Desferrioxamine improves burst-forming unit-erythroid (BFU-E) proliferation in haemodialysis patients

Background: In chronic renal failure, desferrioxamine (DFO) may improve erythropoiesis independent from its aluminium (Al) chelating effect. The mechanism of this action is still unknown. Methods: To verify whether DFO influences proliferation of erythropoietic precursors, we studied 10 patients on chronic haemodialysis, free from malignancies or other haematological diseases, iron deficiency, bone marrow fibrosis, and Al toxicity. Al accumulation was excluded by the DFO test. Peripheral blood samples were drawn for basal burst-forming unit-erythroid (BFU-E) assay. Mononuclear cells were isolated by density gradient centrifugation with Ficoll-Hypaque, and incubated for 15 days with three different experimental conditions: (a) low-dose recombinant human erythropoietin (rHuEpo) (3 U/ml); (b) high dose rHuEpo, (30 U/ml); (c) both DFO (167 &mgr;g/ml) and rHuEpo (3 U/ml). We determined TIBC, transferrin, ferritin, reticulocytes, hypochromic erythrocytes, soluble transferrin receptor (sTR), haemoglobin (Hb), and haematocrit (Hct) at baseline and then every 14 days. Patients received 5 mg/kg DFO infused during the last hour of each dialysis session for 6 weeks; six patients remained in the study for an additional 6 more weeks. BFU-E assays were set up after 6 and 12 weeks of DFO therapy. Results: At baseline DFO had small effect on BFU-E proliferation (33.9±25 vs 30.4±25.9) and high-dose rHuEpo had a significant effect (45.15±27 vs 30.4±25.9, P<0.01). After 6 weeks of DFO therapy a significant increase in BFU-E proliferation was observed in all culture conditions (78.25±32 vs 30.45±25.9 standard culture, P<0.01; 110.9±30 vs45.15±27 high dose rHuEpo, P<0.01; 98.75±32 vs 45.15±27 DFO culture, P<0.01). Moreover, the increase in BFU-E proliferation was significant greater with DFO culture than standard culture (P<0.01). The same trend was found at the third BFU-E assay, performed in only six patients, when all culture conditions showed a further increase of erythroid precursor proliferation. However, the DFO culture was not significantly greater than the standard culture, while the high-dose rHuEpo was significantly greater than the DFO culture. Patients in group 1 (n=10), had a significant increase in reticulocytes (1.5±0.6 vs 1.72±0.3, P<0.01) and of hypochromic erythrocytes (HE) (5.6±5.1 vs 14.4±12.7, P<0.01), while sTR, Epo, Hb, and Hct were only minimally increased. Ferritin decreased significantly (448±224 vs 196±215, P<0.01) and TIBC and transferrin were unchanged. Conclusions: Thus DFO increases erythroid activity by BFU-E proliferation and increases reticulocytes in haemodialysis patients. Such an effect may be related to increased iron utilization. DFO may be a useful tool for anaemic patients with good iron stores and without Al overload. Key words: desferrioxamine; erythroid progenitors; erythropoiesis; haemodialysis      

Stability of access resistance during haemodialysis

Background: Access blood flow (Qac) is considered a useful indicator in the surveillance of haemodialysis access function. However, changes in Qac may be due to changes in blood pressure and/or to changes in access resistance (AR). Methods: Weekly readings of Qac, cardiac output, and arterial blood pressure measured early and late during haemodialysis were obtained in 11 patients for a period of 3 weeks. Qac was determined from thermodilution of extracorporeal blood returning to the patient with reversed placement of blood lines and by measurement of arterial and venous blood temperatures in the extracorporeal circulation. Data are given as mean±SE. Results: Qac dropped as mean arterial pressure (MAP) and total peripheral resistance (TPR) decreased, but increased when MAP and TPR increased. Linear regressions between the change in access flow and the change in MAP (&Dgr;Qac%=0.80*&Dgr;MAP%-1.6, r²=0.39), and the change in TPR (&Dgr;Qac=0.54*&Dgr;TPR%-9.2, r²=0.35) respectively, were significant (P<0.001). Whereas Qac significantly decreased (-8.4±3.3%, P<0.01) during the same treatment. AR remained unchanged (4.7±3.2%; P=NS). AR for all studies was 16.5±1.0 peripheral resistance units (1PRU=2.226 kPa min l^-1). There was a trend for resistance to increase (5.1±2.6%, P=NS) and for flow to decrease (-6.1±2.3%, P=NS) during the 3 weeks of the study. Conclusion: Qac measured during haemodialysis is variable and depends on haemodynamics, but AR is constant. AR is related to the physical structure of the peripheral access. Because of its intradialytic stability AR may be better suited as an indicator of access function.     

High dose enalapril impairs the response to erythropoietin treatment in haemodialysis patients

Background: The resistence to recombinant human erythropoietin (rHuEpo) therapy in haemodialysis (HD) patients has multifactorial aetiologies; erythropoietin insufficiency, dialysis insufficiency, iron deficiency, and secondary hyperparathyroidism. Angiotensin-converting enzyme (ACE) inhibitors induce anaemia in patients with essential hypertension, congestive heart failure, chronic renal insufficiency, and renal transplants. Data exist suggesting that ACE inhibitors impair erythropoiesis in HD patients. Therefore the aim of this study was to investigate the impact of enalapril on rHuEpo requirement. Methods: In the present prospective non-randomized study of 12 months, we compared the effects of enalapril and nifedipine on rHuEpo requirement in 40 hypertensive patients receiving rHuEpo for more than 6 months on maintenance haemodialysis. Twenty normotensive rHuEpo-dependent patients served as a control group. All patients with severe hyperparathyroidism or iron deficiency were excluded. The mean (±SD) haemoglobin concentration was >10 g/dl in all groups. The mean weekly rHuEpo dose increased in the enalapril group (P<0.0001 vs before) and remained constant in the nifedipine and control groups (P=NS vs before). Statistically, there was no differences with regard to iPTH levels, dialysis parameters, iron status, and underlying renal diseases among all groups. Conclusion: High-dose enalapril increases rHuEpo requirement and should be reserved for dialysis patients with hypertension uncontrollable with other antihypertensive medications or dialysis patients with cardiac failure.     

The impact of early normalization of haematocrit by erythropoietin on renal damage in the remnant kidney model

Background: Correction of anaemia in moderate to advanced renal failure is still a matter of debate because of postulated detrimental effects of erythropoietin on the progression of renal damage. Methods: The renal effects of early normalization of haematocrit (Htc) by erythropoietin (rHuEpo) were investigated from the time of 5/6 nephrectomy up to 8 weeks post-intervention in three groups of remnant kidney model rats: untreated controls (CON), rats receiving 100 UI/kg body-wt of rHuEpo i.p. twice a week (EPO), and rats receiving rHuEpo in which periodic phlebotomies maintained Htc similar to the value of the control group (PHL). The latter group was included to evaluate the direct effects of rHuEpo on renal damage, i.e. independent from Htc correction. Results: Two weeks after renal ablation (basal), Htc decreased in CON and PHL (from 49.3±1.4% to 43.2±1.1, P<0.05 and from 49.6±1.1 to 43.3±1.5% P<0.05 respectively), while it remained consistently normal in EPO rats (78.9±1.2% to 48.8±1.5%, P<0.05 vs other groups). Thereafter Htc did not change throughout the remaining period in all groups. At the end of the study, with respect to basal, resting blood pressure increased significantly by the same extent in CON (+13±2%) and EPO rats (+15±5%), while it remained constant in PHL rats. Notably, creatinine clearance significantly decreased in CON (-53±8% vs basal) and EPO (-38±8% vs basal), while it did not change in PHL rats. Likewise the degree of proteinuria as well as renal morphologic alterations and glomerular hypertrophy/sclerosis was similar in CON and EPO rats, and was significantly more severe than in the phlebotomized group. The only difference detected between CON and EPO group was the greater mesangial hypercellularity in rHuEpo-treated rats. Conclusion: In uraemic rats, chronic treatment with rHuEpo aimed at normalization of Htc beginning the early stage of renal failure does not inevitably account for a rise in systemic blood pressure. In addition, neither erythropoietin per se nor the correction of haematocrit accelerates the progression of renal damage when blood pressure remains constant.     

Cytokines and adhesion molecules in renal vasculitis and lupus nephritis

Background: Plasma levels of some pro-inflammatory cytokines and soluble adhesion molecules have been suggested to be useful parameters to assess the activity of antineutrophil cytoplasmic antibody (ANCA)-positive vasculitis and lupus nephritis. We hypothesized that the renal activity of these diseases is better reflected by the urinary excretion and fractional excretion of these molecules. Methods: Plasma levels and urinary excretion of tumour necrosis factor-&agr; (TNF-&agr;), interleukin (IL)-6, IL-8, and the soluble cell adhesion molecules sICAM-1 and sVCAM-1 were measured by enzyme-linked immunosorbent assay (ELISA) in 15 patients with ANCA-positive renal vasculitis (eight active, ANCA-A; six in remission, ANCA-R), six patients with active lupus nephritis (LN), 15 patients with IgA nephropathy (IgAN) and nine healthy subjects. Fractional excretion of selected cytokines and adhesion molecules was also calculated. Results: Patients with ANCA-A had increased urinary excretion and fractional excretion of TNF-&agr; (9.27±3.19% vs 0.58±0.02%, P<0.01), IL-6 (120.79±65.83% vs 1.89±0.34%, P<0.01) and increased fractional excretion of IL-8 (23.34±6.38% vs 2.56±1.07%, P<0.01) and sVCAM-1 (0.81±0.33% vs 0.03±0.02%, P<0.01) compared with controls. Urinary excretion of TNF-&agr; and IL-6 and fractional excretion of TNF-&agr;, IL-6 and IL-8 were higher in ANCA-A than in ANCA-R. Patients with LN had increased plasma TNF-&agr; (20.52±2.01 pg/ml vs 12.33±0.23 pg/ml, P<0.05) and sVCAM-1 (1537.88±276.36 ng/ml vs 692.26±44.42 ng/ml, P<0.05) and increased urinary excretion of TNF-&agr; (2.81±0.51 &mgr;g/mol creat vs 0.98±0.05 &mgr;g/mol creat, P<0.01), IL-8 (35.78±14.03 &mgr;g/mol creat vs 12.46±5.19 &mgr;g/mol creat, P<0.05) and sVCAM-1 (48.98±20.20 &mgr;g/mol creat vs 2.92±1.35 &mgr;g/mol creat, P<0.01) compared with controls. Patients with IgAN had, in comparison with controls only increased plasma TNF-&agr; (18.10±0.57 pg/ml vs 12.33±0.23 pg/ml, P<0.05). Conclusions: Urinary excretion and fractional excretion, but not plasma levels of selected proinflammatory cytokines (TNF-&agr;, IL-6 and IL-8) were increased in patients with active ANCA-positive renal vasculitis, but not in ANCA positive vasculitis in remission. These parameters may be useful to monitor the activity of this disease.     

Effect of endothelin-converting enzyme inhibitors on big endothelin-1 induced contraction in isolated rat basilar artery

Summary.  Introduction: The aim of this study was to investigate whether blocking functional endothelin-converting enzyme (ECE) activity may offer a new approach to inhibit the development of cerebral vasopasm after subarachnoid hemorrhage (SAH) by preventing transformation of big Endothelin-1 (big ET-1) to vasoactive Endothelin-1 (ET-1).  Methods: In vitro, the effect of potential ECE inhibitors was determined by measurement of isometric contractions, induced by big ET-1, in isolated rat basilar arteries. Endothelium intact (E+) and de-endothelialized (E−) segments were examined after pre-incubated with the putative ECE inhibitors: Phosphoramidon (10⁻⁴ M), Captopril (10⁻³ M and 10⁻⁴ M) and [²²D-Val] big ET-1 (16–38) (10⁻⁵ M and 10⁻⁶ M).  Results: Application of 10⁻⁴ M Phosphoramidon resulted in a statistically significant decrease in big ET-1 induced contraction in endothelium intact (E+) and de-endothelialized (E−) segments; 10⁻³ M Captopril in E− segments caused a statistically significant inhibitory effect; 10⁻⁴ M and 10⁻³ M Captopril in E+ segments showed no statistically significant effect; 10⁻⁵ M and 10⁻⁶ M [²²D-Val] big ET-1 (16–38) in E− segments produced no statistically significant effect. The application of 10⁻⁶ M [²²D-Val] big ET-1 (16–38) in E+ segments caused increased contractions as shown by the shift to the left of the concentration-effect curve (CEC).  Conclusion: The present study indicates the existence of functional ECE activity in rat basilar artery, which is different in the endothelium and the smooth muscle layer. This ECE-activity could be inhibited by Captopril and Phosporamidon, suggesting a potency for prevention and therapy of cerebral vasospasm. However, the structural analogue of big ET-1, [²²D-Val] big ET-1 (16–38), was ineffective in reducing big ET-1 induced vasoconstriction and rather increased contraction in E+ vessels. Therefore further studies of the biochemical nature of the functional relevant cerebrovascular ECE activity are required for better understanding and development of other efficient ECE inhibitors. Published online October 31, 2002 Correspondence: Michael Zimmermann M.D., Ph.D., Department of Neurosurgery, University of Frankfurt/Main, Schleusenweg 2-16, 60528 Frankfurt, Germany.     

Haemodynamic changes induced by the correction of anaemia by erythropoietin: role of antiplatelet therapy

BACKGROUND.: In a restrospective study, antiplatelet therapy has been shownto be associated with a decreased incidence of erythropoietin-inducedhypertension. In order to ascertain the role of antiplateletdrugs in the haemodynamic response to the correction of anaemiaby rHuEpo, 18 patients on chronic haemodialysis who startedrHuEpo therapy were prospectively studied. METHODS.: The subjects were randomly assigned to receive or not, one ofthe following antiplatelet drugs: ditazole (3 patients), ticlopidine(3 patients) or aspirin plus dipyridamole (3 patients). Cardiacindex (CI) by echo-Doppler, total peripheral resistance (TPR)and mean arterial pressure (MAP) were determined at baseline10 and 20 weeks following the initiation of rHuEpo therapy.rHuEpo therapy was administered subcutaneously at the same dose(40 U/kg thrice weekly) during the first 10 weeks. Ten uraemicpatients on haemodialysis who had never received rHuEpo therapyserved as the control group. RESULTS.: One patient in the group without antiplatelet drugs discontinuedthe study due to the development of severe hypertension after12 weeks on rHuEpo therapy. There were no significant differencesin the haemodynamic parameters at baseline. At 10 weeks, MAPwas higher in patients without than with antiplatelet drugsor controls untreated with rHuEpo (128.5 ± 28 versus100.6 ± 13.5 versus 98.7 ± 14 mmHg respectively,P = 0.0047), TPR was also higher in patients without antiplateletdrugs than in the 2 other groups (1919 ± 433 versus 1576± 359 versus 1418 ± 324 din.seg.cm^–5 m²respectively, P = 0.0231), but CI did not differ among the threegroups. At 20 weeks, MAP was still higher in patients withoutantiplatelet drugs than in patients with antiplatelet drugsor controls not on rHuEpo therapy respectively (112.9 ±24.6 versus 91.0 ± 9.0 versus 101.7 ± 14.1 mmHgrespectively, P = 0.075), but at this stage TPR and Cl did notdiffer among the three groups. CONCLUSIONS.: These data reinforce the previous observation that antiplatelettherapy may prevent the development of rHuEpo-induced hypertension.     

Norepinephrine-induced blood pressure rise and renal vasoconstriction are not attenuated by enalapril treatment in microalbuminuric IDDM

Background: In non-diabetic subjects, an attenuated systemic norepinephrine (NE) responsiveness may contribute to the mechanisms of action of angiotensin-converting enzyme (ACE) inhibitor treatment. We determined whether ACE inhibitor treatment influences systemic and renal haemodynamic responsiveness to exogenous NE, as well as urinary albumin excretion during NE, in microalbuminuric insulin-dependent diabetic (IDDM) patients, representing a patient category that benefits by strict blood pressure control. Methods: In seven microalbuminuric IDDM patients, systemic and renal responsiveness to NE, infused at individually determined threshold (&Dgr;mean arterial pressure (MAP)=0 mmHg], 20% pressor (&Dgr;MAP=4 mmHg) and pressor (&Dgr;MAP=20 mmHg) doses, were compared before and after 8 weeks treatment with enalapril, 10 mg daily. Blood glucose was clamped at 5 mmol/l and insulin was infused at 30 mU/kg/h. Results: Enalapril decreased MAP (P<0.05) and microalbuminuria (P<0.05), whereas effective renal plasma flow (ERPF) increased (P<0.01) and glomerular filtration rate remained unaltered. The filtration fraction tended to decline (P=0.09). The ACE inhibitor-induced fall in MAP disappeared at NE pressor dose, and the overall mean increase in MAP in response to NE was even higher with than without enalapril (P<0.05). After enalapril, the ERPF remained higher at all NE doses (P<0.05), but the magnitude of the NE-induced fall in ERPF was not altered by ACE inhibition treatment. Overnight urinary albumin excretion fell with ACE inhibition (P<0.05), but this effect was not seen during NE infusion. The angiotensin II/active renin ratio and serum aldosterone levels remained lower with enalapril at all NE doses (P<0.05). <It>Conclusions:Enalapril does not attenuate systemic and renal vascular responsiveness to exogenous NE in microalbuminuric IDDM despite adequate inhibition of the renin-angiotensin-aldosterone system. These findings suggest that the effect of NE on vasoconstriction is not counteracted effectively by ACE inhibition treatment alone.     

Renal artery stenosis: evaluation with colour duplex ultrasonography

Background: Detection of renal artery stenoses (RAS) by means of duplex Doppler ultrasound with direct scanning of the main renal arteries is subject to numerous limitations. Using semiquantitative analysis of the Doppler curve, which can be recorded from intrarenal arteries, it is possible to detect RAs unaffected by the problems of direct Doppler scanning of the renal arteries. Method: Both angiography of the renal arteries and colour duplex ultrasonography (US) of the intrarenal vessels (interlobar arteries) were performed in 214 patients (53.2±15.1 years) with severe arterial hypertension. Angiography was used as 'gold standard' in the diagnosis of RAS and the Doppler results were compared with the subsequent findings on angiography. At angiography, the reduction of diameter >70% was assessed as haemodynamically effective RAS. For the duplex Doppler diagnosis of RAS the following parameters were calculated: (a) resistive index (RI) of each kidney, and (b) side-to-side differences of the resistive indices (&Dgr;RI) between the right and left kidney. Results: Angiography demonstrated 59 RAS (>70%) in 53 patients, including six with bilateral RAS. By means of duplex US we found a significant difference of RI between kidneys with RAS (0.48±0.11) and without RAS (0.63±0.08; P<0.001). In addition, a significant difference of the &Dgr;RI was noted in patients with RAS (24.4%±12.5%) and the controls without RAS (3.6%±2.7%). Using a combination of both RI and &Dgr;RI, threshold values of RI=0.45 resp. &Dgr;RI=8% yields a sensitivity of 92.5% and a specificity of 95.7% in the detection of haemodynamically effective RAS. Conclusion: Colour duplex US with calculation of the RI and &Dgr;RI of intrarenal arteries is a valuable non-invasive test assessing the haemodynamic effects of RAS. Low costs and safety support the use of the Doppler technique in screening for renovascular disease.     

Subacute infusion of physiological doses of parathyroid hormone raises blood pressure in humans

Background. Acute administration of parathyroid hormone (PTH) causes vasodilation and blood pressure decrease in experimental animals. This effect contrasts with the putative role of secondary hyperparathyroidism in the pathogenesis of hypertension of patients with renal failure. Uraemia is characterized by insulin resistance and hyperinsulinaemia. We therefore investigated whether subacute administration of physiological doses of human 1,34-PTH affects blood pressure under conditions of controlled insulin levels (euglycaemic clamp technique) in humans. Methods. In a double-blind cross-over design 10 healthy male subjects received, on two occasions, in random order, for 2 h, either a sham infusion or an infusion of 200 units of 1,34-PTH. Results. Mean ionized calcium concentration increased significantly (P <0.01) within the normal range during euglycaemic hyperinsulinaemia, both with sham infusion (from 1.25 ± 0.04 to 1.29 ± 0.02 mmol/l) and with infusion of 1,34-PTH, but the increase was more marked with 1,34-PTH administration (from 1.26 ± 0.05 to 1.33 ± 0.07). In addition, mean platelet intracellular calcium concentration (by fluorescence spectroscopy) was unchanged with sham infusion (49.9 ± 4.1 versus 50.3 ± 5.0 nmol), but increased significantly (P <0.05; paired t-test) after 1,34-PTH infusion (from 49.8 ± 5.0 to 52.8 ± 5.8). The infusion of 1,34-PTH resulted in a significant (P <0.01) increase in mean MAP (from 84 ± 5 to 88 ± 5 mmHg) as compared with sham infusion (85 ± 4 versus 86 ± 4). The intra-individual changes in intracellular calcium concentration (&Dgr;[Ca²⁺]I) were significantly correlated to the changes in mean MAP (&Dgr;MAP) (r = 0.87, P <0.001). In contrast to blood pressure, insulin sensitivity was not affected by 1,34-PTH infusion (M-value: 7.2 ± 1.6 mg/kg per min) as compared with sham infusion (7.3 ± 1.4). Conclusion. Subacute administration of physiological doses of parathyroid hormone under hyperinsulinaemic conditions significantly affects intracellular calcium and blood pressure in healthy subjects, but does not affect the action of insulin.     

Effects of rHuEpo on cellular proliferation and endothelin-1 production in cultured endothelial cells

BACKGROUND.: Although elevation of blood pressure is considered to be themain adverse effect under rHuEpo therapy in haemodialysis patients,the precise mechanism remains obscure. The direct effect ofrHuEpo on endothelial cells (EC) has been suggested as one ofcontributing factors of rHuEpo-induced hypertension. METHODS.: EC were incubated with various concentrations of rHuEpo (0,1000, 5000, 10000 mU/ml) for up to 7 days, and cell numbers,DNA and protein synthesis by EC and supernatant concentrationsof immunoreactive endothelin-1 (ET) were determined by haemocytometer,³H-thymidine and ³H-leucine incorporation, and RIA, respectively.The effect of rHuEpo on EC proliferation was confirmed by anti-rHuEporabbit antiserum. The effect of cycloheximide or actinomycinD was also examined on the increase in ET production by rHuEpo. RESULTS.: rHuEpo dose-dependently stimulated the proliferation of culturedEC, and this proliferative effect was inhibited by anti-rHuEporabbit antiserum. DNA and protein syntheses by EC were alsoincreased by rHuEpo. The supernatant concentrations of ET culturedwith rHuEpo at 5000 mU/ml or more showed significantly greatervalues than those without rHuEpo and the increase in ET in thesupernatants of media containing 5000 mU/ml rHuEpo was inhibitedby incubation with 0.2 µg/ml actinomycin D or 10 µg/mlcycloheximide. Further, rHuEpo increased DNA synthesis by ECwhich had been cultured in E-BM medium containing 0.5 or 2%FBS for 3 h and which were recultured in E-BM medium containing5% FBS for 15 h. CONCLUSIONS.: rHuEpo directly stimulates EC proliferation as a competencefactor, and it also accelerates endothelin-1 production in associationwith stimulation of DNA and protein syntheses by EC.     

The haematopoietic effect of recombinant human erythropoietin in haemodialysis is independent of the mode of administration (i.v. or s.c.)

Background: Previous studies comparing intravenous (i.v.) and subcutaneous (s.c.) administration of recombinant human erythropoietin (rHuEpo) often did not achieve optimal iron reserve, were restricted to a limited follow-up period (not allowing equilibration) and/or did not exclude the role of other confounding factors. In addition all papers focused on the conversion from i.v. to s.c. Methods: In this study, 30 equilibrated patients on s.c. rHuEpo were randomized into two groups, one converting to i.v. after 6 months of follow-up and one remaining on s.c. rHuEpo. In both groups rHuEpo was administered three times weekly. Only patients completing a further 6 months follow-up were considered for statistical evaluation. Serum ferritin was targeted at 200 ng/ml and haematocrits between 28 and 35% were pursued. Results: The average haematocrit levels before conversion were 31.9±1.1% in the conversion group and 31.4±1.6% at the same time point in the non-conversion group (P-NS). After 6 months haematocrits were 31.5±0.5% in the conversion group and 31.1±0.9% in the non-conversion group (P=NS). Ferritin concentration in the conversion group was 219±49 ng/ml before and 230±83 mg/ml after the conversion. For the non-conversion group ferritin was 224±25 ng/ml and 236±52 ng/ml respectively (P=NS). The weight-standardized average rHuEpo dose per injection remained the same in the conversion group before and after conversion (44.0±1.8 U/kg/injection vs 45.4±4.7 U/kg/injection) P=NS). In the non-conversion group the corresponding rHuEpo doses were 32.9±4.2 U/kg/injection and 39.6±7.0 U/kg/injection respectively (P=NS). There were no differences in serum PTH, aluminium, vitamin B12, folic-acid levels, and intake of co-trimoxazole, ACE inhibitors or theophylline. Conclusion: No changes in rHuEpo dose wee observed after conversion from s.c. to i.v. There were no significant differences between the conversion and non-conversion group. These results are in contrast to some earlier studies suggesting lower rHuEpo requirements in case of s.c. administration. Key words: anaemia; erythropoietin; intravenous erythropoietin; iron; subcutaneous erythropoietin      

Does long-term treatment of renal anaemia with recombinant erythropoietin influence oxidative stress in haemodialysed patients?

Background. Patients with end-stage renal failure undergoing haemodialysis (HD) are exposed to oxidative stress. Increased levels of malondialdehyde (MDA) were demonstrated in plasma of uraemic patients, indicating accelerated lipid peroxidation (LPO) as a consequence of multiple pathogenetic factors. The aim of our investigation was to examine the role of renal anaemia in oxidative stress in HD patients. Methods. MDA and 4-hydroxynonenal (HNE) were measured in three groups of patients undergoing HD: group I comprised eight patients with a blood haemoglobin (Hb) <10 g/dl (mean Hb=8.1±1.3 g/dl), and group II were eight patients with a Hb <10 g/dl (mean Hb=12.4±1.9 g/dl); none of these 16 patients had been treated with human recombinant erythropoietin (rHuEpo). Group III comprised 27 patients with a mean Hb of 10.5±1.6 g/dl after long-term rHuEpo treatment. Results. Mean plasma concentrations of both MDA and HNE were significantly higher (P<0.0001) in all 43 HD patients than in 20 healthy controls (MDA 2.85±0.25 vs 0.37± &mgr;M, HNE 0.32± vs 0.10±0.01 &mgr;M). Comprising the three groups, it was shown that HD patients with a Hb <10 g/dl had significantly higher plasma levels of LPO products (MDA 3.81±0.86 &mgr;M, HNE 0.45±0.07 &mgr;M) than HD patients with a Hb > 10 g/dl (MDA 2.77±0.58 &mgr;M, HNE 0.25±0.05 &mgr;M), and than HD patients treated with rHuEpo (MDA 2.50±0.12 &mgr;M, HNE 0.29±0.03 &mgr;M). Furthermore, an inverse correlation between plasma concentration of LPO products and haemoglobin levels was seen (r=0.62, P<0.0001). Conclusion. Radical generation in HD patients might be caused in part by renal anemia itself. Treatment with rHuEpo may decrease radical generation effectively in HD patients due to the increase in the number of red blood cells and blood haemoglobin concentration. Keywords: erythropoietin; haemodialysis; HNE; lipid peroxidation; MDA; renal anaemia      

[D-Val22]big ET-1[16–38] inhibits endothelin-converting enzyme activity: a promising concept in the prevention of cerebral vasospasm

The aim of this study was to investigate whether the blocking of endothelin-converting enzyme (ECE) activity offers a new approach to inhibiting the development of cerebral vasospasm after subarachnoid hemorrhage (SAH) by preventing transformation of big endothelin-1 (big ET-1) to vasoactive endothelin-1 (ET-1). The effect of potential ECE inhibitors was determined in vitro by measurement of isometric contractions, induced by big ET-1, in isolated rat basilar arteries. Intact and de-endothelialized endothelium (E+ and E-, respectively) segments were examined after preincubation with the putative ECE inhibitors: phosphoramidon (10^–4 M), and [²²D-Val]big ET-1[16–38] (10^–5 M and 10^–6 M). Additionally, the effect of [D-Val²²]big ET-1[16–38] was investigated in rabbits after intracisternal application in order to inhibit the contraction of the basilar artery induced by (2×10^–6 M) big ET-1. Application of 10^–4-M phosphoramidon resulted in a statistically significant decrease in big ET-1-induced contraction in E+ and E- segments; 10^–5-M and 10^–6-M [²²D-Val]big ET-1[16–38] in E- segments produced no statistically significant effect. The application of 10^–6-M [²²D-Val]big ET-1[16–38] in E+ segments caused increased contractions, as shown by the shift to the left of the concentration-effect curve (CEC). In the rabbit group pretreated with [D-Val²²]big ET-1[16–38] (2×10^–5 M) (n=8), the angiographically measured diameter of the basilar artery increased from 0.63±0.12 mm to 0.66±0.12 mm. In the control group (n=8), this diameter decreased from 0.71±0.13 mm to 0.57±0.15 mm. This corresponded to an increase in vessel diameter of 5.24±9.89% in the treatment group and a decrease of 19.54±15.81% in the control group (P=0.002). The present study indicates the existence of functional ECE activity in rat basilar artery, which differs in the endothelium and the smooth muscle layer. These results demonstrate that [D-Val²²]big ET-1[16–38] has a potent ECE-inhibitory effect, preventing cerebral vasospasm in rabbit basilar artery by inhibiting the transformation of big ET-1 to vasoactive ET-1 after intracisternal application in vivo, whereas no inhibitory effect was detectable in rat basilar artery in vitro. Therefore, further studies of the biochemical nature of cerebrovascular ECE activity are required.     

Transfusion and recombinant human erythropoietin requirements differ between dialysis modalities

Background: Before the routine use of recombinant human erythropoietin (rHuEpo), patients dialysed by peritoneal dialysis (PD) received fewer blood transfusions than patients on haemodialysis (HD). We compared transfusion practices in these groups now that the use of rHuEpo has become standard, while controlling for variables known to influence anaemia of end-stage renal disease (ESRD). Maintenance rHuEpo doses were also compared. Methods: Data were examined for 157 HD and 126 PD patients during a 2-year period. Potential confounders included age, gender, albumin, iron deficiency, parathyroid hormone (PTH), underlying renal disease, cormorbid illness, renal transplant, dialysis adequacy and duration. An intent-to-treat analysis was used, with sensitivity analyses to account for change in treatment and transplant. Results: Mean haemoglobin (Hb) was not different (10.47 g/dl for HD, 10.71 G/DL for PD; P=0.45). Mean monthly transfusion rate was higher for HD (0.47 units per month vs 0.19; P<0.01). More HD patients received at least one transfusion (52.9 vs 40.9%; P<0.01). The maintenance rHuEpo dose was higher for HD (7370 U/week vs 5790 U/week; P=0.01). The only factors associated with risk of being transfused were dialysis duration and mode of dialysis (less risk for PD, odds-ratio 0.57; 95% confidence interval 0.35-0.92). Conclusions: Despite the routine use of rHuEpo, HD patients received more blood and rHuEpo than PD patients to achieve the same Hb. No patient factors were identified to account for this difference. The use of fewer transfusions and less rHuEpo in PD represents an advantage over HD in terms of both cost and safety.     

内皮素、内皮素转化酶在自体静脉移植术后内膜增生中的作用

目的探讨内皮素 1(endothelin 1,ET 1)及内源性内皮素转化酶 (endothelinconvertingenzyme ,ECE)在自体静脉移植术后内膜增生中的作用。 方法建立自体静脉移植模型 ,采用反转录聚合酶链反应 (RT PCR)和免疫组织化学方法 ,对ECE、ET 1和增殖细胞核抗原 (proliferatingcellnuclearantigen ,PCNA)在移植静脉的表达情况进行分析。 结果移植术后 6h ,吻合口处静脉中膜即出现PCNA阳性平滑肌细胞 ,并随着时间推移逐渐增高 ,在 1～ 2周左右达到高峰。 2周后 ,PCNA阳性SMC开始减少 ,于术后 8周趋于平稳。移植血管ET 1、PCNA主要在平滑肌细胞中表达。在mRNA水平 ,随着移植术后时间的推移 ,ECE的表达逐渐增高 ,术后 1～ 2周达到高峰 ,在 8周左右趋于平稳 ,ET 1与ECE密切相关 (r =0 975 )。结论ET 1、ECE的动态变化过程具有相关性 ,ECE可能通过ECE→ET 1→SMC通路促进内膜增生。     

Chronic vasopeptidase inhibition restores endothelin-converting enzyme activity and normalizes endothelin levels in salt-induced hypertension.

BACKGROUND: Vasopeptidase inhibition (VPI) represents a new therapeutic principle including both inhibition of angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP). The present study investigated the effect of the vasopeptidase inhibitor omapatrilat on endothelin-1 (ET-1)-mediated vascular function in salt-induced hypertension. METHODS: Dahl salt-sensitive rats (n=6/group) on standard or salt-enriched (4% NaCl) chow were treated for 8 weeks with either omapatrilat (36+/-4 mg/kg/day), captopril (94+/-2 mg/kg/day) or placebo. Aortic and renal artery segments were isolated and suspended in organ chambers for isometric tension recording. Functional endothelin-converting enzyme (ECE) activity was assessed in native segments and after preincubation with omapatrilat. Furthermore, vascular ECE protein levels as well as plasma and tissue ET-1 levels were determined. RESULTS: The increase in systolic blood pressure of salt-fed rats was prevented by omapatrilat and captopril to a comparable degree. In salt-induced hypertension, functional ECE activity (calculated as the ratio of the contraction to big ET-1 divided by the contraction to ET-1) in renal arteries (0.46+/-0.05) and in aorta (0.68+/-0.05) was reduced as compared with control animals (0.9+/-0.05 and 0.99+/-0.04, respectively; P<0.05). While omapatrilat in vitro blunted the response to big endothelin-1 (big ET-1) and diminished ECE activity further (P<0.01 vs native segments), chronic treatment with omapatrilat in vivo restored contractions to ET-1 (120+/-6%) and big ET-1 (98+/-9%) in renal arteries, and therefore normalized renovascular ECE activity. In addition, omapatrilat normalized plasma ET-1 concentrations (12.9+/-1.2 vs 16.6+/-1.4 pg/ml on high salt diet; P<0.05) and renovascular ECE protein levels. CONCLUSIONS: In salt-induced hypertension, vasopeptidase inhibition restores alterations in the endothelin system, such as renovascular ECE activity and responsiveness to ET-1 and big ET-1 with chronic but not acute in vitro application. Thus, the beneficial effects of vasopeptidase inhibition may reflect a resetting of cardiovascular control systems and therefore may be particularly suited to treat hypertension and heart failure.     

Urinary excretion of endothelin-1 in children with absorptive idiopathic hypercalciuria

Urinary excretion of endothelin-1 (ET-1) and plasma ET-1 were measured in 21 children with absorptive idiopathic hypercalciuria (AIH) and 22 controls. The absorptive type of idiopathic hypercalciuria was determined by a calcium loading test. Daily urinary excretion of ET-1 and urinary ET-1/creatinine ratio were significantly increased (P=0.005 and P=0.007, respectively) in patients with AIH (9,274±6,444 pg/24 h and 14.04±9.52 pg/mg, respectively) compared with controls (4,699±2,120 pg/24 h and 7.36±4.71 pg/mg, respectively). Plasma ET-1 levels were significantly lower in patients with AIH (0.84±0.64 pg/ml) than in controls (1.54±0.54 pg/ml, P=0.0001). In conclusion, patients with AIH had increased urinary ET-1 excretion and decreased plasma ET-1 levels. This is most likely due to the decreased reabsorption of ET-1 in the renal tubule and increased renal production.     

Endothelin-1 in children with chronic renal failure

Endothelin-1 (ET-1) was meansured after extraction from plasma of normal adults (5.9±1.9 pg/ml,n=22), normal children (7.1±1.86 pg/ml,n=29), nonhaemodialysed children with chronic renal failure (CRF) (11.1±1.8 pg/ml),n=10), renal graft recipients (9.5±3.4 pg/ml,n=37), haemodialysed children 24 h after a haemodialysis session (20.02±10.9 pg/ml,n=26) and haemodialysed children before and after a haemodialysis session (15.31±10.6 and 13.8±8.5 respectively,n=14). A sensitive and specific radioimmunoassay was used. ET-1 was significantly higher in non-haemodialysed CRF children and in renal graft recipients than in normal children (P<0.001 andP<0.01, respectively) and significantly higher in haemodialysed children when compared with normal children, non-haemodialysed CRF children and renal graft recipients (P<0.001). ET-1 concentrations were similar in normal children and normal adults. ET-1 was inversely correlated with glomerular filtration rate in non-haemodialysed CRF children (r=–0.39,P<0.01) and positively correlated with extracellular volume in haemodialysed children (r=0.435,P<0.03). After haemodialysis, ET-1 increased in 6 and decreased in 8 of the 14 children studied before and after a haemodialysis session.