DR8-DRB1 alleles without using sequence - specific oligonucleotide probes (SSOP) or restriction endonucleases. The entire process after PCR was completed within a few hours. The tested panels were also genotyped for DRB1 gene by the PCR-SSOP method for comparison with results obtained by the PCR-DCP method. Satisfactory coincidence was achieved and it represented how accurately the new system genotyped DRB1 *0802 and DRB1 *0803. PCR-DCP analysis was thus shown to be practical and useful for subtyping of serologically defined DR8 specificities.This work was supported in part by grants-in-aid from the Ministry of Education, Culture and Science, Japan, and research grants from the Ministry of Health and Welfare, Japan

Empagliflozin in Patients with Chronic Kidney Disease

Background The effects of empagliflozin in patients with chronic kidney disease who are at risk for disease progression are not well understood. The EMPA-KIDNEY trial was designed to assess the effects of treatment with empagliflozin in a broad range of such patients. Methods We enrolled patients with chronic kidney disease who had an estimated glomerular filtration rate (eGFR) of at least 20 but less than 45 ml per minute per 1.73 m(2) of body-surface area, or who had an eGFR of at least 45 but less than 90 ml per minute per 1.73 m(2) with a urinary albumin-to-creatinine ratio (with albumin measured in milligrams and creatinine measured in grams) of at least 200. Patients were randomly assigned to receive empagliflozin (10 mg once daily) or matching placebo. The primary outcome was a composite of progression of kidney disease (defined as end-stage kidney disease, a sustained decrease in eGFR to < 10 ml per minute per 1.73 m(2), a sustained decrease in eGFR of & GE;40% from baseline, or death from renal causes) or death from cardiovascular causes. Results A total of 6609 patients underwent randomization. During a median of 2.0 years of follow-up, progression of kidney disease or death from cardiovascular causes occurred in 432 of 3304 patients (13.1%) in the empagliflozin group and in 558 of 3305 patients (16.9%) in the placebo group (hazard ratio, 0.72; 95% confidence interval [CI], 0.64 to 0.82; P < 0.001). Results were consistent among patients with or without diabetes and across subgroups defined according to eGFR ranges. The rate of hospitalization from any cause was lower in the empagliflozin group than in the placebo group (hazard ratio, 0.86; 95% CI, 0.78 to 0.95; P=0.003), but there were no significant between-group differences with respect to the composite outcome of hospitalization for heart failure or death from cardiovascular causes (which occurred in 4.0% in the empagliflozin group and 4.6% in the placebo group) or death from any cause (in 4.5% and 5.1%, respectively). The rates of serious adverse events were similar in the two groups. Conclusions Among a wide range of patients with chronic kidney disease who were at risk for disease progression, empagliflozin therapy led to a lower risk of progression of kidney disease or death from cardiovascular causes than placebo