S. Ringoir

Influence of uraemia and haemodialysis on host defence and infection

Dysfunction of the host defence is one of the major functional disturbances in end-stage renal disease (ESRD) with substantial clinical and socioeconomic implications. The resulting higher susceptibility to infection may lead to life-threatening complications such as sepsis, septic shock, and abcess formation. The immune system defends against infection via a cascade of finely tuned mechanisms, which include (1) the functional involvement of immunocompetent cells, (2) the release of humoral substances upgrading immunocompetence, and (3) the attraction of cells towards regions of infection. All these elements cooperate to achieve ingestion and destruction of infectious agents by phagocytic cells. The effect of uraemia (and its treatment by dialysis) on the immune system will be considered in depth, to formulate therapeutic approaches and preventive measures.

Depressed Phagocytosis in Hemodialyzed Patients: In vivo and in vitro Mechanisms

Infection is a frequent complication and the major cause of death among end-stage renal patients. Polymorphonuclear phagocytes (PMNL) are important in host defense mainly because of bacterial destruction by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-related free radical production following phagocytosis. In this study, hexose monophosphate pathway glycolytic activity, delivering energy to NADPH oxidase, is evaluated in vivo and in vitro, in healthy controls and in dialyzed renal failure patients. Our results show a marked parallel and correlated inhibition in the response to three stimuli for phagocytic activity (Staphylococcus aureus, formyl-methionine-leucine-phenylalanine, phorbol myristic acid) in predialysis samples. These data point to a main suppression of metabolic pathways, possibly beyond protein kinase C. This response is further suppressed at the 15th minute of cuprophane dialysis, for all stimuli studied (-40 to -94%; p < 0.001) except PMA. PMNL response remains intact during dialysis with non-complement-activating dialyzers. In vitro experiments confirm decreased PMNL glycolytic activity after the suspension of cuprophane fragments in normal whole blood. We conclude that polymorphonuclear cell energy delivery to NADPH oxidase is impaired in patients with end-stage renal failure. The impaired response against various stimuli is different in predialysis blood samples compared to samples collected during cuprophane dialysis, and may be related to two different conditions. These events probably contribute to the acquired immune suppression of uremia and the high incidence of infection among dialysis patients.

Factors influencing drug protein binding in patients with end stage renal failure.

SummaryInvestigations were undertaken to evaluate which uraemic solutes decrease drug protein binding. This was done by performing HPLC-fractionation of uraemic biological fluids and studying the effect of addition of a lyophilisate of each fraction to normal plasma containing standard quantities of radiolabelled drugs.From a first study, based only on fractionation of uraemic ultrafiltrate with an HPLC-gradient mainly aimed at elution of hydrophilic compounds, hippuric acid appeared to be a major protein binding inhibitor for theophylline and phenytoin. The problem with this approach was that it did not include the compounds with the most substantial protein binding. Therefore, studies were planned to fractionate deproteinized uraemic sera, but first it was necessary to define which deproteinisation methods gave the highest yields of protein bound ligand.Heat denaturation was found to be one of the most effective deproteinisation methods. When a lyophilisate of uraemic serum, deproteinised by this method, was added to normal plasma, a higher capacity to displace theophylline from protein binding sites was found compared to the effect of an identical volume of an ultrafiltrate of the same samples. Fractionation of the deproteinised sample by HPLC revealed a larger number of fractions able to inhibit drug protein binding.

Antibiotics and energy delivery to the phagocytosis-associated respiratory burst in chronic hemodialysis patients: a comparison of cefodizime and cotrimoxazole.

Twenty-three stabilized chronic uremic patients with no active or recent infection were treated for 10 days with either cefodizime (5 x 2 g intravenously, n = 10) or cotrimoxazole (960 mg orally b.i.d., n = 8) in order to evaluate the effects on the depressed polymorphonuclear metabolic response to phagocytic challenge; a separate group of 5 patients received placebo. Ex vivo evaluation in whole blood of energy delivery to the phagocytosis-associated respiratory burst activity in response to latex and zymosan challenge was determined by measuring hexose-monophosphate shunt NAD(P)H-oxidase-related glycolytic activity. Cefodizime induced a statistically significant increase in the baseline-depressed glycolytic response for both latex and zymosan challenge, in contrast to cotrimoxazole and placebo. Depressed phagocytosis-related metabolic function in hemodialyzed patients was stimulated by cefodizime in recommended therapeutic doses but not by cotrimoxazole, the effect persisting for at least 2 weeks after the end of treatment.

Correlation of a Colorimetric and a HPLC Method for the Determination of Serum Hippuric Acid Concentrations in Uremia

Hippuric acid has been recognized as a potential marker of uremic toxicity in chronic renal failure. However, in most studies, serum hippuric acid concentrations have been determined by sophisticated methods, such as high-performance liquid chromatography. The present study was undertaken to evaluate whether the less complicated colorimetric determination method could replace such methods. Based on 21 different samples, the results obtained by both methods appeared to be correlated to each other in a highly significant way (total hippuric acid: r = 0.99, p less than 0.001; free hippuric acid; r = 1.00, p less than 0.001). Mean total and free hippuric acid concentrations and mean percent protein binding, obtained with both determination methods, were also identical. It is concluded that both the colorimetric method and high-performance liquid chromatography are equally reliable for the study of the concentration of hippuric acid in uremic serum and of its importance as a marker of the clinical and biochemical epiphenomena of uremic toxicity.

Search for optimizing dialysis therapy. I: Acute effects of hemodiafiltration with a highly permeable membrane and a large dose of convection and diffusion

Based on the concept that an optimisation of dialysis therapy might be achieved by increasing the removal of small metabolic substances as well as low molecular weight proteins, hemodialfiltration (HDF) was modified. In 9 ESRD patients, HDF was performed acutely for 4 h, where 60 liters of substitution fluid were infused per patient. Two polysulphone F60 membranes in line were used as hemodialfilters. As compared to conventional hemodialysis (HD), performed with a cuprophane dialyser, HDF resulted in higher extraction indices for small solutes assessed by HPLC. Due to the large amount of convection applied, HDF was followed by significant decreases of low molecular weight proteins ranging from 9.5 kdaltons (iPTH, p less than 0.01) to 17 kdaltons (myoglobin, p less than 0.01). Analysis of the protein pattern of the serum revealed a nonlinear function in the decrease of plasma proteins after HDF. It is concluded that even though the detoxification efficacy of the described HDF method is by far superior to conventional HD in quality and quantity, the efficacy is still far from that of excretory renal function. Thus, to further improve efficacy with respect to the catabolic renal function for low molecular weight proteins, membranes for HDF or hemofiltration barriers but should surpass the sieving properties of the glomerulus in the low molecular protein range.

Two-Pool versus Single-Pool Models in the Determination of Urea Kinetic Parameters

The mathematics used for urea kinetic modeling are currently based on a single-pool distribution of urea throughout the body. In this study, we evaluated which one of a single- or a two-pool model would be more appropriate for the prediction of directly measured urea decay during hemodialysis. A numerical method was used which minimizes the relative root mean square (RMS) error between a calculated single- or two-pool urea decay curve and the measured intradialysis decay in 13 equilibrated dialysis patients. Using a two-pool model, the RMS error was markedly lower (1.27 +/- 0.72%) than the values obtained with a single-pool model, either based on multiple urea concentrations (RMS error 3.14 +/- 1.36%; p < 0.01 vs. two-pool model) or only on pre- and postdialysis urea (RMS error 5.00 +/- 2.38%; p < 0.001). This resulted for the single-pool model in an overall underestimation of urea generation, distribution volume (V) and protein catabolic rate and in an overestimation of Kt/V versus the two-pool model. In individual cases, the difference reached up to 18.7%. Comparison of V calculated from the two-pool model versus V values determined from anthropometric formulae (Watson) resulted in similar mean values (34.05 +/- 4.87 vs. 33.09 +/- 4.19 liters; p = NS), with a weak correlation (n = 13, r = 0.75, p = 0.003). Individual values, however, again differed by up to more than 20%. In conclusion, the use of single-pool kinetic models, as well as of anthropometric estimations of V, should be regarded with care, especially when individual patients are considered instead of groups. The two-pool model follows the directly measured urea decay more exactly which results in substantial differences in calculated kinetic parameters.

Development of Anti-N-Like Antibodies During Formaldehyde Reuse in Spite of Adequate Predialysis Rinsing

The development of anti-N-like antibodies has been demonstrated with formaldehyde reuse of hemodialyzers. It has been recommended to pursue a postrinsing venous effluent formaldehyde concentration in the range of or below 2 to 10 ppm, in order to prevent the development of these anti-N-like antibodies. In the present study, we evaluated formaldehyde reuse in a population of 50 patients and whether the strict control of predialysis effluent formaldehyde concentration below 2 to 3 ppm could prevent the development of anti-N-like antibodies. Five of 50 patients (10%) became positive 6 to 14 months after the start of formaldehyde reuse, indicating that even a careful control of effluent formaldehyde concentration cannot prevent the occurrence of this abnormality.

Hippuric acid as a marker

There are some classical markers of uremic solute retention, such as serum urea and creatinine concentration. These parameters are however not always reliable, especially not in dialysis patients, so that it seemed interesting to us to undertake a multifactorial study, in an attempt to define the most suitable marker molecules in uremia. For this purpose, we used high performance liquid chromatography HPLC as a basic technique.

Influence of feeding, blood sampling method and type of anaesthesia on renal function parameters in the normal laboratory rat

With sophisticated experiments it is necessary to handle laboratory animals many times. To determine the effect of minor handling a series of experiments was performed to measure the impact of fasting, anaesthesia, blood collection method and serum creatinine analysis on renal function. Simple clinical methods to measure renal function parameters such as diuresis, urinary osmolality, urinary creatinine excretion and serum creatinine were used. During fasting a significant increase (P<0.01) in diuresis and a significant decrease (P<0.01) in urinary osmolality were noted. Fasting and anaesthesia have the additional effect of significantly decreasing (P<0.05) urinary creatinine excretion. Blood sampling method also has a significant impact on serum creatinine: venous sampling causes false-positive differences compared with simultaneous arterial sampling.