Effect of epoetin on HO-1 mRNA level and plasma antioxidants in hemodialysis patients.

OBJECTIVE: Patients with renal failure and undergoing hemo- (HD) or peritoneal dialysis are under oxidative stress which is thought to contribute to the long-term complications noted in this patient population. One effect of HD-induced oxidative stress is via red blood cell (RBC) membrane lipid peroxidation leading to RBC destruction and anemia. Interaction of this oxidative stress with epoetin (EPO) treatment to increase RBC number and Hb concentration remains unexplored. PATIENTS AND METHODS: This preliminary study used RT-PCR as well as colorimetric based assay approaches to evaluate the effect of EPO-alpha treatment on markers of oxidative stress in hemodialysis patients. Eighteen patients (12 males, 6 females, age range 45 - 68), were treated with EPO-alpha (Eprex) 50 UI/kg thrice weekly over an 8-month study period. Monocytes were isolated at baseline, then monthly thereafter, monocyte heme-oxygenase-1 (HO-1) and plasma Hb and antioxidant power (AOP) were determined. RESULTS AND CONCLUSIONS: Treatment with EPO increased Hb (9.4 +/- 0.7 g/dl to 10.9 +/- 0.5, mean +/- SD p < 0.001). In addition, both monocyte HO-1 mRNA (0.34 +/- 0.08 vs. 0.59 +/- 0.02 d.u. p < 0.001) and plasma AOP (1,379.8 +/- 175 micromol/l to 1,624 +/- 170, p < 0.04) increased. While AOP changes showed no correlation with other indices, increases in HO-1 and Hb were positively correlated using 2 different measures: delta Hb (peak Hb - baseline Hb) vs. delta HO-1 (peak HO-1 mRNA - baseline HO-1 mRNA) as well as delta Hb(5 months-baseline) vs. delta HO-1 (5 months - baseline) mRNA (r = 0.81, p < 0.001 and r = 0.76, p < 0.001; respectively). In conclusion, the increases upon EPO treatment of both HO-1 gene expression and plasma AOP as well as the significant correlation between delta Hb and delta HO-1 mRNA suggest that EPO treatment reduces oxidative stress via a combination of effects. These could potentially include effects on oxidative stress directly as well as effects on the levels and types of antioxidants present in plasma.

Chemiluminescent and Colorimetric Detection of Erwinia amylovora by Immunoenzymatic Determination of PCR Amplicons from Plasmid pEA29.

A molecular diagnostic technique (polymerase chain reaction enzyme-linked immunosorbent assay [PCR-ELISA]) for detection of Erwinia amylovora was developed. The protocol is based on the immunoenzymatic determination of PCR products. For in vitro amplification, we used previously published primers able to detect the cryptic plasmid pEA29, which is ubiquitous in E. amylovora. Amplicons were labeled with 11-digoxigenin (DIG)-dUTP during the amplification reaction, captured by hybridization to a biotinylated oligonucleotide in streptavidin-coated ELISA microplates, and then detected with anti-DIG-Fab'-peroxidase conjugated antibodies. The specificity of the assay was verified using E. amylovora strains from different host plants and geographical origins in addition to other plant-associated bacteria (either phytopathogenic or saprophytic) belonging to the genera Erwinia, Pseudomonas, and Agrobacterium. In detection threshold experiments with pure cultures, as few as 30 and 3 CFU/reaction tube were detected when the ABTS (colorimetric) and ECL (chemiluminescent) detection assays, respectively, were used. PCR-ELISA coupled with chemiluminescent detection was able to detect as few as 4 × 102 CFU/g of artificially infested pear twigs. The assay was further shown to be suitable for detection of E. amylovora in naturally infected plant organs, and the results were compared to those obtained using standard PCR assays with electrophoretic separation of amplicons.

Why the double-bag system still remains the best technique for peritoneal fluid exchange in continuous ambulatory peritoneal dialysis.

Since March 1979 (the Italian-French-Spanish meeting in Turin), we have been using the double-bag system for peritoneal fluid exchange in patients on continuous ambulatory peritoneal dialysis (CAPD). This technique, subsequently followed by many others because of the advantages to the patients, still represents the best tool in bag-exchange procedure, because it satisfies the following characteristics: single luer-lock connection; flush-before-fill; simple, safe, and aseptic manipulation; short training period; no carrying bag; good patient acceptance; and low incidence of exogenous peritonitis. In 13 years with 237 patients selected for double-bag treatment, we have observed an incidence of 1 episode of peritonitis every 26.6 patient-months. Few clinical CAPD-related complications like hypotension and alterations of Ca-P metabolism were observed, probably as a result of more personalized peritoneal fluid with high Na+ (136 mEq/L) and Ca2+ (3.5 mEqL) concentrations. In the meantime, we have also had available plasticizer-free bags, which eliminated one of the main risk factors in peritoneal sclerosis. The utilization of the plasticizer-free double-bag system, currently adopted by numerous other centers, still remains the best option from a clinical and psychological viewpoint of the patients on CAPD.

Effect of hypertriglyceridemia correction by omega-3 fatty acids on peritoneal transport in continuous ambulatory peritoneal dialysis patients.

Lipid abnormalities, both hypercholesterolemia and particularly hypertriglyceridemia (hyperTg), are common in long-term continuous ambulatory peritoneal dialysis (CAPD) patients. Hyperviscosity and rheological alterations have been proposed as major hemodynamic problems in hyperTg patients. The aim of this study was to evaluate whether a hyperTg correction by employing omega-3 fatty acids (omega-3) affects peritoneal transport. Six hyperTg (> 700 mg/dL) CAPD patients were treated with 2-3 g/day of omega-3 until normal Tg values were achieved. The assessment of peritoneal dialysis efficacy was performed by evaluating the peritoneal equilibration test (PET) before omega-3 supplementation, when normal Tg levels were reached, and 3 weeks after stopping therapy when hyperTg returned. When normal Tg levels were reached, a small but significant improvement of urea and creatinine D/P was noted: 0.85 +/- 0.05 versus 0.93 +/- 0.03 (p < 0.05) and 0.78 +/- 0.03 versus 0.86 +/- 0.05 (p < 0.05), respectively, with negative correlation between D/P of urea and Tg. These preliminary data demonstrate that a hyperTg correction with omega-3 may induce an increase in peritoneal transport of small molecules in CAPD.

Peritoneal sclerosis: role of plasticizers in stimulating interleukin-1 production.

The role of plasticizers (PLS) in inducing water flow inhibition and peritoneal sclerosis has been demonstrated in both in vivo and in vitro studies. Interleukin-1 (IL-1) has been shown to be a regulator of fibroblast proliferation as well as collagenase production. The aim of this study was to evaluate the role of PLS in stimulating mononuclear cell IL-1 secretion. Two cultures containing 10(3) cells/mL were obtained from 14 healthy subjects. One was used as the control, and the other was mixed with diethylhexylphthalate (DEHP) to reach a final concentration of 2.8 x 10(-3) M. After 4 hours the samples were centrifuged, and the supernatants were tested by radioimmunoassay for IL-1 alpha and IL-1 beta. The results showed a significant increase in both IL-1 alpha and IL-1 beta production in DEHP-stimulated cells in comparison to the controls: 42.6 +/- 15.4 versus 29.3 +/- 10 ng/L (p < 0.015) for IL-1 alpha, and 153.6 +/- 55 versus 113.6 +/- 32 ng/L (p < 0.03) for IL-1 beta In conclusion, PLS added to mononuclear cells were able to induce IL-1 secretion. This mechanism could be responsible, at least in part, for the development of peritoneal sclerosis. Thus the employment of plasticizer-free bags should be elective in peritoneal dialysis.

Combined hemodialysis-hemoperfusion in the treatment of secondary hyperparathyroidism of uremic patients.

Hyperparathyroidism and its related symptoms such as bone pain, soft-tissue calcifications and pruritus often get worse during dialysis treatment. We have treated 12 cases among 170 patients on regular dialysis by using coated charcoal (150 g/cartridge) in combination with standard hemodialysis. During a 6-month treatment period, without changing medical therapy and diet regime, the patients reported a marked relief from pruritus. Parathyroid hormone (PTH) levels changed from 552 +/- 86 to 364 +/- 62 pg/ml (p less than 0.001) compared to the pretreatment period, Plasma PO4(3-) changed in the same period from 6.9 +/- 1.8 to 4.6 +/- 1.5 mg/dl (p less than 0.005). The results obtained indicate a relationship between PTH, serum plasma PO4(3-) levels and pruritus. The mechanism which may be involved is that hemoperfusion removes PTH excess by absorption. Our treatment reducing PTH levels resulted in a marked relief from pruritus and other symptoms, suggesting that patients in this condition, before undergoing surgical parathyroidectomy, may be usefully treated with this therapeutic modality.

On-line bioelectric impedance during haemodialysis: monitoring of body fluids and cell membrane status.

We have measured by a computed integrated system (BIA 109, RJL AKERN) the changes of bio-impedance (BI) deriving from a tetrapolar system working on 800 microA, 50 kHz current, in 23 haemodialysed patients. Resistance (R) and reactance (Xc) have been continuously monitored during haemodialysis in each patient. Resistance was strictly inversely correlated to the decrease of body weight (r = 0.82). Also, Xc increased almost constantly. In most of the patients the increase of Xc was proportionally greater than R, resulting in an increase of phase angle (PA). However, Xc showed a transient decrease in response to seven severe symptomatic hypotensive episodes, whereas R maintained the increasing trend, causing a sharp reduction of phase angle. As Xc is an expression of storage of electrical charge by the cells acting as condensers, and phase angle quantifies the active capacitive component in relation to passive electrical resistance, these parameters may be important to evaluate cell membrane function. In fact, the univocal increase of R, Xc and phase angle observed during normal unevenful haemodialysis probably indicates improvement of cellular activities due to the depurative treatment. On the contrary, the transient reduction of Xc and phase angle observed during hypotensive crises may be an expression of cellular distress because of a too rapid ultrafiltration.

Removal of phosphate (Pi) by either bicarbonate dialysis or biofiltration in uremics.

The kinetics of extra and intracellular red blood cell (RBC) Pi and its removal by different therapeutic modalities were evaluated in 30 uremic patients over a 6 mo. period. Acetate hemodialysis alone, combined with hemoperfusion, or associated once a week with plasma-perfusion sessions using an activated bauxite cartridge, bicarbonate dialysis either in single pass or in recirculating system (40 L) and biofiltration, were the depurative treatments employed. The treatments with acetate buffer showed a temporary intracellular shift of Pi at the end of the sessions with post-dialytic plasma Pi rebound. This was not evident with bicarbonate buffer and biofiltration where acidosis was corrected better, and similarly during plasma perfusion treatment because blood pH remained unchanged. These findings may explain the better plasma Pi level at the end of our study with these later therapeutic models compared to acetate dialysis alone or combined with hemoperfusion. In these conditions Pi removal is limited by the correction of acidosis which implies acetate metabolism with ATP activation leading to a transient Pi intracellular influx and a subsequent efflux into the extracellular compartment.

Spontaneous ascites filtration and reinfusion (SAFR) in cirrhotic patients.

Since knowledge about the pathogenesis of hepato-renal syndrome (HRS) is incomplete, the therapy is empiric and supportive. While a number of specific therapeutic measures have been attempted, none has been proved to be of practical value. We describe a very simple technique for concentration of ascitic fluid obtained with spontaneous filtration by gravity. We have been treating with this new device 4 patients affected by HRS with ascites refractory to diuretics. We obtained a rapid disappearance of ascites and improvement in clinical condition. The simplicity and the ease of operation make this technique feasible for repeated chronic ambulatory treatment.