Gut Microbiota and Cardiovascular Uremic Toxicities.

Cardiovascular disease (CVD) remains a major cause of high morbidity and mortality in patients with chronic kidney disease (CKD). Numerous CVD risk factors in CKD patients have been described, but these do not fully explain the high pervasiveness of CVD or increased mortality rates in CKD patients. In CKD the loss of urinary excretory function results in the retention of various substances referred to as "uremic retention solutes". Many of these molecules have been found to exert toxicity on virtually all organ systems of the human body, leading to the clinical syndrome of uremia. In recent years, an increasing body of evidence has been accumulated that suggests that uremic toxins may contribute to an increased cardiovascular disease (CVD) burden associated with CKD. This review examined the evidence from several clinical and experimental studies showing an association between uremic toxins and CVD. Special emphasis is addressed on emerging data linking gut microbiota with the production of uremic toxins and the development of CKD and CVD. The biological toxicity of some uremic toxins on the myocardium and the vasculature and their possible contribution to cardiovascular injury in uremia are also discussed. Finally, various therapeutic interventions that have been applied to effectively reduce uremic toxins in patients with CKD, including dietary modifications, use of prebiotics and/or probiotics, an oral intestinal sorbent that adsorbs uremic toxins and precursors, and innovative dialysis therapies targeting the protein-bound uremic toxins are also highlighted. Future studies are needed to determine whether these novel therapies to reduce or remove uremic toxins will reduce CVD and related cardiovascular events in the long-term in patients with chronic renal failure.

Association of vascular endothelial factors with cardiovascular outcome and mortality in chronic kidney disease patients: a 4-year cohort study.

BACKGROUND: Angiogenic cytokines fms-like tyrosine kinase-1(sFlt-1) and placental growth factor (PlGF) are associated with increased risk for cardiovascular disease (CVD) in the general population. In this study we examine the association between these vascular endothelial factors and atherosclerosis, cardiovascular outcome, and mortality in chronic kidney disease (CKD) patients. METHODS: Serum level of PlGF and sFlt-1 were measured in 301 patients with CKD, who were followed for up to 4 years. Primary outcomes were CV events and all-cause mortality. Carotid-intima media thickness (CIMT) was used as marker of atherosclerosis. Kaplan-Meier survival curves and the Cox proportional hazard model were used to assess the association of biomarkers and clinical outcomes. RESULTS: Mean (SD) PlGF and sFlt-1 were 5.45 ng/ml (3.76) and 68.6 (28.0) pg/ml, respectively. During the follow up time, 60 patients (19.9%) experienced CV events and 22 patients (7.3%) died. Compared with low PlGF, patients with PlGF above median level had higher CV events (12.7% vs. 27.2%, p = 0.002) and mortality (2.0% vs. 12.6%, p < 0.001). The associations of PlGF and sFlt-1 with CV events were not statistically significant in the fully adjusted model. Higher PlGF was associated with greater death risk (HR = 5.22, 95% CI: 1.49-18.33, p = 0.01), which was robust to adjustment for sFlt-1 and other risk factors. Elevated sFlt-1 level was also an independent predictor of mortality (HR 3.41, 95% CI: 1.49-9.51, p = 0.019). CONCLUSION: In CKD patients not yet on dialysis, higher serum level of PlGF and sFlt-1 are associated with increased mortality, but not CV events.

Examining associations of circulating endotoxin with nutritional status, inflammation, and mortality in hemodialysis patients.

OBJECTIVE: Lipopolysaccharide or endotoxin constitutes most part of the outer portion of the cell wall in the gram-negative bacteria. Subclinical endotoxemia could contribute to increased inflammation and mortality in hemodialysis (HD) patients. Endotoxin level and clinical effect are determined by its soluble receptor sCD14 and high-density lipoprotein. We examine the hypothesis that endotoxin level correlates with mortality. METHODS: In this cohort study, endotoxin levels were measured in 306 long-term HD patients who were then followed up for a maximum of 42 months. Soluble CD14 and cytokines levels were also measured. RESULTS: The mean (±SD) endotoxin level was 2.31 ± 3.10 EU/mL (minimum: 0.26 EU/mL, maximum: 22.94 EU/mL, interquartile range: 1.33 EU/mL, median: 1.27 EU/mL). Endotoxin correlated with C-reactive protein (r = 0.11, P < .04). On multivariate logistic regression analysis, high body mass index and low high-density lipoprotein (HDL) cholesterol levels were associated with higher endotoxemia (endotoxin below or above of median). In multivariate Cox regression analysis adjusted for case-mix and nutritional/inflammatory confounders, endotoxin levels in the third quartile versus first quartile were associated with a trend toward increased hazard ratio for death (hazard ratio: 1.83, 95% confidence interval: 0.93 to 3.6, P = .08). CONCLUSIONS: In this HD cohort, we found associations between endotoxemia and C-reactive protein, body composition, and HDL. Moderately high endotoxin levels tended to correlate with increased mortality than the highest circulating endotoxin level. Additional studies are required to assess the effect of endotoxemia on mortality in dialysis population.

Beyond genetics: epigenetic code in chronic kidney disease.

Epigenetics refers to a heritable change in the pattern of gene expression that is mediated by a mechanism specifically not due to alterations in the primary nucleotide sequence. Well-known epigenetic mechanisms encompass DNA methylation, chromatin remodeling (histone modifications), and RNA interference. Functionally, epigenetics provides an extra layer of transcriptional control and plays a crucial role in normal physiological development, as well as in pathological conditions. Aberrant DNA methylation is implicated in immune dysfunction, inflammation, and insulin resistance. Epigenetic changes may be responsible for 'metabolic memory' and development of micro- and macrovascular complications of diabetes. MicroRNAs are critical in the maintenance of glomerular homeostasis and hence RNA interference may be important in the progression of renal disease. Recent studies have shown that epigenetic modifications orchestrate the epithelial-mesenchymal transition and eventually fibrosis of the renal tissue. Oxidative stress, inflammation, hyperhomocysteinemia, and uremic toxins could induce epimutations in chronic kidney disease. Epigenetic alterations are associated with inflammation and cardiovascular disease in patients with chronic kidney disease. Reversible nature of the epigenetic changes gives a unique opportunity to halt or even reverse the disease process through targeted therapeutic strategies.

Activation of caspase-3 in the skeletal muscle during haemodialysis.

BACKGROUND AND OBJECTIVE: Muscle atrophy in end-stage renal disease (ESRD) may be due to the activation of apoptotic and proteolytic pathways. We hypothesized that activation of caspase-3 in the skeletal muscle mediates apoptosis and proteolysis during haemodialysis (HD). MATERIALS AND METHODS: Eight ESRD patients were studied before (pre-HD) and during HD and the findings were compared with those from six healthy volunteers. Protein kinetics was determined by primed constant infusion of L-(ring (13)C(6) ) Phenylalanine. RESULTS: Caspase-3 activity in the skeletal muscle was higher in ESRD patients pre-HD than in controls (24966·0 ± 4023·9 vs. 15293·3 ± 2120·0 units, P<0·01) and increased further during HD (end-HD) (37666·6 ± 4208·3 units) (P<0·001). Actin fragments (14 kDa) generated by caspase-3 mediated cleavage of actomyosin was higher in the skeletal muscle pre-HD (68%) and during HD (164%) compared with controls. The abundance of ubiquitinized carboxy-terminal actin fragment was also significantly increased during HD. Skeletal muscle biopsies obtained at the end of HD exhibited augmented apoptosis, which was higher than that observed in pre-HD and control samples (P<0·001). IL-6 content in the soluble fraction of the muscle skeletal muscle was increased significantly during HD. Protein kinetic studies showed that catabolism was higher in ESRD patients during HD compared with pre-HD and control subjects. Muscle protein catabolism was positively associated with caspase-3 activity and skeletal muscle IL-6 content. CONCLUSION: Muscle atrophy in ESRD may be due to IL-6 induced activation of caspase-3 resulting in apoptosis as well as muscle proteolysis during HD.

MDR1 hypermethylation contributes to the progression of neuroblastoma.

MDR1 hypermethylation plays an important role in pathogenesis and progression of neuroblastoma tumors. This hypothesis was tested by studying the methylation status of MDR1 gene promoter in neureoblastoma biopsy specimens during the progression of tumor from stage-1 to stage-4. Results of our findings demonstrate an inverse correlation between the methylation status of MDR1 promoter and MDR1 expression during the progression of disease from stage-1 to stage-4 as determined by methylation specific PCR (MSP) and RT/PCR analyses. The results of the RT/PCR and MSP analyses were validated by quantitative Real Time PCR analysis. Data from present study suggest that transcriptional inactivation of MDR1 gene due to increased MDR1 promoter methylation may be a contributing factor in pathogenesis and progression of neuroblastoma tumors, and may be used in designing an effective treatment therapy to neuroblastoma patients.

Invasive aspergillosis causing small bowel infarction in a patient of carcinoma breast undergoing chemotherapy.

BACKGROUND: To report a 45 year old lady presenting with proximal jejunal gangrene due to invasive Aspergillosis. The patient was undergoing adjuvant chemotherapy for advance carcinoma of breast (Stage IV). METHODS: The patient was referred to our surgical emergency for acute abdominal symptoms for 6 hours. Histopathology revealed bowel wall necrosis and vascular invasion by Aspergillus Fumigatus. Postoperative recovery was uneventful and the patient received Amphotericin-B (1 mg/kg/day) for invasive aspergillosis. Invasive pulmonary aspergillosis was confirmed by isolating Aspergillus Fumigatus from bronchoalveolar lavage and by a positive circulating galactomannan test (ELISA Assay). RESULTS: Detailed history revealed dry cough and two episodes of haemoptesis for 2 weeks. Haemogram and counts revealed anemia and neutropenia. Plain X-ray of the abdomen showed multiple air fluid levels and ultrasound of the abdomen revealed distended bowel loops. On exploration small bowel was found to be gangrenous. The patient was successfully managed by supportive treatment and conventional intravenous Amphotericin-B for 2 weeks. The lady was discharged one week after completion of antifungal therapy and one month later she underwent toilet mastectomy. The lady came to follow up for 1 year and she is currently under hormone therapy. CONCLUSION: With the emergence of new and powerful immunosuppressive, anticancer drugs and potent antibiotics the survival of transplant and critically ill patients has remarkably increased but it has shown a significant rise in the incidence of invasive opportunistic fungal infections. We conclude hat the diagnosis of invasive gastrointestinal aspergillosis may be considered in a neutropenic patient with acute abdominal symptoms.

Inhibition of DNA methyltransferase reverses cisplatin induced drug resistance in murine neuroblastoma cells.

BACKGROUND: Acquired drug resistance is a major obstacle to the successful treatment of neuroblastoma by chemotherapy. Recent studies from our laboratory have demonstrated that drug-induced alterations in DNA methylation play an important role in this process. METHODS: The reversal of resistance to cisplatin in murine neuroblastoma (MNB) was induced by inhibition of DNA methyltransferase activity. MNB cells overexpressing DNA methyltransferase activity (Dnmt3a or Dnmt3b) were established by stable co-transfection of wild type MNB cells with plasmids containing Dnmt3a or Dnmt3b cDNA. Cytotoxic response (IC50), total DNA methyltransferase activity and expression of Dnmt3a or Dnmt3b methyltransferase were determined in Dnmt3a or Dnmt3b transfected MNB cells, respectively. RESULTS: These data demonstrated that total DNA methyltransferase activity was increased to 3-fold above controls (P<0.001) in cisplatin resistant MNB cells, 3-fold in Dnmt3a and 4-fold in Dnmt3b transfected MNB cells. Western blot and RT-PCR data confirmed a corresponding increase in Dnmt3a and 3b expression in cisplatin resistant and transfected cells when compared with control MNB cells (P<0.001). MNB clones overexpressing Dnmt3a or Dnmt3b proteins were resistant to cisplatin treatment (10(-6) M). Incubation of cisplatin resistant, Dnmt3a or Dnmt3b overexpressing MNB cells with 5'-azacytidine (5'-azaC), a methylation inhibitor (2.5 microM) significantly decreased DNA methyltransferase activity, expression of Dnmt3a and Dnmt3b proteins and mRNA levels of cisplatin resistant, Dnmt3a and Dnmt3b transfected MNB cells. Cytotoxicity studies using the MTT assay demonstrated that the sensitivity of cisplatin resistant, Dnmt3a and Dnmt3b overexpressing MNB cells to cisplatin was increased 10-fold (P<0.001) following 5'-azaC treatment. CONCLUSIONS: These findings suggest that the overexpression of DNA methyltransferase is associated with a cisplatin resistant phenotype in MNB cells that may or may not be true in animal studies or in the clinical setup. Thus, DNA methylation plays a central role in onset of drug resistance in cisplatin resistant neuroblastoma cells in vitro.

Coactivator PRIP, the peroxisome proliferator-activated receptor-interacting protein, is a modulator of placental, cardiac, hepatic, and embryonic development.

Nuclear receptor coactivator PRIP (peroxisome proliferator-activated receptor (PPAR gamma)-interacting protein) and PRIP-interacting protein with methyltransferase activity, designated PIMT, appear to serve as linkers between cAMP response element-binding protein-binding protein (CBP)/p300-anchored and PBP (PPAR gamma-binding protein)-anchored coactivator complexes involved in the transcriptional activity of nuclear receptors. To assess the biological significance of PRIP, we disrupted the PRIP gene in mice by homologous recombination. Mice nullizygous for PRIP died between embryonic day 11.5 and 12.5 (postcoitum) due in most part to defects in the development of placenta, heart, liver, nervous system, and retardation of embryonic growth. Transient transfection assays using fibroblasts isolated from PRIP(-/-) embryos revealed a significant decrease in the capacity for ligand-dependent transcriptional activation of retinoid X receptor alpha and to a lesser effect on PPAR gamma transcriptional activity. These observations indicate that PRIP like PBP, CBP, and p300 is an essential and nonredundant coactivator.

Differential expression of DNA-methyltransferases in drug resistant murine neuroblastoma cells.

Neuroblastoma tumors frequently become drug resistant during the process of chemotherapy resulting in unfavorable clinical outcomes. Development of sustained drug resistance in neuroblastoma is a major problem in successful treatment. To explore the role of DNA-methyltransferases (Dnmt) in acquired drug resistance of neuroblastoma, the present investigation was carried out to study the expression of Dnmtl, Dnmt3a, and Dnmt3b in drug resistant murine neuroblastoma cells, in an in vitro model system. We have analyzed the expression of Dnmtl, Dnmt3a, and Dnmt3b methyltransferases in wild type and drug resistant murine neuroblastoma cells by using Western blot, immunofluorescence microscopy, semiquantitative and quantitative real time RT-PCR analyses. The present investigation demonstrates that total Dnmt enzymatic activity was increased two-fold (P < 0.001) with a 33% increase in global DNA methylation rate in drug resistant cells. Results of the Western blot, immunofluorescence microscopy, RT-PCR, and quantitative real time RT-PCR analysis demonstrated that Dnmt1 and Dnmt3b expression increased significantly (P < 0.001) in drug resistant cells when compared with wild type cells. Dnmt3a expression did not reveal any change between wild type and drug resistant cells. These findings suggest that Dnmtases are differentially expressed in drug resistant murine neuroblastoma cells and overexpression of Dnmtl and Dnmt3b may contribute towards loss of function of the growth regulatory or tumor suppressor genes by methylation of their 'CpG' region and subsequently silencing of their expression. The products of these methylated genes may, thus, confer a high level of drug resistant phenotype in drug resistant neuroblastoma cells.