follistatin system during hemodialysis: Role of heparin

Kidney International, Vol. 64 (2003), pp. 2229–2237

DIALYSIS – TRANSPLANTATION

Activation of hepatocyte growth factor/activin A/follistatin system during hemodialysis: Role of heparin JACEK BORAWSKI, BEATA NAUMNIK, and MICHAL MYS´ LIWIEC Department of Nephrology and Internal Medicine, Medical University, Bialystok, Poland

Activation of hepatocyte growth factor/activin A/follistatin system during hemodialysis: Role of heparin. Background. Hepatocyte growth factor (HGF), activin A, and follistatin compose an organotrophic system that may be modulated by heparin. We prospectively studied the effects of unfractionated heparin (UFH) versus low-molecular-weight heparin (LMWH) enoxaparin-anticoagulated hemodialysis on plasma levels of the cytokines. Methods. The factors were measured by immunoassays in 25 chronic hemodialysis patients at the start and at 10 and 180 minutes of the hemodialysis procedure anticoagulated with bolus enoxaparin. Then, the patients were randomized to either receive a bolus and infusion of UFH or to continue LMWH, and were reexamined after 12 weeks. Results. Predialysis HGF and follistatin were increased (both P < 0.0001), while activin A was normal in hemodialysis patients. Baseline HGF directly correlated with activin A in hemodialysis subjects (P = 0.004). In healthy controls, it was positively associated with follistatin (P = 0.001). Both HGF and activin A were markedly increased at each interval of enoxaparin-anticoagulated hemodialysis, and follistatin was increased at 10 minutes (all P < 0.0001). The early increments in HGF and follistatin directly depended on the dose of enoxaparin (both P < 0.030). Remarkably, the rise in activin A was inversely associated with the predialysis level of the cytokine (P < 0.0001). The actions of UFH resembled those of LMWH, although the releasing effects on the growth factors were not dose-dependent. The switch from LMWH to UFH resulted in a significant increase in over-dialysis HGF, a fall in follistatin, and no change in activin A. Conclusion. HGF/activin A/follistatin system is activated and disturbed in chronic hemodialysis patients, including depletion of tissue stores of activin A. Type and dose of heparin used during hemodialysis procedures profoundly influence this pleiotropic system, and may thus modulate vital body functions and course of critical diseases.

Hepatocyte growth factor (HGF) is an intrinsic repair factor with morpho-, mito-, and motogenetic as well as Key words: activin A, enoxaparin, follistatin, hemodialysis, heparin, hepatocyte growth factor. Received for publication March 30, 2003 and in revised form June 3, 2003 Accepted for publication July 14, 2003
C

2003 by the International Society of Nephrology

antiapoptotic effects on almost every cell of the body [1, 2]. The expression of HGF in mesenchymal cells and its c-Met receptor in epithelial cells, including vascular endothelium and erythroid progenitors, is activated by the local tissue damage and, interestingly, by the injury to distant organs. Then, the released cytokine acts in a complex auto-, para-, and, remarkably, endocrine- and concentration-dependent manner [1, 2]. Recent experimental studies [3, 4] showed that some of the regenerative effects of HGF in the kidney and lung were strongly inhibited by activin A, a pluripotent cytokine of the transforming growth factor-b (TGF-b) superfamily [5–9]. Furthermore, follistatin, a principal antagonist of activin A [5–9], reversed the inhibitory effects of activin A on the HGF-induced renal tubulogenesis [3]. These studies indicate that HGF, activin A, and follistatin compose an organotrophic system [3, 4]. Blood HGF levels in patients undergoing maintenance hemodialysis are several-fold higher than in healthy subjects [10–13]. They are directly predictive of incident cardiovascular mortality [12], severity of carotid arteriosclerosis [12] and left ventricular hypertrophy [14], prevalence of cardiovascular disease and viral hepatitis [15], closely associated with activated inflammatory [12, 15], antioxidant [13] and liver-reparative responses [13, 16], and with less anemia [12, 15]. The increase in HGF in chronic hemodialysis patients is thus regarded as an important part of the reparative reaction against the most common disorders. Studies of activin A and follistatin in this specific population are scarce; only low activity and normal blood concentrations of activin A [17] and increased follistatin levels [18] were separately reported so far. Circulating HGF levels strikingly increase during hemodialysis sessions, largely [10, 11] but not exclusively [11] due to heparin administered as a blood anticoagulant. The mechanism may include displacement of HGF from cell-surface and extracellular matrix proteoglycans [19], prolonged lifetime of the circulating HGF/heparin complexes [20, 21], and an enhanced production of the cytokine [22, 23]. Recently, heparin was also reported to remarkably increase blood activin A and follistatin 2229

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levels in patients undergoing cardiovascular procedures, likely by similar mechanisms [24]. It is thus conceivable that heparin is an under-appreciated modulator of the HGF/activin A/follistatin system. These pleiotropic effects of heparin may be of particular importance in patients undergoing maintenance hemodialysis. We aimed to determine, for the first time, the relations between plasma levels of HGF, activin A, and follistatin in chronic hemodialysis patients and healthy subjects, the over-dialysis profiles of the growth factors during procedures employing low-molecular-weight heparin (LMWH) enoxaparin, and to compare the effects of unfractionated heparin (UFH)- and LMWHanticoagulated hemodialysis treatments on plasma HGF/activin A/follistatin system in a prospective controlled study.

METHODS Patients We enrolled 25 patients (15 men, 10 women; mean age, 59.3 ± 13.3 years) who had been undergoing maintenance hemodialysis for a median period of 40.4 months (range 2.0 to 162 months). All the participants had been anticoagulated with LMWH enoxaparin (Clexane
, Bellon Rhone-Poulenc ˆ Rorer, Montrouge, France) during hemodialysis for a minimum of 2 months prior to the study. Primary renal diseases were chronic glomerulonephritis (N = 8), interstitial nephritis (N = 6), polycystic kidney disease (N = 3), hypertensive nephropathy (N = 1), secondary amyloidosis (N = 1), acute renal failure (N = 1), and unknown (N = 5). Exclusion criteria were severe liver disease, malignancy, diabetes, human immunodeficiency virus (HIV) infection, treatment with immunosuppressive, contraceptive, lipid-lowering, antiplatelet or nonsteroidal anti-inflammatory drugs, and any recent acute inflammatory or infectious diseases. The patients were dialyzed for 4 to 5 hours 3 times weekly using the double-needle technique, native arteriovenous fistulas, standard bicarbonate buffer, low-flux modified cellulose dialyzers, and machines with ultrafiltration controllers. Before each hemodialysis session, the extracorporeal circuit was rinsed with 1000 mL of 0.9% saline containing 2.0 IU/mL of UFH (Heparin; Biochemie, Kundl, Austria). Then, LMWH enoxaparin was administered as a single bolus of 40 mg (20 to 60 mg) via the first-access needle. The effective dose of enoxaparin was 0.68 ± 0.20 mg/kg (1 mg = 100 IU of antifactor Xa activity). It was established on the basis of the following clinical guidelines: no visible fibrin clots in the arterial and venous bubble traps during hemodialysis, no clotted filters after hemodialysis, and no bleeding from the fistula puncture sites after compression [25, 26].

Eighteen age- and gender-matched healthy volunteers served as controls for determination of plasma HGF, activin A, and follistatin levels. Study protocol Plasma HGF, activin A, and follistatin levels in 25 patients anticoagulated with enoxaparin were measured predialysis (T0 ) and at 10 minutes (T10 ) and 180 minutes (T180 ) after the beginning of hemodialysis. The subjects were next randomized either to continue enoxaparin (N = 13) or to receive UFH (N = 12). The study was designed to keep the heparin doses, other hemodialysis prescriptions and pharmacologic treatment stable during the follow-up. The study protocol was approved by the Clinical Research and Ethics Committee of the Medical University of Bialystok, and all patients and controls gave their informed consent. UFH was injected as a bolus of 2500 IU (1500 to 3500 IU) (42.1 ± 9.2 IU/kg) into the arterial line just prior to the start of hemodialysis, and followed by a continuous infusion of 3750 IU (2500 to 4000 IU) (57.8 ± 12.4 IU/kg) via a syringe pump. The total dose of UFH per hemodialysis was 6333 ± 1115 IU (99.9 ± 18.9 IU/kg). The infusion was started just after the bolus, and stopped 1 hour before the scheduled end of hemodialysis. The UFH dosage had been individually titrated on the basis of whole blood activated partial thromboplastin time (WBAPTT) and established during the first three sessions after randomization. The goal was to attain an approximately twofold prolongation of WBAPTT at both 30 and 120 minutes of hemodialysis compared with baseline. Twenty-three patients were reexamined after a 3-month interval. In the 12 patients who had been randomized to UFH, the over-dialysis plasma HGF, activin A, and follistatin levels were compared to those obtained when they were anticoagulated with enoxaparin. Eleven out of the 13 subjects maintained on hemodialysis treatment with enoxaparin completed the study. Two patients died out-hospital during the follow-up. Laboratory procedures Fasting blood was drawn into ethylenediaminetetraacetate (EDTA)-coated vacutainers during a midweek morning hemodialysis: at T0 from the access (before heparinization), and at T10 and T180 from the predialyzer port after slowing the blood flow to 100 mL/min for 1 minute. In healthy controls, fasting morning blood was obtained without stasis from the antecubital vein punctured with a 19-gauge needle. Plasma was prepared by centrifugation at 3000g for 20 minutes at room temperature within 30 minutes of collection, aliquoted, and stored at −40◦ C until assays. Plasma HGF, activin A, and follistatin levels were determined using solid-phase enzyme-linked

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immunosorbent assay (ELISA) kits purchased from R&D Systems, Inc., Minneapolis, MN, USA (Quantikine
; cat. no. DHG00, DFN00, and DAC00, respectively). The assays quantify human-specific factors; their mean minimum detectable levels are 0.04 ng/mL, 2.3 pg/mL, and 29 pg/mL, respectively. The diluent provided with the activin A kit dissociates the activin A/follistatin complex in the biologic samples; the assay is thus capable of quantifying total activin A. The follistatin assay detects the 288, 300, and 315 amino acid polypeptides. All the measurements were performed in duplicate using a 400 SFC photometer (SLT-Labinstruments, Groding/Salzburg, ¨ Austria) and calibrated using provided recombinant human reference samples and standards. For calculations of the results, a computer and a curve-fitting program were used. The within- and between-assay coefficients of variations were <8%. The T180 values were corrected for hemoconcentration according to plasma protein levels. WBAPTT was measured using an automated coagulation system and reagents from BioMerieux ´ (Marcy-l’Etoile, France). Statistical analysis Shapiro-Wilk W test of normality was used for data distribution analysis. The normally distributed data were presented as mean ± 1 SD, and the skewed data as median (full range). Intergroup comparisons were performed with nonparametric Mann-Whitney U test, Kruskal-Wallis analysis of variance (ANOVA) by ranks and median test, and with chi-squared analysis, when appropriate. Intragroup comparisons were performed with nonparametric Friedman ANOVA by ranks and Wilcoxon tests. Bivariate correlations between continuous variables were assessed using Spearman regression; those between dichotomous and continuous factors by quasi-Newton logistic analysis. All statistical tests were two-sided, and P < 0.05 was considered significant. Analyses were performed with Statistica software (version 6.0, StatSoft, Tulsa, OK, USA). RESULTS HGF/activin A/follistatin in hemodialysis patients and healthy subjects Predialysis plasma HGF and follistatin levels in hemodialysis patients were markedly higher than in healthy controls (Table 1). Plasma activin A did not differ between the groups. In the patients, baseline HGF directly correlated with activin A (r = 0.549, P = 0.004) (Fig. 1A); in the controls, HGF was associated with follistatin (r = 0.699, P = 0.001) (Fig. 1B). Other relations between the markers were non-significant in either group (all P > 0.168). They were also not associated with either gender (all P > 0.473) or age (all P > 0.246). In the

Table 1. Plasma hepatocyte growth factor (HGF), activin A, and follistatin in patients undergoing enoxaparin-anticoagulated hemodialysis and in healthy subjects

HGF ng/mL Activin A pg/mL Follistatin pg/mL

Hemodialysis patients

Healthy subjects

P value

1.84 (0.66–6.20) 143 ± 81.7 3285 ± 1070

0.61 ± 0.17 111 ± 67.1 1323 ± 403

<0.0001 0.242 <0.0001

Blood samples were obtained at the onset of a morning midweek hemodialysis session in the patients (before heparinization), and in a fasting morning state in the controls. Normally and not normally distributed data are presented as mean ± 1 SD and median (range). Two-tailed P by nonparametric Mann-Whitney U test between the groups.

patients, no significant effects of dialysis vintage (all P > 0.098) or cause of renal failure (all P > 0.185) on the markers were found. HGF/activin A/follistatin during LMWH hemodialysis Plasma HGF levels significantly changed over enoxaparin HD (v 2 ANOVA = 50.0, P< 0.0001) (Fig. 2A). They increased by a mean of 1631% to 28.7 ng/mL (15.3 to 154 ng/mL) at T10 , and by a median of 377% to 8.21 ng/mL (2.73 to 98.3 ng/mL) at T180 compared with T0 (both P < 0.0001). HGF levels were lower at T180 than at T10 (P < 0.0001). Plasma activin A levels followed the same pattern (v 2 ANOVA = 41.2, P< 0.0001) (Fig. 2B). At T10 , they increased by a median of 139% to 357 ± 128 pg/mL. At T180 , activin A levels were 203 ± 67.4 pg/mL, lower than at T10 (P < 0.0001), but still elevated by a median of 36% compared with the T0 values (all P < 0.0001). Plasma follistatin levels also significantly changed over hemodialysis (v 2 ANOVA = 39.1, P < 0.0001) (Fig. 2C). At T10 , they increased by a median of 53% to 5054 ± 1733 pg/mL (P < 0.0001); at T180 , follistatin concentrations were 3211 ± 853 pg/mL and not different from those at T0 (P = 0.536). The percentage increments in HGF (r = 0.496, P = 0.012) (Fig. 3A) and follistatin (r = 0.436, P = 0.029) (Fig. 3B) at T10 directly correlated with the dose of enoxaparin, and with one another (r = 0.500, P = 0.011) (Fig. 3C). In case of HGF, the dose dependency was still evident at T180 (r = 0.401, P = 0.047). The increases in activin A at both T10 (r = −0.702, P < 0.0001) (Fig. 3D) and T180 (r = −0.816, P < 0.0001) strongly and inversely correlated with the T0 levels. Effects of a change from LMWH to UFH As shown in Table 2, plasma HGF levels in 12 patients switched to UFH increased by 48% at T0 , by 23% at T10 , and by 45% at T180 compared with the prerandomization values (Fig. 4A). In contrast, over-dialysis plasma activin A levels did not change (Fig. 4B). Follistatin decreased by 46% at T0 , by 30% at T10 , and by 36% at T180

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B

A

0.90 Hepatocyte growth factor, ng/mL

HD patients 4.5

r = 0.549 P = 0.004 3.0

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Healthy subjects

r = 0.699 P = 0.001

0.45

0.30 0

100 200 300 Activin A, pg/mL

160 120 80 40 0 0 10 180 HD time, minutes

600

1200 1800 2400 Follistatin, pg/mL

B

A Activin A, pg/mL

Hepatocyte growth factor, ng/mL

0.75

600 450 300 150

Fig. 1. Relationships between plasma hepatocyte growth factor (HGF) and activin A or follistatin levels in hemodialysis patients (HD) (A) and healthy subjects (B). Blood was obtained predialysis from the arteriovenous fistula in the patients, and by venepuncture in the controls. The correlations remained significant after exclusion of the outliers: two patients with highest baseline HGF (A) r = 0.437, P = 0.037, and one healthy subject with follistatin of 2519 pg/mL (B) r = 0.643, P = 0.005.

C Follistatin, pg/mL

Hepatocyte growth factor, ng/mL

6.0

8000 6000 4000 2000

0

0 10 180 HD time, minutes

(Fig. 4C). The increase in HGF at T0 did not correlate with the respective fall in follistatin (P = 0.276). Plasma follistatin levels at T0 in the UFH-anticoagulated patients were still higher than in healthy controls (P = 0.022). In the 11 patients who completed the LMWH arm of the study, the cytokines remained unchanged (Table 2). Since baseline HGF levels in these subjects were marginally higher at randomization compared to those in the 12 patients assigned to UFH (Table 2), additional analyses were performed. After adjustment for the above baseline HGF values, the percentage increments in overdialysis HGF in the patients switched to UFH remained statistically different compared with the unchanged cytokine in the LMWH subjects (all P < 0.05). The groups did not differ with regard to baseline concentrations of the other factors studied, demographic, clinical, or laboratory characteristics (data not shown). No bleeding, thrombotic, or infectious complications occurred

0 10 180 HD time, minutes

Fig. 2. Case profiles of plasma hepatocyte growth factor (HGF) (A), activin A (B), and follistatin (C) levels during enoxaparinanticoagulated hemodialysis (HD). Blood was obtained at the start (before enoxaparin administration) and at 10 and 180 minutes of the hemodialysis session. The cytokines were markedly increased over-dialysis (Wilcoxon P < 0.0001) except of plasma follistatin, which returned to baseline at 180 minutes.

during the follow-up. Predialysis leukocytes, hematocrit, platelets, C-reactive protein, fibrinogen, albumin, liver enzymes, delivered dialysis dose, ultrafiltration, and dry body weight did not change in either group (data not shown). None of the patients required adjustment of the heparin doses, hemodialysis prescriptions or pharmacologic treatment. HGF/activin A/follistatin during UFH hemodialysis The over-dialysis response profiles of the growth factors under UFH were qualitatively similar to those under LMWH (Table 2). Plasma HGF levels increased by a mean of 1182% at T10 , and by a mean of 515% at T180 ; activin A by a median of 168% and 70%, respectively; follistatin by 54% at T10 (all P< 0.0001). The percentage increase in HGF at T10 under UFH was lower than the previous one under enoxaparin in the 11 respective

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B

A

C

250

250

200

200

2400

r = 0.496 P = 0.012 1800

r = 0.436 P = 0.029

150 100

150 100

r = 0.500 P = 0.011 Enoxaparin

1200 50 600

50

0 0.3

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600 1200 1800 2400 3000 ∆ Hepatocyte growth factor, %

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∆ Follistatin, %

∆ Follistatin, %

∆ Hepatocyte growth factor, %

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Predialysis activin A, pg/mL

r = −0.937 P < 0.0001

Unfractionated heparin

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Predialysis activin A, pg/mL

Fig. 3. Variables influencing intradialytic hepatocyte growth factor (HGF), activin A, and follistatin release. The  symbol denotes the change in plasma cytokine levels at 10 minutes of hemodialysis compared with baseline (T10 to T0 ). The associations were significant even after deleting the single outlier with maximal  HGF (A) r = 0.430, P = 0.036 and  follistatin (B) r = 0.393, P = 0.046 and (C) r = 0.442, P = 0.031 or two patients with  activin A above 1174% (D) r = −0.617, P = 0.002.

subjects (1182% vs. 1636%, P = 0.028). The other over-dialytic changes were not significantly different vs. prerandomization. By contrast to enoxaparin, the percentage increments in the markers did not correlate with either total, bolus or infusion dose of UFH (all P > 0.113). As for LMWH, the percentage increase in activin A both at T10 (r = −0.937, P < 0.0001) (Fig. 3E) and T180 (r = −0.853, P = 0.0004) inversely correlated with the T0 levels. DISCUSSION The novel findings of this study are that plasma HGF, activin A, and follistatin compose a system which is remarkably altered in maintenance hemodialysis patients compared with healthy subjects, the growth factors are repeatedly activated during hemodialysis procedures lead-

ing to depletion of heparin-releasable stores of activin A, and that the type and dose of heparin used during hemodialysis profoundly influence this pleiotropic system. Our results are consistent with the previous reports of increased blood HGF [10–13] and follistatin [18], and normal activin A levels [17] in chronic hemodialysis patients. The associations between the factors were, however, studied for the first time. We found that plasma HGF was directly correlated with activin A in hemodialysis patients and, by contrast, with follistatin in healthy controls. The reason for this discrepancy is not clear. Hypothetically, it may be due to the nature of an inflammatory response where activin A is one of the earliest and strongest but short-lived reactants, while follistatin increases slowly over hours and inhibits activin A through a short-loop feedback mechanism [7].

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Table 2. Over-dialysis plasma hepatocyte growth factor (HGF), activin A, and follistatin levels in patients anticoagulated with enoxaparin or unfractionated heparin during hemodialysis procedures Before randomization 0 minutes HGF ng/mL Activin A pg/mL Follistatin pg/mL

1.65 (0.66–5.68) 122 ± 55.9 3117 ± 1170

HGF ng/mL Activin A pg/mL Follistatin pg/mL

2.02 (1.10–6.20) 154 ± 98.0 3494 ± 1074

10 minutes

After 12 weeks 180 minutes

Enoxaparin (N = 12) 23.4 (15.4–75.0) 8.27 (3.11–17.0) 331 ± 110 213 ± 70.1 4342 (2885–8557) 2531 (2114–4247) Enoxaparin (N = 11) 29.6 (15.5–154) 7.83 (2.73–98.3) 313 ± 134 167 ± 65.1 5018 ± 1781 3068 (2441–5009)

0 minutes

10 minutes

180 minutes

Unfractionated heparin (N = 12) 2.42 (0.95–13.0)a 28.7 (13.2–85.0)c 13.8 (8.41–36.4)a 103 ± 48.7 300 ± 107 223 ± 67.6 1677 (1031–8869)c 3038 (1533–13475)c 1616 (635–4124)b Enoxaparin (N = 11) 2.22 (0.87–5.82) 30.8 (12.9–147) 9.09 (3.89–86.3) 136 ± 58.0 287 ± 114 187 ± 66.6 3121 (1028–8669) 5128 (2118–12123) 3261 ± 1004

Data are shown as mean ± 1 SD and median (range) depending on their normal or skewed distribution, respectively. Differences by Wilcoxon signed-rank test between patients switched from enoxaparin to unfractionated heparin. Mean ± 1 SD value of baseline HGF was 1.85 ± 1.29 ng/mL in patients randomized to unfractionated heparin (UFH), and 2.49 ± 1.42 ng/mL in patients assigned to UFH; two-tailed P by Mann-Whitney U test = 0.061. a < P 0.0005; b P < 0.005; c P < 0.05.

Moreover, intermittent hemodialysis patients are not only exposed three times a week to an acute inflammatory stimulus but also to a high dose of heparin. As shown in the current study, this repeatedly administered heparin profoundly activates and disarranges plasmatic HGF/activin A/follistatin system in hemodialysis patients. To our knowledge, the magnitude of the increments in the growth factors during LMWH enoxaparinanticoagulated hemodialysis sessions cannot be compared to any other moieties studied in this clinical setting so far. The early increase in circulating HGF was, in fact, comparable to that in healthy volunteers in response to intravenous LMWH [27, 28]. In hemodialysis patients, plasma HGF levels were also found to increase more than 20-fold after 15 minutes of the session [11, 16], and remain elevated postdialysis [10, 11, 16] and even 24 hours later [16]. Unfortunately, the type and dose of used heparin were either not specified [11, 16] or the regimen consisted of a fixed bolus dose of UFH [10]. Although the over-dialytic increase in circulating HGF may be partially due to activation of mononuclear cells and fibroblasts [11], this and previous studies [10, 11, 15] prove the causal role of heparin. Namely, the extent of the over-dialytic HGF release in our patients was directly associated with the dose of LMWH enoxaparin; in another report, it was much more pronounced during a standard than heparin-free hemodialysis [11]; in healthy subjects, the increase in HGF was directly associated with the plasma antifactor Xa activity as a surrogate of heparin concentration [27]. Explanation for these effects is derived from experimental studies where heparin was proved to bind and displace HGF from heparan sulfate proteoglycans on the cell surface and the extracellular matrix [19], form a complex with the cytokine, and thus prolong its lifetime in the circulation [20, 21], and enhance HGF production by influencing posttranscriptional mechanisms [22, 23]. Notably, this heparin-released circulating HGF was highly bioactive

[11, 27], and its specific pharmacokinetics was maintained even during prolonged heparin treatment [27]. The marked increase in circulating activin A and follistatin during heparin-anticoagulated hemodialysis is reported for the first time. The sole related study showed a several-fold rise in the cytokines occurring within minutes after a single intravenous injection of UFH in patients undergoing cardiovascular procedures, and no change in the markers under placebo administration [24]. The mechanisms of the increase in circulating activin A and follistatin by heparin are their displacement from the cell surface proteoglycans [29] and the dissolution of the complex [30]. In confirmation, the extent of the early rise in plasma follistatin in our patients, similarly to that in HGF, was directly dependent on the dose of enoxaparin. By contrast, the increments in activin A were strongly and, unexpectedly, inversely associated with the baseline levels of the cytokine rather than the heparin dose. These negative associations were highly significant during both LMWH- and UFH-anticoagulated hemodialysis procedures. From a pharmacokinetic point of view, such regression pattern indicates exhaustion of the reaction due to depletion of the heparin-releasable pool of a substance. Its validity was recently proved for tissue factor pathway inhibitor, which, similarly to activin A, remains assembled with heparan sulfate glycosaminoglycans. Depletion of this coagulation inhibitor during repeated heparin administration attenuates the anticoagulant effect of the therapy [31]. Thus, our current findings indicate that three times a week administration of enoxaparin or UFH in maintenance hemodialysis patients induces depletion of heparin-releasable stores of activin A. Furthermore, the normal predialysis concentrations of activin A, together with its increased inhibitor follistatin, suggest that the biologic effects of activin A are suppressed in these patients. The consequences of this event are difficult to predict since the systemic effects of activin A are not yet fully understood. Generally, the cytokine inhibits proliferation and differentiation of various cell types, induces

Borawski et al: HGF/activin A/follistatin in hemodialysis

UFH Hepatocyte growth factor, ng/mL

Hepatocyte growth factor, ng/mL

A Enoxaparin 80

60

40

20

0

80

60

40

20

0 0

10

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0

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Activin A, pg/mL

Activin A, pg/mL

600

450

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C

12,000 Follistatin, pg/mL

Follistatin, pg/mL

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9000

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9000

6000

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0 10 180 HD time, minutes

0

0 10 180 HD time, minutes

Fig. 4. Case profiles of over-dialysis plasma hepatocyte growth factor (HGF) (A), activin A (B), and follistatin (C) levels in patients switched from enoxaparin to unfractionated heparin (UFH) anticoagulation. HGF was increased, activin A was unchanged and follistatin was decreased at each time point while on UFH (Table 2). The increments in HGF were significant even after deleting the patient with HGF of 85.0 ng/mL at 10 minutes of the UFH procedure (A). Exclusion of the subject with 10 minutes activin A of 577 pg/mL while on UFH (B) did not change the results.

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their apoptosis, and prevents carcinogenesis [5]. Some of these effects are of particular importance in chronic hemodialysis patients who commonly present with cardiovascular disease, chronic inflammation, viral hepatitis, anemia, impaired immunologic function, and osteodystrophy. Notably, bioactive activin A was detected in the atherosclerotic plaque, inhibited propagation of vascular endothelial cells, stimulated proliferation of smooth muscle cells, and enhanced differentiation of monocytes into macrophages [6]. All these actions may promote atherogenesis and arteriosclerosis. On the other hand, the cytokine was also reported to inhibit foam cell formation [32] and stabilize the atherosclerotic plaque [33]. The fact that activin A antagonizes some regenerative actions of HGF in the kidney and lung [3, 4] suggests, however, that its depletion may augment the protective effects of HGF on the cardiovascular system [2]. The role of activin A in inflammation is not fully elucidated either; it seems to be proinflammatory in acute episodes and anti-inflammatory in a chronic condition [7]. By contrast, induction of apoptosis in hepatocytes by activin A is wellestablished [5, 34]. The liver protection yielded by the simultaneous inhibition of activin A and up-regulation of HGF by heparin is thus more explicit. The depletion of activin A may, however, exacerbate uremic anemia since the cytokine enhances proliferation and maturation of erythroid progenitors [8]. This effect is likely to be counterbalanced by the concomitant enhancement of the erythropoiesis-stimulating HGF [1, 2, 12, 15]. The suppression of activin A may also improve generation and function of B lymphocytes [8]. On the other hand, it may harmfully promote heparin-induced osteoporosis since activin A is a potent bone-healing agent [9]. The issue that activin A depletion may propagate carcinogenesis is worrisome [5], especially in view of HGF being also implicated in this process [1, 2]. Taken together, a novel and attractive hypothesis emerges that heparin used during hemodialysis procedures is not only a mere anticoagulant but also an agent that modulates the course of the most common diseases in this population. The results of our prospective controlled study employing traditional regimens of LMWH and UFH [25, 26] are in line with intravenous UFH producing higher HGF levels than intravenous LMWH in healthy subjects [27, 28]. The difference may be explained by a larger molecular size and degree of sulphation of UFH accounting for its greater binding to both endothelial cells [31] and HGF molecules [19], and by a lower clearance of circulating HGF/UFH complexes compared to those with LMWH [20, 21]. The consequences of HGF up-regulation under UFH anticoagulation may be important because circulating HGF is a principal mediator of the angiogenic [35] and intriguing liver-regenerative effects of heparin [23, 36]. Interestingly, endogenous heparin derived from degranulating mast cells in the course of thrombosis also

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stimulates the release of HGF [37], which may then be used for neoangiogenesis and repair of injured blood vessels. The clinical significance of circulating HGF measured after heparin administration was recently established by the CAPTURE Study [38]. The cytokine was a direct and independent predictor of improved long-term survival in the general population patients with previous acute coronary syndromes, likely as a result of enhanced formation of collateral blood vessels. The results are in contrast to those in chronic hemodialysis patients where a high HGF was a negative predictor of survival [12]. The discrepancy may, however, represent another example of “reverse epidemiology” of cardiovascular risks in the hemodialysis population [39], where an increased HGF reflects the augmented reparative response in the most diseased subjects [12–16]. The present increase in plasma HGF after conversion from LMWH to UFH may be partially counteracted by the fall in plasma follistatin. The reason for this behavior of follistatin is currently not known. These and other under-appreciated effects of UFH and LMWH on numerous pleiotropic heparin-binding growth factors [40] deserve further detailed studies in maintenance hemodialysis patients. The issue is of special significance because these subjects are exposed to heparin for years and often for a lifetime.

CONCLUSION Heparin used for blood anticoagulation during hemodialysis procedures is an important modulator of the newly described system of pluripotent growth factors such as HGF, activin A, and follistatin. Type and dose of this heparin may have a profound impact on vital body functions and progression of critical diseases in chronic hemodialysis patients.

ACKNOWLEDGMENTS This work was funded by the Medical University, Bialystok, Poland (grant 4-54456 for Dr. Borawski) and the Polish National Research Council (grant 6P05B 06420 for Dr. Naumnik). The authors were not financially supported by the manufacturer of either heparin. Reprint requests to Dr. Jacek Borawski, Department of Nephrology and Internal Medicine, Medical University, 14 Zurawia St., 15-540 Bialystok, Poland. E-mail: [email protected]

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