Moshe Zlotnik

Involvement of adenosine in the antiinflammatory action of ketamine

Background:Ketamine is an anesthetic drug. Subanesthetic doses of ketamine have been shown to reduce interleukin-6 concentrations after surgery and to reduce mortality and the production of tumor necrosis factor α and interleukin 6 in septic animals. Similarly, adenosine was shown to reduce tumor necrosis factor α and mortality of septic animals. The aim of this study was to determine whether adenosine mediates the antiinflammatory effects of ketamine. Methods:Sepsis was induced in mice by lipopolysaccharide or Escherichia coli inoculation. Leukocyte recruitment and cytokine concentrations were used as inflammation markers. Adenosine concentrations were assayed by high-performance liquid chromatography, and the involvement of adenosine in the effects of ketamine was demonstrated by adenosine receptor agonists and antagonists. Results:Ketamine markedly reduced mortality from sepsis, leukocyte recruitment, and tumor necrosis factor-α and interleukin-6 concentrations. Ketamine administration in mice and rats was associated with a surge at 20–35 min of adenosine in serum (up to 5 &mgr;m) and peritoneal fluid. The adenosine A2A receptor agonist CGS-21680 mimicked the effect of ketamine in peritonitis, whereas the A2A receptor antagonists DMPX and ZM 241385 blocked its antiinflammatory effects. In contrast, A1 and A3 receptor antagonists had no effect. ZM 241385 reversed the beneficial effect of ketamine on survival from bacterial sepsis. Conclusions:The current data suggest that the sepsis-protective antiinflammatory effects of ketamine are mediated by the release of adenosine acting through the A2A receptor.

Anti-Inflammatory Preconditioning by Agonists of Adenosine A1 Receptor

Background Adenosine levels rise during inflammation and modulate inflammatory responses by engaging with four different G protein-coupled receptors. It is suggested that adenosine exhibits pro-inflammatory effects through its A1 receptor (A1R), and anti-inflammatory effects through A2A receptor (A2AR). Therefore, understanding of the mechanisms that govern adenosine receptor regulation may advance treatment of various inflammatory disorders. We previously reported that peak A1R expression during leukocyte recruitment, is followed by a peak in A2AR during inflammation resolution. Principal Findings Here, we examined whether A1R activation sequentially induces A2AR expression and by this reverses inflammation. The effect of adenosine on A1R mediated A2AR expression was examined in peritoneal macrophages (PMΦ) and primary peritoneal mesothelial cells (PMC) in vitro. Induction of A2AR was inhibited by pertussis toxin (PTX) and partly dependent on A2AR stimulation. Administration of A1R agonists to healthy mice reduced A1R expression and induced A2AR production in PMC. Mice that were preconditioned with A1R agonists 24 hours before E. coli inoculation exhibited decreased TNFα and IL-6 sera levels and reduced leukocytes recruitment. Preconditioning was blocked by pretreatment with A1R antagonist, as well as, or by late treatment with A2AR antagonist, and was absent in A2AR−/− mice. Conclusions Our data suggest that preconditioning by an A1R-agonist promotes the resolution of inflammation by inducing the production of A2AR. Future implications may include early treatment during inflammatory disorders or pretreatment before anticipated high risk inflammatory events, such as invasive surgery and organ transplantation.

Circulating cell-free DNA in hemodialysis patients predicts mortality

BACKGROUND Circulating cell-free DNA (CFD) appears following cell damage and DNA release, and increases in hemodialysis (HD) patients particularly following HD. We hypothesized that CFD is an integrative marker of tissue damage and can be an independent predictor for all-cause mortality in HD patients. METHODS In a prospective study, CFD levels before and after HD were evaluated in 31 chronic HD patients with no acute disease, using the reported rapid non-cumbersome inexpensive fluorometric assay developed in our laboratory. Follow-up levels were assessed at 18 months in 22 patients. All-cause mortality was a primary endpoint. RESULTS During 42 months of follow-up, 13 of the 31 (41.9%) patients died. The decedents were older than the survivors (mean age 69.9 versus 61.5 years, P = 0.06), but did not differ in end-stage renal disease (ESRD) duration, gender, albumin and hemoglobin, diabetes mellitus and weight. Post-dialysis CFD levels were significantly lower in survivors (median 688 versus 880 ng/mL, P = 0.01). The sensitivity and specificity of CFD levels of 850 ng/mL to predict 42 months (3.5 years) mortality were 73 and 75%, respectively, and the area under the receiver-operating characteristic curve was 0.77 [95% confidence interval (CI) 0.60-0.94]. The Cox proportional hazard regression model showed that CFD higher than 850 ng/mL adjusted for age, ESRD duration, weight and creatinine (stepwise model) was highly predictive of all-cause death with a hazard ratio of 8.0 (95% CI 2.3-28.5, P = 0.001). CONCLUSIONS Post-dialysis CFD level is an independent predictor of all-cause mortality in patients undergoing HD. We propose that CFD detection is an inexpensive applicable tool for identifying patients at risk and their follow-up.

Association Between Renal Injury and Reduced Interleukin-15 and Interleukin-15 Receptor Levels in Acute Kidney Injury

Interleukin (IL)-15 serves as a survival factor for a broad array of cells. Renal cells express both IL-15 and its receptor (IL-15R); however, the role of IL-15 in the kidney is yet to be determined. We examined IL-15 and IL-15R levels in sepsis-related renal injury, ischemia-reperfusion injury (IRI), and cisplatin-induced nephrotoxicity. To test the anti-apoptotic effect of IL-15, Bcl-2/Bax mRNA levels were assessed in kidneys of IL-15Ralpha(-/-) mice and in IL-15-stimulated renal epithelial cells (RECs). In addition, RECs were exposed to cisplatin and apoptosis was evaluated by TUNEL staining, caspase-3 activity, and cell cycle analysis. Intrarenal IL-15 levels decreased 24 h after initiation of all three examined pathologies by 5.8-fold (sepsis), 11-fold (IRI), and 23-fold (cisplatin-induced nephrotoxicity). Further experiments revealed that while addition of rIL-15 (1 ng/mL) to wild-type (WT) RECs increased Bcl-2/Bax ratio by 2-fold, kidneys of IL-15Ralpha(-/-) mice exhibited 4-fold lower Bcl-2/Bax ratio compared to WT mice. Accordingly, IL-15 lowered the apoptotic rate in cisplatin-treated cultured REC, and IL-15Ralpha(-/-) renal cells exhibited a higher rate of cisplatin-induced apoptosis. Furthermore, IL-15 levels negatively correlated with BUN of cisplatin-treated mice (R = -0.69, P = 0.003), suggesting that a decline in renal-derived IL-15 is detrimental to renal cell survival and kidney function during pathological stress.

Blocking adenosine A2A receptor reduces peritoneal fibrosis in two independent experimental models

BACKGROUND Long-term peritoneal dialysis (PD) is associated with peritoneal fibrosis and loss of function. It has been shown that activation of the adenosine A(2A) receptor (A(2A)R) promotes tissue repair, wound healing and extracellular matrix (ECM) production. We have previously shown that adenosine is a potent regulator of inflammation in the peritoneum. In the current study, we explored the role of adenosine and the A(2A)R in two experimental models. METHODS Collagen deposition was evaluated in primary peritoneal fibroblasts following treatment with an A(2A)R agonist and antagonist. In addition, peritoneal fibrosis was induced by i.p. injection of either chlorhexidine gluconate for 2 weeks or 4.25% glucose peritoneal dialysis fluid (PDF) for 1 month. The development of fibrosis was compared between wild-type (WT) and WT mice treated with caffeine (an A(2A)R antagonist) in drinking water or between (A(2A)R(+/+)) mice and A(2A)R-deficient mice (A(2A)R(-/-)). RESULTS Adenosine or the A(2A)R agonist CGS21680 stimulated collagen production by peritoneal fibroblasts in vitro and A(2A)R antagonists (ZM241385 and caffeine) blocked this effect. Consistent with these results, caffeine-treated WT or A(2A)R(-/-) mice had reduced submesothelial thickness, collagen deposition and mRNA levels of fibroblast-specific protein (FSP-1) and connective tissue growth factor (CTGF). In addition, treatment with caffeine in vitro and in vivo diminished A(2A)R and A(2B)R mRNA levels induced by CG or PDF while it upregulated A(1)R levels. CONCLUSION Our data suggest that adenosine through its A(2A)R promotes peritoneal fibrosis and therefore should be considered as a target for pharmacological intervention.

The effects of hemodialysis on blood glutamate levels in chronic renal failure: Implementation for neuroprotection ☆

PURPOSE The purpose of the present study is to investigate whether hemodialysis (HD) is effective in lowering blood glutamate levels. In addition, we examined the effect of HD on glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) levels in the blood and described the rate and pattern of blood glutamate clearance during HD. MATERIALS AND METHODS Blood samples were taken from 45 patients with stage V chronic kidney disease immediately after initiation of HD and hourly, for a total of 5 blood samples. Samples were sent for determination of glutamate, glucose, GOT, GPT, hemoglobin, hematocrit, urea, and creatinine levels. A blood sample from 25 healthy volunteers without chronic renal failure was used as a control for the determination of baseline blood levels of glutamate, GOT, and GPT. RESULTS Glutamate and GPT levels in patients on HD were higher at baseline compared with healthy controls (P < .001). In the first 3 hours after HD, there was a decrease in blood glutamate levels compared with baseline levels (P < .00001). At the fourth hour, there was an increase in blood glutamate levels compared with the third hour (P < .05). CONCLUSIONS Hemodialysis may be a promising method of reducing blood glutamate levels.

Sporadic culture-negative peritonitis in peritoneal dialysis patients--absence of endotoxin in dialysate.

Background: Indiscriminate use of broad-spectrum antibiotics in peritonitis may have either unwanted side effects or contribute to the development of antibiotic resistance. It is tempting to use broad-spectrum antibiotics in cases of culture-negative peritonitis. This study examines whether Gram-negative agents have to be considered in the management of culture-negative peritonitis. Gram-negative agents are manifested by endotoxin easily detected by the Limulus amebocyte lysate (LAL) test. Methods: 138 episodes of Gram-negative and culture-negative peritonitis have been retrospectively analyzed; episodes of Gram-negative peritonitis were controls. Correlation between LAL and culture results was compared between the two groups. The LAL test was performed using a commercial kit by incubating a mixture of dialysate effluent and LAL reagent at 37°C. Development of a stable solid clot was considered positive. Results: In controls, 80 out of 117 Gram-negative peritonitis were LAL positive (68%). None of the 21 culture-negative episodes was LAL positive. In 7 recurrences of Gram-negative peritonitis, the LAL test turned from negative to positive but in none of the recurrences of culture-negative peritonitis. The difference in correlation was highly significant. Conclusions: Gram-negative organisms do not seem to be involved in sporadic culture-negative peritonitis. In episodes of peritonitis in which bacteriologic cultures stay negative for 48 h, initial coverage of Gram-negative organisms may be dropped.

Regulation of adenosine system at the onset of peritonitis

BACKGROUND Adenosine, a potent regulator of inflammation, is produced under stressful conditions due to degradation of ATP/ADP by the ectoenzymes CD39 and CD73. Adenosine is rapidly degraded by adenosine deaminase (ADA) or phosphorylated in the cell by adenosine kinase (AK). From four known receptors to adenosine, A(1) (A(1)R) promotes inflammation by a G(i)-coupled receptor. We have previously shown that A(1)R is up-regulated in the first hours following bacterial inoculation. The aim of the current study is to characterize the inflammatory mediators that regulate adenosine-metabolizing enzymes and A(1)R at the onset of peritonitis. METHODS Peritonitis was induced in CD1 mice by intraperitoneal injection of Escherichia coli. TNFalpha and IL-6 levels were determined in peritoneal fluid by enzyme-linked immunosorbent assay. Adenosine-metabolizing enzymes and the A(1)R mRNA or protein levels were analyzed by quantitative PCR or by Western blot analysis, respectively. RESULTS We found that CD39 and CD73 were up-regulated in response to bacterial stimuli (6-fold the basal levels), while AK and ADA mRNA levels were down-regulated. Cytokine production and leukocyte recruitment were enhanced (2.5-fold) by treatment with an A(1)R agonist (2-chloro-N(6)-cyclopentyladenosine, 0.1 mg/kg) and reduced (2.5-3-fold) by the A(1)R antagonist (8-cyclopentyl-1, 3-dipropylxanthine, 1 mg/kg). In contrast to lipopolysaccharide, IL-1, TNF and IFNgamma, only low IL-6 levels (0.01 ng/ml), in the presence of its soluble IL-6R (sIL-6R), were found to promote A(1)R expression on mesothelial cells. In mice, administration of neutralizing antibody to IL-6R or soluble gp130-Fc (sgp130-Fc) blocked peritoneal A(1)R up-regulation following inoculation. CONCLUSION Bacterial products induce the production of adenosine by up-regulation of CD39 and CD73. Low IL-6-sIL-6R up-regulates the A(1)R to promote efficient inflammatory response against invading microorganisms.

Severe Coombs'-negative autoimmune hemolytic anemia in a kidney transplant patient.

Background/Aim: Few cases are found in the literature regarding autoimmune hemolytic anemia which is Coombs’ test positive in kidney transplant patients, although hemolytic uremic syndrome due to cyclosporin and FK506 has been well described. In the following, we describe a case of severe life-threatening Coombs’ test negative autoimmune hemolytic anemia after kidney transplantation. Methods: Soon after undergoing renal transplantation, the patient presented with hemolytic anemia. Kidney biopsy, routine Coombs’ test, gel filtration and flow-cytometric assay were undertaken. Results: Kidney biopsy ruled out hemolytic uremic syndrome; although Coombs’ test and gel filtration assay were negative, flow cytometry revealed circulating antierythrocytic autoantibodies. Conclusions: Our findings indicate that flow cytometry may be an efficient method in the diagnosis of hemolysis of unknown origin in transplant patients. We further hypothesize that the underlying mechanism of autoimmune hemolytic anemia is related to the passenger B lymphocytes in the graft.

Successful Treatment of Secondary Hyperparathyroidism in Hemodialysis Patients with Oral Pulse 1-Alpha-Hydroxycholecalciferol Therapy

We have used high-dose oral pulse therapy with 1 alpha-hydroxycholecalciferol (1 alpha-OH-D3) to treat 40 hemodialysis patients suffering form secondary hyperparathyroidism. Forty patients with intact parathyroid hormone (PTH) levels of > 150 pg/ml were treated with 4 micrograms oral 1 alpha-OH-D3 twice weekly for 1 year. The mean PTH level was 515 +/- 50 pg/ml prior to treatment, which fell to 191 +/- 42 pg/ml after 6 months of treatment (p < 0.00001), and to 164 +/- 39 pg/ml after 12 months of treatment. Patients with very high PTH levels (> 800 pg/ml) suppressed less well than patients with lower levels (150-300 pg/ml). The therapeutic end point of PTH < 100 pg/ml was achieved in 23 patients (58%). The main side effect of the treatment was hypercalcemia, but this was symptomatic in only 3 patients, all above the age of 70 years. In summary, oral high-dose pulse therapy with 1 alpha-OH-D3 was highly effective in suppressing PTH levels in hyperparathyroid hemodialysis patients, and side effects were relatively few.