Abstract
Background. Dialysis patients are at high risk of vascular/cardiovascular complications with multifactorial pathogenesis, and pregnancy-associated plasma protein A (PAPP-A) is one of the new markers related to cardiovascular risk. Because hemodiafiltration (HDF) is supposed to be better for cardiovascular status, the aim of this study was to describe whether it has any advantage concerning changes of PAPP-A and related molecules during the session in comparison with hemodialysis (HD). Methods. The studied group consisted of 20 chronic hemodialysis patients. In each patient, PAPP-A and related parameters—IGFBP-4 (insulin like growth factor binding protein), IGF-I (insulin like growth factor), and two MMPs (matrix metalloproteinases)-2 and 9—were determined both during a single online HDF session (high-flux polysulfone membrane HF80, postdilution) and during a single HD session (low-flux polysulfone membrane F6, F7) at time 0 (start), 15 min, 120 min, and 240 min (end) of the session. Results. PAPP-A, elevated at baseline in dialysis patients, changes significantly both during HDF and HD without significant differences between these two procedures (mean levels during HDF were 24.3, 53.9, 24.3, and 27.3 mIU/L). It increases more than two-fold from 0 to 15 min of the session (p < 0.001) and then decreases until the end of the session (p < 0.001). MMP-2 decreased slightly during both sessions (p < 0.001), and changes of other molecules were only minimal. Conclusion. A single HDF session compared to HD has no advantage in the decrease of PAPP-A and other tested molecules, all of them related to cardiovascular risk. Studies aimed at a long-term effect of both procedures on these parameters would be needed to further evaluate these therapeutical strategies.
INTRODUCTION
Dialysis patients are at high risk of vascular/cardiovascular complications with multifactorial pathogenesis. Several studies have shown that pregnancy-associated plasma protein A (PAPP-A) is one of the new markers of cardiovascular risk in general population.Citation[1–4] Additionally, it is elevated at baseline in chronic hemodialysis patientsCitation[5],Citation[6] and might be associated with the prognosis of these patients.Citation[7] PAPP-A is a metalloproteinase that cleaves IGFBP-4 (insulin-like growth factor binding protein), an inhibitor of IGF-I (insulin-like growth factor I) action and thus increases the bioavailability of IGF-I.Citation[8] This way, it can possibly contribute to the pathogenesis of atherosclerosis, as IGF-I induces the migration of smooth-muscle cells and is important for monocyte chemotaxis and the activation and release of cytokines within the atherosclerotic lesion.Citation[9],Citation[10]
In addition to PAPP-A, other metalloproteinases (MMPs, matrix metalloproteinases) are involved in the pathogenesis of atherosclerosis, the acceleration of its progression, and the destabilization of plaque.Citation[11] MMPs—mainly MMP-2 and 9—play a role in vascular tissue remodeling by degrading the extracellular matrix and thus contributing to the weakening of the vascular wall and plaque disruption.Citation[12],Citation[13] MMP-9 is elevated in acute coronary syndromes, and its plasma levels were described as a new predictor of cardiovascular mortality in patients with coronary artery disease in the general population.Citation[14]
PAPP-A first increases during hemodialysis (HD)Citation[15],Citation[16] and than decreases to levels usually measured before the dialysis session; however, its explanation is difficult, as it is a high molecular weight protein. Its behavior might be associated with a complex biological response to the extracorporeal procedure, which starts immediately after the contact of blood with the artificial surfaces and includes leukocytes and complement activation, the release of enzymes stored intracellularly, as well as the interaction of molecules with the membrane and possibly their removal. Because hemodiafiltration (HDF) is supposed to be better for cardiovascular status, the aim of this study was to describe whether it has any advantage concerning the changes of PAPP-A and related molecules during the session in comparison with hemodialysis.
SUBJECTS AND METHODS
Patients
The studied group consisted of 20 chronic hemodialysis (HD) patients (9 men and 11 women), mean age 54 ± 15 years. Patients were dialyzed three times a week for four hours, and their dialysis treatment lasted for three years (range 1–17 years). In the last five months (2–19 months), half of these patients were treated with HDF. All patients fulfilled the criteria of adequate dialysis (Kt/V >1.2 according to Daugirdas II formula). Causes of renal failure were as follows: glomerulonephritis in five cases, interstitial nephritis in nine cases, polycystic kidney disease in four cases, and diabetic nephropathy and hypertensive nephropathy in one case each. Their residual diuresis was 500 ml (0–600 ml). Their albumin was 41 (39–43) g/L; hemoglobin, 112 (106–119) g/L; and hematocrit, 33.4 % (31.7–35.7), measured before the dialysis session after the long interval. Patients received an average weekly erythropoietin dose of 60 IU/kg body weight. The majority of patients had hypertension well controlled with antihypertensive drugs (monotherapy or a combination of two drugs). Two of the patients were type 2 diabetics on oral antidiabetics or diet, five patients had ischemic heart disease, and three patients were treated for dyslipidemia. All patients were in stable clinical status and had no acute cardiac problems, and there were no signs of acute infection. Current drug regimes were not altered throughout the study.
Design of the Study
Each patient was tested during a four-hour HD with low flux polysulfone membranes (F6 1.3 m2 or F7 1.6 m2, Fresenius Medical Care, Bad Homburg, Germany) and a four-hour postdilution on-line hemodiafiltration (HDF) with high flux polysulfone membranes (HF80, 1.8 m2 Fresenius Medical Care, Bad Homburg, Germany). The amount of substitution fluid was 18750 ± 2693 mL/4 hours (78.1 ± 11.2 mL/min). Dialysis was performed with a bicarbonate dialysis solution, blood flow rate was 300 mL/min, and dialysate flow rate was 500 mL/min. Patients received heparin during dialysis at a dose of 6100 ± 1500 IU (two-thirds of the dose as bolus after the start and the rest as bolus in the middle of the procedure), and mean ultrafiltration rate per session was 1.9 kg. Endotoxin in dialysate was below the detection limit (LAL – limulin amebocyte lysate test, performed once in three months).
Blood samples from the arterio-venous fistula (arterial line) were drawn before dialysis (time 0) and at 15 min, 120 min and 240 min (end) of the session. Blood was centrifuged for 10 min at 1450 g, and plasma was frozen at −80°C. An analysis of the samples was performed within six months.
The study was approved by local Institutional Ethical Committee (approval No 26/03), and all patients have given their informed consent prior to entering the study. The study is registered as a clinical trial in The Cochrane Renal Group Registry (http://www.cochrane-renal.org/dbsearch.php), and its ID number is CRG110500021.
Laboratory Methods
PAPP-A Assay
PAPP-A was measured by TRACE (Time Resolved Amplified Cryptate Emission) technology based on non-radiating energy transfer. A commercial kit for PAPP-A determination (BRAHMS GmbH, Berlin, Germany) contains two different monoclonal antibodies—one conjugated with europium cryptate and the other with fluorescent agent XL 665. The antigen (PAPP-A) present in serum samples is sandwiched between two conjugates. The fluorescent signal measured during the formation of the antigen-antibody complex by the KRYPTOR analyser (BRAHMS GmbH, Berlin, Germany) is proportional to the antigen concentration.
Other Parameters
MMP-2, MMP-9, and IGFBP-4 were assessed with standard ELISA (enzyme linked immunosorbent assay) kits (RD Systems, Minneapolis, Minnesota, USA) according to the manufacturer's protocol. IGF-I was measured with standard immunoradiometric assay (IRMA, Immunotech, Marseille, France). Routine biochemical parameters were determined with standard clinical-chemistry methods recommended by IFCC (International Federation of Clinical Chemistry) with automated analyzers.
Statistical Analysis
Results are expressed as medians and interquartile ranges. All parameters were evaluated as measured and additionally corrected for the albumin content due to hemoconcentration. IGF-I and IGFBP-4 were judged per se and also together, as IGFBP-4 binds IGF-I and the degradation of IGFBP-4 increases the IGF-I bioavailability. Due to their inverse relationship we have calculated the IGFBP-4 × IGF-I product.
Two factors ANOVA (analysis of variance) was used to determine the influence of the type of procedure and the effect of time. ANOVA repeated measures with post-tests (Tukey-Kramer test) for normally distributed data and Friedman ANOVA with post-tests (Dunn´s multiple comparison test) for non-normally distributed data were used for analyzing the effect of time. Mann-Whitney test and unpaired t-test were used for analyzing the effect of the procedure. Results were considered as statistically significant at p < 0.05.
RESULTS
Results are shown in . All measured values were corrected for the albumin content due to hemoconcentration; the corrected results are shown in .
Table 1 PAPP-A and related parameters during hemodiafiltration and hemodialysis
Table 2 PAPP-A and related parameters during hemodiafiltration and hemodialysis. Correction for albumin content due to hemoconcentration
PAPP-A changes significantly during both HDF and HD with no significant differences between these two procedures. It increases more than two-fold from 0 to 15 min of the session (p < 0.001) and then decreases until the end of the session (p < 0.001).
Other studied molecules showed only moderate changes in both sessions. MMP-2 decreased slightly during both procedures (p < 0.001), again, without significant differences between HDF and HD. On the other hand, MMP-9 decreased slightly only during HD; however, the difference between HDF and HD was not statistically significant.
IGFBP-4 levels remained stable during both procedures but differed significantly between HDF and HD. This difference was given by different levels before the sessions (lower levels during HDF). Changes of IGF-I were minimal as well; however, a significantly higher decrease of this molecule was observed during HDF after correction for the albumin content. Higher levels of IGF-I were measured during HDF, but the difference compared to HD was not statistically significant. The values IGFBP-4 × IGF-I product were nearly identical both before HDF and HD and followed similar trends during both procedures (see ).
Figure 1 Changes of IGFBP-4 × IGF-1 product during hemodiafiltration and hemodialysis. Abbreviations: HD = hemodialysis, HDF = hemodiafiltration, IGF-1 = insulin-like growth factor-1, IGFBP-4 = insulin-like growth factor binding protein-4.
Taken together, a significant effect of both HDF and HD was demonstrated on pregnancy-associated plasma protein A in the initial phase of the extracorporeal procedure and describe only moderate changes of related molecules during both procedures.
DISCUSSION
In this study, PAPP-A was demonstrated as a marker of cardiovascular risk increases at the beginning of both HDF and HD with polysulfone membranes and then decreases without significant differences between these two procedures. Changes of molecules related to PAPP-A (i.e., IGFBP-4 and IGF-I) both linked to PAPP-A action, and MMP-2 and 9 as other zinc-dependent metalloproteinases are much less pronounced.
It was shown that PAPP-A increases during dialysis independently of the material of the dialysis membrane—polysulfone membranes in the present study, as well as diacetate cellulosic and polyamide membranes in a previous oneCitation[15]—and this increase can be observed as early as within 15 minutes of HD. Other factors than membrane biocompatibility are probably responsible for the increase, such as a release of PAPP-A from complexes or storage, or possibly a puncture of the fistula. Because the increase is observed after heparin application, the possible effect of heparin and its role in redistribution of PAPP-A should be taken into consideration.Citation[17] A slight increase of PAPP-A was also shown one hour after the start of HD with polysulfone membranes;Citation[16] however, these levels were lower than in the 15th min in the present study, suggesting that subsequently PAPP-A begins to decrease rapidly. The fate of PAPP-A in circulation in non-pregnant individuals still remains unclear. In pregnant women, PAPP-A exists in a heterotetrameric complex with eosinophilic major basic protein,Citation[18] and this complex is very stable.Citation[19] In patients with unstable plaque, PAPP-A was cleared rather quickly by some unknown mechanism;Citation[20] this is probably also the case in dialysis patients. These results show a relatively short half-life of PAPP-A, at least during extracorporeal elimination. As the convective and/or diffusive removal of this molecule due to its large molecular weight (400 kDa) is not suspected, other factors must be involved. Metabolic pathways (i.e., the degradation into several fragments by proteinases and the formation of complexes with other proteins or redistribution to tissues, possibly binding to heparan sulfate) in its place seem to be of particular importance. The destruction due to the interaction with the dialysis membrane and adsorption to the membrane should be taken into consideration as well. Types of the procedure and predominating transport mechanisms (convective transport during HDF and diffusive one during HD) do not differ in affecting the levels of PAPP-A, as shown in this single-session study. However, it should be further investigated whether this is true also for long-term usage of HDF, as it has a beneficial effect on cardiovascular risk profiles (i.e., inflammation, oxidative stress, and lipid profiles, as recently demonstratedCitation[21]), and PAPP-A is associated both with inflammatory and oxidative stress parameters.Citation[5],Citation[6]
Although IGFBP-4 and IGF-I are closely related to PAPP-A, their changes during both procedures (per se as well as evaluated simultaneously) were only moderate and did not reflect the changes of PAPP-A. The significant increase of PAPP-A was only transient and probably did not affect these parameters. IGF-I with a low molecular weight (7.5 kDa) and small IGFBPs were expected to clear through the dialyzers; on the contrary, and in accordance with a study in 5 patients performed with high permeability polyacrylonitrile AN-69 membranes,Citation[22] that was not the case. Their levels were very stable both during HDF and HD, perhaps stabilized by some unknown biological mechanisms, and a more pronounced decrease of IGF-I was observed only at the end of HDF after correction for the albumin content.
Unlike PAPP-A, changes of other metalloproteinases were only moderate. Ebihara et al.Citation[23] described higher MMP-9 mRNA in HD patients, and its levels were similar at the start and end of HD. In this study, a slight decrease of MMP-9 is shown during HD but not HDF, which could be related to transport mechanisms and adhesion to the membrane; however, a chance finding cannot be fully excluded. A significant reduction of MMP-9 can be achieved by LDL apheresis, as shown in diabetic dialysis patients where MMP-9 was associated with arteriosclerosis obliterans.Citation[24] On the contrary, MMP-2, also related to cardiovascular risk, decreased during both procedures, with neither of them having any advantage.
One can conclude that a single HDF session compared to HD has no advantage in the decrease of PAPP-A and other tested molecules, all of them related to cardiovascular risk. Studies aimed at the long-term effect of both procedures on these parameters would be needed to further evaluate these therapeutical strategies.
ACKNOWLEDGMENT
The study was supported by grant IGA MH CZ NR/8094-3. The authors are thankful to Dr. J. Soukupová for technical assistance and to Ing. Míková for statistical consultations.
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