A study on the impact of CYP2C19 genotype and platelet reactivity assay on patients undergoing PCI

AbstractBackgroundA thorough understanding of the patient's genotype and their functional response to a medication is necessary for improving event free survival. Several outcome studies support this view particularly if the patient is to be started on clopidogrel due to the prevalence of clopidogrel resistance. Such guided therapy has reduced the incidence of Major Adverse Cardiac Events (MACE) after stent implantation.MethodsBetween August 2013 and August 2014, 200 patients with coronary artery disease undergoing percutaneous coronary intervention (PCI) were prescribed any one of the anti-platelet medications such as clopidogrel, prasugrel or ticagrelor and offered testing to detect CYP2C19 gene mutations along with a platelet reactivity assay (PRA). Intended outcome was modification of anti-platelet therapy defined as either dose escalation of clopidogrel or replacement of clopidogrel with prasugrel or ticagrelor for the patients in clopidogrel arm, and replacement of ticagrelor or prasugrel with clopidogrel if those patients were non-carrier of mutant genes and also if they demonstrated bleeding tendencies in the ticagrelor and prasugrel arms.ConclusionClopidogrel resistance was observed to be 16.5% in our study population. PRA was useful in monitoring the efficacy of thienopyridines. By having this test, one can be safely maintained on clopidogrel in non-carriers, or with increased dose of clopidogrel in intermediate metabolizers or with newer drugs such as ticagrelor or prasugrel in poor metabolizers. Patients on ticagrelor and prasugrel identified as non-carriers of gene mutations for clopidogrel metabolism could be offered clopidogrel resulting in economic benefits to the patients. Patients at high risk of bleeding were also identified by the PRA

Kidney injury molecule-1 is up-regulated in renal epithelial cells in response to oxalate in vitro and in renal tissues in response to hyperoxaluria in vivo.

Oxalate is a metabolic end product excreted by the kidney. Mild increases in urinary oxalate are most commonly associated with Nephrolithiasis. Chronically high levels of urinary oxalate, as seen in patients with primary hyperoxaluria, are driving factor for recurrent renal stones, and ultimately lead to renal failure, calcification of soft tissue and premature death. In previous studies others and we have demonstrated that high levels of oxalate promote injury of renal epithelial cells. However, methods to monitor oxalate induced renal injury are limited. In the present study we evaluated changes in expression of Kidney Injury Molecule-1 (KIM-1) in response to oxalate in human renal cells (HK2 cells) in culture and in renal tissue and urine samples in hyperoxaluric animals which mimic in vitro and in vivo models of hyper-oxaluria. Results presented, herein demonstrate that oxalate exposure resulted in increased expression of KIM-1 m RNA as well as protein in HK2 cells. These effects were rapid and concentration dependent. Using in vivo models of hyperoxaluria we observed elevated expression of KIM-1 in renal tissues of hyperoxaluric rats as compared to normal controls. The increase in KIM-1 was both at protein and mRNA level, suggesting transcriptional activation of KIM-1 in response to oxalate exposure. Interestingly, in addition to increased KIM-1 expression, we observed increased levels of the ectodomain of KIM-1 in urine collected from hyperoxaluric rats. To the best of our knowledge our studies are the first direct demonstration of regulation of KIM-1 in response to oxalate exposure in renal epithelial cells in vitro and in vivo. Our results suggest that detection of KIM-1 over-expression and measurement of the ectodomain of KIM-1 in urine may hold promise as a marker to monitor oxalate nephrotoxicity in hyperoxaluria

Genome wide analysis of differentially expressed genes in HK-2 cells, a line of human kidney epithelial cells in response to oxalate.

Nephrolithiasis is a multi-factorial disease which, in the majority of cases, involves the renal deposition of calcium oxalate. Oxalate is a metabolic end product excreted primarily by the kidney. Previous studies have shown that elevated levels of oxalate are detrimental to the renal epithelial cells; however, oxalate renal epithelial cell interactions are not completely understood. In this study, we utilized an unbiased approach of gene expression profiling using Affymetrix HG_U133_plus2 gene chips to understand the global gene expression changes in human renal epithelial cells [HK-2] after exposure to oxalate. We analyzed the expression of 47,000 transcripts and variants, including 38,500 well characterized human genes, in the HK2 cells after 4 hours and 24 hours of oxalate exposure. Gene expression was compared among replicates as per the Affymetrix statistical program. Gene expression among various groups was compared using various analytical tools, and differentially expressed genes were classified according to the Gene Ontology Functional Category. The results from this study show that oxalate exposure induces significant expression changes in many genes. We show for the first time that oxalate exposure induces as well as shuts off genes differentially. We found 750 up-regulated and 2276 down-regulated genes which have not been reported before. Our results also show that renal cells exposed to oxalate results in the regulation of genes that are associated with specific molecular function, biological processes, and other cellular components. In addition we have identified a set of 20 genes that is differentially regulated by oxalate irrespective of duration of exposure and may be useful in monitoring oxalate nephrotoxicity. Taken together our studies profile global gene expression changes and provide a unique insight into oxalate renal cell interactions and oxalate nephrotoxicity

Supercritical Fluid Extract of Putranjiva roxburghii Wall. Seeds Mitigates Fertility Impairment in a Zebrafish Model

Putrajeevak (Putranjiva roxburghii Wall.; synonym Drypetes roxburghii (Wall.) Hurus) seeds have been used since ancient times in the treatment of infertility in the Ayurvedic system of medicine in India. In this study, the oil component of Putrajeevak seeds (PJSO) was extracted using the supercritical fluid extraction (SCFE) method using liquid CO2 and the constituents were analyzed using gas chromatography-flame ionized detectorand high-performance thin-layer chromatography. PJSO contained trace amounts of β-sitosterol with oleic and linoleic acids as the major fatty acid constituents. Male and female zebrafish were mutagenized with N-ethyl-N-nitrosourea (ENU) and fish that produced less than 20 viable embryos were selected for the study. SCFE oil extracts from the P. roxburghii seeds were used in this study to reverse fertility impairment. The mutant fish were fed with PJSO for a period of 14 days and the rates of fertility, conception, and fecundity were determined with wild-type healthy fish as a breeding partner. Treatment with PJSO increased the ovarian follicle count as well as the number of mature eggs, while reducing the number of ovarian cysts. Sperm count as well as sperm motility were greatly enhanced in the ENU-mutagenized male zebrafish when treated with PJSO. The results obtained in this study demonstrate the effectiveness of P. roxburghii seed oil in reversing impaired fertility in both male and female zebrafish models

Sequences of primers used in RT-PCR.

<p>Sequences of primers used in RT-PCR.</p

Exposure of HK-2 cells to oxalate results in changes in global gene expression.

<p>(A) Levels of gene expression are color coded to depict high to low expression and the numbers represent percentages of genes at each expression level. Representative images from duplicate experiments are shown. (B) Quantitative representation of translational suppression following exposure to Oxalate. (C) Quantitative analysis of differential gene expression in HK2 cells following oxalate exposure. Each data point represents mean of two individual experiments.</p

Exposure to oxalate modulates signaling pathways in HK-2 cells.

<p>Changes in the expression of genes involved in various signaling pathways upon exposure of HK-2 cells to oxalate, as determined by Affymetrix gene chip analysis. (A) Changes in gene expression of genes involved in signal transduction pathways after exposure to oxalate. (B) Heat map and cluster diagram represent changes in expression of genes that constitute the Mitogen Activated Protein Kinase (MAPK) pathway. Each data point is representative of the median expression level of two independent gene chip arrays. The Cluster diagram and dendogram was generated using BRB-Array Tools software with a minimum fold change less than 20% of expression data and have at least a 1.5 fold change in either direction from the gene's median expression value.</p