John D. Williams

Peritoneal Dialysis Fluid Inhibition of Polymorphonuclear Leukocyte Respiratory Burst Activation Is Related to the Lowering of Intracellular pH

In order to elucidate the mechanism of peritoneal dialysis fluid inhibition of cell functions, laboratory-prepared fluids were used to investigate the specific influences of low pH and high lactate concentration on neutrophil viability, phagocytosis, respiratory burst activation and leukotriene B4 (LTB4) generation. In the absence of any reduction of viability, respiratory burst activation, stimulated by serum-treated zymosan (STZ), was significantly inhibited by fluids of low pH containing high concentrations of sodium lactate. Neither low pH nor lactate concentration alone, however, caused significant suppression of this parameter of cell activation. Under the same conditions, the phagocytosis of STZ was partially inhibited in a lactate- and pH-dependent manner. In contrast, the generation of LTB4 in response to STZ was unaffected by pH and lactate concentration. The incubation of polymorphonuclear leukocytes (PMN) in fluids containing 35 mM lactate at pH 5.2 resulted in an immediate and profound lowering in intracellular pH ([pH]i) which was not observed in lactate-containing fluids at neutral pH or at low pH in the absence of lactate. We postulate that the critical lowering of [pH]i in PMN, caused by the combination of high lactate concentration and low pH of the dialysis fluids, is responsible for the observed inhibition of respiratory burst activation. It is also possible that under these conditions, the lactate ion acts as a proton carrier across the cell membrane following the [H+] gradient. The time course of this [pH]i change suggests that host defence mechanisms may be impaired following short-time exposure to unused dialysis fluid prior to its equilibration in vivo.

Macrophages and Mesothelial Cells in Bacterial Peritonitis

Research in recent years has examined the mechanisms underlying cellular host defence in the peritoneal cavity. These studies have established that the resident cells of the peritoneal cavity, the peritoneal macrophages (PM phi) and the mesothelial cells (HPMC) contribute to the initiation, amplification and resolution of peritoneal inflammation. Ex vivo measurements of intra-peritoneal inflammatory mediators during peritonitis has elucidated the time courses for the generation of proinflammatory, chemotactic and anti-inflammatory cytokines and have identified that their secretion occurs largely within the peritoneum. These studies provide evidence that both PM phi- and HPMC-derived mediators are directly involved in controlling inflammation. It has been widely accepted that resident PM phi form the first line of defence against peritoneal infection, a more contemporary view would suggest that the direct or indirect (via secreted pro-inflammatory cytokines) interaction between PM phi and HPMC is pivotal to the activation and subsequent amplification of the peritoneums response to infection. Whilst the site of these interactions is unknown, considerable evidence suggests that it occurs on the surface of the mesothelium, where invading micro-organisms may colonize. In this respect Staphylococcal exoproducts can directly activate HPMC cytokine synthesis. Once the inflammatory response is initiated, recent evidence suggests, that mesothelial cells upon activation by PM phi-derived IL-1 beta and TNF-alpha, are capable of amplifying inflammation and generating signals (via the creation of a gradient of chemotactic cytokines, IL-8, MCP-1 and RANTES) for the recruitment of leukocytes into the peritoneum. This process is also facilitated via the cytokine driven up-regulation of adhesion molecule expression (ICAM-1 and VCAM-1) on HPMC. Much less is understood about the mechanisms by which inflammation is resolved, although the secretion of anti-inflammatory molecules (IL-6, IL-1ra and soluble TNF-p55/75) by receptors by PM phi and HPMC may be important in the process. The existence of a peritoneal cytokine network controlling inflammation is now well established, within this the interaction of PM phi and HPMC appears to play a pivotal role in the hosts response to peritoneal infection.

The complement‐inhibiting protein, Protectin (CD59 antigen), is present and functionally active on glomerular epithelial cells

Protectin (CDS9 antigen) is a 20‐kD phosphatidyl‐inositol‐linked membrane protein that inhibits formation of the membrane attack complex (MAC) of complement on homologous cells. Although the antigen has been identified in a number of human tissues, until recently a functional role had been demonstrated only in circulating cells. Using immunofluorescence techniques we have shown the presence of protectin on human glomerular epithelial cells (GEC) in culture and on GEC, tubular epithelial cells and endothelial cells in frozen sections of normal human renal cortex. In addition, we present evidence that this protein functions in protection of GEC from homologous complement: cultured cells incubated with the Fab2 fragment of a monoclonal anti‐protectin antibody were markedly more susceptible to killing by homologous serum than were cells in the absence of Fab2 anti‐protectin. These findings suggest that this protein may be important in the maintenance of glomerular integrity in vivo, and may be of relevance in certain renal diseases.

Effect of peritoneal dialysis on cytokine production by peritoneal cells.

Over the past few years it has become increasingly clear that the peritoneal membrane contributes significantly to inflammatory processes occurring in the peritoneal cavity. The increasing use of continuous ambulatory peritoneal dialysis has focused attention of both the role of the mesothelium in peritoneal host defence as well as assessing the importance of the infusion of dialysis fluids on modulation of peritoneal cell function. The current review will examine our increasing knowledge of the peritoneal host defense mechanisms, particularly the involvement of the peritoneal membrane, and the mesothelium in particular, and its interaction with peritoneal macrophages. We will discuss our present knowledge of how cell viability and the secretion of inflammatory and regulatory mediators (cytokines) by the mesothelial cells are modulated following exposure to peritoneal dialysis fluid in vitro and in vivo.

Renal Transplant Artery Rupture Secondary to Candida Infection

We report a case of a diabetic renal transplant patient with candiduria who developed repeated elevations in serum creatinine concentration due to an intermittently obstructing candida ball in the urinary tract. After unsuccessful attempts to place a nephrostomy tube for local irrigation and debulking, the renal graft artery ruptured secondary to extensive perirenal candida infection. Percutaneous nephrostomy may increase the risks of renal and extrarenal tissue infection with the potential for fatal complications such as infection of the vascular anastomosis.

Decreased Degradation of Collagen and Fibronectin following Exposure of Proximal Cells to Glucose

Background/Aims: Thickening and reduplication of the tubular basement membrane has been reported as an early event in diabetic nephropathy. The aim of the work outlined here was to examine the effects and mechanisms involved in the modulation of renal proximal tubular type-IV collagen and fibronectin turnover by glucose. Methods: The effect of glucose on type-IV collagen and fibronectin generation was studied by exposure of primary cultures of human renal proximal tubular cells (HPTC) to elevated D-glucose concentrations. Subsequently the mechanism of modulation of fibronectin generation was examined in a polarised system utilising the porcine proximal tubular cell line LLC-PK1 grown on porous tissue culture inserts. Results: Incubation of confluent growth-arrested HPTC with 25 mM D-glucose led to the accumulation of both type-IV collagen and fibronectin. This increase was not dependent on new gene transcription for either protein. Exposure of HPTC to 25 mM D-glucose also led to the induction of tissue inhibitor of metalloproteinases (TIMP-1 and TIMP-2) and also gelatinase A. There was, however, a net decrease in overall gelatinolytic activity. Incubation of confluent monolayers of LLC-PK1 cells grown on tissue culture inserts with 25 mM D-glucose on either their apical or basolateral aspect led to fibronectin accumulation seen only in the basolateral compartment. Under these experimental conditions, we can demonstrate polyol pathway activation, and furthermore the increase in fibronectin concentration in response to glucose was inhibited by the aldose reductase inhibitor sorbinil. Fibronectin accumulation was also demonstrated following both apical and basolateral addition of 1 mM sorbitol, but not following the addition of 25 mM galactose to either aspect of the cells. Conclusions: These data demonstrate that the glucose-induced accumulation of type-IV collagen and fibronectin was associated with alterations in the degradative pathway of these matrix components. In addition fibronectin generation in response to glucose was non-polar in terms of application of glucose, but polar in terms of fibronectin accumulation. The mechanisms of glucose-induced modulation of fibronectin were mediated by polyol pathway activation, and more specifically related to the metabolism of sorbitol to fructose.

Host defence and effects of solutions on peritoneal cells

It is general experience that some patients receiving long-term peritoneal dialysis suffer from repeated episodes of peritonitis while others remain relatively free of this complication. It has been suggested that differences in the ability of individual subjects to resist infection, i.e., their host defence, are at least partly responsible for this clinical observation. This hypothesis was strengthened by the report by Verbrugh et al. on the potential efficacy of peritoneal macrophages and opsonins in the antibacterial defence of such patients [1]. In this chapter we review the available evidence on the various components of host defences of peritoneal dialysis patients including the effect of the dialysate.

Effects of a selective adenosine A1 receptor antagonist on the development of cyclosporin nephrotoxicity.

1 The clinical application of cyclosporin as an immunosuppressive agent is limited by its nephrotoxicity. 2 The effect of FK453, a selective A1‐receptor antagonist, administered twice daily to rats at a dose of 100 mg kg−1 was assessed on the development of nephrotoxicity induced by cyclosporin (10 mg kg−1 i.p. daily) administered for 14 days. The effects of nifedipine administered twice daily (0.3 mg kg−1 s.c.) for 14 days, on cyclosporin nephrotoxicity were also studied. 3 Cyclosporin induced a 46.58% and 35.78% decline in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) respectively and a reduction of 16.69% in filtration fraction (FF). Co‐administration of FK453 resulted in falls of 30.5%, 18.59% and 14.7% in GFR, ERPF and FF respectively, the former two significantly less than the falls seen with cyclosporin (CyA) alone (P < 0.05 vs CyA, ANOVA). 4 Nifedipine appeared to have a more pronounced protective effect resulting in a decline of only 20.91% in GFR, with no significant change in ERPF (increase of 0.93%) when co‐administered with CyA. 5 These observations indicate adenosine plays a minor role in the pathophysiology of CyA nephrotoxicity.

Leukotriene B4 generation by human monocytes and neutrophils stimulated by uropathogenic strains of Escherichia coli

The generation of the 5-lipoxygenase product, leukotriene B4 (LTB4) by human mononuclear phagocytes (monocytes) following incubation with 25 different uropathogenic strains of Escherichia coli correlated with the haemolytic activity of the strains (r = 0.572, P less than 0.01). LTB4 generation by human neutrophils (PMN), however, was unrelated to this haemolytic potential (r = 0.164). In contrast, both prelabelled monocytes and PMN were stimulated by haemolytic strains of E. coli and by haemolytic culture supernatants to release significant amounts of [3H]arachidonic acid. There was a significant correlation between haemolytic activity and [3H]arachidonic acid release generated by individual strains from monocytes (r = 0.804, P less than 0.001) and PMN (r = 0.888, P less than 0.001). In addition, nonhaemolytic strains but not their culture supernatants were capable of causing slow release of both [3H]arachidonic acid and LTB4 from PMN and mononuclear cells. These results suggest that both the possession of haemolytic activity, and the direct interaction of bacteria with the leukocyte surface are mechanisms by which uropathogenic strains of E. coli may cause the release and metabolism of arachidonic acid. In addition, there was synergistic augmentation by nonhaemolytic bacteria of the PMN LTB4 response to haemolytic culture supernatants or to low doses of the calcium ionophore A23187. These results support an ionophore-like mechanism for the activation of the cell by haemolysin. LTB4 generation by PMN incubated with haemolytic supernatants was also augmented by particulate zymosan in a manner dependent on the dose of zymosan, suggesting that the direct interaction of E. coli with PMN may involve an activation mechanism similar to that for zymosan. These results demonstrate differing responses of peripheral mononuclear cells and PMN from the same donors to identical strains of E. coli and suggest that the generation of the potent chemotactic agent LTB4 in response to E. coli infection in vivo need not depend solely on the elaboration of cytotoxic haemolysins by individual strains.

The influence of net surface charge on the interaction of uropathogenic Escherichia coli with human neutrophils

Escherichia coli strains, grown to suppress fimbrial expression, synthesised enhanced quantities of polysaccharide capsule, which significantly lessened their binding to heparin sepharose columns. In the presence of poly-L-lysine, these strains were strongly retained on the columns confirming their highly anionic nature. Uropathogenic strains of E. coli expressing type 1 fimbrial adhesins activated the respiratory burst, the degranulation response and the release of leukotrienes from human neutrophils (PMN) to a significantly greater extent than the same strains grown in a medium to suppress this fimbrial expression. The addition of the poly-cation poly-L-lysine, however, selectively increased neutrophil activation in response to these non-fimbriate strains. This dose-dependent effect was reversed by the addition of heparin suggesting a mechanism dependent on surface charge. The results of this study suggest that non-specific mechanisms involving the neutralisation of surface charge, in addition to specific receptor and adhesin mediated events could affect neutrophil activation at sites of infection.