Reduced Deposition of Aluminium in Trabecular Bone of Uraemic Rats Treated with Dihydroxylated Vitamin D Metabolites

The rate of aluminium accumulation in bone may be related tothe presence of vitamin D metabolites. The present study investigatedthe effect of 1,25(OH)₂D₃ (24 pmol/d s.c.) and 24R,25(OH)₂D₃(480 pmol/day), combined or alone, on the deposition of aluminium(119 µmol/kg per day) in bone of uraemic rats during concomitantparenteral administration of aluminium for 9 weeks. Bone histomorphometryof trabecular bone revealed a severe low-turnover osteodystrophyin aluminium-treated uraemic rats, as evidenced by a decreasein osteoblastic osteoid surfaces and mineral apposition rates.1,25(OH)₂D₃ as well as 24R,25(OH)₂D₃ decreased stainable bonealuminium and the aluminium content of trabecular bone and,in parallel, the number of osteoblasts and osteoclasts increased.Additional treatment with one or both vitamin D metabolites14 days prior to the aluminium load further improved these results.Despite these effects, dynamic histomorphometric parametersremained suppressed and osteoidosis persisted. Serum PTH concentrationswere significantly elevated in aluminium-loaded uraemic ratstreated with 24R,25(OH)₂D₃ alone compared to controls. In conclusion,administration of 1,25(OH)₂D₃ or 24R,25(OH)₂D₃ reduces the accumulationof aluminium in trabecular bone in uraemic rats and preventssome of its excess toxicity. The mechanism of action may bedifferent for either vitamin D metabolite; however, combinedtreatment does not result in further reduction of the accumulationrate of aluminium in bone in this model.     

Enhanced Gastrointestinal Absorption of Aluminium in Uraemia: Time Course and Effect of Vitamin D

The present study examines the time course of aluminium absorptionin uraemic rats vs controls and investigates the effect of vitaminD. Following an oral load of 410 µmol aluminium there wasa significant increase in the urinary excretion rate of aluminiumas early as 60 min in uraemic rats. Compared with controls thisincrease was significantly greater in uraemic animals and maximumexcretion rates (77±49 vs pre-load 2±1 nmol Al/h)were achieved after 2 h. When vitamin-D-deficient rats with normal renal function werecompared with vitamin-D-replete controls, the latter excreteda significantly greater amount of the oral dose of aluminiumin their urine (727±361 vs 359±l40nmol Al/5d;P<0.02) and the post-load increase in the serum aluminiumconcentration was more pronounced in the vitamin-D-replete animals. Aluminium administered i.v. resulted in similar urinary aluminiumexcretion rates in both groups. In uraemicrats, however, regardlessof their vitamin D status, adminis tration of 1,25(OH)₂D₃ hadno effect on the amount of urinary aluminium excretion afteroral or i.v. loads. These findings suggest that although in rats with normal renalfunction aluminium absorption appears to be partly vitamin Ddependent, 1,25(OH)₂D₃ does not further augment the enhancedgastrointestinal absorption of aluminium in uraemia.     

Failure of a daily haemofiltration programme using a highly permeable membrane to return beta 2-microglobulin concentrations to normal in haemodialysis patients.

In an attempt to return to normal serum beta 2-microglobulin levels in a group of seven ESRD patients, a programme of daily HF with highly permeable AN69 membranes was undertaken. Pre-HF beta 2-M serum levels stabilized after 4 days at 20 mg/l, only 40% lower than the initial concentration. A total of 985 +/- 20 mg beta 2-M was removed over the week. The beta 2-M release rate averaged 97 micrograms/min with a broad range of values (63-128 micrograms/min). beta 2-M release peaked at 602 micrograms/min 1 h after the end of the HF session before returning to baseline by 12 h post-HF. We conclude that a return to normal blood beta 2-M concentrations in ESRD patients seems quite unrealistic despite a highly intensive extracorporeal therapy. Therefore other therapeutic alternatives have to be designed to prevent or cure beta 2-M amyloidosis.     

Prevention of osteitis fibrosa, aluminium bone disease and soft-tissue calcification in dialysis patients: a long-term comparison of moderate doses of oral calcium +/- Mg(OH)2 vs Al(OH)3 +/- 1 alpha OH vitamin D3

Since 1980, moderately large doses of oral calcium (80 +/- 35 mmol/day as CaCO3 +/- calcium polystyrene sulphonate), in association if necessary with Mg(OH)2 (2.5 +/- 1 g/day), with a reduction in the dialysate Mg concentrations from 0.75 to 0.375 mmol/24 h, have replaced A1(OH)3 as phosphate binders in our centre. A1(OH)3 was previously given to our haemodialysis patients in association with small doses of Ca CO3 (less than or equal to 3 g/day) and if necessary with 1 alpha OH vitamin D3. To compare the long-term efficacy of this new approach with the former one in the prevention of renal osteodystrophy and soft-tissue calcification, 32 current patients were selected on the basis of at least 24 months of treatment in our centre and availability of a yearly bone survey (profile of lumbar spine and anteroposterior view of the pelvis, shoulders and hands). A group of 30 patients treated before 1980 were then selected on the same criteria and matched for age, sex, and duration on dialysis. Linear calcifications of the anterior and posterior walls of the aorta in front of L2, L3, L4 and on the lateral walls of the iliac and femoral arteries were measured and the para-articular calcifications and subperiosteal resorptions of the hands evaluated. The initial extent and the subsequent increase of the ocular and para-articular calcification were comparable in the two groups. Plasma alkaline phosphatase was stable in the normal range in both groups, as was plasma concentration of calcium. Plasma phosphate was slightly elevated (1.7 mmol/l) but stable and comparable in the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo prostanoid formation during acute renal allograft rejection

Vasoconstriction during acute renal allograft rejection maybe regulated by increased formation of vasoactive prostanoids.To address this hypothesis we investigated the biosynthesisof thromboxane (Tx)A₂, a potent vasoconstrictor and plateletagonist, of prosta-cyclin (PGI₂), a vasodilator and plateletantagonist, and of prostaglandin (PG)E₂, a mediator of saltand water excretion, in nine children with 12 acute rejectionepisodes, prospectively during the first 7 weeks after renaltransplantation. We used physicochemical analysis of stableurinary prostanoid index metabolites. Rejection crises wereassociated with an increase in TxB₂ excretion from baselinemedian 9.2 (range 1.9–18.6) ng/h/1.73m² to 21.2 (range10.0–133.0) ng/h/1.73m² (P<0.005) during acute rejectionepisodes. Methylprednisolone pulse therapy resulted in a partialreduction, but not normalization of TxB₂ excretion. Urinary2,3-dinor-TxB₂ was slightly stimulated during allograft rejection,urinary 1 l-dehydro-TxB₂ did not change significantly. RenalPGI₂ and PGE₂ biosynthesis remained essentially unchanged. Incontrast to acute graft rejection, patients with chronic graftrejection and those with stable graft function on differentimmunosuppressive regimens with or without cyclosporin A didnot present stimulated renal TxA₂ formation. Increased renalTxA₂ formation in acute renal allograft rejection is likelyto mediate vasoconstriction and potentiate the loss of renalblood flow and glomerular filtration rate, in the absence ofan adequate response of the renoprotective prostanoids PGI₂and PGE₂.     

A shift in the Th(1)/Th(2) ratio accompanies the clinical remission of systemic lupus erythematosus in patients with end-stage renal disease.

BACKGROUND: Patients suffering from systemic lupus erythematosus (SLE) with renal involvement often show remission of systemic clinical activity after progression to end-stage renal disease (ESRD). SLE is characterized by predominantly humoral, T-helper (Th)(2)-mediated autoimmune responses. Since ESRD induces a state of immunodeficiency that affects the balance of Th cell subsets, we hypothesized that a Th(1) shift induced by ESRD leads to clinical remission of SLE. METHODS: Using single-cell measurement of intracellular cytokines by flow cytometry after polyclonal stimulation with PMA/ionomycin, helper cell profiles were analysed in SLE patients with preserved renal function and in SLE patients with ESRD, from both isolated peripheral blood mononuclear cells (PBMC) and whole blood. RESULTS: Using the whole-blood assay, patients with SLE and preserved renal function showed a predominance of Th(2) cells compared to healthy controls (patients, Th(1)/Th(2) ratio 6.0+/-1.0 vs controls, 9.0+/-1.0; P<0.05). In contrast, SLE patients with ESRD have significantly more Th(1) cells (36.8+/-5.0%) than those without ESRD (23.4+/-3.6%; P<0.05). This results in an enhancement of the Th(1)/Th(2) ratio to 12.1+/-2.6, which is not significantly different from healthy controls. These data were confirmed using a PBMC-based assay. CONCLUSIONS: SLE patients with preserved renal function show a bias in the differentiation of Th cells towards Th(2). Once ESRD occurs, the Th(1)/Th(2) ratio normalizes. This may contribute to the remission of Th(2)-mediated autoimmune diseases such as SLE.     

G-protein beta(3)-subunit C825T genotype and nephropathy in diabetes mellitus.

BACKGROUND: Recent studies have identified a novel polymorphism (C825T) of the gene encoding the beta(3) subunit of heterotrimeric G proteins (G:beta(3)) which is associated with enhanced activation of G-proteins and appears to be more common in hypertensive patients and possibly contributes to decreased kidney allograft survival. METHODS: In the present study we examined the relationship between this genetic variant, type 1 and type 2 diabetes and renal complications of diabetes in 1008 Caucasian patients recruited from an outpatient diabetes clinic and four dialysis centres. We also studied 1940 healthy controls. RESULTS: After multivariate adjustment and in univariate statistics, the G:beta(3) 825TT genotype was not associated with a significantly enhanced risk of diabetes or renal complications. CONCLUSIONS: These findings indicate that the G:beta(3) 825T allele apparently does not contribute to the development of diabetes or associated renal complications in patients with type 1 or type 2 diabetes mellitus.     

Biochemical characterization of serum and urinary beta 2 microglobulin in end-stage renal disease patients.

Since the identification of beta 2 microglobulin (beta 2-M) in haemodialysis-associated amyloidosis, the biochemical characterization of the different forms of beta 2-M has been sought by several groups. New beta 2-M isoforms (pI 5.1 and lower) have been identified in amyloid deposits, and it has been suggested that they are of pathogenetic importance. The finding of N-terminal proteolysed beta 2-M in amyloid deposits prompted the hypothesis that proteolysis would render beta 2-M more amyloidogenic. Finally, a 'novel beta 2-M' (pI 5.2) with a single amino acid replacement (Asn by Asp at position 17) has been reported as possibly specific for patients with dialysis associated amyloidosis, and consequently proposed as 'the amyloidogenic' form. We purified beta 2-M from serum of a newly haemodialysed patient and from urine of a transplanted patient in the early recovery period. Both patients were clinically amyloid free. Three pure isoforms were obtained from serum (pI 5.7, 5.3, and 5.1) and only two from urine (5.7 and 5.3). Further purification of each isoform was obtained by HPLC in a C4 column. Sequence analysis showed that all isoforms had an intact N-terminus. Tryptic digestion of the serum isoforms was performed after alkylation with iodoacetic acid and the peptides were isolated by HPLC in a C18 column. The 5.3 and 5.1 isoforms had identical peptide patterns with the appearance of an early peak missing in the 5.7 form. The sequence of this peptide showed a replacement of the D 42 (Asp 42) by N (Asn) after K41 (Lys 41).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of autonomic neuropathy on ventilatory response to progressive hypercapnia in dialysis patients

The impact of autonomic neuropathy (common in patients on haemodialysis)on ventilatory response to hypercapnia has been studied. We investigated cardiac reflex tests in 20 patients on chronichaemodialysis (8 patients were found with and 12 without neuropathyof the autonomic nervous system). Using the hyperoxic CO₂-rebreathingmethod (according to Read), we tested the above-mentioned twogroups of patients and compared them with 14 healthy controlsubjects. Accumulation of CO₂ in blood with hyperoxic CO₂ rebreathingstimulates central chemoreceptors, and therefore causes a progressiverise in minute ventilation. In patients with autonomic neuropathy (n=8), ventilatory responseto increasing pCO₂ was significantly lower than that in thecontrols (1.7±0.3 versus 3.2±0.5 l/min/mmHg, P<0.001).On the other hand ventilatory response in patients without autonomicdamage (n=12) showed no significant difference when comparedto controls (3.1±0.8 l/min/mmHg). There were no differencesin lung function, arterial blood gas analysis, blood chemistry,duration on dialysis, and demographic data when comparing thepatients with and those without autonomic damage. Our analysis shows different patterns of ventilatory responseto increasing pCO₂ in patients on haemodialysis. Autonomic neuropathyhas to be considered when rebreathing tests are interpreted.The clinical relevance of these findings needs further investigation.     

Effects of the adenosine A1 receptor inhibitor FK 838 on proximal tubular fluid output in rats.

BACKGROUND: Adenosine A(1) receptor blockade has been suggested as a treatment in conditions with sodium and fluid retention because it increases urinary Na(+) excretion and increases proximal tubular fluid output. In the present study, we examine the time course for the renal responses to adenosine A(1) receptor blockade in order to investigate whether the effects may be prolonged and not just temporary. METHODS: The acute effects of the adenosine A(1) receptor inhibitor FK 838 on segmental tubular Na(+) handling were examined by a renal clearance technique in conscious chronically instrumented rats. Lithium clearance (C(Li)) was used as a clearance marker of proximal tubular fluid output. RESULTS: Acute adenosine A(1) receptor inhibition did not affect the glomerular filtration rate (GFR) significantly. In contrast, the inhibition led to significant increases in C(Li) (from 290+/-28 to 431+/-28 microl/min/100 g), fractional Li(+) excretion (FE(Li)) (from 33+/-2 to 47+/-3%) and fractional Na(+) excretion (FE(Na)) (from 0.44+/-0.07 to 2.03+/-0.42%). Sodium excretion, expressed as a fraction of proximal tubular fluid output (C(Na)/C(Li)), rose from 1.3+/-0.2 to 4.2+/-0.4%, suggesting that the natriuretic effect was supported by inhibition of distal nephron Na(+) reabsorption. All values returned to baseline values during the clearance study and thereby indicated that neither proximal tubular fluid output nor urinary sodium excretion remained elevated for a prolonged time. CONCLUSION: It is concluded that in conscious unstressed rats, acute adenosine A(1) receptor inhibition by FK 838 led to a significant natriuresis that was caused by inhibition of proximal tubular Na(+) reabsorption, possibly with a contribution from inhibition of distal nephron Na(+) reabsorption. The increased proximal tubular fluid output and the increased urinary Na(+) excretion returned to baseline values during the clearance study, indicating that none of these effects of adenosine A(1) blockade were long lasting.     

Potent suppression of the parathyroid glands by hydroxylated metabolites of dihydrotachysterol(2).

BACKGROUND: Dihydrotachysterol(2), a licensed pharmaceutical, is hydroxylated to 25-hydroxydihydrotachysterol(2) (25(OH)DHT(2)) and 1 alpha,25-dihydroxydihydrotachysterol(2) (1 alpha,25(OH)(2)DHT(2)) in man. We have compared the biological activity of these metabolites with calcitriol and the 'non-calcaemic' analogue, 22-oxacalcitriol (OCT) in bovine parathyroid cell cultures and in rats. METHODS: The effect of each sterol on parathyroid hormone (PTH) secreted by primary bovine parathyroid cells was measured. High-performance liquid chromotography and gas chromotography-mass spectrometry were used to investigate in vitro 25(OH)DHT(2) metabolism. Rats were given a single intraperitoneal injection or five daily injections of each sterol, and changes in ionized calcium and PTH were measured. RESULTS: In vitro, all sterols suppressed PTH significantly. Calcitriol and OCT were of similar potency, but 1 alpha, 25(OH)(2)DHT(2) and 25(OH)DHT(2) required higher concentrations to suppress PTH equally. We were unable to detect metabolism of 25(OH)DHT(2) to 1 alpha,25(OH)(2)DHT(2) in vitro. In rats, a single dose of 0.5 microg/rat of calcitriol increased ionized calcium at 30 and 40 h (statistically significant at 48 h). 50 microg of OCT and 1 alpha,25(OH)(2)DHT(2) did not cause significant hypercalcaemia at 48 h, although 1 alpha,25(OH)(2)DHT(2) caused hypercalcaemia at 30 h. In contrast, 50 microg of 25(OH)DHT(2) caused hypercalcaemia at 48 h but not at 30 h. Five daily doses of 0.001 microg/rat of calcitriol caused a significant rise in calcium and a 50% fall in PTH. OCT and 1 alpha,25(OH)(2)DHT(2) at 0.025 and 0.5 microg/rat respectively caused similar suppression of PTH but without hypercalcaemia. CONCLUSION: 1 alpha,25(OH)(2)DHT(2) and 25(OH)DHT(2) are potent suppressors of PTH in vitro and in vivo. 25(OH)DHT(2) may be active by virtue of its pseudo-1 alpha-hydroxyl group. Hypercalcaemia caused by a single dose of 1 alpha,25(OH)(2)DHT(2) appeared to be more transient than calcitriol. Five daily doses of 1 alpha, 25(OH)(2)DHT(2) and OCT could achieve 50% suppression of PTH without significant increments in ionized calcium. In contrast, suppression of PTH by calcitriol was associated with significant increments in ionized calcium. These data suggest that like OCT, 1 alpha, 25(OH)(2)DHT(2) can dissociate calcaemic actions from parathyroid-suppressing actions in a manner that may be therapeutically useful.     

In subtotally nephrectomized rats 22-oxacalcitriol suppresses parathyroid hormone with less risk of cardiovascular calcification or deterioration of residual renal function than 1,25(OH)2 vitamin D3.

BACKGROUND: Although it effectively suppresses parathyroid hormone (PTH) secretion, vitamin D [1,25(OH)(2)D(3)] therapy often causes tissue calcification over the long term. In patients on chronic dialysis, cardiovascular calcification is clearly linked to an unfavourable prognosis. In pre-dialysis patients, renal calcification of the kidney leads to the deterioration of renal function. METHODS: We compared the propensities of 22-oxacalcitriol (OCT), with lesser calcaemic action, and 1,25(OH)(2)D(3) for producing their potential side effects in rats: (i) metastatic calcification of heart and aorta, and (ii) renal dysfunction with nephrocalcinosis, using the same effective doses for hyperparathyroidism. OCT (1.25 and 6.25 micro g/kg) or 1,25(OH)(2)D(3) (0.125 and 0.625 micro g/kg) solutions were administered intravenously to subtotally nephrectomized (SNX) rats three times weekly for 2 weeks. RESULTS: Despite the suppression of PTH to comparable levels, the calcification of the hearts, aortas and kidneys in the 1,25(OH)(2)D(3)-treated group was significantly greater than in the OCT-treated group. Of interest was that, in the OCT (6.25 micro g/kg) group, the degree of calcification in hearts, aortas and kidneys were distinctly lower than those in the 1,25(OH)(2)D(3) (0.125 micro g/kg) group despite the comparable serum Ca x Pi products. Therefore, there may be different mechanisms behind the calcifications resulting from OCT and 1,25(OH)(2)D(3). Deterioration of renal function, tubular changes, and atypical hyperplasia of proximal tubules associated with calcification were more severe in the 1,25(OH)(2)D(3)-treated group than in the OCT-treated group. CONCLUSIONS: These results indicate that OCT may be an effective agent for the suppression of PTH with a lesser risk of cardiovascular calcification or deterioration of residual renal function.     

Tissue Uptake of 125I-{beta}2-Microglobulin({beta}2-M) in Anephric Animals in the Presence or Absence of Aluminium Intoxication

In long-term haemodialysis patients a new type of amyloidosiscomposed of ß₂-microglobulin (ß₂-M) hasrecently been described. The amyloid deposition has a particularpredilection for articular structures. In the pathogenesis ofthis complication markedly elevated plasma ß₂-M concentrations,such as those observed in anuric patients, have a role. However,other as yet ill-defined factors must also be implicated, possiblecandidates being aluminium intoxication and the widely usedregenerated cellulose (cuprophan) membrane. In the present experimentalstudy, we examined tissue distribution of exogenous ß₂-Mafter i.v. injection of ¹²⁵I-ß₂-M to bilaterally nephrectomisedrats. One hundred and twenty minutes after injection, most radioactivityremained in the vascular compartment. The accumulation in tissueswas weak, and no predilection for a particular tissue becameapparent. Interestingly, chronically aluminium-overloaded, acutelyanephric rats accumulated a significantly greater amount of¹²⁵I-ß₂-M in their spleens than anephric rats withoutprior aluminium intoxication. We then attempted to induce ß₂-M amyloid depositionin rats and mice, some of whom had undergone chronic aluminiumintoxication and subcutaneous implantation of regenerated cellulosefragments for various periods of time. They were subsequentlymade anephric to obtain high plasma ß₂-M concentrations.None of the animals developed ß₂-M amyloidosis inspleen, liver, skin and mechanically altered joint synovium. In conclusion, chronic aluminium intoxication enhances splenicaccumulation of exogenous ¹²⁵I-ß₂-M in anephric rats.The factors required to form ß₂-M-amyloidosis in vivohave still to be defined.     

Prostaglandin E1: a new agent for the prevention of renal dysfunction in high risk patients caused by radiocontrast media? PGE1 Study Group.

BACKGROUND: Acute renal failure following the administration of radiocontrast media (RCM) is a complication found especially in patients with impaired renal function. Within the limits of a pilot study, the objective was to (a) show the effectiveness and compatibility of prostaglandin E(1) (PGE(1)=Alprostadil) in preventing acute renal failure in patients with elevated levels of serum creatinine and (b) to identify the most appropriate PGE(1)-dose. METHODS: 130 patients with renal impairment (serum creatinine >/=1.5 mg/dl) were included in the study prior to intravascular RCM injection. The patients received one of three different doses of PGE(1) (10, 20, or 40 ng/kg bodyweight/min) or placebo (physiologic sodium chloride solution) intravenously over a time period of 6 h (beginning 1 h prior to RCM application). Serum creatinine was measured 12, 24, and 48 h post RCM-application and creatinine clearance was determined with two 12 h collection periods, as well as one 24 h collection within 48 h post RCM administration. Adverse events during PGE(1) administration were recorded. RESULTS: In the placebo group, the mean elevation of serum creatinine was markedly higher (0.72 mg/dl) 48 h after RCM administration compared with the three PGE(1) groups (0.3 mg/dl in the 10 ng/kg/min group, 0. 12 mg in the 20 ng/kg/min group, and 0.29 mg/dl in the 40 ng/kg/min group). No clinically relevant changes were seen regarding the creatinine clearance in the four groups examined. CONCLUSIONS: Results from this pilot-study suggest that intravenous PGE(1) may be used efficaciously and safely to prevent RCM-induced renal dysfunction in patients with pre-existing impaired renal function.     

{beta}2-Microglobulin Kinetics During Haemodialysis and Haemofiltration

Since the identification of ß₂-microglobulin as amajor component of ‘dialysis amyloid’, concern aboutits removal by different dialysis methods has been raised. Haemodialysiswith regenerated cellulose membranes increases serum ß₂-microglobulinby 10–15%. Serial measurements show a very early increaseduring cuprophan haemodialysis, the mechanism of which is asyet unknown. After cuprophan haemodialysis, serum values returnto the initial pretreatment concentrations by the time of thenext haemodialysis. In contrast to regenerated cellulose, dialysiswith polycarbonate lowers serum ß₂-microglobulin by8%, and dialysis with polysulphone by 53%. As opposed to cuprophan,after polysulphone haemodialysis the serum concentrations havenot returned to the initial pretreatment levels within 48 h.Comparison of ß₂-microglobulin removal using the samepolysulphone membrane for haemodialysis and haemofiltrationshows that ß₂-microglobulin is more effectively removedby convection than by diffusion when both treatment modes arematched for blood flow and urea clearance. Therefore, in contrast to haemodialysis with regenerated cellulosemembranes, where a transient, intradialytic release of ß₂-microglobulinis induced, significant removal is observed using, higher permeablemembranes. These findings may have implications for the generationof ‘dialysis amyloid’.     

Partial Purification and Characterisation of a Renal Growth Factor from Plasma of Uninephrectomised Rats

Renal growth factor activity was extracted from plasma of adultuninephrectomised rats and partially purified by gel filtrationand anion-exchanger FPLC. It induced a maximal stimulation ofmouse DNA synthesis in vivo at 1.75 µg/mouse. In addition,renal growth factor was found to maximally stimulate DNA synthesisin LLC-PK₁ cells at 150 ng/ml. This maximal response was thenfound to decrease with higher doses of renal growth factor,in vivo and in vitro. The apparent molecular weight of renalgrowth factor was estimated to be 17 K–22 K by gel filtration.It was found to be resistant to heat and to trypsin, but labileto reduction with dithiothreitol.     

Determinants of Plasma {beta}2 Microglobulin Concentration: Possible Relation to Membrane Biocompatibility

Beta₂ microglobulin (ß₂m) concentrations were measuredby radioimmunoassay in the serum of haemodialysed patients.ß₂m was higher in males (n=48) than in females (n=26),i.e. 40.3±10.1 mg/l (SD) vs 31.2±8.0, P<0.01).ß₂m was not significantly higher in patients withbone cysts (37.7±11.4 mg/l vs 37.0±10.0), butmedian duration of dialysis was significantly (P<0.01) longerin patients with bone cysts (90 vs 57 months). ß₂mwas lower in patients maintained on dialysis for less than 1year and whose residual urine volume was greater than 0.1 litreper day. During one single session of dialysis, using cuprophanemembranes, ß₂m increased acutely at 15 min and hadrisen by 32.4% at the end of the dialysis session, more thancould be explained by haemoconcentration. In contrast, ß₂macutely decreased by 38.7% during a single session using polysulphonemembranes and the steady state predialysis values were lowerby 37.1% after two weeks intermittent haemodialysis with polysulphonemembranes. After re-exposure to cuprophane serum ß₂mincreased to the original value. It is concluded that ß₂m concentrations on dialysisare a function of residual urinary volume, sex, and type ofmembrane used. Data are consistent with effective removal ofß₂m by membranes with high cut-off.     

Serum {alpha}2-macroglobulin in haemodialysis patients: baseline and kinetic studies

The pathogenesis of dialysis related amyloidosis remains unresolveddespite the identification of ß₂-microglobulin (ß₂M)as the major protein constituent, as well as other proteinsbeing present in the deposits. Among the latter we have assessedthe serum concentrations of ₂-macroglobulin (₂M) both in thebaseline stage and during the haemodialysis (HD) procedure.We have also assessed the influence of the membrane on ₂M kinetics. Fifteen HD patients with histologically proven dialysis-relatedamyloidosis (DRA group) and 15 HD patients clinically and radiologicallyconsidered dialysis-related amyloidosis free (control group)were included in the baseline study. Blood was sampled the daybefore the second dialysis of the week and ₂M, ß₂Mand ₁, antitrypsin were determined along with the routine biologicalanalysis of these patients. Serum ₂M was greater in dialysis-relatedamyloidosis than in control patients (t = 2.35; P<0.026).Serum ß₂M was similar in both groups. The serum ₂Mand ß₂M correlated in patients with dialysis-relatedamyloidosis (r = 0.64; P<0.01), while no correlation wasfound in controls (r = 0.17; NS). Stepwise analysis taking thepresence of dialysis-related amyloidosis as the dependent variableretained the serum ₂M concentration as the first variable inthe model (F = 4.4; partial r = 0.38; P<0.046). The sameproteins were determined in another group of seven patients,before and hourly during HD as well as 2 and 8 h after the endof HD during nine consecutive dialyses (3 cycles of 3 HD eachusing AN69 and cuprophane membranes in a crossover design).Serum ₂M significantly increased from hour 3 and continued toincrease 2 hours post-HD (+11% and +9% with AN69 and cuprophanerespectively; P<0.001). Total proteins peaked at hour 4 (+4% and +3% P<0.01) and decreased after HD. Serum ß₂Msignificantly decreased with AN69 HD ( – 29% P<0.001)and remained unchanged during cuprophane HD. In conclusion, significant increases in serum ₂M are observedimmediately after and during the early post-dialysis periods,regardless of the membrane used. Further, serum ₂M correlateswith ß₂M only in patients with dialysis-related amyloidosis,and this variable was retained in the multivariate regressionanalysis to predict dialysis-related amyloidosis. Although thebaseline results require confirmation with larger studies, wepostulate that the present results are of relevance for dialysis-relatedamyloidosis pathogenesis since ₂M, previously identified indialysis related amyloid deposits, is closely related to acute-phasereactant proteins, and interacts with the main infiltratingcells of the deposits (macrophages). ₂M modifications couldrepresent a new manifestation of the inflammatory response tothe haemodialysis procedure.     

Plasma transforming growth factor beta(1) and platelet activation: implications for studies in transplant recipients.

BACKGROUND: Evidence from animal models supports the hypothesis that dysregulated transforming growth factor beta(1) (TGF beta(1)) expression plays a role in chronic allograft rejection, the progression of diabetic nephropathy and fibrotic glomerulopathies. However, more evidence is required to support this hypothesis in man, and the current literature concerning blood TGF beta(1) levels in clinical studies is highly confused. We have investigated: (i) the hypothesis that the widespread practice of activating clinical samples prior to measurement of TGF beta(1) is detecting the platelet-released pool of TGF beta(1), artefactually generated on venepuncture and unrepresentative of the real circulating in vivo TGF beta(1) pool; and (ii) the effect of different immunosuppressive drugs on apparent TGF beta(1) plasma levels. METHODS: The effect of two different venepuncture procedures on plasma TGF beta(1) was compared in 10 healthy volunteers, one procedure designed to minimize platelet activation and the other representing standard venepuncture practice in a clinic situation. Blood samples from 52 renal transplant recipients on either cyclosporine or tacrolimus immunosuppression were taken by standard venepuncture to investigate the effect of immunosuppressive drugs on plasma TGF beta(1). Plasma TGF beta(1) and beta thromboglobulin were measured by ELISA. RESULTS: Among 10 healthy volunteers who underwent two different methods of venepuncture, eight of 10 had undetectable levels of TGF beta(1) (<100 pg/ml) under conditions that minimize platelet activation. In contrast, all 10 paired plasma samples collected by vacutainer had measurable TGF beta(1) (median 7.70 ng/ml, interquartile range 5.87-13.64 ng/ml) following acid/ urea activation. The median beta TG level (a measure of platelet degranulation) was 0.71 microg/ml (interquartile range 0.53-1.19 microg/ml) in the special collections compared with 3.39 microg/ml (interquartile range 2.27-4.33 microg/ml) in the vacutainer samples (P=0.0029). Among 52 allograft recipients there was a significantly higher mean TGF beta(1) level in plasma from patients on cyclosporine therapy compared with patients on tacrolimus (28,090+/-26,860 pg/ml vs 7173+/-10 610 pg/ml, respectively; P<0.002). Mean plasma beta TG levels were also significantly higher during cyclosporine therapy compared with tacrolimus (8.14+/-5.54 microg/ml vs 3.66+/-3.32 microg/ml, respectively; P<0.002). However, when TGF beta(1) values were corrected for the degree of platelet activation (by factoring with beta TG) there was no significant difference between TGF beta(1) levels on cyclosporine or tacrolimus (4117+/-2993 pg/microg beta TG vs 2971+/-658 pg/microg beta TG, respectively; P=0.294). CONCLUSIONS: To avoid erroneous hypotheses concerning TGF beta(1) and perpetuating confusion in the literature over levels in health and disease, it is imperative that proper internal controls for platelet activation are used. The effects of experimental treatments and drugs on platelet biology must be rigorously controlled when attempting to measure and interpret plasma levels of TGF beta(1) in clinical practice.     

Dialysis Arthropathy, {beta}2-Microglobulin and the Effect of Dialyser Membrane

Patients with dialysis arthropathy had the greatest mean serumß₂-microglobulin (59.5 mg/l) but there was no thresholdconcentration of ß₂-microglobulin above which allpatients developed dialysis arthropathy. Haemodialysis patientswithout dialysis arthropathy and patients on continuous ambulatoryperitoneal dialysis (CAPD) also had grossly elevated valuesof ß₂-microglobulin (47.9 mg/l and 30.7 mg/l respectively).There was a significant positive correlation between durationof treatment and serum ß₂ for the patients treatedby haemodialysis, but this was not the case for patients onCAPD. There was a significant negative correlation between residualurinary volume and serum ß₂-microglobulin for thepatients on haemodialysis without dialysis arthropathy, andalso for patients on CAPD. This was not true for the patientswith dialysis arthropathy. Both duration of treatment and residualurine volume correlated with serum ß₂-microglobulin,and therefore an analysis of covariance was used to take accountof this in comparing the groups. This showed that there wasno difference between serum ß₂-microglobulin in haemodialysispatients with and without dialysis arthropathy. However, CAPDpatients had a significantly lower corrected mean serum ß₂-microglobulinHaemodialysis with cuprophane membranes was associated withan increase in ß₂-microglobulin of 11.5%, whereashaemodialysis with polycarbonate was associated with a decreaseof 6.8% at 6 h. Our results provide circumstantial evidencethat repeated haemodialyses with cupro phane membranes may predisposelong-term haemo dialysis patients to dialysis arthropathy. CAPDpatients have lower ß₂-microglobulin concentrationsand may be less likely to develop dialysis a Long-term prospectivestudies are needed to confirm these assertions.