Prevention and treatment of renal osteodystrophy in children on chronic renal failure : European guidelines Childhood renal osteodystrophy -LRB- ROD -RRB- is the consequence of disturbances of the calcium-regulating hormones vitamin D and parathyroid hormone -LRB- PTH -RRB- as well as of the somatotroph hormone axis associated with local modulation of bone and growth cartilage function . The resulting growth retardation and the potentially rapid onset of ROD in children are different from ROD in adults . The biochemical changes of ROD as well as its prevention and treatment affect calcium and phosphorus homeostasis and are directly associated with the development of cardiovascular disease in pediatric renal patients . The aims of the clinical and biochemical surveillance of pediatric patients with CRF or on dialysis are prevention of hyperphosphatemia , avoidance of hypercalcemia and keeping the calcium phosphorus product below 5 mmol2/l2 . The PTH levels should be within the normal range in chronic renal failure -LRB- CRF -RRB- and up to 2 -- 3 times the upper limit of normal levels in dialysed children . Prevention of ROD is expected to result in improved growth and less vascular calcification . Introduction Chronic renal failure is associated with specific abnormalities of skeletal homeostasis , commonly called renal osteodystrophy -LRB- ROD -RRB- , which if not treated appropriately during the critical phases of skeletal growth can result in bone deformities and a disturbed growth pattern . The main factors for the development of ROD are disturbances in the calcium phosphate homeostasis , in vitamin D and parathyroid hormone metabolism as well as alterations in the somatotroph axis , i.e. , on the endocrine and paracrine levels . In recent years it has been recognized that the spectrum of renal bone disease covers ` high - ' as well as ` low-turnover ' conditions . As a consequence of chronic renal failure itself and of the treatment of renal bone disease , high plasma phosphate levels and an elevated calcium phosphorus product are common . These are important risk factors for the development of vascular calcification and cardiovascular morbidity and mortality in young adults who have been on renal replacement therapy since childhood -LSB- 1 , 2 -RSB- . Because aluminium-containing phosphate binders are no longer indicated in children , aluminium-related osteopathy is not considered in these recommendations . The European Pediatric Peritoneal Working Group -LRB- EPPWG -RRB- was established in 1999 by pediatric nephrologists with a major interest in peritoneal dialysis and has , among others , published guidelines on chronic and acute peritoneal dialysis -LSB- 3 , 4 , 5 , 6 -RSB- . The group incorporates pediatric nephrologists from 12 European countries . One of the functions of the group is to establish expert guidance in important clinical areas associated with chronic renal failure and dialysis -LSB- now the European Pediatric Dialysis Working Group -LRB- EPDWG -RRB- -RSB- in conjunction with other members of the multidisciplinary team . These guidelines were initiated and discussed at meetings of the group and developed by e-mail discussion to develop consensus of opinion based upon cumulative clinical experience and reported studies . Recommendations Recommendation 1 Clinical , biochemical and radiological markers of renal bone disease should be monitored regularly . The clinical markers of renal bone disease to be prevented are signs of overt rickets , slipped femoral epiphysis and disturbances of growth . The biochemical markers are plasma phosphate , calcium , alkaline phosphatase , bicarbonate and intact parathyroid hormone -LRB- PTH -RRB- . The minimal frequency of measurements -LRB- and target ranges -RRB- for biochemical markers in a stable phase vary according to renal function -LRB- Table 1 -RRB- . If the patient has active ROD , additional blood samples may be required . Table 1 Frequency of measurements for biochemical and radiological markers of renal osteodystrophyMarker1Frequency of measurement -LRB- every x month -RRB- TargetGFRGFRGFR < 15 , dialysis59 -- 3029 -- 15 -LRB- Evidence -RRB- Calcium or631Normal range -LRB- corrected calcium -RRB- 2Ionized calciumNormal range2Phosphate631Normal range for age bandCalcium phosphorus product631 ≤ 5.0 mmol2/l2Target range 3.3 -- 4.4 mmol2/l2 , 3Alkaline phosphatase631Normal range for age bandSerum bicarbonate/base excess631Normal range , at least : bicarbonate > 22 mmol/lBase excess > − 5 mmol/lIntact PTH/whole PTH631Normal range in moderate CRF -LRB- GFR > 29 ml/min/1 .73 m2 -RRB- Up to 2 -- 3 times upper limit of normal in advanced CRF or on dialysis25 - -LRB- OH -RRB- vitamin D3As indicated4As indicated4As indicated4 > 20 ng/lLeft hand and wrist X-ray6 -- 12No radiological signs of hyperparathyroidismNo Looser zones or osteopeniaCalcium : mmol/l in mg/dl : × 4 , phosphate mmol/l in mg/dl : × 3.0969 ; calcium phosphorus product : mmol2/l2 in mg2/l2 : × 12.387 . 2Corrected calcium -LRB- mg/dl -RRB- = measured calcium concentration -LRB- mg/dl -RRB- + 0.8 × -LSB- 4-measured albumin concentration -LRB- g/dl -RRB- -RSB- ; corrected calcium -LRB- mmol/l -RRB- = measured calcium concentration -LRB- mmol/l -RRB- +0.2 × -LSB- 4-measured albumin concentration -LRB- g/dl -RRB- -RSB- . 3Depending on age . 4Only in patients with suspected vitamin D deficiency Plasma calcium must be adjusted for albumin levels -LRB- = corrected calcium -RRB- or by measuring ionized -LRB- free -RRB- calcium , in patients with hypoalbuminemia or acid-base disorders . Ionized calcium values are often available on blood gas analysis systems with calcium-sensitive electrodes , but correct values are only obtained in validated conditions . Alkaline phosphatase as a marker for osteoblast activity has particular importance in both low and high-turnover bone disease with elevated or low alkaline phosphatase serum levels , respectively . PTH should be monitored monthly in advanced CRF -LRB- GFR < 15 ml/min/1 .73 m2 -RRB- because of its rapid changes . Different assays are available for the measurement of PTH . In principle , intact PTH -LRB- 1 -- 84 -RRB- -LRB- iPTH -RRB- should be measured because it is believed to represent the active hormone . For this purpose , two-site assays were developed at the end of the 1980s -LSB- 7 -RSB- . However , PTH fragments accumulating in end-stage renal disease may also show biological activity . Furthermore , recent research using plasma samples from healthy and renal patients analyzed by HPLC and the iPTH assay demonstrated cross-reactivity of the intact PTH-assay with a PTH-fragment -LRB- PTH7 -- 84 -RRB- . Therefore , new assays were developed that detect only PTH molecules with a complete N-terminal end , called `` whole-PTH '' assays -LSB- 8 -RSB- . In adult and pediatric dialysis patients , these `` whole PTH '' assays yielded 30 -- 60 % lower PTH levels than the intact PTH assay . Despite theoretical considerations , no significant improvement in the distinction between the different forms of renal bone disease using the `` whole PTH '' assays has yet been demonstrated in clinical practice -LSB- 9 , 10 , 11 , 12 -RSB- . Radiological signs of renal bone disease , which alone are not sensitive enough to indicate therapy-adaptations , include signs of hyperparathyroidism and growth zone lesions . Periosteal resorption zones and metaphyseal changes are the most obvious signs . In late adolescent and young adult patients , Looser zones as specific signs of osteomalacia may be found . The sites and expression of bone lesions are age-dependent according to the age-dependent growth and remodeling at different sites of the skeleton . In pre-school children , the metaphysis of the distal radius and ulna may present only minor abnormalities , whereas more severe lesions may be seen at the upper and lower femoral epiphysis . Adynamic bone disease shows no specific radiological changes . However , extraosseous calcifications , fractures or osteopenia may indicate adynamic bone disease . Histological evaluation of bone biopsy specimens remain the `` gold standard '' in assessment of renal osteodystrophy . However , due to its invasive nature , bone biopsies are not performed in clinical practice , but they remain an important tool for research -LSB- 12 -RSB- . Recommendation 2 Metabolic acidosis should be corrected -LRB- evidence -RRB- . Chronic metabolic acidosis leads to increased bone resorption and inhibits endochondral bone formation in animal experiments -LSB- 13 , 14 -RSB- . Infants with isolated chronic metabolic acidosis show growth retardation -LSB- 14 -RSB- . In adult hemodialysis patients , negative aspects of chronic metabolic acidosis on metabolism have been reported -LSB- 15 -RSB- . Therefore , metabolic acidosis should be corrected by a stepwise approach : The first step is optimization of the dialysis regimen both for HD - and PD-patients -LSB- 4 , 15 -RSB- . Since the introduction of HCO3-based PD solutions -LSB- 16 -RSB- , metabolic acidosis is a less frequent problem . In combination with a daytime-dwell , patients may even be at risk for alkalosis -LSB- 17 -RSB- . However , if these adaptations do not result in correction of the metabolic acidosis , sodium bicarbonate should be administered orally . Whenever possible , formulations that dissolve in the small intestine should be used . The use of sodium citrate increases the risk of aluminium absorption -LSB- 18 , 19 -RSB- . Acidosis should be corrected to the normal range of the local laboratory . Recommendation 3 The plasma-phosphate level should be kept within the normal age-specific range -LRB- evidence -RRB- . In children with chronic renal failure , hyperphosphatemia is observed at GFR levels below 40 ml/min/1 .73 m2 -LSB- 20 -RSB- and almost always in children on dialysis . Hyperhosphatemia has several deleterious effects on PTH secretion -LSB- 21 , 22 -RSB- , parathyroid cell proliferation -LSB- 23 -RSB- and soft tissue -LRB- vascular -RRB- calcification -LSB- 1 , 2 -RSB- . Increased phosphate levels stimulate PTH secretion in vitro -LSB- 24 -RSB- and in vivo -LSB- 21 , 25 -RSB- independent of calcium and 1,25 -LRB- OH -RRB- 2 vitamin D3 levels . This effect of phosphate on PTH secretion occurs via post-transcriptional processes by regulating pre-pro-PTH mRNA stability -LSB- 26 -RSB- . Furthermore , a reduction of serum-phosphate results in a parallel reduction of PTH levels without change in serum calcium -LSB- 27 -RSB- . Hyperphosphatemia accelerates parathyroid cell proliferation -LSB- 23 -RSB- , which can result in nodular hyperplasia and severe hyperparathyroid bone disease necessitating parathyroidectomy -LRB- see below -RRB- . Increased plasma phosphate levels have a profound effect on soft tissue and vascular calcification , which are often observed in young patients on or even after dialysis -LSB- 1 , 2 , 28 -RSB- , increasing the risk of cardiovascular morbidity and mortality . Block et al. -LSB- 29 -RSB- found an increased risk of death for serum phosphate exceeding 2.09 mmol/l in adult patients ; other studies described a much lower phosphate level -LRB- ~ 1.4 -- 1.6 mmol/l -RRB- at which a higher incidence of calcifications was found -LSB- 1 , 30 , 31 -RSB- . The histology of the calcification pattern is quite distinct from the process of atherosclerosis . In renal patients , extensive calcification of the tunica media occurs even after short periods of dialysis , as shown by investigations of the epigastric artery in adult patients -LSB- 32 -RSB- . Experimental work gave evidence for an active role of phosphate in this process as phosphate induces osteoblastic marker followed by calcification in cultured smooth muscle cells -LSB- 33 -RSB- . Dialysis patients are at risk of heart failure . Although this is a multifactorial process , it was demonstrated in animal experiments that high plasma phosphate levels per se accelerated cardiac fibrosis -LSB- 34 -RSB- . Recommendation 4 If plasma phosphate is elevated , phosphate intake should be limited to the recommended levels . Dietary intake of protein and as a consequence of phosphate in western countries usually exceeds the recommended intake at least in adolescent dialysis patients . Therefore , dietary counseling should be performed by a trained dietician -LSB- 35 -RSB- . If the dietary records show a protein intake above the recommended level for pediatric dialysis patients , the patients and their parents should be trained to reduce their phosphate intake . This may start with the reduction of dairy products and/or substitution with special low-phosphorus products and a reduction of meat intake . Recommendation 5 In case of hyperphosphatemia , the dialysis efficacy should be optimized -LRB- evidence -RRB- . Phosphate is slowly transported via the peritoneal membrane into the dialysate -LSB- 36 -RSB- . The dialysate to plasma ratio -LRB- D/P -RRB- for phosphate is volume and time related ; after 4 h in standard peritoneal equilibration tests , this ratio reaches the value of 0.5 -- 0.6 , which is a much lower ratio than for urea , which is usually nearly 1 . Therefore , an increase in phosphate removal should consider not only an increase in dwell volume to 1,000 -- 1,400 ml/m2 BSA , but also dwell time optimization , avoiding a too short dwell time . A daytime dwell should be added . The details of a dialysis regimen optimization has been described elsewhere -LSB- 5 -RSB- . However , due to the low clearance of phosphate achievable even with an optimized PD regimen , dietary phosphate restriction and the use of oral phosphate binders are almost always necessary . The dialytic phosphate removal kinetic during a hemodialysis session differs from urea kinetic : after an early initial drop the phosphatemia decrease only slowly -LSB- 37 , 38 -RSB- . Therefore , it is admitted that the hemodialysis phosphate purification capacity is directly impacted by the duration of the dialysis session , because the phosphate shift from the intracellular to the vascular compartment is time-dependent . Therefore , longer dialysis sessions -LSB- 39 -RSB- or daily dialysis sessions -LSB- 40 , 41 , 42 -RSB- offer a unique , powerful dialysis method for optimal phosphate purification . The hemodialysis modality , HD , HF or HDF , and the type of the synthetic membrane used have only a limited impact on dialytic phosphate removal -LSB- 38 -RSB- , whereas the dialysate composition in terms of glucose and bicarbonate concentration are presumed to be of importance -LSB- 42 , 43 , 44 -RSB- , modulating a shift of intracellular phosphate trapped in the cells , where it is not available for dialysis exchanges . Recommendation 6 For control of hyperphosphatemia , aluminium-free phosphate binders should be administered -LRB- evidence -RRB- . Phosphate binders are necessary to reduce phosphate absorption from the gut . Ca-containing phosphate binders , i.e. , calcium carbonate -LRB- CaCO3 , elemental calcium content 40 % -RRB- or calcium acetate -LRB- CaAc , elemental calcium content 25 % -RRB- should be used as the first line . No data are published on the efficacy and safety of calcium acetate/magnesium carbonate compound phosphate binders in pediatric patients . The upper intake level of elemental calcium is suggested to be 2,500 mg/day for healthy children above 4 years of age -LSB- 19 -RSB- . Whereas for adult dialysis patients the DOQI guidelines suggest to limit the elemental calcium intake to 2,000 mg/day , no safe upper level can be given for the pediatric age band . However , a positive calcium balance in the range of +200 -- +300 mg in the growing skeleton should be maintained . In contrast , a too high calcium load should be avoided , because one of the identified risk factors for soft tissue calcification in pediatric CRI patients was cumulative calcium intake -LSB- 2 , 24 -RSB- . Calcium-containing phosphate binders are started at approximately 500 mg per 200 mg phosphate content of the diet -LRB- 0 -- 1 years , 1 -- 2 × 500 mg ; 1 -- 4 years , 2 -- 3 × 500 mg/day ; 5 -- 8 years , 3 -- 4 × 500 mg/day ; 9 -- 18 years , 5 × 500 mg -RRB- . An alternative approach is a start dose of approximately 50 mg/kg/day , which is well below the doses given in clinical studies . Phosphate binders are then adjusted to normalize serum-phosphate and calcium . CaCO3 can be crushed to fine powder , or a 10 % solution can be used for administration in infants , often via a feeding tube . The phosphate binders should be taken with meals , because fecal excretion of phosphate was higher when calcium acetate was given with meals instead of in between meals -LSB- 45 -RSB- . Biochemically , CaAc has a higher phosphate binding capacity , which is independent of the pH. The higher efficacy of CaAc , calculated on a weight basis compared to CaCO3 , for phosphate control was also shown in clinical practice in adult and pediatric patients -LSB- 46 , 47 -RSB- . Unfortunately , compliance of the patients with the intake of phosphate binders is often poor and should be checked regularly . It may be helpful if the patient is involved in selecting the flavor and size of the phosphate binder and has regular contact with the dietician at clinic appointments . Snacks during the day are often a source of additional phosphate and require additional phosphate binders . Patients treated with Ca-containing phosphate binders and active vitamin D metabolites are particularly at risk for the development of hypercalcemia . Therefore , in case of hypercalcemia , active vitamin D metabolites should be stopped -LRB- see recommendation 12 -RRB- . If hypercalcemia , the most common side-effect of Ca-containing phosphate binder therapy , persists or the calcium phosphorus product exceeds 5.0 mmol2/l2 with the use of Ca-containing phosphate binders , the calcium content in the dialysis fluid should be reduced -LSB- 5 -RSB- . Furthermore , the dose of the calcium-containing phosphate binders should be reduced whenever possible or they should be replaced by Ca - and aluminium-free phosphate binders , because epidemiological studies have shown a direct relationship between serum phosphate and calcium phosphorus product on mortality -LSB- 29 , 48 -RSB- . The only commercially available aluminium - and calcium-free phosphate binder is Sevelamer . This compound is usually taken orally as capsules , but anecdotally can be delivered via enteral tubes by dissolving the capsule in 5 ml water -LRB- instruction Fa genzyme -RRB- . Aluminium-containing phosphate binders or calcium citrate increase intestinal aluminium absorption and should not be used in pediatric dialysis patients . An alternative phosphate binder that is available is lanthanum carbonate , which has a high affinity for phosphate , and is minimally absorbed in the intestine . In a randomized study in adult patients , lanthanum carbonate controlled plasma phosphate levels well and induced less adynamic bone disease than CaCO3 -LSB- 49 -RSB- . However , no long-term data on the effect of lanthanum on bone , on which surface lanthanum can accumulate -LSB- 50 -RSB- , and its safety profile for use in children are available yet . Recommendation 7 Vitamin D deficiency should be avoided -LRB- evidence -RRB- . In early renal failure , an increase of PTH correlates positively with 25 -LRB- OH -RRB- - vitamin D3 levels -LSB- 51 -RSB- . Renal synthesis of 1,25 -LRB- OH -RRB- 2 D3 is impaired in chronic renal disease . However , extrarenal cells , i.e. , macrophages and osteoblasts , are also capable of 1,25 -LRB- OH -RRB- 2 D3 production -LSB- 52 -RSB- . In contrast to the kidney , the extra-renal synthesis is strictly substrate dependent . It has been shown that supplementation with vitamin D3 in elderly CRF patients with 25 -LRB- OH -RRB- - vitamin D3 levels between 20 and 50 pg/ml decreased PTH serum concentrations -LSB- 53 -RSB- . Furthermore , 25-OH-vitamin D3 , but not 1,25 -LRB- OH -RRB- 2 D3 , improved muscular function and phosphate content -LSB- 54 -RSB- . Therefore , vitamin D deficiency should be prevented . Recommendation 8 Marked hyperparathyroidism should be prevented in children with CRF prior to dialysis -LRB- evidence -RRB- . Due to the risk of persisting bone disease and the development of parathyroid adenoma , an increase in PTH above normal to slightly elevated levels should be prevented in children with CRF . Therefore , low doses of active vitamin D metabolites should be given in time . In adult patients this treatment regimen has resulted in controlled iPTH without negative aspects -LSB- 55 -RSB- . In children with moderate renal failure -LRB- GFR > 30 ml/min/1 .73 m2 -RRB- , normal levels of PTH in association with strictly controlled phosphate levels are associated with a normal ratio of intact PTH to `` whole '' PTH and normal levels of alkaline phosphatase , indicating a physiological PTH secretion and unremarkable bone turnover -LSB- 10 -RSB- . In such patients , slight catch-up growth with PTH levels at the upper limit of normal was reported -LSB- 56 -RSB- . In a sub-group analysis , improved growth was restricted to patients with enteral feeding tubes . Recommendation 9 PTH levels should be kept at two to three times the upper limit of the normal range in end-stage renal disease -LRB- evidence -RRB- . Grossly elevated PTH concentrations -LRB- > four times the upper normal range -RRB- in the presence of normal or high serum calcium and high alkaline phosphatase are almost always associated with high-turnover bone disease -LSB- 56 , 57 -RSB- . The exception is intermittent therapy with high-dose active vitamin D metabolites , because 1,25 -LRB- OH -RRB- 2 vitamin D3 directly inhibits osteoblastic function and proliferation . Hyperphosphatemia can occur in hyperparathyroid bone disease because of excessive bone resorption . However , in pediatric dialysis patients low and even normal levels of PTH are reported to be associated with low turnover bone disease -LSB- 57 -RSB- . A PTH concentration higher than in healthy subjects is needed to stimulate bone turnover due to resistance of the skeleton to PTH in advanced renal failure . If PTH falls into the -LRB- low -RRB- normal range , the risk of hypercalcemia increases -LSB- 58 -RSB- . Hyperphosphatemia and hypercalcemia can develop in low turnover bone disease because the skeleton is unable to take up enough phosphate and calcium . Low bone turnover or adynamic bone disease is increasingly observed in adult -LSB- 59 -RSB- as well as in pediatric PD patients -LSB- 56 -RSB- . Risk factors for adynamic bone disease are high calcium intake -LRB- Ca-containing phosphate binders -RRB- , therapy with high doses of active vitamin D metabolites , peritoneal dialysis using dialysis fluid with high calcium content and age -LRB- adolescents after the growth spurt -RRB- . Biochemically , hypercalcemia and/or PTH levels within or below the normal range are indicative of low turnover bone disease -LSB- 56 , 58 -RSB- , which may adversely affect growth in dialysed children -LSB- 60 -RSB- . Therefore , active vitamin D therapy should be reduced or stopped if PTH falls below the low normal range -LSB- 57 , 59 , 61 -RSB- . If PTH remains below normal despite normal calcium and phosphate levels , the dialysate should be changed to low calcium solutions -LRB- 1.0 or 1.25 mmol/l -RRB- to stimulate PTH secretion -LSB- 61 -RSB- , if this is not already used as the standard solution -LSB- 5 -RSB- . Recommendation 10 If PTH is elevated in children with CRF or if PTH is elevated more than two to three times normal in the presence of Pi < 2 mmol/l in dialyzed children , active vitamin D metabolites should be administered orally -LRB- evidence -RRB- . Treatment with active vitamin D metabolites results in biochemical and/or histological improvement in patients with high-turnover bone disease . Doses of 1,25 -LRB- OH -RRB- 2 vitamin D3 or 1α - -LRB- OH -RRB- vitamin D3 usually range from 0.1 µg/day to 0.75 µg/day , with a starting dose of 20 -- 40 ng/kg . High doses often induce hypercalcemia , necessitating the reduction or discontinuation of vitamin D therapy -LRB- see hypercalcemia -RRB- . As to the mode of administration of the active vitamin D metabolites , intravenous or oral therapy , have the same efficacy -LSB- 63 -RSB- . Therefore , the oral route should be preferred in children on dialysis . Doses should be given in the evening , because fewer episodes of hypercalcemia have been reported compared to taking the vitamin D metabolites in the morning -LSB- 64 -RSB- . Intermittent therapy was shown to be effective to suppress elevated PTH serum levels -LSB- 59 -RSB- . However , recent data indicate that large intermittent 1,25 -LRB- OH -RRB- 2 vitamin D3 doses adversely affect bone turnover -LSB- 57 , 61 , 65 -RSB- and chondrocyte activity -LSB- 66 -RSB- resulting in low turnover bone disease and in reduced growth -LSB- 65 , 66 , 67 , 68 -RSB- . In addition , episodes of hypercalcemia occur with similar frequency with the intermittent or oral administration of vitamin D metabolites -LSB- 63 , 69 -RSB- ; furthermore , the intermittent mode of administration was not shown to be more effective in suppressing PTH levels in adult -LSB- 70 -RSB- and pediatric -LSB- 63 -RSB- patients in prospective , randomized trials . For the above mentioned reasons , intermittent high-dose vitamin D therapy should be avoided in pediatric patients . Three newer vitamin D analogs , so called `` non-hypercalcemic '' vitamin D analogs -LRB- doxercalciferol , paricalcitol and 22-oxa-calciferol -RRB- , have been introduced in clinical use in adult patients with secondary hyperparathyroidism due to CRF . Despite their ability to induce less hypercalcemia in animal experiments , only one clinical study with a head-to-head comparison of the new vitamin D analogue -LRB- paricalcitol -RRB- is available demonstrating a borderline reduction of hypercalcemia or a decrease in calcium phosphorus product -LSB- 71 -RSB- . No data are available for their use in children . Agents that specifically enhance the sensitivity of the calcium-sensing receptor , called calcimimetics , used in combination with active vitamin D metabolites in adult dialysis patients resulted in a persistent decrease of PTH levels without elevated calcium phosphorus product -LSB- 72 -RSB- . If plasma Ca or Pi is high in the presence of elevated PTH , the calcimimetics may become first choice . However , no data for pediatric patients are available . Recommendation 11 Treatment with growth hormone should not be started in the presence of severe hyperparathyroid bone disease . It is well established that the uremic growth retardation is at least in part due to disturbed pulsatile secretion of growth hormone as well as a peripheral growth hormone resistance -LSB- 73 -RSB- . After correction of metabolic acidosis and normalization of caloric intake , administration of recombinant human growth hormone increases growth velocity resulting in catch-up growth and in improved final height -LSB- 74 -RSB- . Growth hormone directly stimulates growth cartilage proliferation and metabolism and osteoblast activity -LSB- 75 , 76 -RSB- . This growth promoting effect of GH was suspected to increase the metaphyseal instability induced by severe hyperparathyroidism . In hyperparathyroid bone disease , i.e. , osteitis fibrosa , a fibrous layer is formed at the metaphyseal junction of the growth plate with the bone tissue . This can lead to epiphyseal slipping resulting in gross deformities of the affected long bones -LSB- 77 -RSB- . Therefore , parathyroid hormone , as well as calcium and phosphate levels , should be treated towards the recommended normal ranges prior to administration of GH . However , in a recent prospective study , no increased frequency of epiphyseal slipping associated with the use of growth hormone was reported -LSB- 78 -RSB- . After the start of GH administration , an increase of PTH levels was observed -LSB- 79 , 80 -RSB- . Recommendation 12 In case of hypercalcemia , active vitamin D metabolites and calcium-containing phosphate binders should be stopped and dialysate changed to low calcium solutions -LRB- evidence -RRB- . Hypercalcemia and elevated calcium phosphorus product -LRB- > 5.0 mmol2/l2 -RRB- should be corrected promptly -LRB- Fig. 1 -RRB- . Vitamin D metabolites should be discontinued and in case of persisting hypercalcemia the dialysate calcium decreased to low calcium dialysate . Laboratory values should be controlled weekly until improvement is seen . One exception is hypercalcemia due to severe hyperparathyroidism and high-turnover bone disease . In addition , in patients with residual renal function , loop diuretics , i.e. , furosemide may increase calcium excretion . If hypercalcemia is not corrected despite the discontinuation of active vitamin D metabolites , calcium-containing phosphate binders should be replaced by calcium-free phosphate binders to reduce the calcium load . Hypercalcemia is reported in up to 25 % of patients taking calcium-containing phosphate binders -LRB- -LSB- 1 , 29 -RSB- , see recommendation 6 -RRB- . Fig. 1 Clinical algorithm for treatment of elevated calcium phosphorus product in children with CRF . Ca-cont calcium containing ; Ca-free calcium-free ; phos-binder phosphate binder ; vit D vitamin D ; ADBD adynamic bone disease Recommendation 13 The calcium phosphorus product should be kept within the normal range , at least below 5.0 mmol2/l2 -LRB- 60 mg2/dl2 -RRB- -LRB- evidence -RRB- . One of the risk factors for cardiovascular morbidity was shown to be an elevated calcium phosphorus product above 5.0 mmol2/l2 -LRB- see also recommendation 2 -RRB- . If the calcium phosphorus product is elevated above this level , phosphate and/or calcium levels should be reduced . This may be achieved by an increase in phosphate binders , reduction/stopping of active vitamin D metabolites and use of low calcium -LRB- Ca + + 1.0 -- 1.25 mmol/l -RRB- dialysate . Recommendation 14 Parathyroidectomy has to be considered in case of severe , therapy-refractory hyperparathyroidism with radiological signs in combination with hypercalcemia and/or elevated calcium phosphorus product -LRB- evidence -RRB- . Severe hyperparathyroidism may no longer react to even high doses of 1,25 -LRB- OH -RRB- 2 vitamin D3 and reduction of phosphate levels . The clinical observations in adults show that patients with at least one parathyroid gland larger than 0.5 cm3 or 1.0 cm in diameter usually do not respond to active vitamin D metabolites -LSB- 81 , 82 -RSB- . Therefore , persisting grossly elevated PTH levels in the presence of high-dose active vitamin D metabolites , radiological signs of hyperparathyroidism on wrist X-ray and/or high serum calcium and normal phosphate and/or elevated calcium phosphorus product indicate the need for surgical parathyroidectomy . Medical parathyroidectomy by alcohol injection was described in adult patients -LSB- 83 -RSB- , but to our knowledge not in pediatric patients . Parathyroid hormone stimulates bone turnover and regulates calcium homeostasis , both of which are of utmost importance in the growing skeleton . Therefore , subtotal parathyroidectomy or autotransplantation of parathyroid tissue is recommended in children or adolescents , avoiding hypoparathyroidism -LRB- -LSB- 84 -RSB- , review -RRB- . However , these procedures carry the risk of recurrence of severe parathyroid tissue hyperplasia . The use of the new calcimimetic agents -LSB- 85 -RSB- may help to prevent the most severe hyperparathyroidism and therefore modify the approach to that condition .

O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

chronic renal failure
secondary hyperparathyroidism
vitamin d
parathyroidectomy


The

management

of

anemia

in

pediatric

peritoneal

dialysis

patients

Anemia

is

common

in

chronic

renal

failure

Guidelines

for

the

diagnosis

and

treatment

of

anemia

in

adult

patients

are

available

With

respect

to

the

diagnosis

and

treatment

in

children

on

peritoneal

dialysis

the

European

Pediatric

Peritoneal

Dialysis

Working

Group

-LRB-

EPPWG

-RRB-

has

produced

guidelines

After

a

thorough

diagnostic

work-up

treatment

should

aim

for

a

target

hemoglobin

concentration

of

at

least

g/l

This

can

be

accomplished

by

the

administration

of

erythropoietin

and

iron

preparations

Although

there

is

sufficient

evidence

to

advocate

the

intraperitoneal

administration

of

erythropoietin

most

pediatric

nephrologists

still

apply

erythropoietin

by

the

subcutaneous

route

Iron

should

preferably

be

prescribed

as

an

oral

preparation

Sufficient

attention

has

to

be

paid

to

the

nutritional

intake

in

these

children

There

is

no

place

for

carnitine

supplementation

in

the

treatment

of

anemia

in

pediatric

peritoneal

dialysis

patients

The

treatment

of

anemia

in

chronic

kidney

disease

has

been

summarized

in

the

NKF/DOQI

guidelines

-LSB-

-RSB-

which

have

recently

been

updated

-LSB-

-RSB-

European

best

practice

guidelines

have

also

been

recently

published

-LSB-

-RSB-

These

published

guidelines

pay

no

or

very

limited

attention

to

the

special

situation

in

children

The

European

Pediatric

Peritoneal

Dialysis

Working

Group

-LRB-

EPPWG

-RRB-

was

established

in

by

pediatric

nephrologists

with

a

major

interest

in

peritoneal

dialysis

and

has

among

other

things

published

guidelines

on

commencing

elective

chronic

peritoneal

dialysis

-LSB-

-RSB-

One

of

the

functions

of

the

group

is

to

establish

expert

guidance

in

important

clinical

areas

associated

with

peritoneal

dialysis

in

conjunction

with

other

members

of

the

multidisciplinary

team

These

guidelines

were

initiated

and

discussed

at

meetings

of

the

group

and

developed

by

e-mail

discussion

to

develop

a

consensus

of

opinion

based

upon

cumulative

clinical

experience

and

reported

studies

The

present

guidelines

apply

to

the

management

of

anemia

in

pediatric

peritoneal

dialysis

patients

Definition

of

anemia

More

than

in

adult

patients

hemoglobin

and

hematocrit

values

are

age

dependent

in

children

-LSB-

-RSB-

-LRB-

Tables

-RRB-

Also

concentrations

of

ferritin

transferrin

and

iron

are

dependent

on

age

Early

iron

deficiency

may

be

diagnosed

by

an

increase

in

hypochromic

red

blood

cells

-LSB-

-RSB-

The

technique

for

measuring

this

parameter

is

not

available

everywhere

Since

children

with

renal

failure

generally

do

not

have

underlying

co-morbidity

contributing

to

anemia

it

is

obvious

that

in

children

on

peritoneal

dialysis

normal

hematocrit

levels

should

be

aimed

for

-LRB-

opinion

-RRB-

The

work-up

of

the

anemic

child

is

not

different

from

the

work-up

that

is

advocated

for

adult

patients

by

the

DOQI

guidelines

although

the

importance

of

a

stool

test

for

occult

blood

may

be

less

useful

in

this

age

group

-LSB-

-RSB-

-LRB-

opinion

-RRB-

Iron

deficiencyClinical

historyAssessment

of

nutritional

statusHemoglobin

and

hematocritRed

blood

cell

indicesReticulocyte

countIron

parametersSerum

ironTable

Reference

ranges

of

anemia

parameters

in

children

-LSB-

-RSB-

hemoglobin

and

hematocrit

-LRB-

M

male

F

female

-RRB-

Hemoglobin

-LRB-

g/dl

-RRB-

Hematocrit

-LRB-

-RRB-

years

Reference

ranges

of

anemia

parameters

in

children

-LSB-

-RSB-

ferritinFerritin

-LRB-

ng/ml

-RRB-

months

Reference

ranges

of

anemia

parameters

in

children

-LSB-

-RSB-

iron

-LRB-

M

male

F

female

-RRB-

Iron

-LRB-

μg/dl

-RRB-

Reference

ranges

of

anemia

parameters

in

children

-LSB-

-RSB-

transferrinTransferrin

-LRB-

mg/dl

-RRB-

After

this

work-up

has

been

completed

iron

and/or

erythropoietin

therapy

should

be

initiated

to

obtain

a

target

hemoglobin

concentration

of

at

least

g/dl

-LRB-

hematocrit

-RRB-

although

there

is

a

tendency

to

increase

this

target

-LSB-

-RSB-

-LRB-

opinion

-RRB-

A

target

hemoglobin

concentration

of

at

least

g/l

should

be

aimed

for

in

children

on

peritoneal

dialysis

-LRB-

opinion

-RRB-

Erythropoietin

Most

experience

has

been

obtained

with

subcutaneous

administration

of

recombinant

human

erythropoietin

-LSB-

-RSB-

Erythropoietin

is

available

as

erythropoietin

alpha

and

beta

Erythropoietin

alpha

and

beta

differ

noticeably

in

their

formulation

excipients

No

clear

prescription

schedules

for

starting

erythropoietin

are

available

for

children

and

schedules

provided

in

the

literature

vary

greatly

It

seems

reasonable

to

start

with

a

subcutaneous

dosage

of

U/kg

body

weight

or

times

per

week

In

some

selected

patients

-LRB-

particularly

if

started

in

the

predialysis

period

-RRB-

a

lower

frequency

or

dosage

can

be

attempted

In

studies

in

adult

patients

once

weekly

subcutaneous

administration

of

erythropoietin

was

effective

for

the

treatment

of

renal

anemia

-LSB-

-RSB-

It

is

well

known

that

younger

children

need

relatively

more

erythropoietin

than

older

ones

-LSB-

-RSB-

-LRB-

evidence

-RRB-

Maintenance

recommendations

vary

from

U/kg/week

for

a

child

with

a

weight

of

kg

to

U/kg/week

for

a

child

with

a

weight

of

kg

-LSB-

-RSB-

-LRB-

opinion

-RRB-

Each

child

will

need

the

dosage

titrated

to

achieve

the

target

hemoglobin

concentrations

For

the

titration

of

erythropoietin

the

DOQI

guidelines

can

be

used

-LSB-

-RSB-

If

the

increase

in

hematocrit

after

initiation

of

erythropoietin

therapy

is

less

than

over

a

to

period

the

dose

should

be

increased

by

If

the

absolute

rate

of

increase

in

hematocrit

after

initiation

of

erythropoietin

therapy

or

after

a

dose

increase

exceeds

per

month

the

weekly

dose

of

erythropoietin

should

be

decreased

by

Side

effects

of

erythropoietin

therapy

are

rare

increased

clotting

tendency

hypertension

and

seizures

are

to

be

considered

the

consequence

of

the

therapeutic

effect

rather

than

an

adverse

effect

of

the

preparation

Nevertheless

blood

pressure

should

be

carefully

monitored

during

therapy

Recently

attention

was

drawn

to

a

severe

side

effect

of

erythropoietin

therapy

pure

red

blood

cell

aplasia

due

to

the

occurrence

of

neutralizing

antierythropoietin

antibodies

-LSB-

-RSB-

Subsequent

investigations

showed

that

this

was

in

most

cases

associated

with

subcutaneously

administered

erythropoietin

alpha

from

one

brand

although

some

cases

have

also

been

described

using

the

other

brand

available

on

the

market

The

application

of

erythropoietin

beta

is

associated

with

a

much

lower

incidence

of

this

severe

side

effect

In

many

countries

the

subcutaneous

administration

of

erythropoietin

alpha

is

discouraged

at

present

The

administration

of

darbepoetin

alpha

is

not

associated

with

pure

red

blood

cell

aplasia

but

it

should

be

remembered

that

experience

with

this

drug

is

still

limited

Subcutaneous

administration

of

drugs

is

psychologically

distressing

especially

for

children

-LRB-

opinion

-RRB-

The

use

of

ultrafine

needles

and

special

injection

pens

may

help

to

alleviate

the

upset

In

adult

peritoneal

dialysis

patients

a

noncompliance

with

erythropoietin

administration

was

reported

in

between

and

of

patients

-LSB-

-RSB-

For

children

no

data

on

erythropoietin

are

available

but

from

clinical

practice

it

is

known

that

subcutaneous

administration

is

frightening

and

a

source

of

conflict

between

child

and

caregivers

Limited

data

are

available

on

the

compliance

with

another

drug

which

has

to

be

administered

subcutaneously

recombinant

human

growth

hormone

Noncompliance

was

reported

to

be

between

and

-LSB-

-RSB-

In

one

study

it

was

reported

that

noncompliance

increased

significantly

from

at

year

to

at

years

-LSB-

-RSB-

Intravenous

administration

is

a

more

expensive

alternative

but

will

rarely

be

applied

in

pediatric

peritoneal

dialysis

patients

-LSB-

-RSB-

Despite

some

initial

discouraging

but

still

frequently

cited

reports

in

the

literature

erythropoietin

can

be

administered

very

well

by

the

intraperitoneal

route

-LSB-

-RSB-

If

erythropoietin

is

administered

in

a

small

volume

of

dialysis

fluid

-LRB-

bags

are

commercially

available

-RRB-

bioavailability

is

similar

to

that

after

subcutaneous

administration

of

the

same

amount

of

hormone

-LSB-

-RSB-

-LRB-

evidence

-RRB-

Mean

dosage

needed

for

maintaining

the

target

hematocrit

decreased

from

to

U/kg/week

if

the

drug

was

administered

in

a

dialysis

bag

during

the

daytime

-LSB-

-RSB-

In

a

more

recent

study

in

patients

on

nightly

intermittent

peritoneal

dialysis

mean

dosage

was

U/kg/week

-LSB-

-RSB-

Dialysis

adequacy

is

a

major

factor

of

concern

in

such

a

regimen

KT/V

urea

was

in

the

group

studied

However

in

some

patients

the

application

of

intraperitoneal

erythropoietin

may

be

limited

by

the

inability

to

obtain

adequate

dialysis

-LRB-

opinion

-RRB-

In

patients

not

achieving

adequate

dialysis

the

daytime

period

should

be

used

for

additional

dialysis

exchanges

In

this

category

that

period

will

not

be

available

for

intraperitoneal

erythropoietin

therapy

Peritonitis

frequency

does

not

need

to

increase

using

intraperitoneal

erythropoietin

the

two

pediatric

studies

reporting

peritonitis

rates

mention

one

episode

every

and

treatment

months

respectively

-LSB-

-RSB-

One

early

study

in

children

was

broken

off

because

of

a

high

peritonitis

rate

-LSB-

-RSB-

possibly

this

was

due

to

insufficient

training

of

the

caregivers

Recently

an

erythropoietin

analogue

was

developed

-LRB-

darbepoetin

alpha

or

NESP

novel

erythropoiesis

stimulating

protein

-RRB-

which

is

a

hyperglycosylated

erythropoiesis-stimulating

protein

with

a

presumed

threefold

longer

half-life

than

erythropoietin

in

man

-LSB-

-RSB-

-LRB-

evidence

-RRB-

In

children

a

two

to

fourfold

longer

half-life

was

reported

-LSB-

-RSB-

The

pharmacokinetics

when

administered

intravenously

and

subcutaneously

appear

to

be

the

same

in

adult

and

pediatric

patients

A

randomized

comparative

study

of

darbepoetin

and

erythropoietin

in

pediatric

patients

with

chronic

or

end-stage

renal

disease

is

just

starting

There

are

no

data

available

with

respect

to

the

intraperitoneal

administration

of

darbepoetin

The

development

of

an

orally

active

agonist

of

the

erythropoietin

receptor

will

be

an

interesting

future

feature

-LSB-

-RSB-

Erythropoietin

resistance

may

be

due

to

a

number

of

causes

InfectionHyperparathyroidismMalnutritionHemolytic

disordersFolate

or

vitamin

deficiencyUnderdialysisVitamin

C

deficiencyACE

inhibitorsAnti-erythropoietin

antibodies

Since

there

is

an

excellent

review

of

these

in

the

adult

guidelines

-LSB-

-RSB-

and

they

are

not

essentially

different

in

children

they

are

not

discussed

in

detail

here

In

children

responding

poorly

to

erythropoietin

therapy

special

emphasis

should

be

put

on

the

possible

contribution

of

inflammation

or

hyperparathyroidism

-LSB-

-RSB-

The

possible

occurrence

of

pure

red

cell

aplasia

due

to

the

development

of

neutralizing

antierythropoietin

antibodies

has

been

discussed

before

Erythropoietin

should

be

administered

by

the

subcutaneous

or

intraperitoneal

route

in

children

on

peritoneal

dialysis

-LRB-

evidence

-RRB-

Although

there

is

sufficient

evidence

to

advocate

the

intraperitoneal

administration

of

erythropoietin

most

pediatric

nephrologists

still

apply

erythropoietin

by

the

subcutaneous

route

in

their

peritoneal

dialysis

patients

Iron

Iron

supplementation

is

indicated

in

virtually

all

pediatric

patients

with

renal

anemia

who

are

treated

with

erythropoietin

According

to

the

DOQI

guidelines

transferrin

saturation

should

be

maintained

above

and

serum

ferritin

concentration

above

ng/ml

-LSB-

-RSB-

-LRB-

evidence

-RRB-

There

is

no

reason

to

anticipate

that

these

guidelines

should

be

different

for

children

and

for

adults

-LRB-

opinion

-RRB-

It

may

be

difficult

to

maintain

sufficient

iron

stores

in

children

on

peritoneal

dialysis

using

oral

iron

preparations

only

Iron

supplements

should

be

given

to

prevent

iron

deficiency

and

to

maintain

adequate

iron

stores

A

dosage

of

mg/kg

body

weight

per

day

is

recommended

and

administered

in

two

to

three

divided

doses

either

or

h

after

food

Iron

supplements

should

not

be

added

to

the

infant

formula

or

nutrition

supplements

-LSB-

-RSB-

-LRB-

evidence

-RRB-

If

possible

they

should

be

prescribed

with

vitamin

C

to

enhance

absorption

-LRB-

opinion

-RRB-

However

it

is

important

not

to

oversupplement

with

vitamin

C

for

risk

of

increased

oxalate

formation

Iron

supplements

should

ideally

not

be

taken

with

cereals

and

legumes

tannins

-LRB-

tea

cocoa

chocolate

-RRB-

and

dairy

products

as

these

interfere

with

absorption

Micronutrient

supplements

should

also

be

prescribed

following

individualized

dietary

assessment

and

should

account

for

the

potential

peritoneal

dialysis

losses

of

folic

acid

and

vitamins

C

and

Compliance

with

oral

iron

preparations

for

micronutrient

supplements

may

be

difficult

and

must

be

reinforced

by

both

medical

and

dietetic

staff

-LSB-

-RSB-

Parenteral

iron

preparations

commonly

used

in

patients

on

hemodialysis

are

more

difficult

to

apply

in

children

on

peritoneal

dialysis

After

the

recent

approval

of

iron

gluconate

iron

sucrose

and

iron

saccharate

the

application

of

iron

dextran

should

also

be

abandoned

in

the

United

States

-LSB-

-RSB-

-LRB-

evidence

-RRB-

Recommended

dosage

is

mg

iron

per

kg

per

week

for

intravenous

iron

sucrose

treatment

-LSB-

-RSB-

Before

starting

intravenous

therapy

the

administration

of

a

test

dosis

is

recommended

It

is

clear

however

that

the

intravenous

administration

of

iron

preparations

is

cumbersome

and

oral

administration

is

preferred

-LSB-

-RSB-

-LRB-

opinion

-RRB-

In

rare

cases

for

example

with

noncompliance

with

oral

medication

intermittent

intravenous

administration

will

be

indicated

Limited

but

positive

experience

has

been

obtained

with

the

intraperitoneal

administration

of

iron

dextran

both

in

rats

and

men

-LSB-

-RSB-

Reports

on

the

intraperitoneal

administration

of

iron

gluconate

or

iron

sucrose

are

lacking

Iron

should

preferably

be

prescribed

as

an

oral

preparation

-LRB-

evidence

-RRB-

In

rare

cases

intermittent

intravenous

administration

will

be

indicated

Carnitine

Several

studies

in

adult

patients

suggest

that

intravenous

supplementation

with

l-carnitine

reduces

requirements

for

erythropoietin

by

-LSB-

-RSB-

-LRB-

opinion

-RRB-

Published

data

on

the

effect

of

l-carnitine

supplementation

on

the

treatment

of

anemia

in

children

are

very

scarce

One

study

showed

an

increase

in

the

hematocrit

by

in

two

children

on

hemodialysis

with

l-carnitine

supplementation

without

modification

of

erythropoietin

dosage

-LSB-

-RSB-

Another

study

in

children

on

dialysis

of

whom

five

were

on

peritoneal

dialysis

showed

no

beneficial

effect

of

oral

supplementation

with

l-carnitine

on

erythropoietin

requirement

-LSB-

-RSB-

There

is

no

precise

place

for

carnitine

supplementation

in

the

treatment

of

anemia

in

pediatric

peritoneal

dialysis

patients

-LRB-

opinion

-RRB-

O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O B O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B I O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O B O O O O O O O O O O O B O O O O O O B O O O B O O O O B O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O B I O O O O O O O O O O O O O O O B O O O O O O B O O O O O O B I O O B B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O B O B I O O O B O O O B O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O B I O O O O O O B O O B O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O B O O O O O O O O B I O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O B I O O O O O

peritoneal dialysis
children
erythropoietin
iron
carnitine


Interactions of Shiga-like toxin with human peripheral blood monocytes The cytotoxic effect of Shiga-like toxin -LRB- Stx ; produced by certain Escherichia coli strains -RRB- plays a central role in typical hemolytic uremic syndrome -LRB- HUS -RRB- . It damages the renal endothelium by inhibiting the cellular protein synthesis . Also , the monocyte has a specific receptor for Stx but is not sensitive for the cytotoxic effect . In this work , monocytes were studied as a potential transporter for Stx to the renal endothelium . Coincubation of isolated human monocytes loaded with Stx and target cells -LRB- vero cells and human umbilical vascular endothelial cells -RRB- were performed . Transfer was determined by measuring the protein synthesis of target cells and by flow cytometry . Furthermore , the effect of a temperature shift on loaded monocytes was investigated . Stx-loaded monocytes reduced the protein synthesis of target cells . After adding an antibody against Stx , incomplete recovery occurred . Also , adding only the supernatant of coincubation was followed by protein synthesis inhibition . Stx detached from its receptor on the monocyte after a change in temperature , and no release was detected without this temperature shift . Although the monocyte plays an important role in the pathogenesis of HUS , it has no role in the transfer of Stx . Introduction Hemolytic uremic syndrome -LRB- HUS -RRB- is a clinical syndrome consisting of three characteristic features : hemolytic anemia , thrombocytopenia , and acute renal failure -LSB- 1 -RSB- . In the new classification of HUS , infections due to Shiga-like toxin -LRB- Stx -RRB- - producing bacteria belong to the category `` etiologically advanced '' -LSB- 2 -RSB- . This work focuses on the form in which Stx-producing Escherichia coli is the most common pathogen -LSB- 1 -RSB- . It can produce several types of Stx , of which Stx1 , Stx2 , and Stx2c are most frequently associated with HUS -LSB- 3 , 4 -RSB- . Stx plays a crucial role in the pathogenesis because of its cytotoxic effect on the renal endothelium . Both renal tubular epithelial cells and glomerular visceral epithelial cells -LRB- podocytes -RRB- are also sensitive to the toxic effect of Stx -LSB- 5 , 6 -RSB- . It can inhibit the protein synthesis of these cells after specifically damaging the ribosomal RNA -LSB- 7 -RSB- . However , the question of how this toxin is targeted mainly to the kidney remains unsolved . Stx was never detected in the serum of patients , but it was detected in renal biopsy material of patients with HUS -LSB- 8 -RSB- . As a specific treatment for HUS is still lacking , more insight into the transport of this toxin might lead to new intervention strategies . After oral ingestion of the bacteria through contaminated food or water , the noninvasive bacteria adhere to the intestinal epithelial cells of the distal small bowel and colon . This leads to a rearrangement of the morphology of the cells and initiates inflammation -LSB- 9 , 10 -RSB- . Bacterial flagellin plays an important role in this process -LSB- 11 -RSB- . Stx can probably reach the circulation because of active transport in these cells and also passively after damage to the intestinal cells -LSB- 12 -RSB- . Subsequently , it has to be transported in the circulation to reach its primary target , the renal endothelium . It is very tempting to look at the blood cells as a carrier for the toxin . Stx can bind to a specific receptor , which is a globotriaosylceramide -LRB- Gb3 , Pk Antigen , CD77 -RRB- -LSB- 13 -RSB- . This receptor is present on renal endothelial cells but also on blood cells . Stx binding has been described on red blood cells -LSB- 14 -RSB- , B lymphocytes -LSB- 15 -RSB- , and platelets , which also have an additional binding possibility -LRB- glycolipid , band 0.03 -RRB- -LSB- 16 -RSB- . Several groups showed the existence of a specific binding of Stx on monocytes -LSB- 17 , 18 , 19 -RSB- . After binding to its receptor , Stx can be internalized . Whereas in epithelial cells the toxin follows the retrograde transport route and becomes cytotoxic , in monocytes it is targeted to the lysosomes and will get degraded -LSB- 19 -RSB- . During this transport , the monocyte becomes activated . This will lead to an increase of transcription factors , such as nuclear factor kappa B -LRB- NF-κB -RRB- and activator protein 1 -LRB- AP-1 -RRB- , and an upregulated production of cytokines such as interleukin -LRB- IL -RRB- -1 β , tumor necrosis factor -LRB- TNF -RRB- - α , IL-6 , and IL-8 -LSB- 17 , 20 -RSB- . These events will have a pro-inflammatory effect . We postulated that , as the monocyte has a specific receptor , it might also function as a carrier to transport Stx to the renal endothelium . To investigate this hypothesis , Stx was loaded to isolated monocytes from healthy donors and coincubated with target cells -LSB- vero cells and human umbilical cord venous endothelial cells -LRB- HUVEC -RRB- -RSB- . The level of transfer was determined by measuring the protein synthesis of these target cells and the transfer of fluorescein isothiocyanate -LRB- FITC -RRB- - labeled B subunit of Stx1 with flow cytometry . Materials and methods Materials Stx2 was kindly provided by Dr. M. Karmali -LRB- Public Health Agency of Canada , Ontario , Canada -RRB- . FITC-labeled Stx1B subunit and 125I-Stx1B subunit were a gift from Dr. L. Johannes -LRB- Institut Curie , Paris , France -RRB- . Stx1B subunit is a useful tool for studying binding in monocytes -LSB- 19 -RSB- . It is the binding part of the toxin , whereas the enzymatic A subunit will only stimulate the uptake of the toxin and does not affect binding -LSB- 21 -RSB- . Vero cell medium consists of M199 -LRB- Gibco ; Paisley/UK -RRB- , fetal calf serum -LRB- FCS , Greiner Bio-One ; Kremsmunster/Austria -RRB- , penicillin/streptomycin -LRB- Gibco , Paisley/UK -RRB- , and glutamine -LRB- MP Biomedicals ; Eschwege/Germany -RRB- . HUVEC medium is made of M199 , human serum -LRB- HS ; Cambrex ; Walkersville/USA -RRB- , newborn calf serum -LRB- NBCS ; Gibco , Paisley/UK -RRB- , penicillin/streptomycin , glutamine , heparine -LRB- Leo Pharma BV , Breda/The Netherlands -RRB- , and endothelial-cell growth factor -LSB- 22 -RSB- . Ethylenediamine tetraacetic acid -LRB- EDTA -RRB- tubes were purchased from BD Vacutainer -LRB- Alphen aan de Rijn/The Netherlands -RRB- . The MACS kit for negative selection of monocytes was provided by Miltenyi Biotec -LRB- Bergisch Gladbach/Germany -RRB- . Hank 's balanced salt solution -LRB- HBSS -RRB- was ordered from MP Biomedicals -LRB- Eschwege/Germany -RRB- . Human serum albumin -LRB- HSA -RRB- from Sanquin -LRB- Amsterdam/The Netherlands -RRB- and porcine gelatin from Fluka -LRB- Neu-Ulm , Germany -RRB- was used . Trichloroacetic acid and TNF-α was obtained from Sigma-Aldrich Chemie B.V. -LRB- Zwijndrecht/The Netherlands -RRB- . The antibody against Stx2 -LRB- TMA-15 -RRB- is well characterised -LSB- 23 -RSB- . It was a kind gift from Dr. Yamagami from the Department of Biomedical Research from the Teijin Institute , Tokyo , Japan . 3H-leucine and Ficoll-paque PLUS was purchased from Amersham Biosciences -LRB- Uppsala , Sweden -RRB- . Culture plates were ordered from Corning Inc. -LRB- Corning , USA -RRB- . Culture of vero cells and HUVEC Vero cells -LRB- renal epithelial cells of the African green monkey -RRB- were grown to confluency on 24-well plates -LRB- ordered from ATCC ; Middlesex , UK -RRB- . These cells have a high basal expression of Stx-receptor CD77 . HUVEC were isolated , and these cells were grown to confluence on gelatin-coated 24-well plates -LSB- 24 -RSB- . Every 2 days , fresh medium was added to the cells . In contrast , HUVEC were preincubated for 24 h with TNF-α -LRB- 10 ng/ml -RRB- to upregulate the expression of CD77 . Isolation of monocytes and loading with Stx2 Fresh venous blood -LRB- 20 ml -RRB- from 40 healthy donors was collected into EDTA tubes . Monocytes were isolated by negative selection using antibody-labeled beads -LRB- CD3 , CD7 , CD16 , CD56 , and CD123 -RRB- . After centrifugation of blood over Ficoll -LRB- 20 min 400 g without break at room temperature -RRB- , the interphase -LRB- containing monocytes , lymphocytes , and platelets -RRB- was collected . Platelets were removed by centrifugation -LRB- 200 g 10 min at room temperature -RRB- before adding the beads . The purity of monocytes after the magnetic isolation was -LRB- as determined by flow cytometry -RRB- 80 -- 85 % . After the isolation , the monocytes were resuspended in HBSS with 1 % HSA and placed on ice . In every experiment , monocytes from one donor were used . To load the monocytes with Stx2 , the toxin was added to a concentration of 10 nM during a time period of 3 h -LSB- 17 -RSB- . The cells remained on ice . After proper washing to remove all unbound Stx2 , the cells were resuspended in vero cell or HUVEC medium -LRB- in which HS and NBCS were substituted by FCS -RRB- . Monocytes stayed viable after this loading , as determined by trypan blue exclusion . Coincubation Stx2-loaded monocytes and target cells These experiments were performed in two different experimental settings : with transfer of loaded monocytes from 4 ° to 37 ° , and without change of temperature . To start with the first setting , the Stx2-loaded monocytes were added to a monolayer of target cells -LSB- HUVEC -LRB- n = 10 -RRB- or vero - cells -LRB- n = 9 -RRB- -RSB- in a concentration of 1 × 106 per well . This was performed during 24 h at 37 °C . For comparison , also monocytes without Stx were used . To determine the specificity of the effect of Stx2 , the loaded monocytes were preincubated with a well-characterized antibody against Stx2 -LRB- TMA-15 , 1 μg/ml , approximately 150 × excess -RRB- . All experiments were performed in duplicate . To measure the transfer of Stx2 from the monocyte , the protein synthesis of the target cells was determined by adding 3H-Leucine -LRB- 0.67 μCi/ml -RRB- . Subsequently , intracellular proteins were precipitated by treatment with trichloroacetic acid -LRB- TCA -RRB- , and the radioactivity was measured in a liquid scintillation counter . In the other experimental setting , monocytes were loaded in a similar way with FITC-labeled Stx1B subunit ; 1 × 106-loaded monocytes were coincubated with 1.25 × 105 vero cells in suspension for 3 h at 4 °C -LRB- n = 5 -RRB- while being continuously rotated . At this temperature , bias due to internalization of the toxin could be avoided -LSB- 19 , 25 -RSB- . After 3 h , the presence of Stx1B subunit FITC on the vero cells was determined by flow cytometry . For every experiment , at least 1,000 cells were measured . Study of supernatant Stx-loaded monocytes To study the effect of the supernatant of Stx2-loaded monocytes , it was collected after 16 h of coincubation on vero cells -LRB- n = 11 -RRB- . The supernatants were centrifuged to remove possible monocytes and added again to fresh vero cells . Protein synthesis was measured by adding 3H-leucine and measuring the incorporation after 24 h of incubation at 37 °C . Also , the antibody against Stx2 was used to determine a possible effect of free Stx2 . Furthermore , monocytes were loaded with 125I-Stx1B subunit to investigate whether it could be released after a change in temperature from 4 ° to 37 °C -LRB- n = 3 -RRB- . For each experiment , between 3 and 4 × 106 monocytes are loaded with 200 nM Stx1B subunit -LRB- 3,600 cpm/ng protein -RRB- . After loading , unbound Stx1B subunit was removed by centrifugation . Subsequently , the monocytes were placed at 37 °C for 2 h . They were then centrifuged , and cells and supernatant were measured separately for the presence of 125I-Stx1B with a gamma counter . This was compared with the amount of binding to the monocytes before the temperature change . The experiment was performed in duplicate . Statistics All data presented are expressed as a range with the median . Significance of increase or decrease of protein synthesis compared with controls was analyzed using the Wilcoxon signed ranks test . The statistical level of significance was defined as P < 0.05 . Results Coincubation Stx2-loaded monocytes and target cells To investigate whether monocytes can transfer Stx2 to target cells -LRB- vero cells and HUVECs -RRB- , Stx2-loaded monocytes were added to a monolayer of these cells . After coincubation , protein synthesis of vero cells and HUVEC was measured . If the Stx2 was transferred to the target cells , there was protein synthesis inhibition , as this is the biological effect of Stx2 in both types of target cells . After 24 h of coincubation at 37 °C , in both cell types , there was protein synthesis inhibition -LRB- Fig. 1 -RRB- . In vero cells , greater protein synthesis reduction was measured than in HUVEC . Directly adding 10 nM Stx2 to vero cells reduced protein synthesis to 5.5 % -LRB- data not shown -RRB- . When an antibody against Stx2 was added to the monocytes before coincubation with vero cells , protein synthesis inhibition was partly restored -LRB- Fig. 2 -RRB- . Only in one out of five experiments was there complete recovery . Fig. 1Coincubation of Shiga-like toxin -LRB- Stx -RRB- 2-loaded monocytes with target cells -LSB- vero cells and human umbilical cord venous endothelial cells -LRB- HUVEC -RRB- -RSB- . To investigate whether monocytes could transfer Stx2 , toxin-loaded monocytes were coincubated with vero cells -LRB- n = 9 -RRB- and HUVEC -LRB- n = 10 -RRB- . This led to protein synthesis inhibition , as could be determined with the incorporation of 3H-leucine . The coincubation with unloaded monocytes was used as a control and set at 100 % . ** P < 0.01 Fig. 2Effect of the addition of an antibody against Shiga-like toxin -LRB- Stx -RRB- 2 . The monocytes of five healthy donors were coincubated with vero cells -LRB- Stx2-loaded monocytes , white bar ; Stx2-loaded monocytes with Stx2 antibody , black bar -RRB- . Addition of the antibody against Stx2 led to partial protein synthesis recovery in four donors and complete recovery in one donor . Coincubation with unloaded monocytes was used as a control and set at 100 % In equal experiments performed with HUVEC -LRB- n = 3 -RRB- , TMA-15 also partly prevented protein synthesis inhibition -LRB- data not shown -RRB- . Study of supernatant Stx-loaded monocytes Because the addition of an antibody against Stx2 was not sufficient for complete protein synthesis recovery in four out of five tested donors , we investigated the possible presence of an additional inhibitor in the supernatant after 16 h of coincubation . This was performed by re-adding the supernatant of coincubated Stx2-loaded monocytes and a monolayer of vero cells to new vero cells . Figure 3 shows that this incubation again led to an inhibitory effect on vero-cell protein synthesis in contrast to the supernatant of unloaded monocytes . But this reduction was less than with direct coincubation . The effect could be partly blocked by adding the antibody against Stx2 . This means that there is unbound Stx2 present in the supernatant . Next , we investigated whether the temperature change of the Stx-loaded monocytes from 4 ° to 37 °C could lead to toxin release . For this reason , we loaded the monocytes with 125I-Stx1B subunit . The amount of radioactivity on or inside the monocytes was measured before and after incubation of the cells for 2 h at 37 °C -LRB- Table 1 -RRB- . Vero cells were used as a control . In vero cells as in monocytes , the toxin was released from its receptor after the incubation . Fig. 3Addition of supernatants coincubation to vero cells . After coincubation of Shiga-like toxin -LRB- Stx -RRB- - loaded and unloaded monocytes , supernatants were collected and re-added to fresh vero cells -LRB- n = 11 -RRB- . The supernatant from the toxin-loaded monocytes induced , again , protein synthesis inhibition . This inhibition could be partially restored with a Stx2 antibody -LRB- AB -RRB- . The incubation of supernatant with unloaded monocytes was used as a control and set at 100 % . ** P < 0.01 Table 1Amount of 125I - Shiga-like toxin -LRB- Stx -RRB- 1B subunit on cells after change of temperatureStx-loaded cellsBefore 4 → 37 °C -LRB- cpm -RRB- After 4 → 37 °C -LRB- cpm -RRB- Vero cells1 ,034,383671,060 Monocytes donor 11,437427 Monocytes donor 21,070374 Monocytes donor 31,121677 cpm counts per minute Transfer of Stx to target cells without change in temperature A change in temperature from 4 ° to 37 °C released Stx from its receptor on the monocyte . To study again the possibility of Stx transfer from monocyte to target cells , we loaded isolated monocytes with Stx1B subunit labeled with FITC . Coincubation of these monocytes with vero cells in suspension without a change in temperature did not result in a transfer of the B subunit to the vero cells -LRB- n = 5 , Fig. 4 -RRB- . Fig. 4Transfer of Shiga-like toxin -LRB- Stx -RRB- - loaded monocytes to vero cells without temperature shift -LRB- white : normal monocytes ; black : Stx-loaded monocytes ; grey : vero cells -RRB- . The x-axis represents the intensity of the fluorescein isothiocyanate -LRB- FITC -RRB- signal , the y-axis represents the number of cells . a FITC-labeled Stx1B subunit was bound to monocytes from a healthy donor , showing an increase in intensity . b Coincubation of vero cells and unloaded monocytes ; note the high basal signal of vero cells . c After 2 h of coincubation with vero cells in suspension at 4 °C , there was no transfer of the FITC signal . The cells remained in position Discussion After coincubation of Stx2-loaded monocytes with target-cells -LRB- vero cells and HUVEC -RRB- , protein synthesis inhibition could be detected . As the biological effect of Stx is protein synthesis inhibition , a transfer was expected . This effect could not be due to the presence of monocytes , because it is well described that Stx has no inhibitory effect on peripheral blood monocytes -LSB- 17 -RSB- . However , the addition of an antibody against Stx only partly restored the inhibition . We hypothesized that in parallel to transfer of Stx2 , a possible additional inhibiting factor was present in the supernatant during the experiment . To further investigate this possibility , the supernatant of this coincubation was re-added to fresh vero cells . Again , there was protein synthesis inhibition , which could partly be decreased by blocking with the Stx2 antibody . The conclusion was made that there another inhibitory factor needed to be present -LRB- possibly released by activated monocytes -RRB- , but unbound Stx2 was also present -LSB- 26 -RSB- . Apparently , Stx1B subunit is released from its receptor when there is shift of temperature from 4 ° to 37 °C , as shown in this study and also by Ramegowda and Tesh -LSB- 18 -RSB- . This finding stresses the caution that must be taken when drawing conclusions from in vitro experiments performed at 4 °C . In Fig. 5 , all findings are schematically summarized . Fig. 5Schematic summary of performed experiments . a Monocytes loaded with Shiga-like toxins -LRB- Stx -RRB- at 4 °C . b After shifting Stx-loaded monoytes to target cells at 37 °C , Stx was released from its receptor . This led to protein synthesis inhibition of target cells . However , transport could not be excluded -LRB- but this was performed in experiments without a shift in temperature -RRB- . Probably , some toxin was internalized and monocytes became activated . This could lead to cytokine and proteases production -LRB- possible cytotoxic factors -RRB- . c When the supernatant is re-added to new target cells , there are released Stx and secreted products present . This induced , again , protein synthesis inhibition As these in vitro experiments were not suitable to investigate a possible transfer , experiments were performed without a change of temperature . No transfer of the binding part of the toxin could be detected in this setting . Experiments could not be performed at 37 °C because the toxin is internalized by the monocytes during 3 h of incubation . Because of these in vitro experiments , we believe that the monocyte can not function as a transporter for Stx in the circulation . However , monocytes still seem to play an important role in pathogenesis . As a component of the innate immune system , they play a central role in immunity and inflammation . Fernandez et al. showed that patients in the acute period of HUS have monocytes with phenotypic -LRB- reduced expression of CD14 , CD64 , and CD11b -RRB- and functional -LSB- decreases lipopolysaccharide -LRB- LPS -RRB- - induced TNF-α production and Fcγ-dependent cytotoxicity -RSB- differences compared with healthy children and acute uremic children -LSB- 27 -RSB- . Some unanswered questions remain . What is the inhibitory factor released by monocytes after loading with Stx ? Why is there still unbound Stx2 present in the supernatant after 16 h of coincubation ? It is surprising that Stx2 is not completely bound and internalized by the numerous receptors on monocytes and vero cells . Also , how Stx is transported in the circulation remains unsolved . Kimura et al. described that serum amyloid P -LRB- SAP -RRB- can bind Stx2 and function as a neutralizing factor -LSB- 28 -RSB- . However , in humans , there was no correlation between circulating SAP and the development of HUS -LSB- 29 -RSB- . As renal endothelial damage is already present after the occurrence of clinical symptoms of HUS , it is very important to develop efficacious early prevention . Understanding the mechanism in which the toxin is specifically targeted to the kidney can lead to novel intervention strategies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B O O O O O O O O O O O O O O O O B I O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

monocyte
hemolytic uremic syndrome
acute renal failure
huvec
vero cells