Bradykinin and nitric oxide generation by dialysis membranes can be blunted by alkaline rinsing solutions.
BACKGROUND
Bradykinin (BK) generation following the first contact of blood with the dialysis materials is thought to enhance hypersensitivity reactions (HSRs). Some of the effects of BK are mediated by nitric oxide (NO). We have recently reported that the pH of diluted blood modulates the kinin system. The present study was aimed to investigate the role of the pH of culture media and filter-washing solutions and BK and NO generation, either in vitro and ex vivo.


METHODS
BK was measured by a specific enzyme-linked immunosorbent assay (ELISA), and NO synthase (NOS) activity by 3H-citrulline production after incubation with 3H-arginine and nitrites by using the Griess reagent. In in vitro experiments, NOS activity was detected in endothelial cells (ECs) cultured with graded BK concentrations at various pH values. Blood from 30 patients in regular dialysis was ex vivo circulated in one single passage through minifilters prerinsed with pH 7 or pH 8 phosphate buffer (PB) solutions. The out-flowing blood was tested for BK and nitrite content and was incubated with cultured ECs to evaluate its capacity to modulate NOS activity.


RESULTS
BK induced in vitro a dose-dependent increase in NOS activity of ECs, which was mediated by tyrosine kinase phosphorylation. NO generation was enhanced at pH 7.2, which remained unchanged at pH 7.6. In ex vivo experiments, blood out-flowing after one passage on filters washed with pH 7 PB solutions had increased BK levels (P < 0.0001), increased nitrites (P < 0.05), and enhanced EC NOS activity (P < 0. 05) in comparison to data found when filters were washed with pH 8 PB. Only when the filters were rinsed with a solution at pH 7 did PAN DX and AN69 membranes show a distinct BK generation capability, and cuprophane a peculiar capability to enhance NOS. Such effects were prevented when dialyzers were prerinsed with pH 8 PB. Multiple regression analysis showed that the pH of the uremic blood was the driving factor for BK and NOS activation (r = 0.54, P < 0.02).


CONCLUSIONS
BK and NO generation are modulated by environmental pH. Rinsing the blood and dialysate compartments of filters with an alkaline solution prior to use may mitigate the activation of mediators likely to be involved in some HSRs.
Coppo
R
R
Nephrology and Dialysis Department, Regina Margherita Hospital, Torino, Italy. nefrologia@oirsantanna.piemonte.it
Amore
A
A
Cirina
P
P
Scelfo
B
B
Giacchino
F
F
Comune
L
L
Atti
M
M
Renaux
J L
JL
D000138:Acidosis / Q000378:metabolism; D000468:Alkalies / Q000494:pharmacology*; D000818:Animals; D001784:Blood Gas Analysis; D001920:Bradykinin / Q000096:biosynthesis* / Q000494:pharmacology; D002460:Cell Line; D003877:Dermatitis, Contact / Q000378:metabolism / Q000517:prevention & control; D015314:Dialysis Solutions / Q000494:pharmacology*; D004727:Endothelium / Q000166:cytology / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008567:Membranes, Artificial; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012044:Regression Analysis; D006435:Renal Dialysis / Q000379:methods*; D012377:Rodentia
D000468:Alkalies; D015314:Dialysis Solutions; D008567:Membranes, Artificial; D009573:Nitrites; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D011505:Protein-Tyrosine Kinases; D001920:Bradykinin