Importance of the bradykinin–nitric oxide synthase system in the hypersensitivity reactions of chronic haemodialysis patients

Rosanna Coppo and Alessandro Amore

Nephrology, Dialysis and Transplantation Department, Regina Margherita Hospital, Torino, Italy

Keywords: AN69; bioincompatibility; bradykinin; filter rinsing solutions; hypersensitivity reactions; nitric oxide

Incidence of hypersensitivity reactions

The syndrome of hypersensitivity reactions (HSRs) occurs within the first 20 min of the haemodialysis (HD) session, with protean presentation and variable clinical expression ranging from mild to life-threatening symptoms, including nausea, faintness, itching, flushing, swelling of tongue or throat, wheezing or bronchospasm, hypotension and in most severe cases, irreversible shock.

Its impact on patients is likely to be underestimated. The frequency of severe episodes was reported in 1982 to be rather low (0.3/1000 patients/year with flat sheet dialysers) [1] but it has been recently estimated in a US survey to involve 1000 patients/year [2], highlighting this syndrome's unpredictability of appearance and severity.

An analysis of the factors that might increase the risk of HSR is necessary for appropriate prevention.

Enhancing factors and pathophysiology of HSRs

Attention has been focused on several possible enhancing factors, including ethylene oxide (Eto) residues, bacterial lipopolysaccharide fragments and dialysis capillary geometry. The overlap of the clinical presentation of the HSR syndrome with the symptoms described following contact-phase activation has led to the hypothesis of bradykinin (BK) generation due to blood–membrane interaction. By impairing kinin degradation angiotensin-converting enzyme inhibitors (ACE-I) could further increase BK to pathological levels, allowing the full development of the clinical feature. This hypothesis could explain the high frequency of HSRs after using the AN69 polyacrylonitrile membrane, particularly in patients undergoing treatment with ACE-I [3].

BK exerts its vasodilating effect via increased production and release of nitric oxide (NO), which is the pivotal mediator [4]. NO is a short-lived gas produced by a specific enzyme, NO synthase (NOS), which increases cytosolic cGMP in smooth-muscle vascular cells, leading to vasodilatation [5]. Endothelial cells are equipped with two isoforms of NOS. The constitutive isoform (cNOS) produces small physiological puffs of NO, which play a role in maintaining vascular tone, while the inducible isoform (iNOS) is synthesized de novo and releases high and sustained amounts of NO, which may lead to acute severe vasodilatation and shock or, after chronic repeated stimulations, to vascular sclerosis as reported by our group [6].

Modulation of enhancing factors

It has been recently demonstrated that the contact-phase system activation possibly induced by the dialysis membrane's electronegativity is a pH-dependent and dilution-dependent phenomenon, and that in diluted plasma kallicrein generation can be inhibited by keeping the pH value over 7.4 [7]. We found that in cultured endothelial cells, purified BK induced a dose-dependent increase in NOS activity which was mediated by the phosphorylation of two tyrosine kinases of 60 and 145 kDa respectively [8]. The enzyme was greatly modulated by the pH of culture medium. In fact, cells cultured at pH 7.2 increased their NOS activity after 10 min and this increase lasted for 6 h, while NOS activity remained unchanged when cells were cultured at pH 7.6.

These in vitro observations may be of clinical relevance. In routine dialysis practice the blood compartment of the dialyser is primed with saline in preparation for a treatment. The dialysate compartment is generally rinsed with bicarbonate containing dialysis fluid following priming. During rinsing and immediately after the start of dialysis, an equilibration between blood and dialysate compartment takes place. First, a transfer of CO2 occurs from the bicarbonate in the dialysis solution to blood, leading to a greater magnitude of change in pCO2 than in plasma bicarbonate and to a further acidification of diluted blood. Hence, immediately after the start of dialysis, the pH of the diluted blood can meet the conditions for BK generation and NOS activation.

The hypothesis of a modulation of both BK and NOS activity by environmental pH was further proved by ex vivo experiments performed by our group [8]. Experimental devices and conditions were designed to provide a model for the selective investigation of the crucial event in the generation of HSRs i.e. the initial contact between diluted blood and dialysis material. Blood of uraemic patients was circulated in one single passage through mini-dialysers made of various membranes. Before the test run, mini-dialysers were successively rinsed by phosphate buffer (PB) solutions at either pH 7 or 8, circulating through the blood and dialysate compartments. Subsequently, the dialysate compartment was clamped.

Three minutes after connecting each patient, outflowing blood was tested for pH, BK, and NO systems. The mean levels of BK generated in the blood of uraemic patients passed through mini-dialysers were significantly higher when filters were pre-rinsed with pH 7 than with pH 8 washing solutions. BK generation was distinctly elevated in the blood that had circulated through classic polyacrylonitrile membranes—PAN DX and AN69®—when filters were washed with pH 7 PB, while it was almost completely blunted when filters were washed with pH 8 PB. Blood contact with triacetate, polysulphone, or Cuprophan membranes induced only a slight and non-significant release of BK when washed with either pH 7 or 8 PB solutions.

NOS activity in cultured endothelial cells incubated with blood after one single passage through mini-filters was higher when filters had been previously washed with pH 7 than pH 8 solutions. NOS activity of blood passed on AN69 membrane was increased only when filters were washed with pH 7 solutions, while it remained unchanged with pH8 PB washes. NOS activity was distinctly enhanced by Cuprophan, and pH 8 solutions blunted the increase. This effect was probably due to a complement-dependent activation, irrespective of BK pathway. In a sham dialysis model with bicarbonate buffer using healthy donors' blood, we found that Cuprophan strongly enhanced NOS activity, as early as after 5 min of circulation [9,10]. The enhanced NOS activity was due to TNF released by leukocytes following complement activation, as hypothesized by Hakim et al. [11].

The comparison of these two series of data suggests that blood pH is critical for enhancing a BK-mediated NOS activation when AN69 membranes are used. Even though Cuprophan does not activate the BK system, it enhances NOS activity by a complement-mediated pathway, which can be modulated by the pH of the washing solution as well.

Hence, the membrane type was a discriminating factor for BK and NOS activation and the pH of blood at the outflow of mini-dialyser was the main factor governing the activation of these significantly related variables.

Possible attempts to avoid HSRs

We recently undertook a multicentre retrospective analysis of HSRs in several Italian centres, focusing on the precautions adopted by nephrologists to avoid recurrences and determining whether a rinsing procedure which maintains the pH of the diluted blood above 7.4 may prevent HSRs [12].

HSRs were reported in 54 patients on home dialysis treatment between January 1995 and June 1997. Our survey confirmed a higher frequency of HSRs in patients using AN69 (72% of the reported HSRs) compared with other dialysis membranes, particularly when ACE-I was being administered at the same time (44% of the patients). However, the HSRs reported represent only a small percentage of the total number of patients treated with AN69 membrane and/or an association of AN69 and ACE-I. Hence, the presence of co-factors modulating the biological pathway and leading to the full clinical picture of this syndrome is conceivable. No relationship was found with anti Eto antibodies. The highest incidence of HSRs actually occurred with dialysers sterilized by physical methods. Backfiltration of some fragments of the bacterial cell wall from dialysate to blood was not a major mechanism, since most patients used sterile washing solutions.

HSR prevention modalities varied considerably among centres. Two-thirds of the nephrologists did not modify dialysis prescription but tried to prevent the acidic environment of the patient's diluted blood at the first contact with the dialysis device, using an alkaline rinsing procedure (BioPrime® with alkaline solution). This procedure allows a greater stability of the blood pH during the first few minutes of dialysis: rinsing with saline always leads to blood pH values lower than 7.4, while rinsing with a bicarbonate solution (0.167 mol/l (NaHCO3), pH 7.6, pCO2 <=80 mmHg) leads to values higher than 7.4, which was the threshold limit in vitro of contact-phase system activation in diluted blood.

None of the patients enrolled in this study developed new episodes of HSRs. ACE-I was withdrawn in several cases. However, interestingly, in some cases the combination of AN69 membrane and ACE-I treatment was kept unchanged, while only the alkaline washing procedure was introduced. In none of these cases was a recurrence of HSRs observed.

Conclusions

The observations made in in vitro and ex vivo experiments suggest that attention should be paid to the pH and pCO2 of the solutions used to rinse dialysis filters before clinical use. These revised procedures provide a significant protection against BK release induced by AN69 and PAN DX dialysis membranes and against the increase of endothelial cell NOS activity stimulated by Cuprophan membrane.

Even though we have gained better knowledge on HSRs over the last few years and the interactions between gas analysis of diluted blood, bradykinin, and NO have become clearer than previously, it is likely that contact-phase activation is only one of the processes involved in HSRs, and that other mechanisms and/or modulating factors are involved, though perhaps only in a minority of cases.

The simple procedure of rinsing with sodium bicarbonate—containing solutions may be useful to mitigate the activation of mediators likely to be involved in HSRs.

Notes

Correspondence and offprint requests to: Rosanna Coppo MD, Divisione Nefrologia e Dialisi, Ospedale Regina Margherita, Piazza Polonia 94, I10126 Torino, Italy. Back

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