Human Peritoneal Dialysate Research Articles

The Iron Biology Status of Peritoneal Dialysis Patients May Be a Risk Factor for Development of Infectious Peritonitis.

Background:Infectious peritonitis is a clinically important condition contributing to the significant mortality and morbidity rates observed in peritoneal dialysis (PD) patients. Although some of the socioeconomic risk factors for PD-associated peritonitis have been identified, it is still unclear why certain patients are more susceptible than others to infection.Methods:We examined the molecular components of human peritoneal dialysate (HPD) in an attempt to identify factors that might increase patient susceptibility to infection. Characterization studies were performed on initial and follow-up dialysate samples collected from 9 renal failure patients on PD.Results:Our in vitro data showed that peritonitis-causing bacteria grew differently in the patient dialysates. Proteomic analysis identified an association between transferrin presence and infection risk, as peritoneal transferrin was discovered to be iron-saturated, which was in marked contrast to transferrin in blood. Further, use of radioactive iron-labeled transferrin showed peritoneal transferrin could act as a direct iron source for the growth of peritonitis-causing bacteria. We also found catecholamine stress hormones noradrenaline and adrenaline were present in the dialysates and were apparently involved in enhancing the growth of the bacteria via transferrin iron provision. This suggests the iron biology status of the PD patient may be a risk factor for development of infectious peritonitisConclusions:Collectively, our study suggests transferrin and catecholamines within peritoneal dialysate may be indicators of the potential for bacterial growth in HPD and, as infection risk factors, represent possible future targets for therapeutic manipulation.

Growth and siderophore production of staphylococci in human peritoneal dialysate.

Although activity of iron uptake system (IUS) was thought to play an important role in staphylococcal growth in human peritoneal dialysate (HPD) solution, siderophore production, one of the well-known IUS, was not yet detected directly in HPD solution. Therefore, we tried to detect siderophore production directly in HPD solution by using a newly developed chrome azurol S (CAS) agar diffusion assay and to investigate the effect of IUS activity on bacterial growth in HPD solution. According to the susceptibility test for streptonigrin and the productivity of siderophore in the iron-deficient (ID) medium, Staphylococcus aureus ATCC 6538 strain and Staphylococcus epidermidis clinical isolate had higher IUS activity and grew better than S. aureus ATCC 25923 strain in the ID medium. These bacteria did not grow and produce siderophore in the unused chronic ambulatory peritoneal dialysis solution. However, these bacteria grew and produced siderophore in the HPD solution. Moreover, S. aureus ATCC 25923 strain with lower activity of IUS grew poorly and produced smaller amount of siderophore in HPD compared to S. aureus ATCC 6538 strain and S. epidermidis clinical isolate with higher activity of IUS like in the ID medium. To the best of our knowledge, this is the first report that siderophore production is directly detected in the HPD by CAS agar diffusion assay. These results indicated that activity of IUS plays an important role in bacterial growth in the HPD solution and pathogenesis of continuous ambulatory peritoneal dialysis peritonitis.

The Staphylococcus aureus and Staphylococcus epidermidis transferrin-binding proteins are expressed in vivo during infection.

Staphylococci express a 42 kDa cell-wall-associated protein which functions as a receptor for the mammalian iron-binding glycoprotein transferrin. To determine whether this transferrin-binding protein (TBP) is expressed during infection, Staphylococcus aureus and Staphylococcus epidermidis were grown in vivo in chambers implanted intraperitoneally in rats. SDS-PAGE and Western blotting of cell wall proteins prepared from staphylococci recovered directly from the chambers revealed the presence of both the TBP and bacterial-surface-associated rat transferrin. To obtain evidence for the in vivo expression of the staphylococcal TBPs in humans, sera and human peritoneal dialysate (HPD) from non-infected patients undergoing continuous ambulatory peritoneal dialysis (CAPD) and sera from healthy human volunteers were screened for anti-TBP antibodies. Western immunoblots revealed that three out of ten samples from the latter group, seven out of ten HPD samples and ten of ten CAPD patient serum samples contained antibodies to the TBP of both S. aureus and S. epidermidis. To gain further insights into the appearance of TBP antibodies, HPD samples were collected over time from CAPD patients whose HPD samples taken immediately after catheter insertion lacked anti-TBP antibodies. In two of these patients, each of whom experienced an episode of peritonitis due to S. epidermidis or Staphylococcus hominis, antibodies to the TBP appeared in the HPD collected immediately post-infection. To determine whether such TBP antibodies were capable of blocking interactions between transferrin and its staphylococcal receptor, HPD immunoglobulin fractions were purified using protein A-Sepharose beads. In competition assays, these immunoglobulins blocked the binding of 125I-labelled transferrin both to whole bacteria and to the isolated 42 kDa TBPs of S. aureus and S. epidermidis. These provide evidence to show that staphylococcal TBPs are expressed in vivo during infection.

A Labile Sodium Pump Inhibitor From The Peritoneal Dialysate of Hypertensive Renal Failure Patients: Estimates of Potency

Estimations of concentration of the labile sodium pump inhibitor isolated from human peritoneal dialysate were made using supercritical fluid chromatography coupled to flame ionization detection to determine the quantity of this factor in half of a purified preparation of the factor compared to the bioactivity of the other half in different assays. Ouabain was used for comparison. The labile factor appeared to be 30 times more effective than ouabain against canine renal [Na,K]ATPase. Moreover, this same factor appeared to be $1,000 times more potent than ouabain in causing vascular smooth muscle contraction. The differences between this labile sodium pump inhibitor and ouabain most likely reflect their respective binding affinities. The assay differences in half maximal response to the labile sodium pump inhibitor may be due to differences in sodium pump alpha isoform sensitivity

Inorganic pyrophosphatase-labelled enzyme immunoassay for β 2-microglobulin

β 2-Microglobulin was purified from human peritoneal dialysate by ultrafiltration, gel chromatography and DEAE-high performance chromatography. Anti-β 2-monoclonal antibodies were developed in mice and a pair of the antibodies was utilized to develop an enzyme-linked immunosorbent assay (ELISA) kit for the analyte with utilizing a new enzyme label, inorganic pyrophosphatase (EC 3.6.1.1). The sensitivity of the assay was 0.6 μg/l (3×SD) and the assay was linear up to absorbance values of around 2.0. No hook effect occurred in any putative concentrations of β 2-microglobulin in serum. The precision of the assay of one run varied within 5–7% CV and the interassay precision was 2.8–8.6% CV, while the recovery was 99.2±6.0%. Excellent correlation occurred against an established radioimmunoassay method. All the used reagents were storable for a minimum of 1 year at +4°C. It was decided that inorganic pyrophosphatase is a label of choice for ELISA.

Specificity of the volume-sensitive sodium pump inhibitor isolated from human peritoneal dialysate in chronic renal failure

Compromised renal function predisposes to volume-dependent hypertension. Increased plasma levels of a sodium pump inhibitor as a possible pathogenetic factor have been demonstrated by many investigators in such patients, but efforts to identify the responsible agent have led to many, diverse candidates. Our premise in this study is that candidacy must depend on the satisfaction of rigorous criteria, including a specific action of the agent on the sodium pump. These criteria included reversibility, concentration dependence, receptor mediation, and an action at the appropriate step in the enzyme cycle. These criteria were applied to a potent [Na,K]ATPase inhibitor we have identified in the peritoneal dialysate of patients with chronic renal failure, present only during extracellular fluid volume expansion, the levels of which are correlated with the blood pressure rise that results from excessive NaCl and water intake. In microsomes that contained both [Na,K]ATPase and other ATPases, this candidate inhibited only the Na and K dependent, ouabain-sensitive ATPase. It displaced ouabain from the cardioglycoside binding site and its binding was linked to inhibition. Inhibition was produced by slowing the pump's dephosphorylation step, the exact action of all cardioglycosides. Finally, the candidate cross reacted with a digoxin Fab fragment and this Fab reversed its inhibition of [Na,K]ATPase. Together, these experiments demonstrate that the PD candidate specifically, and reversibly, inhibits the sodium pump via the cardioglycoside binding site, and hence, meets this crucial criterion for candidacy.

Continuous ambulatory peritoneal dialysis-associated peritonitis as a model device-related infection: phenotypic adaptation, the staphylococcal cell envelope and infection

During the development of infection, pathogens are translocated from one body site to another and so must readily adapt to changing environmental conditions. The influence of host environment on bacterial behaviour and virulence gene expression is, however, often overlooked. Environmental signals such as temperature, pH and nutrient (especially iron) availability which inform pathogens of their living conditions thus contribute to both bacterial survival and virulence. In the context of medical device-associated infections such as peritonitis in continuous ambulatory peritoneal dialysis (CAPD) patients, the pathogenesis of infection is related to the ability of the infecting organism to multiply, to adhere to catheter polymers and host tissues and to evade host defences. Coagulase-negative staphylococci (CNS) such as Staphylococcus epidermidis are commonly responsible for CAPD-associated peritonitis. Although staphylococci cannot grow in commercial peritoneal dialysate solutions, these fluids are modified during dialysis and become enriched by a plasma ultrafiltrate which can support bacterial growth. Given that growth environment exerts considerable influence on bacterial behaviour, the physiology of CNS cultured in vitro in a model system employing pooled human peritoneal dialysate and in vivo in implanted peritoneal chambers in the rat has been investigated. Using such models marked variation in surface physicochemistry, antibiotic susceptibility and adherence to catheter polymers has been observed. This plasticity is clearly reflected in the cell envelope phenotype of CNS, the study of which has recently lead to the discovery of a staphylococcal receptor for the iron-binding serum glycoprotein, transferrin.

Staphylococci express a receptor for human transferrin: identification of a 42-kilodalton cell wall transferrin-binding protein

Staphylococcus aureus and the coagulase-negative staphylococci are commonly responsible for peritonitis in renal patients undergoing continuous ambulatory peritoneal dialysis. To simulate growth conditions in vivo, staphylococci isolated from peritoneal infections were cultured in used human peritoneal dialysate (HPD). Immunoblotting experiments using cell wall preparations from these staphylococci revealed the presence of the host iron-binding glycoprotein transferrin bound to S. aureus, S. epidermidis, S. capitis, S. haemolyticus, and S. hominis but not to S. warneri or S. saprophyticus. Similar results were obtained by incubating broth-grown staphylococci with human transferrin, although, in contrast to S. aureus, the coagulase-negative staphylococci bound more transferrin after growth in iron-restricted broth. To determine whether the staphylococci express a saturable specific receptor for human transferrin, the interaction of human 125I-transferrin with the staphylococci was examined. Both S. aureus and S. epidermidis bound the radiolabelled iron-saturated ligand in a time- and concentration-dependent manner. From competition binding assays, the affinity (Kd) and number of receptors were estimated for S. epidermidis (Kd, 0.27 microM; 4,200 receptors per cell) and S. aureus (Kd, 0.28 microM; 4,200 receptors per cell). S. epidermidis but not S. aureus receptor activity was partially iron regulated. Human apotransferrin and iron-saturated transferrin and rabbit and rat transferrins competed equally well for the staphylococcal receptor. Bovine and porcine transferrins and ovotransferrin as well as human and bovine lactoferrins were much less effective at competing with human transferrin. Treatment of whole staphylococci with protease abolished transferrin binding, indicating the involvement of cell surface protein. Western blots (immunoblots) of cell wall preparations probed with human transferrin revealed the presence of a 42-kDa transferrin-binding protein common to both S. aureus and S. epidermidis. On Western strip blots, the binding of human transferrin to this protein was blocked by labelled human transferrin but not by albumin, immunoglobulin G, or bovine transferrin or ovotransferrin. To assess the conservation of the 42-kDa transferrin-binding protein, cell wall proteins of S. epidermidis, S. haemolyticus, S. capitis, S. hominis, S. warneri, and S. saprophyticus were Western blotted and probed with human transferrin. Only S. warneri and S. saprophyticus lacked the 42-kDa wall protein, consistent with their inability to bind transferrin. These data show that the staphylococci express a specific receptor for human transferrin based at least in part on a common 42-kDa cell wall protein.

Cell envelope proteins of Staphylococcus epidermidis grown in vivo in a peritoneal chamber implant.

Staphylococcus epidermidis was grown in vivo in chambers implanted intraperitoneally in rats. The cell wall and cytoplasmic membrane protein profiles of the in vivo-grown organisms were compared with those of S. epidermidis grown in vitro in nutrient broth (NB), in iron-restricted NB, or in pooled human peritoneal dialysate (HPD). Compared with growth in broth and in common with growth in HPD, growth in vivo in chambers resulted in the repression of many S. epidermidis wall proteins, with proteins of 27, 42, 54, and 70 kDa predominating. Growth in vivo also resulted in the induction of two iron-repressible cytoplasmic membrane proteins of 32 and 36 kDa, which were also present in staphylococci grown in HPD and in iron-restricted NB. Immunoblotting experiments revealed that in sera taken 21 days after inoculation of the intraperitoneal chambers, the predominant antibody response to cell envelope proteins was directed against the 32- and 36-kDa iron-repressible membrane proteins.

Variation in the expression of cell envelope proteins of coagulase-negative staphylococci cultured under iron-restricted conditions in human peritoneal dialysate

Strains of coagulase-negative staphylococci (CNS), including Staphylococcus epidermidis, S. hominis, S. warnerii, S. simulans, S. capitis, S. haemolyticus and S. saprophyticus, were isolated from patients with continuous-ambulatory-peritoneal-dialysis-related peritonitis. The cell wall and cytoplasmic membrane protein profiles of CNS strains cultured in either nutrient broth (NB) or pooled human peritoneal dialysate (HPD) were compared. Some interspecies variation in both the wall and membrane protein profiles was noted. However, the cell wall protein profiles of HPD-grown CNS strains differed markedly from those cultured in NB. Growth in HPD resulted in a marked reduction in the total number of cell-wall-associated proteins but up to three antigenically related proteins in the 40-56 kDa range, depending on the species, predominated. Growth in HPD also resulted in the induction of two iron-repressible cytoplasmic membrane proteins (IRMPs) of 32 and 36 kDa in S. epidermidis. Other CNS strains only appeared to express a single IRMP, which varied in molecular mass from 32 to 36 kDa. Whilst the IRMP in these CNS strains showed considerable antigenic homology with the 32 kDa IRMP, the S. warneri IRMP showed cross-reactivity with both the 32 and 36 kDa IRMPs of S. epidermidis. Immunoblotting experiments revealed that whilst the CNS cell wall proteins were poorly immunogenic, the IRMPs were the immunodominant CNS protein antigens, reacting strongly with antibodies present in HPD. This finding provides evidence to suggest that the IRMPs are expressed in vivo during infection.

Characterization of cell envelope proteins of Staphylococcus epidermidis cultured in human peritoneal dialysate

The cell envelope protein profiles of Staphylococcus epidermidis cultured in used human peritoneal dialysate (HPD) differed markedly from those of cells cultured in nutrient broth. Compared with broth-grown cells, many cell wall proteins were repressed in HPD, although three proteins of 42, 48, and 54 kDa predominated and an iron-repressible 130-kDa protein was induced. Growth in HPD also resulted in expression of two cell membrane proteins of 32 and 36 kDa which were iron repressible. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis using monospecific polyclonal antisera raised against the 32- and 36-kDa proteins revealed considerable antigenic and molecular mass homology among 12 S. epidermidis isolates from patients with continuous ambulatory peritoneal dialysis-related peritonitis. The 32-kDa antiserum also cross-reacted with a 32-kDa S. aureus cell membrane protein. Immunoblots of S. epidermidis cell walls and membranes were also probed with normal human serum and serum and HPD from continuous ambulatory peritoneal dialysis patients. While the cell wall proteins of S. epidermidis appeared to be relatively poorly immunogenic, the 32- and 36-kDa membrane proteins reacted strongly with antibodies present in each of the body fluids evaluated. These results suggest that the highly conserved 32- and 36-kDa iron-repressible proteins are expressed during growth in vivo and may be involved in iron transport, since all 12 S. epidermidis strains examined also produced iron chelators.

Influence of carbon dioxide on growth and antibiotic susceptibility of coagulase-negative staphylococci cultured in human peritoneal dialysate

Used peritoneal dialysis fluid was collected from patients undergoing continuous ambulatory peritoneal dialysis, and its pH and composition were assessed after incubation in either air or air with 5% CO2. Precipitation of calcium, magnesium, phosphate, and proteins occurred in the dialysis fluid incubated in air at 37 degrees C and was associated with a mean pH increase of 1.23 U. Incubation of dialysis fluid in air with 5% CO2 prevented precipitation and maintained pCO2 and pH levels at those found physiologically. Coagulase-negative staphylococcal strains isolated from patients with peritonitis tended to grow less well in dialysis fluid incubated in air than in dialysis fluid incubated in the carbon dioxide-enriched atmosphere. MICs of cefuroxime, ciprofloxacin, and vancomycin for seven strains of coagulase-negative staphylococci in dialysis fluid were markedly affected by atmosphere type (16 of 21 MICs). Of these 16 atmosphere-dependent MICs, 14 were at least fourfold higher in air than in air with 5% CO2.

Protein antigens of Staphylococcus epidermidis grown under iron-restricted conditions in human peritoneal dialysate

Comparison of the whole cell protein profiles of Staphylococcus epidermidis grown in pooled human peritoneal dialysate (HPD) with those of cells grown in nutrient broth (NB) revealed proteins of 27, 39, 45, 54 and 98 kDa which were absent or poorly expressed in NB-grown cells. IgG, but not transferrin, was detected bound to the surface of bacteria grown in HPD. Immunoblotting experiments revealed that IgG antibodies present in pooled HPD recognised staphylococcal protein antigens of 16, 27, 35, 39, 45, 54 and 98 kDa. The 16-, 35- and 39-kDa antigens which were associated with the cytoplasmic membrane were repressed following growth in NB or in HPD supplemented with excess iron.

Protein antigens of Staphylococcus epidermidis grown under iron-restricted conditions in human peritoneal dialysate

Comparison of the whole cell protein profiles of Staphylococcus epidermidis grown in pooled human peritoneal dialysate (HPD) with those of cells grown in nutrient broth (NB) revealed proteins of 27, 39, 45, 54 and 98 kDa which were absent or poorly expressed in NB-grown cells. IgG, but not transferrin, was detected bound to the surface of bacteria grown in HPD. Immunoblotting experiments revealed that IgG antibodies present in pooled HPD recognised staphylococcal protein antigens of 16, 27, 35, 39, 45, 54 and 98 kDa. The 16-, 35- and 39-kDa antigens which were associated with the cytoplasmic membrane were repressed following growth in NB or in HPD supplemented with excess iron.