Plasma Met-Enkephalin and Leu-Enkephalin in Chronic Renal Failure

Plasma met-enkephalin and leu-enkephalin has been measured ina group of 28 patients with chronic renal failure, to discoverwhether these opioids are affected by standard haemodialysisand haemofiltration. Met-enkephalin was markedly higher (P<0.001) in uraemic patientsthan in a group of 13 normal subjects, and was directly relatedto plasma creatinine (r=0.60; P<0.01) and to plasma urea(r=0.36; P=0.06). In contrast, leuenkephalin was suppressedin uraemic patients (P<0.001). Met-enkephalin fell slightlybut significantly (P<0.02) after both haemodialysis and haemofiltration;however, on average it remained at concentrations four timeshigher than normal. No changes in plasma leu-enkephalin wereobserved after haemodialysis and haemofiltration. The cause(s)of the altered plasma concentrations of these opioid substancesremains to be clarified.     

Pentosidine, carotid atherosclerosis and alterations in left ventricular geometry in hemodialysis patients

OBJECTIVE: To assess the relationship between advanced glycation end products (AGE) and cardiovascular damage in end-stage renal diseases. METHODS: Ninety-one hemodialysis patients who had been on dialysis treatment for at least six months were recruited for the study. Each patient underwent echocardiography and an echo-color Doppler study of the carotid arteries. We measured plasma pentosidine and related it to intima media thickness, atherosclerotic plaques and parameters of left ventricular geometry. RESULTS: Pentosidine was higher in patients treated by low-flux dialysis (31.0+/-16.6 pmol/mg protein) than in those treated by high-flux dialysis (25.4+/-7.6 pmol/mg protein), but this difference was of marginal statistical significance (P=0.08). On multivariate analysis, plasma IgG (beta=0.24, P=0.02) was the only independent correlate of plasma pentosidine. Intima media thickness and the number of atherosclerotic plaques were unrelated to plasma pentosidine. Mean wall thickness (beta=0.18, P<0.05), relative wall thickness (beta=0.20, P<0.05) and left ventricular end-diastolic volume (beta= -0.23, P<0.01) were independently related to plasma pentosidine. CONCLUSIONS: Pentosidine, a reliable marker of "carbonyl stress", is unrelated to intima media thickness and to the number of atherosclerotic plaques, but it is related to alterations in heart geometry. These data suggest that the effect of carbonyl stress on the cardiovascular system is complex and that the effects of AGE on the heart may be dissociated from those on the arterial system.     

Renal accumulation of pentosidine in non-diabetic proteinuria-induced renal damage in rats.

BACKGROUND: Advanced glycation end-products (AGEs) contribute to the pathogenesis of diabetic glomerulopathy. The role of AGEs in non-diabetic renal damage is not well characterized. First, we studied whether renal AGE accumulation occurs in non-diabetic proteinuria-induced renal damage and whether this is ameliorated by renoprotective treatment. Secondly, we investigated whether renal AGE accumulation was due to intrarenal effects of local protein trafficking. METHODS: Pentosidine was measured (by high-performance liquid chromatography) in rats with chronic bilateral adriamycin nephropathy (AN), untreated and treated with lisinopril. Age-matched healthy rats served as negative controls. Secondly, we compared renal pentosidine in mild proteinuric and non-proteinuric kidneys of unilateral AN and in age-matched controls at 12 and 30 weeks. Intrarenal localization of pentosidine was studied by immunohistochemistry. RESULTS: Renal pentosidine was elevated in untreated AN (0.14+/-0.04 micromol/mol valine) vs healthy controls (0.04+/-0.01 micromol/mol valine, P<0.01). In lisinopril-treated AN, pentosidine was lower (0.09+/-0.02 micromol/mol valine) than in untreated AN (P<0.05). In unilateral proteinuria, pentosidine was similar in non-proteinuric and proteinuric kidneys. After 30 weeks of unilateral proteinuria, pentosidine was increased in both kidneys (0.26+/-0.10 micromol/mol valine) compared with controls (0.18+/-0.06 micromol/mol valine, P<0.05). Pentosidine (AN, week 30) was also increased compared with AN at week 12 (0.16+/-0.06 micromol/mol valine, P<0.01). In control and diseased kidneys, pentosidine was present in the collecting ducts. In proteinuric kidneys, in addition, pentosidine was present in the brush border and cytoplasm of dilated tubular structures, i.e. at sites of proteinuria-induced tubular damage. CONCLUSION: Pentosidine accumulates in non-diabetic proteinuric kidneys in damaged tubules, and renoprotective treatment by angiotensin-converting enzyme (ACE) inhibitors inhibits AGE accumulation, supporting a relationship between abnormal renal protein trafficking, proteinuria-induced tubular damage and tubular pentosidine accumulation. Future studies, applying specific AGE inhibitors, should be conducted to provide insight into the pathophysiological significance of renal AGEs in non-diabetic renal disease.     

Comparative Effects of Haemodialysis and Haemofiltration on Plasma Atrial Natriuretic Peptide

The effects of 4 h haemodialysis (15 patients) or 4 h haemofiltration(five patients) on plasma concentrations of atrial natriureticpeptide (ANP) were compared by means of a sensitive radioreceptorbinding assay, and related to accompanying changes in body weight,blood pressure and plasma renin activity. Before dialysis, plasmaANP concentrations were considerably elevated: haemodialysisgroup 10–484 pmol/l (mean 156 pmol/l); haemofiltrationgroup 72–320 pmol/l (mean 170 pmol/l). Although plasmaconcentrations of ANP fell markedly with treatment in both groups:post-haemodialysis 2–187 pmol/l (mean 67 pmol/l); post-haemofiltration47–135 pmol/l (mean 79 pmol/l), after treatment it remainedabove the normal range in 14 of the 20 patients. Pretreatmentplasma ANP was related to systolic blood pressure (r=0.459;P<0.05) but bore no relationship to mean or diastolic bloodpressure, or plasma renin activity. The fall in plasma ANP concentrationduring treatment correlated with the postural blood pressuredrop after dialysis (r=0.505; P<0.05), but was unrelatedto changes in weight or plasma renin activity with haemodialysisor haemofiltration. Plasma ANP concentrations rose rapidly againin the 60 min after dialysis treatment, without change in bodyweight. These results show that high levels of biologically active ANPcirculate in end-stage renal disease. The fact that these arenot reduced to normal by haemodialysis or haemofiltration, despiterestoration to normovolaemic or hypovolaemic state, suggeststhat the increased levels of ANP in end-stage renal failureare due to both hypervolaemia and other factors, which may includeoccult cardiac dysfunction and loss of renal clearance.     

Advanced glycated end-products (AGE) during haemodialysis treatment: discrepant results with different methodologies reflecting the heterogeneity of AGE compounds.

BACKGROUND: There has been much recent interest in accumulation of advanced glycation end-products (AGE) in uraemic patients. Analysis of AGE has been difficult, because commonly used methodologies, i.e. immunodetection assays or fluorescence measurements, reflect group reactivity and are not specific for chemically defined substances. Some investigators measured individual AGE compounds, e.g. pentosidine, carboxymethyllysine, pyrraline or imidazolone, but a systematic assessment of known compounds using specific HPLC methods in diabetic and non-diabetic end-stage renal disease (ESRD) patients during treatment has not been performed. METHODS: For the present study, the concentrations of early and late products of the Maillard reaction in plasma and ultrafiltrate were monitored during high-flux dialysis sessions in diabetic and non-diabetic patients. AGE were analysed by fluorescence spectroscopy and size exclusion chromatography with fluorescence detection. Specific HPLC methods were used to quantify the Amadori product fructoselysine and the AGE compounds pentosidine and pyrraline in acid or enzymatic hydrolysates. RESULTS: Using size exclusion chromatography, we confirmed a similar fluorescent peak distribution for diabetic and non-diabetic ESRD patients. Main fractions were found at approximately 70, approximately 14 and <2 kDa, confirming results obtained by other authors. In diabetic patients, the fluorescence intensity of the low molecular weight fraction was higher. Uraemic patients differed from controls mainly by the fluorescence of the low molecular weight fraction. The peak spectrum in ultrafiltrates was similar to that in plasma regarding low molecular weight fractions and the 14 kDa peak, but no protein-bound fluorescence was found at 70 kDa. HPLC analysis revealed a significant reduction of plasma pentosidine during high-flux dialysis in non-diabetic (from 9.1+/-5.1 to 8.5+/-4.7 pmol/mg protein; P<0.05) and diabetic patients (from 10.0+/-9.1 to 6.8+/-4.0 pmol/mg protein; P<0.05). In contrast, plasma fructoselysine showed only a non-significant trend to decrease in diabetic (from 3.24+/-0.88 to 3.05+/-0.77 nmol/mg protein) and non-diabetic patients (from 2.69+/-0.52 to 2.56+/-0.50 nmol/mg protein). Pyrraline, a nonfluorescent late AGE product derived from reaction of 3-deoxyglucosone with lysine, could not be detected (detection limit approximately 40 pmol/mg protein). Comparing HPLC and size exclusion analysis, it was found that pentosidine accumulated in the range of low molecular weight substances and was removed by high-flux dialysis. CONCLUSIONS: High-flux dialysis reduces the plasma concentration of fluorescent AGE compounds, i.e. pentosidine, but the Amadori product fructoselysine is not removed, indicating that this compound is protein associated.     

Influence of hemodialysis membrane type on pentosidine plasma level, a marker of "carbonyl stress".

Influence of hemodialysis membrane type on pentosidine plasma level, a marker of "carbonyl stress." BACKGROUND: The accumulation of advanced glycation end products (AGEs) in uremia has been ascribed to the retention of carbonyl precursors of AGEs. Pentosidine plasma level has been identified as a surrogate marker of carbonyl precursors ("carbonyl stress"). The influence of hemodialysis (HD) membrane type and residual diuresis on carbonyl stress has not been studied. METHODS: We measured protein-linked and free plasma pentosidine (a surrogate marker of carbonyl stress) by high-performance liquid chromatography in patients on HD with low-flux cellulose (N = 29), high-flux polysulfone (PS; N = 57), polymethylmethacrylate (PMMA) (N = 25), and AN69 (N = 15). RESULTS: Both protein-linked and free pentosidine were similar on low-flux cellulose, high-flux PMMA, and AN69, but were lower (P < 0.01) on high-flux PS. Pentosidine levels were virtually identical on Fresenius and Asahi PS in Japanese and Belgian patients. By multivariate analysis, only the type of HD membrane and residual diuresis proved to be independent determinants (P < 0.001) of pentosidine levels. During a single HD session, the clearance of free pentosidine was similar with all membranes. In three patients who were switched from AN69 to PS, the protein-linked pentosidine level dropped to the control level after resumption of the AN69 membrane. CONCLUSIONS: Both HD membrane type and residual diuresis are independent determinants of pentosidine plasma level, which is a marker of carbonyl stress.     

Concentration of dimethyl-L-arginine in the plasma of patients with end-stage renal failure

Abstract. N^GN^Gdimethyl-L-arginine (asymmetric dimethyl-L-arginineADMA) and N^GN^G dimethyl-L-arginine (symmetric dimethyl-L-arginine;SDMA) are naturally occurring analogues of L-arginine, the substratefor nitric oxide (NO) synthesis. ADMA is a potent inhibitorof NO synthesis, and accumulates in the plasma of patients withrenal failure. However the precise concentration of ADMA andSDMA in renal patients is still controversial. This study wasperformed to measure plasma ADMA and SDMA concentrations bytwo different HPLC techniques in nine healthy controls and 10uraemic subjects, and to investigate the effects of haemodialysis.In controls, the mean (±SEM) plasma concentrations ofADMA and SDMA were 0.36±0.09 and 0.39±0.05 µmol/lrespectively, yielding an ADMA/SDMA ratio of 1.2± 0.17.In uraemic patients, the plasma concentrations of ADMA and SDMAwere 0.9±0.08 µmol/l (P<0.001 compared to controls)and 3.4±0.3 µmol/l (P<0.001 compared to controls)with an ADMA/SDMA ratio of 0.27±0.015 (P<0.001). Inthe course of one 4 h haemodialysis session, ADMA concentrationsdecreased from 0.99±0.13 to 0.77±0.3 µmol/land SDMA concentrations from 3.38±0.44 to 2.27±0.21µmol/l. The plasma ADMA/creatinine ratio tended to increasefrom 1.26±0.20 x 10^–3 to 2.01±0.41 x 10^–3It is concluded that there is a modest (3-fold) but definiteincrease in plasma ADMA concentration in uraemic patients comparedto controls. SDMA accumulates to a greater degree (8-fold increase)and more closely parallels creatinine concentration than ADMA.The change in the ADMA/SDMA ratio is not accounted for by greaterrenal or dialysis clearance of ADMA, and, even though alternativeexplanations are not excluded, greater metabolism of ADMA thanSDMA is the most likely explanation. Although small in magnitude,the increase in ADMA concentration might be biologically significant.     

A sensitive and specific ELISA for plasma pentosidine.

BACKGROUND: Advanced glycation end products are formed by non-enzymatic glycation and oxidation reaction. Pentosidine is a well-known and characterized structure among them, and has been implicated in the pathogenesis of complications associated with chronic renal failure and long-term dialysis, such as dialysis-related amyloidosis and atherosclerosis. METHODS: We established a highly sensitive and specific competitive enzyme-linked immunosorbent assay (ELISA) for plasma pentosidine and applied it to large numbers of plasma samples including haemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD) patients. We compared their plasma pentosidine levels determined by the competitive ELISA with those determined by high-performance liquid chromatographic (HPLC) assay currently used. RESULTS: The plasma pentosidine levels determined by the ELISA were correlated well with those determined by sophisticated instrumental HPLC assay, both in non-diabetic and diabetic dialysis patients. Both analyses yielded comparable results, with over 8-fold higher plasma pentosidine levels in HD and CAPD patients, irrespective of the presence or absence of diabetes, as compared to normal subjects and non-uraemic diabetic patients. CONCLUSIONS: The competitive ELISA will provide a rapid and convenient determination of plasma pentosidine content and thus be useful to assess the carbonyl stress in uraemic patients.     

Involvement of oxidative stress in the accelerated formation of pentosidine in patients with end-stage renal failure

SUMMARY: Advanced glycation end products (AGEs) have been found to accumulate in the amyloid deposits, skin and plasma of haemodialysis patients (HD), implicating the possible involvement of AGE-modified protein in pathogenesis in dialysis-related amyloidosis. Pentosidine, an AGE cross-link, is a specific marker for AGEs. Plasma pentosidine levels in HD patients were increased dramatically. In the present study, plasma pentosidine, fructoselysine, advanced oxidation protein products (AOPP) and glutathione peroxidase (GSHPx) levels were measured to elucidate the role of oxidative stress in pentosidine formation in nondiabetic HD patients. Plasma pentosidine did not correlate with fructoselysine; plasma AOPP levels were significantly higher than those in normal subjects (201.45 ± 57.93 vs. 55.91 ± 6.57 μmol/L, P <0.001) and correlated positively with plasma pentosidine in HD patients ( r =0.52, P <0.005); plasma GSHPx levels were significantly lower than those in normal subjects (168.40 ± 65.08 vs. 348.87 ± 86.10 U/I, P <0.001) and correlated negatively with plasma pentosidine ( r =0.54, P <0.001) in HD patients. Decreased GSHPx levels may lead to the accumulation of hydrogen peroxide. These findings implicate the involvement of oxidative stress in the accelerated formation of pentosidine in uraemia and suggest that pentosidine could be considered as an oxidative stress biomarker to estimate the degree of oxidative-stress-mediated protein damage.     

Influence of dialysis modalities on serum AGE levels in end-stage renal disease patients.

BACKGROUND: The accumulation of advanced glycation end-products (AGEs) in end-stage renal disease (ESRD) influenced by dialysis modalities is of current interest. Highly permeable membranes in haemodialysis or haemofiltration should be able to eliminate circulating AGEs as well as their AGE precursors more efficiently. METHODS: In our study, 10 non-diabetic and 10 diabetic ESRD patients were on haemodialysis with low-flux membranes (LF) followed by a cross-over haemodialysis with high-flux or super-flux polysulfone membranes (HF, SF) for 6 months each. We measured the protein-bound pentosidine and free pentosidine serum levels by high-performance liquid chromatography (HPLC) as well as the serum AGE peptide, AGE-beta(2)-microglobulin and beta(2)-microglobulin concentrations, using ELISA assays. RESULTS: All parameters investigated were significantly higher in dialysis patients than in healthy subjects. The reduction rates during a single dialysis session were found to be higher using the SF than those obtained with the HF (free pentosidine 82.4+/-7.3 vs 76.6+/- 8.7%; AGE peptides 79.7+/-7.7 vs 62.3+/-14.7%; AGE-beta(2)-microglobulin 64.0+/-16.5 vs 45.4+/-17.7%; beta(2)-microglobulin 70.5+/-5.6 vs 58.2+/-6.0%). The protein-bound pentosidine levels remained constant over the respective dialysis sessions. In the 6-month treatment period with the SF, decreased pre-dialysis serum levels of protein-bound pentosidine, free pentosidine and AGE peptides were observed in non-diabetics and diabetics as compared with values obtained with the LF. The respective pre-dialysis AGE-beta(2)-microglobulin concentrations decreased insignificantly, whereas those of beta(2)-microglobulin were significantly lower. Using the HF dialyser, only moderate changes of the parameters measured were noted. CONCLUSION: Treatment with the biocompatible polysulfone SF dialyser seems to be better suited to lower serum AGE levels and to eliminate their precursors.     

On-line urea kinetics in haemodiafiltration

BACKGROUND.: Calculation of Kt/V and assessment of nutrition have so farbeen dependent upon off-line urea measurements of blood or dialysatesamples. Here we describe a biosensor for on-line urea measurementduring haemodiafiltration. METHODS.: The biosensor consisted of a cartridge containing covalentlylinked urease placed between two conductivity cells. The biosensorwas placed on the outlet line of a haemofilter in series witha dialyser in order to obtain an aliquot of plasma ultrafiltratefor on-line measurement of urea. RESULTS.: Urea nitrogen concentrations were highly correlated to the difference() in conductivity measured by the two conductivity cells bothin aqueous solutions (in-vitro studies, y=–6.676+32.12x,R²=0.998, P<0.0001) and in ultrafiltrates (ex-vivo studies,y=–6.7+32.01x, R²=0.98, P<0.00001). conductivity washighly reproducible (% variation: 0.8–5.3%) and stable(maximal % variation at 150 mg/dl after 180 min: 0.9±0.3vs initial values). The intradialytic plasma water urea profilewas obtained in 10 haemodialysis patients. To study recirculation,the plasma water urea profile was analysed before and 3 minafter stopping the dialysate flow. The pre- and post-stoppedflow ratio (1.21±0.1, mean±1 SD) was superimposableto conventional blood sampling data (opposite arm venous/arterial:1.22±0.11) and allowed correction for recirculation.A novel approach to urea kinetic modelling was described andused to reliably project end-dialysis and post-dialysis reboundurea concentration as early as 90 min. Projected (29.2±10.4g) or measured (29.8±10.5 g) net urea removal was highlycorrelated with the amount of urea collected in the total spentdialysate (29.7±10.6 g) (R²=0.99, R²=0.97 respectively). CONCLUSIONS.: These results indicate that on-line, real-time analysis of ureakinetics may provide information on delivery of adequate dialysisin high-efficiency techniques.     

PHARMACOKINETICS OF GLYCOPYRRONIUM IN URAEMIC PATIENTS

We studied the pharmacokinetics of glycopyrronium 11 uraemicpatients undergoing cadaveric renal transplantation and in sevenASA I control patients undergoing general surgery. Glycopyrronium4 µg kg^–1 was given i.v. before induction of anaesthesia.Blood and urine samples were collected for up to 24 h for measurementof glycopyrronium concentrations using a radioreceptor assay.Volume of distribution in the elimination phase (V^ß)was similar in both groups, the elimination half-life was longer (P <0.05), area underthe plasma concentration-time curve (AUC) larger (P <0.01)and plasma clearance (Cl) smaller (P < 0.01) in the uraemicpatients. In 3 h, mean 0.7 (range 0–3) % and 50 (21–82)% of glycopyrronium was excreted in the urine in the uraemicand healthy patients, respectively (P <0.001). The 24-h renalexcretion was 7 (0–25) % in uraernic and 65 (30–99)% in control patients (P <0.001). We conclude that the eliminationof glycopyrronium is severely impaired in uraemic patients.     

Pancreatic Beta-cell Function in CAPD

Pancreatic beta-cell function was evaluated in uraemic patientsby measuring beta-cell peptides in the peripheral blood afterintravenous glucagon (1 mg) stimulation. Patients in chronicrenal failure, patients on haemodialysis, and both new and establishedsubjects on continuous ambulatory peritoneal dialysis (CAPD)(10in each group) were studied and compared to 8 healthy controls.Fasting glucose (3.6–4.4 mmol/1) and insulin concentrations(9.5–11.7 mU/1) were normal and did not differ betweenthe uraemic groups, but c-peptide concentrations were markedlyincreased in uraemia (1.84–2.38 nmol/1) compared to controls(0.48 nmol/1). Following glucagon stimulation an exaggeratedblood glucose response with delayed glucose peak was observed,while the peak insulin response to glucagon was normal; however,the return to basal concentrations was delayed in uraemia. Thec-peptide response was also exaggerated and peak concentrationsin uraemic subjects (3.0–4.3 nmol/1) were significantlygreater than controls (1.5 nmol/1). The response of CAPD patientswas similar to those on haemodialysis and non-dialysed uraemicpatients. The abnormalities seen were due to uraemia, and CAPDtreatment had no specific adverse effect on beta-cell function.Thus, from this data there was no evidence that CAPD per seis detrimental to beta-cell integrity.     

Creatinine kinetic modelling: a simple and reliable tool for the assessment of protein nutritional status in haemodialysis patients

While the mathematical modelling of urea kinetics is in wideuse for evaluating treatment adequacy and protein nutritionin dialysis patients, the kinetics of creatinine generationin dialysis patients has been relatively unexplored. In thisstudy creatinine kinetic modelling as a clinical tool was investigatedin a group of 90 patients treated by haemodialysis (n=20), haemodiafiltration(60), haemofiltration (7), or biofiltration (3) over a 6–36-monthperiod. A single pool model of creatinine kinetics was employedto obtain monthly values of creatinine distribution space andcreatinine appearance rate. Extrarenal creatinine degradationrate, estimated using a clearance of 0.038 l/kg/24 h as suggestedby Mitch and co-workers, was added to creatinine appearancerate in urine and dialysate to calculate a corrected creatinineindex (CI). Extrarenal degradation accounted for 12 ±2% of CI. CI was higher in males (22.4 ± 4.5 mg/kg/24h) than females (19.8 ± 4.8) and decreased with age,falling off more sharply for the female group (CI=29.9–0.185.age,R=0.72) than the males (CI=24.1–0.030.age, R=0.31). CIwas found to correlate strongly with protein catabolic ratedetermined by urea kinetic modelling (CI=8.84+10.91.PCR). Lowor reduced CI was associated in this study group with severemalnutrition status and high mortality rate. CI is suggestedas a strong predictor of patient morbidity and mortality.     

Uraemic toxic retention solutes depress polymorphonuclear response to phagocytosis

Previous studies from our laboratory have demonstrated thatthe activity of the hexose mono-phosphate shunt (HMS) pathwayin phagocytosisrelated respiratory burst is disturbed in end-stagerenal disease. To determine whether uraemic solute retentionis responsible for this defect the HMS-path was evaluated bymeasurements of glucose-1-C¹⁴ utilization and determinationof ¹⁴CO₂ production in polymorphonuclear cells (PMNLs), suspendedin normal plasma or uraemic biological fluids. Normal PMNLs,while suspended in normal or uraemic plasma, were stimulatedwith either latex, zymosan or Staph. aureus; CO₂ generation(measured as DPM/10³ PMNL, normal versus uraemic plasma) wasdepressed in uraemic plasma in response to latex (from 43±5to 20±3), zymosan (from 72±8 to 47±4) (P<0.01),and Staph aureus (from 73±17 to 47±8 DPM/10³ PMNL)(P<0.05). The degree of inhibition was similar for each stimulus.To characterize the substances responsible for this defect wefractionated uraemic plasma ultrafiltrate by polarity-basedsemipreparative C₁₈ reversed phase HPLC and found a decreasedresponse to Staph. aureus in the presence of fraction 2 (from102±13 to 23±10 DPM/10³ PMNL, P<0.05), andin fractions 8 and 11 (lowest value in fraction 8, 54±14DPM/10³ PMNL, P<0.05 versus control). The pattern of HPLCelution on a gradient from 100% formiate (pH 4.0) to 100% methanolindicates that there are at least two chemically distinguishablegroups of compounds, one hydrophilic (in fraction 2), and onelipophilic (in fractions 8 and 11). We conclude that uraemicbiological fluids contain factors that inhibit HMS activityrelated to phagocytosis, and that at least two groups of componentswith different characteristics are involved.     

Compliance and effects of nutritional treatment on progression and metabolic disorders of chronic renal failure

Low-protein, low-phosphorus diets (LPD) are prescribed to patientswith chronic renal failure (CRF) to slow down the rate of progressionof CRF and to control uraemic symptoms. A satisfactory adherenceof patients to the prescribed diet is needed to meet these twogoals. We studied the compliance of CRF patients to a LPD providingdaily (per kg body weight) 0.3 g protein, 3–5 mg phosphorus,35 kcal, and supplemented with essential amino-acids and ketoanalogues.Forty CRF patients were studied for 23.3±10.8 months(range 12–45). Compliance to LPD was evaluated by dietaryinquiry and protein intake estimated from urinary urea excretion.According to compliance to LPD, patients were retrospectivelyassigned to the compliant (n=27) or the non-compliant (n=13)group. GFR measured by the urinary clearance of [⁵¹Cr]-EDTAwas identical in the two groups at the start of the study: compliantpatients 15.7±5.3 ml/mn, non-compliant patients 15.4±5.9ml/mn. The decrease of GFR was – 0.08±0.22 ml/minper month in compliant patients versus –0.31±0.37ml/min per month in non-compliant patients (P<0.02). Theseresults were not demonstrated if the progression of CRF wasassessed by the linear regressions over time of creatinine clearanceor the reciprocal of creatinine. Serum bicarbonate, serum phosphorusand PTH levels were corrected by LPD in compliant patients (P<0.005 for all parameters) but not in non-compliant patients.These results suggest that evaluation of compliance is necessaryto assess the response of CRF patients to LPD, whether the aimis to slow the progression of CRF or to control its metabolicconsequences. A beneficial effect of compliance to LPD was demonstratedupon these two goals.     

Uraemic medium causes endothelial cell dysfunction characterized by an alteration of the properties of its subendothelial matrix

Uraemic patients suffer from haemorrhagic disorders and acceleratedatherosclerosis. To evaluate the possible role of the vesselwall in these haemostatic alterations associated with uraemia,we investigated the effect of a uraemic milieu on human endothelialcell (EC) cultures and the reactivity of the extracellular matrices(ECM) generated by these cells towards platelets. EC cultureswere exposed to a pool of sera (20% in the culture medium) obtainedeither from uraemic patients or from normal donors, and thefollowing parameters were evaluated: (1) EC viability (trypanblue exclusion test); (2) von Willebrand factor (vWF) levelsin supernatants and associated with ECM; (3) the reactivityof EC and EC-derived ECM towards platelets, measured ‘exvivo’ under flow conditions (5 min, wall shear rate 800s^–1); and (4) ultrastructure of the ECM. The viabilityof EC cultured in the presence of uraemic sera was similar tocontrols. Platelet interaction with ECM generated by EC exposedto uraemic sera was significantly reduced (P<0.05). Thisdecrease was mainly related to a reduction in platelet adhesion(9.8 ± 1.9% vs 16.7±1.8% in controls, P<0.02).VWF levels in supernatants and associated with ECM were similarto controls. Ultrastructural analysis of the ECM generated byEC exposed to uraemic sera revealed a deficient matrix. An increasedremoval of EC was observed in experiments in which EC culturedin the presence of uraemic sera were perfused with citratedblood. These results indicate that a uraemic milieu induces quantitativeand qualitative changes in the vascular subendothelium, characterizedby a less intrincate network of fibrils, as well as a decreasedattachment of EC and reduced thrombogenicity to the ECM. Thesechanges may represent another mechanism which contributes tothe haemostatic dysfunction observed in uraemic patients.     

Renal proximal tubular metabolism of protein-linked pentosidine,an advanced glycation end product

BACKGROUND: Pentosidine, an advanced glycation end product, accumulates in plasma proteins of uremic patients. Its fate is, however, yet to be fully understood. METHODS: Three cell lines, JTC-12 (proximal tubular cells), MDCK (distal tubular cells), and BALB3T3 (nonrenal cells), were cultured in a double chamber system and were exposed to uremic serum, and the contents of protein-linked pentosidine derived from uremic sera were determined in each medium by HPLC assay. The presence of pentosidine in the cytoplasm of these cells was assessed by immunoperoxidase staining. RESULTS: When the apical cell membrane was exposed to uremic serum (fortified in the upper chamber), the contents of protein-linked pentosidine in the upper medium decreased by up to 30% after 24- and 48-hour incubations of JTC-12 cells but not of other cells. On the other hand, the contents of protein-linked pentosidine in the lower medium did not change. By contrast, exposure of the basolateral cell membrane of the three cell lines to uremic serum (fortified in the lower chamber) did not change the contents of protein-linked pentosidine both in the upper and lower medium after a 24-hour incubation. Pentosidine was detected immunohistochemically in the cytoplasm of JTC-12 cells, but not of BALB3T3 and MDCK cells, the apical membranes of which were exposed to uremic sera for 8 h. The immunoreaction disappeared 48 h after exposure. Pentosidine was not detected in the cytoplasm of JTC-12 cells, the basolateral membranes of which were exposed to uremic sera. The relevance of the in vitro results to humans was demonstrated by immunohistochemical studies in normal human kidney tissues showing that pentosidine was identified in the proximal renal tubules. CONCLUSION: These results suggest that the proximal tubular cells play a role in the disposal of plasma pentosidine.     

Improvement of Anaemia With Desferrioxamine in Haemodialysis Patients

We have prospectively investigated the effect of desferrioxamine(DFO)administration (2 g i.v. after every haemodialysis session for6 months) on the normocytic and normochromic anaemia of sevenhaemodialysis patients. None had either clinical or analyticaldata characteristic of chronic aluminium intoxication. At theend of DFO therapy, the haematocrit had increased from 20.5±2.7%to 30.4±7.7% (P< 0.005), and the transfusional requirementsdecreased from 3.5±2.2 units (range 1–8 units)in the 6 months prior to DFO, to 0.7±0.9 units (range0–2 units) during DFO administration (P<0.01). No transfusionwas required during the second half of the DFO therapy period.Serum ferritin decreased from 1059±532 nmol/l (2649±1331ng/ml) to 507±403 nmol/l (1268±1008 ng/ml) (P<0.025).Two months after DFO withdrawal the haematocrit value fell significantlyto 22.2±1.6% (P<0.01). DFO therapy was restarted inone patient at a lower dose (1 g i.v. after every haemodialysissession) and an increase of haematocrit from 23.8% to 40.2%was again observed after 3 months of treatment. The toleranceto DFO was excellent. We conclude that DFO therapy should beconsidered in haemodialysis patients with severe anaemia andincreased blood transfusion requirements.     

Plasma pentosidine is associated with inflammation and malnutrition in end-stage renal disease patients starting on dialysis therapy

Pentosidine is an advanced glycation end-product (AGE), formed by glycosylation and oxidation, that accumulates markedly in end-stage renal disease (ESRD). It has been speculated that AGE and carbonyl stress contributes to long-term complications such as cardiovascular disease (CVD) in ESRD patients. This study determined plasma levels of pentosidine as well as the presence of inflammation (CRP > or = 10 mg/L), clinical CVD (CVD(clin)), and malnutrition (subjective global assessment [SGA] > 1) in a cohort of 191 ESRD patients, median age of 55 yr (range, 23 to 70 yr) and median GFR = 7 ml/min (range, 2 to 17 ml/min), close to start of renal replacement therapy. Fifty-one elderly subjects, median age of 82 yr (range, 71 to 110 yr), with mild renal impairment, median GFR = 67 ml/min (range, 38 to 113 ml/min), were also studied for comparative analysis of plasma pentosidine. The plasma pentosidine content was elevated in all patients compared with the levels in the elderly subjects and were negatively correlated with GFR both in the ESRD patients (Rho = -0.24; P < 0.01; n = 159) and in the elderly subjects (Rho = -0.31; P < 0.05). Moreover, the plasma pentosidine content was correlated with age in the ESRD patients (Rho = 0.26; P < 0.001) and in the elderly subjects (Rho = 0.44; P < 0.001). The 63 malnourished ESRD patients (35%) had a significantly higher (P < 0.05) median plasma pentosidine than the well-nourished patients (39 versus 27 pmol/mg albumin). Similarly, 73 inflamed patients (38%) had a significantly higher (P < 0.001) median pentosidine content compared with 118 non-inflamed patients (37 versus 24 pmol/mg albumin). Also, the plasma pentosidine content showed weak but significant positive correlations with CRP (Rho = 0.28; P < 0.0001), fibrinogen (Rho = 0.23; P < 0.01; n = 126), IL-6 (Rho = 0.22; P < 0.01; n = 169), and soluble vascular cellular adhesion molecule-1 (Rho = 0.38; P < 0.001; n = 74). On the other hand, no significant differences in plasma pentosidine content were noted between the patients with and those without CVD(clin) (32 versus 27 pmol/mg albumin, respectively). Analyses of all-cause mortality, by Kaplan-Meier, showed that mortality was not linked to the plasma pentosidine content. Moreover, survival analysis by the Cox regression model showed that age (P < 0.001), diabetes mellitus (P < 0.01), malnutrition (P < 0.01), and CVD(clin) (P < 0.01) independently predicted poor outcome, whereas an elevated plasma pentosidine content did not. The present study shows that an elevated plasma pentosidine content in ESRD patients is significantly associated with both inflammation and malnutrition and confirms that low residual renal function and high age further contribute to an increased plasma pentosidine content. However, in this small cohort, the plasma pentosidine content did not predict outcome. Thus, accumulation of plasma pentosidine is unlikely to be an appropriate clinically useful marker to predict mortality in ESRD patients.