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Short-term growth hormone treatment and microcirculation: Effects in patients with chronic kidney disease
Microvascular Research 78 (2009) 246–252
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Microvascular Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y m v r e
Regular Article
Short-term growth hormone treatment and microcirculation: Effects in patients with chronic kidney disease Richard Nissel a,1, Dagmar-Christiane Fischer a,⁎,1, Andreas Puhlmann a, Birgit Holdt-Lehmann b, Andrea Mitzner c, Michael Petzsch d, Thomas Körber d, Michael Tieβ e, Reinhard Schmidt c, Dieter Haffner a a
Department of Pediatrics, University Children's Hospital, Rembrandtstrasse 16/17, 18057 Rostock, Germany Institute of Clinical Chemistry and Laboratory Medicine, University of Rostock, Germany Department of Nephrology, University of Rostock, Rostock, Germany d Department of Cardiology, University of Rostock, Rostock, Germany e Health Care Center, Dialysis Association North, Rostock, Germany b c
a r t i c l e
i n f o
Article history: Received 27 January 2009 Revised 21 April 2009 Accepted 26 May 2009 Available online 3 June 2009 Keywords: Endothelial dysfunction Chronic kidney disease Growth hormone treatment Nailfold capillaroscopy Microcirculation Venous occlusion plethysmography Cardiac index
a b s t r a c t Endothelial dysfunction is common in patients with chronic kidney disease (CKD) and contributes significantly to the high long-term cardiovascular morbidity and mortality. The short-term cardiovascular effects of recombinant human growth hormone (rhGH) in CKD patients (stages III–V) and healthy controls (n = 15 each) were explored in a single-center, non-randomized pilot study. Subjects were investigated before, after a 7 day treatment with rhGH, and after a 7 day wash-out period. Microcirculation was assessed by nailfold capillaroscopy and leg strain gauge plethysmography. Echocardiography was performed and serum concentrations of IGF-I and IGF-binding protein-3 (IGFBP-3) were determined. Before the start of rhGH therapy, mean post-ischemic maximum flow velocity of erythrocytes (VRBC) and leg blood flow (LBF) in CKD patients were significantly reduced to 68% and 75% of that seen in controls, whereas VRBC and LBF under resting conditions were comparable. Treatment with rhGH significantly increased VRBC and LBF under resting conditions. Whereas maximum post-ischemic VRBC was improved by rhGH in patients and controls, maximum post-ischemic LBF increased in controls only. This was paralleled by a non-significant reduction of total vascular resistance, and increased heart rate and cardiac index. In conclusion, CKD patients respond to short-term rhGH treatment with significantly improved capillary blood flow, whereas only minor effects on total peripheral resistance and cardiac output were noted. © 2009 Elsevier Inc. All rights reserved.
Introduction Endothelial dysfunction is an early manifestation of the atherogenic process in patients with chronic kidney disease (CKD) and appears to be a strong predictor of the overall increased cardiovascular mortality in these patients (Briese et al., 2006; Brown et al., 1994; Kruger et al., 2006; London et al., 2002; London et al., 2004). The mechanisms by which uremia promotes cardiovascular complications are poorly understood. Apart from conventional risk factors and metabolic aberrations typically associated with chronic renal failure (e.g. oxidative stress, chronic (micro-) inflammation, and disturbed mineral metabolism), chronic uremia negatively affects endothelial signaling and the physiological activity of endogenous growth hormone (GH) (Briese et al., 2006; Cannata-Andia et al., 2006; Köhler et al., 2005). GH is of major importance for maintenance of cardiovascular and endothelial function in humans and GH deficiency has been associated with increased carotid intima-media thickness (cIMT) and endothelial dysfunction (Lanes
⁎ Corresponding author. Fax: +49 381 494 7044. E-mail address: dagmar-christiane.fi[email protected] (D.-C. Fischer). 1 These authors contributed equally to this study and should both be considered as first authors. 0026-2862/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.mvr.2009.05.006
et al., 2005; Thum and Bauersachs, 2006; Thum et al., 2007). In fact, resistance to GH due to a diminished expression of the GH receptor together with an impairment of the respective downstream signal transduction cascades is a hallmark of uremia (Rabkin et al., 2005). As a consequence, secretion and thus physiological activity of insulin-like growth factor I (IGF-I) decreases. This is further aggravated by the concomitant increase in circulating IGF-binding proteins competing with the IGF-I receptor (Mahesh and Kaskel, 2008; Rabkin et al., 2005; Zheng et al., 2005). Furthermore, endothelial cells are endowed with high affinity receptors for IGF-I and IGF-I exerts a vasodilatory action mediated by endothelial NO-synthase/cycloxygenase (eNOS/COX) pathways in conduit arteries and most likely by endothelium-derived hyperpolarizing factors (EDHFs) in microvessels (Bar et al., 1988; Oltman et al., 2000; Thum et al., 2007; Wu et al., 1994). Reduced availability of and/or reduced response to NO/prostacyclin (PGI2) and EDHF of conduit arteries and resistant vessels have been accused as major contributors to the uremia-associated endothelial dysfunction (Köhler et al., 2005; Passauer et al., 2005a,b). Supplementation of GH in GH-deficient patients has been shown to decrease cIMT, to improve endothelial function and the cardiovascular risk profile (Lanes et al., 2005; Twickler et al., 2000). Furthermore, a recent study on 6-months rhGH therapy in CKD patients on maintenance hemodialysis revealed
R. Nissel et al. / Microvascular Research 78 (2009) 246–252
beneficial effects with respect to nutritional state, quality of life and the serum profile of cardiovascular risk markers (Feldt-Rasmussen et al., 2007). We performed a non-randomized pilot study to investigate the effects of short-term rhGH treatment (1 week) with respect to endothelial and cardiac function in non-malnourished CKD patients in comparison to age- and sex-matched controls. Materials and methods Study protocol The study (registered at http://eudract.emea.europa.eu; EudraCT, No.: 2006-000016-25) was designed as a single-center, nonrandomized pilot study (patients and healthy controls) and gained approval from the Ethics Committee of the University of Rostock. All patients and controls gave written informed consent.
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Clinical examinations Patients and controls were examined before onset of rhGH treatment (t0), after 7 days of rhGH treatment (t1), and after a 7 day wash-out period (t2). At these time points, investigation of serum marker indicative for GH activity was paralleled by assessment of endothelial and cardiovascular function. In particular, nailfold video-microscopy, venous occlusion plethysmography, measurement of carotid intimamedia thickness (cIMT), and echocardiography were performed. At each time point, study participants were examined after an overnight fasting period (at least 8 h) in the morning and an interval of at least 24 h after the last dialysis session. Patients took their regular morning medication regardless of clinical examinations but were not allowed to smoke before finalization of the individual investigations. At t1, examinations were done within 12 to 15 h after the last rhGH dosing. Vascular properties
Patients and controls Inclusion criteria: adult CKD patients on conservative treatment with a glomerular filtration rate (GFR) b 60 ml/min/1.73m2 (CKD stage III/IV), or undergoing hemodialysis treatment (CKD stage V) were eligible for this study. Exclusion criteria: a history of diabetes mellitus, familial hyperlipidemia, malignant tumors or alcohol abuse as well as pregnancy or signs of lower extremity varicose veins were exclusion criteria. A total of 15 patients (CKD stage III/IV: n = 6; CKD stage V: n = 9; mean time on dialysis: 3.42 ± 2.54 years; 95% CI: 1.46–5.37; mean GFR in patients on conservative treatment: 16.2 ± 6.0 ml/min/1.73m2, 95% CI: 10.4–22.2) were enrolled and the baseline clinical characteristics are given in Table 1. The primary renal diseases were chronic glomerulonephritis (n = 4), autosomal-dominant polycystic kidney disease (n = 3), interstitial nephritis (n = 2), and juvenile nephronophthisis (n = 2). In the remaining four patients renal disease was due to IgA nephropathy, IgM nephropathy, renal artery stenosis and obstructive uropathy, respectively. To reduce variability within the population, 15 healthy control persons were selected from over 80 volunteers by matching them pairwise for sex, age, smoking habits and usage of oral contraceptives in female patients (Table 1). Study medication Patients and healthy controls received rhGH (Genotropin(, Pfizer, Berlin, Germany) for 7 days. The drug was administered every evening in a dosage of 1.33 mg/m2 body surface area (approximately 30 μg/kg) by subcutaneous injection. Concomitant medications are summarized in Table 1 and were not changed during the study period.
Intravital video capillary microscopy was used to examine blood flow in nailfold capillaries. A light microscope (reflectance mode; total magnification 80×) equipped with a green cold light source and a video imaging unit was employed essentially as described (Jung et al., 2001; Schumann et al., 1996). In brief, erythrocyte velocity was measured after the patient has had adapted to room temperature (22 °C) and the skin temperature was at least 27.4 °C. The arm selected for investigations was positioned at heart level and gently immobilized using a vacuum pillow. The fourth finger of the right hand (left hand, when right arm was the one with the hemodialysis fistula) was fixated with the nailfold area under the microscope. For analysis of the registered images (video camera: Philips CCP VCM 6250; recorder: Panasonic AG7350) the CapImage software package (Version 6.01; Zeintl, Heidelberg, Germany) was used. Per capillary, erythrocyte velocity was measured at least 10 times per minute to compensate for the vasomotion related rhythmic fluctuations and the average of these measurements represents the erythrocyte velocity (VRBC) under resting conditions. A pneumatic occlusion cuff was placed around the upper arm and arterial occlusion was achieved by inflating the cuff to 300 mm Hg for a period of 3 min. During post-ischemic reactive hyperemia erythrocyte velocity was recorded until deceleration to pre-ischemic velocity occurred and the maximum erythrocyte velocity after cessation of ischemia was determined. Representative examples for VRBC as a function of time under resting conditions and after ischemia are given in Fig. 1. Capillary density was determined essentially as described (Gasser and Bühler, 1992). Capillary microscopy was performed by the same trained investigator (BHL) and within the same temperaturecontrolled room throughout the study. The intra-individual coefficient of variation was ≤20% for determination of VRBC.
Table 1 Baseline clinical characteristics of CKD patients and healthy controls.
Gender Age (range, 95% CI) Body mass index Serum albumin Smoking Blood pressure systolic diastolic Medication Contraceptives Antihypertensive drugsa Lipid lowering drugsb Folic acid 25-hydroxyvitamin D Calcitriol
CKD patients
Controls
(Male/Female) [years] [kg/m2] [g/l] (yes/no) [pack years] (mm Hg) (mm Hg)
9/6 43.3 ± 17.6 (range: 18–69; 95% CI: 33.6–53.1) 24.6 ± 4.3 (CI: 22.2–27.0) 36.8 ± 4.5 (CI: 34.3–39.3) 4/11 1.8 ± 3.6 (CI: 0.1–4.1) 128 ± 17 (CI: 118–137) 76 ± 14 (CI: 69–84)
9/6 40.2 ± 12.7 (range: 21–69; 95% CI: 33.2–47.2) 24.5 ± 3.2 (CI: 22.7–26.3) 39.0 ± 3.5 (CI: 37.0–40.9) 4/11 2.7 ± 6.7 (CI: 0.1–6.9) 121 ± 13 (CI: 114–128) 75 ± 9 (CI: 70–80)
(yes/no) (yes/no) (yes/no) (yes/no) (yes/no) (yes/no)
1/5 11/4 6/9 3/12 0/15 13/2
2/4 0/15 0/15 0/15 0/15 0/15
Data are presented as mean ± SD, 95% CI are given in brackets. a Calcium channel antagonists, β-adrenoreceptor antagonists, α-adrenoreceptor antagonists, ACE inhibitors, and diuretics. b Fluvastatin in three, simvastatin in two and lovastatin in one patient.
p-value 0.5795 0.9456 0.1512 1.0000 0.6720 0.2383 0.7807 1.0000 b 0.0001 0.0084 0.2235 1.0000 b 0.0001
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Serum concentrations of asymmetric dimethyl-arginine (ADMA) were determined with an enzyme immunoassay (DLD, Hamburg, Germany). Determination of nitrate and nitrite as surrogate markers for NO was done with a modified Griess procedure (Nitric oxide assay kit, Assay Designs, Ann Arbor, USA) after removal of proteins by means of ultrafiltration through a 10,000 MWCO filter (VectaSpin Micro, Whatman, Dassel, Germany). Assays were performed essentially as described by the manufacturer and all samples were assayed in duplicate. Dynamic blood viscosity was assessed by use of a Reverse Flow Viscosimeter (Holdt et al., 2005). The coefficients of variation were below 10% for determination of blood viscosity, ADMA and NO, respectively. Statistical analysis Fig. 1. Recording of post-ischemic reactive hyperemia by nailfold capillaroscopy. Erythrocyte velocity measured in a healthy control (●\●) and in a CKD patient (○- -○) under resting conditions and after induction of ischemia is shown. The maximal erythrocyte velocity (Vmax) is indicated.
Venous occlusion plethysmography was performed on both legs with the Compactus device (Gutmann-Medizinelektronik, Germany) as described previously (Briese et al., 2006). Subjects were laying with legs and ankles supported above heart level. A pneumatic occlusion cuff was placed around each upper leg and connected with an automatic inflator (Compactus; Gutmann-Medizinelektronik, Germany). Mercury strain gauges were placed on the largest part of the lower legs distal to the knee. After calibration, venous occlusion was achieved by cuff inflation to 60 mm Hg for the measurement of baseline leg blood flow (LBF, expressed in ml/100 ml/min). After inflating the occlusion cuffs for a period of 5 min to at least 50 mm Hg above the systolic pressure (or at maximum 180 mm Hg), reactive hyperemic blood flow was measured every 10 s for 100 s. The post-ischemic peak flow (PIPF) was defined as the highest flow measured during this period expressed in absolute values (ml/100 ml/min). The total post-ischemic flow during this period (TPIF) was defined as the area under the plethysmography curve (AUC) (Briese et al., 2006). All examinations were done after an acclimatization period (ca. 20 min) within the same temperature-controlled room and by the same trained observer (RN). The intra-individual coefficient of variation was ≤15%. Carotid intima-media-thickness (cIMT) was assessed by highresolution B-mode ultrasound using a EUB-525 Duplex Scanner (Hitachi, Tokyo/Japan) equipped with a 12-MHz linear array transducer and echo-tracking system (Pignoli et al., 1986). Four measurements in each of the two carotid arteries were performed. The mean was calculated and used for further analysis. Blood pressure and heart rate were measured by the standard sphygmomanometric method after the patients has been in the supine position for at least 10 min preceding standard and pulsed Doppler-echocardiographic examinations at time of enrollment and after 7 days of rhGH treatment. Left ventricular end-diastolic diameter, thickness of interventricular septum and left ventricular posterior wall as well as stroke volume and cardiac output were measured (Lanes et al., 2005; Sahn et al., 1978). Left ventricular mass, relative wall thickness, left ventricular mass index for body surface area and total peripheral resistance were calculated according to standard procedures (Ganau et al., 1992).
Results are given as mean± standard deviation if not indicated otherwise. When appropriate, range and/or 95% confidence interval (CI) are shown. Normal distribution of the data was assessed by the Shapiro– Wilks test. The longitudinal changes in cardiovascular and biochemical data were evaluated by repeated measure ANOVA including a withinsubject factor (t0; t1; t2) and, a between-subject factor (patients and controls). Pairwise comparisons between time points were performed using the CONTRAST option of the general linear models procedure in the SAS-software (v 8.2). A Bonferoni–Holm adjustment was made to account for multiple testing. Differences in the distribution of specific attributes (e.g. smoking/non-smoking, use of drugs) in the treatment groups were analyzed by Chi-square Test. A p-valueb 0.05 was considered as statistically significant. Results Baseline characteristics of CKD patients and controls are presented in Table 1. At time of enrollment (t0), patients showed well controlled hypertension, and no signs of malnutrition, i.e. normal BMI and serum albumin levels.
Laboratory methods Blood was obtained prior to plethysmography and was stored on ice until delivered to the laboratory, where preparation of serum and plasma took place. Samples were stored at −20 °C until analysis. GFR was calculated according to the “Modification of Diet in Renal Disease Study Prediction Equation” (MDRD) (Levey et al., 1999). Homocysteine, IGF-I and IGFBP-3 were determined by immunoassay-procedures combined with either fluorescence-induced polarization or chemiluminescence (FPIA, IMx, Abbott, Wiesbaden Germany and Immulite 1000, Siemens, Bad Nauheim, Germany). The molar ratio of IGF-I and IGFBP-3 was calculated essentially as described by Morimoto (Morimoto et al., 2005).
Fig. 2. Effect of rhGH treatment on cutaneous microcirculation in CKD patients and controls. CKD patients and healthy controls were investigated before (t0), after 7 days of rhGH treatment (t1), and after a 7-day wash-out period (t2). At these time points, erythrocyte velocity (VRBC) under resting conditions (A), and post-ischemic maximum erythrocyte velocity (Vmax; B) were measured and data are given as mean ± SEM. Significant differences between patients and controls (⁎) as well as significant changes compared to baseline (#) are indicated.
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6.24); controls, 7.5 ± 0.85 loops/mm (95% CI: 7.00–7.99); p b 0.001). Whereas VRBC under resting conditions was rather similar in patients and controls, the maximum post-ischemic VRBC was only about 68% of that seen in controls (Fig. 2). After 1 week of rhGH therapy (t1) VRBC under resting conditions and post-ischemia was significantly enhanced in CKD patients and controls (each p b 0.05 versus baseline; Fig. 2). At the end of the seven day wash-out period VRBC determined at either condition was still significantly higher in controls compared to pretreatment values (each p b 0.05 t2 versus t0; Fig. 2). By contrast, this was not seen in CKD patients. Capillaroscopy revealed no significant differences between CKD patients on conservative treatment and on hemodialysis (data not shown). Venous occlusion plethysmography At baseline, LBF at rest was comparable in both groups (p = 0.7431; Fig. 3A). Mean post-ischemic peak flow (PIPF) and total post-ischemic flow (TPIF) in CKD patients were approximately 75% of the values determined in controls (patients versus controls, each p b 0.05 Figs. 3B and C). After 1 week of rhGH therapy (t1) patients and controls presented with a significantly enhanced LBF at rest (+34% and +55% in CKD patients and controls, respectively; each p b 0.01 versus baseline). The mean PIPF remained rather constant throughout the study period (Fig. 3B) and only healthy subjects responded to rhGH therapy with a slight, but significant increase in TPIF (Fig. 3C). At the end of the wash-out period (t2), LBF at rest was still higher compared to the pretreatment values, but was already lower than at the end of the treatment period. However, this effect reached significance in controls only. At the same time (t2) CKD patients presented with slightly enhanced values for PIPF and TPIF (Figs. 3B and C). There were no significant differences between CKD patients on conservative treatment compared to those on hemodialysis with respect to LBF during the study period (data not shown). Cardiac function
Fig. 3. Data obtained by venous occlusion plethysmography in CKD patients and controls. CKD patients and healthy controls were investigated before (t0), after 7 days of rhGH treatment (t1), and after a 7-day wash-out period (t2). At these time points, leg blood flow at rest (LBF; A), post-ischemic peak flow (PIPF; B) and total post-ischemic flow (TPIF; calculated as the area under the curve of LBF; C) were measured. Data are given as mean ± SEM. Significant differences between patients and controls (⁎) as well as significant changes compared to baseline (#) are indicated.
Intravital video capillary microscopy Nailfold capillary density was significantly reduced in CKD patients compared to controls (patients, 5.57 ± 1.16 loops/mm (95% CI: 4.90–
CKD patients showed significantly increased interventricular septum diameter, LV wall thickness, LV mass index, systolic blood pressure, and cIMT (each p b 0.05; Table 2). RhGH significantly increased heart rates by a mean of 15% and 13% in patients and controls, respectively (each p b 0.01; Fig. 4A). This resulted in a concomitant increase in cardiac output and cardiac index which reached statistical significance in controls but not in CKD patients (Fig. 4B). The increased cardiac index, together with an unchanged blood pressure, resulted in a slight decrease of systemic vascular resistance (patients, −9%, p = 0.09; controls, −14%, p b 0.05; Fig. 4C). There were no significant differences between CKD patients on conservative treatment compared to those on hemodialysis with respect to cardiac function (data not shown).
Table 2 Doppler-echocardiographic data, blood pressure, and carotid intima-media-thickness (cIMT) in CKD patients and controls at baseline and after 7 days of rhGH treatment. CKD patients LV end-diastolic diameter Interventricular septum LV posterior wall LV mass index Relative wall thickness Single stroke volume Cardiac output Systolic BP Diastolic BP cIMT
[cm] [cm] [cm] [g/m2] [ml] [l/min] [mm Hg] [mm Hg] [mm]
Controls
Baseline
rhGH
Baseline
RhGH
5.07 ± 0.60 (4.74–5.40) 1.18 ± 0.28 (1.02–1.34)⁎ 1.12 ± 0.23 (0.99–1.25)⁎ 128 ± 50 (100–156)⁎ 0.44 ± 0.08 (0.40–0.49) 75.0 ± 13.6 (67.0–82.5) 4.81 ± 0.97 (4.34–5.24) 128 ± 17 (118–137) 76 ± 14 (69–84) 0.80⁎ ± 0.21 (0.68–0.91)
5.01 ± 0.55 (4.66–5.36) 1.23 ± 0.27 (1.06–1.39)⁎ 1.13 ± 0.18 (1.01–1.24)⁎ 117 ± 51 (86–148) 0.45 ± 0.07 (0.41–0.50)⁎
4.97 ± 0.42 (4.74–5.20) 1.00 ± 0.08 (0.95–1.05) 0.98 ± 0.09 (0.93–1.03) 93 ± 13 (86–100) 0.40 ± 0.05 (0.37–0.42) 68.9 ± 16.1 (58.3–76.6) 4.23 ± 0.86 (3.69–4.64) 121 ± 13 (114–128) 75 ± 9 (70–80) 0.67 ± 0.09 (0.63–0.72)
4.96 ± 0.44 (4.72–5.20) 0.99 ± 0.10 (0.94–1.05) 0.97 ± 0.10 (0.91–1.02) 91 ± 15 (83–100) 0.39 ± 0.05 (0.36–0.42) 69.6 ± 13.8 (60.2–76.2) 4.88 ± 1.16 (4.21–5.55)⁎⁎
71.0 ± 13.1 (61.8–83.8) 5.33 ± 1.25 (4.56–6.28) 132 ± 13 (125–139)⁎ 77 ± 10 (71–83) n.d.
CKD = chronic kidney disease; LV = left ventricular; BP = blood pressure; cIMT = carotid intima-media-thickness; n.d. = not done. ⁎ = significant difference patients vs. controls (p b 0.05). ⁎⁎ = significant difference vs. baseline (p b 0.05); data are presented as mean ± SD, 95% CI are given in brackets.
122 ± 11 (115–128) 75 ± 8 (71–80) n.d.
498.9 ± 177.9⁎⁎(400.3–597.4) 7.61 ± 3.78⁎⁎(5.52–9.71) 0.26 ± 0.10⁎⁎(0.21–0.32) 35.5 ± 17.2(27.0–48.5) 0.76 ± 0.25(0.63–0.89) 9.06 ± 2.07(7.9–10.2)
Washout (t2) rhGH (t1)
602.9 ± 254.6⁎⁎(461.9–744.0) 8.46 ± 2.47⁎⁎(7.09–9.83) 0.27 ± 0.16⁎⁎(0.18–0.36) 90.8 ± 44.8⁎(66.0–115.6) 0.98 ± 0.22⁎(0.86–1.10) 20.1 ± 6.2⁎,⁎⁎(16.6–23.7) 195.5 ± 84.1⁎(148.9–242.0) 6.53 ± 1.98⁎(5.43–7.63) 0.11 ± 0.05(0.08–0.14) 107.9 ± 42.5⁎(84.4–129.8) 0.93 ± 0.23(0.80–1.06) 24.9 ± 9.0⁎(19.7–30.2) [μmol/l] [μmol/l] [μmol/l]
[ng/ml] [mg/l]
221.5 ± 110.6(160.3–282.8) 7.47 ± 2.68⁎(5.98–8.95) 0.11 ± 0.04(0.09–0.13) 93.6 ± 47.8⁎(67.1–120.0) 1.05 ± 0.27⁎(0.90–1.20) 25.6 ± 8.9⁎(20.7–30.5)
Controls
Baseline (t0) Baseline (t0)
Washout (t2) rhGH (t1) CKD patients
IGF = Insulin-like growth factor; BP = binding protein; NO = nitric oxide; ADMA = asymmetric dimethyl-arginine. Data are presented as mean ± SD and 95% CI are given in brackets. ⁎ = significant difference patients vs. controls (p b 0.05). ⁎⁎ = significant difference vs. baseline (p b 0.05).
Fig. 4. Cardiac function in CKD patients and controls. Assessment of cardiac function was performed before (t0), and after 7 days of rhGH treatment (t1). Heart rate (A), cardiac index (B) and total peripheral resistance (TPR) (C) are given as mean ± SEM. Significant differences between patients and controls (⁎) as well as significant changes compared to baseline (#) are indicated.
Table 3 Biochemical markers of GH activity and endothelial function in CKD patients and healthy controls before, after 7 days of rhGH treatment, and after a seven day wash-out period.
Before onset of treatment mean serum level of IGF-I was slightly and mean serum level of IGFBP-3 was markedly higher in CKD patients compared to controls (each p b 0.01), whereas the molar ratio of IGF-I/IGFBP-3 was similar in both groups (p = 0.8593; Table 3). At the end of the 7-day treatment period, mean serum concentrations of IGF-I were about three times higher than at time of enrollment. This was accompanied by a less pronounced increase of the IGFBP-3 concentration and approximately doubling of the molar IGF-I/IGFBP-3 ratio (each p b 0.01). During the 7-day treatment period, serum concentration of urea decreased from 18.9 ± 4.3 mmol/l (95% CI: 16.5–21.3) to 14.3 ± 5.6 mmol/l (95% CI: 11.2–17.4) in CKD patients and from 4.2 ± 1.3 mmol/l (95% CI: 3.4–4.9) to 3.1 ± 1.0 mmol/l (95% CI: 2.6–3.7) in controls (each p b 0.05). Serum concentrations of NO, ADMA, and homocysteine were significantly higher in CKD patients compared to controls (each p b 0.02; Table 3). Whereas NO and ADMA levels remained constant throughout the study, homocysteine levels were significantly lowered after rhGH in CKD patients. No significant changes in hematocrit, dynamic blood viscosity, and body weight, or side effects (e.g. fluid retention) were noted in both treatment groups during the study period (data not shown).
130.2 ± 54.7(99.9–160.5) 4.09 ± 0.92(3.58–4.60) 0.12 ± 0.04(0.09–0.14) 41.1 ± 25.3(25.8–52.6) 0.80 ± 0.25(0.67–0.93) 10.4 ± 3.4(8.5–12.2)
Systemic effects of rhGH
170.6 ± 65.2⁎⁎(134.5–206.7) 4.81 ± 1.53⁎⁎(3.97–5.66) 0.13 ± 0.05(0.11–0.16) 41.8 ± 20.7(28.3–52.2) 0.82 ± 0.18(0.68–0.97) 9.84 ± 3.16(8.1–11.6)
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IGF-I IGFBP-3 IGF-I/IGFBP-3 molar ratio NO ADMA Homocysteine
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Discussion Growth hormone has long been known to maintain and to mediate cardiovascular function at the level of blood vessel reactivity and cardiac structure (Cittadini et al., 2002; Farkas et al., 2005; Hana et al., 2002; Irving et al., 2002). A relative GH deficiency is a common feature of chronic uremia and endothelial dysfunction is an early manifestation of the atherogenic process in CKD patients. Furthermore, endothelial dysfunction turned out to be a strong predictor of the overall increased cardiovascular mortality in these patients (Briese et al., 2006; Brown et al., 1994; Cupisti et al., 2000a; Cupisti et al., 2000b; Farkas et al., 2005; Jung et al., 2001; Kruger et al., 2006; London et al., 2002; London et al., 2004; Rossi et al., 2008; ShamimUzzaman et al., 2002; Sigrist and McIntyre, 2008). In the current study, nailfold capillaroscopy and venous occlusion plethysmography were used to evaluate rhGH effects on microcirculation in non-malnourished CKD patients with well controlled hypertension. RhGH increased capillary blood flow under resting conditions as well as during reactive hyperemia in CKD patients and healthy subjects. In addition, a significantly increased leg blood flow was noted in all study participants under resting conditions. PIPF and TPIF showed only small and non-significant changes in CKD patients, whereas in healthy controls, TPIF was significantly increased after 1 week of rhGH treatment. This discrepancy with respect to the results obtained by capillaroscopy and plethysmography is most likely due to the fact that the latter allows no discrimination between skin microcirculation and skeletal blood flow, whereas capillaroscopy measures skin microcirculation only. Interestingly, in both groups an improved endothelial function was noted even 1 week after cessation (i.e. at t2) of treatment, albeit this effect reached statistical significance in healthy subjects only (Figs. 2 and 3). At the same time, the systemic IGF-I levels as well as the IGFBP-3 concentration were still significantly elevated in controls (Table 3). Thus, we have to consider that the rhGH induced stimulation of IGF-I and the related binding proteins lasted longer in controls compared to CKD patients. It remains to be elucidated, whether this is due to an attenuated release of IGF-I from cellular storages after withdrawal of exogenous rhGH or reflects sensibilization of the GH–IGF-I axis secondary to the administration of rhGH. This hypothesis is indirectly supported by the known GH-resistance in CKD patients, which presented 1 week after withdrawal of rhGH with only slightly and non-significantly elevated IGF-I and IGFBP-3 levels (Rabkin et al., 2005). It is still a matter of discussion, whether rhGH directly or via IGF-I mediates endothelial function, but either of both mediators preferentially activate the eNOS/COX pathways leading to release of NO and prostanoids, respectively. Whereas the vasodilatory effect of NO and prostanoids is at maximum in conduit arteries, EDHFs came into play with decreases in vessel diameter being almost solely responsible for vasodilation in capillaries (Bar et al., 1988; Li et al., 2008; Oltman et al., 2000; Thum et al., 2007; Wu et al., 1994). Therefore, our results suggest that rhGH may affect the release of EDHFs and that this effect is predominant under the condition of chronic uremia. In both study groups the seven-day treatment with rhGH had beneficial effects on the cardiac index and total peripheral resistance. All of these findings are most likely related to the rhGH induced enhancement of microcirculation, rather than to functionally relevant cardiac growth and/or change in cardiac structure (Ito et al., 1993; Turner et al., 1988). This point of view is supported by a recent study showing, that a four-week rhGH treatment in healthy subjects was associated with functional but not with structural adaptation of the cardiovascular system (Cittadini et al., 2002). The overall lower impact of rhGH on cardiovascular and endothelial function in CKD patients in the present study is most probably due to the global GH insensitivity in uremia (Mahesh and Kaskel, 2008; Rabkin et al., 2005). Data on short-term cardiovascular effects of rhGH in CKD patients are grossly lacking. Instead, the effects of rhGH therapy on endothelial and/or cardiovascular function have been investigated in healthy
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subjects, in patients with GH deficiency and in patients with congestive heart failure (Capaldo et al., 2001; Farkas et al., 2005; Hana et al., 2002; Irving et al., 2002; Lanes et al., 2005; Møller et al., 1989; Napoli et al., 2003; Oomen et al., 2002; Twickler et al., 2000). Results of these studies indicate that the beneficial effects of rhGH therapy on vascular function are likely to be, at least in part, due to an improved availability of NO. This concept is especially appealing for two reasons. Firstly, chronic uremia has been associated with an impaired generation of NO due to inhibition of synthesis as well as activation of trapping reactions, leading to overall diminished systemic concentrations of NO (Passauer et al., 2005a; Schmidt and Baylis, 2000). Homocysteine and ADMA are thought to impair NOdependent vasodilation, to increase smooth muscle cell proliferation, and to enhance platelet dysfunction (van Guldener et al., 2007). Furthermore, elevated ADMA and homocysteine levels have been associated with atherosclerotic disease, left ventricular dysfunction, and cardiovascular mortality in uremic patients (van Guldener et al., 2007; Zoccali et al., 2002). Secondly, elegant in-vivo and in-vitro experiments point to acute and direct NO-mediated vascular effects of rhGH in healthy subjects (Li et al., 2008; Napoli et al., 2003). Obviously, our data on the serum concentrations of NO and ADMA do not fit into these concepts. Our CKD patients presented with systemic NO concentrations about twice of those seen in healthy subjects, whereas quite similar concentrations of ADMA, a potent inhibitor of eNOS, were determined in both groups. Therefore, we had to consider, that in our patients the NO pathway was already fully activated at time of enrollment. This is in line with a previous report on functional investigation of the NO pathway in uremic patients (Passauer et al., 2000). In this study, evidence was obtained that rather the availability of NO under baseline conditions but its generation on demand is impaired. Since our functional assessment of vascular function indicated parallel (and positive) effects in both cohorts, we expected to see also an increase of the circulating NO concentrations. To our surprise, this was not the case. Whereas for CKD patients the fairly constant levels of NO fit well to the hypothesis of an already fully activated NOgenerating system, the situation is less clear in healthy controls. Beside the technical problems related to the direct quantitative determination of NO the design of our study was quite different from those described recently (Baylis and Vallance, 1998; Li et al., 2008; Napoli et al., 2003). Whereas patients and controls received rhGH as a bolus immediately before functional assessment, we administered rhGH over a period of 7 days and used apparently non-invasive assessment of the endothelial function timely apart from rhGH administration. Thus, it could well be, that we missed the acute and transient effects of rhGH on NObioavailability but detected the overall improvement of capillary endothelial vasodilation. One may speculate, whether the induction of NO is in fact a very rapid and transient effect, probably mediated by direct interaction between rhGH and the GH receptor, whereas the induction of EDHFs occurs later and their biological activity may last longer. Furthermore, it remains to be elucidated whether rhGH mediates EDHFs directly or via IGF-I. Our study has several limitations. The design of the study was open and not randomized and the number of patients was rather low, leaving a possibility for bias. Considering the relatively short time period, the fact that all patients were kept on constant medication during the study, and the complete remission of the vasodilatory effects in CKD patients after the wash-out period, a time-dependent bias seems unlikely. However, whether the positive effects on microcirculation in CKD patients will last during longer treatment periods remains to be elucidated. In conclusion, treatment with rhGH significantly increased microcirculation under resting conditions in CKD patients and healthy subjects. This was paralleled by a significantly increased heart rate, reduction of total vascular resistance, and an increased cardiac index in controls, whereas the latter two findings did not reach statistical significance in CKD patients. If rhGH treatment is able to modulate
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cardiovascular morbidity and mortality in CKD patients must be addressed in carefully designed long-term trials. Disclosure Clinical and biochemical studies, data interpretation and statistical analyses were performed by RN, DCF, BH, AP, and DH. RN and DH received lecture fees, travel fees, and/or grant support from Pfizer. Acknowledgments We thank the patients and healthy volunteers participating in this study for their cooperation and Anja Rahn, Birgit Salewski, and Birgit Jasper for outstanding technical assistance. This study was supported by Pfizer and the FORUN program of the Medical Faculty of Rostock University. References Bar, R.S., et al., 1988. IGF receptors in myocardial capillary endothelium: potential regulation of IGF-I transport to cardiac muscle. Biochem. Biophys. Res. Commun. 152, 93–98. Baylis, C., Vallance, P., 1998. Measurement of nitrite and nitrate levels in plasma and urine—what does this measure tell us about the activity of the endogenous nitric oxide system? Curr. Opin. Nephrol. Hypertens. 7, 59–62. Briese, S., et al., 2006. Arterial and cardiac disease in young adults with childhood-onset end-stage renal disease—impact of calcium and vitamin D therapy. Nephrol. Dial. Transplant. 21, 1906–1914. Brown, J.H., et al., 1994. Comparative mortality from cardiovascular disease in patients with chronic renal failure. Nephrol. Dial. Transplant. 9, 1136–1142. Cannata-Andia, J.B., et al., 2006. Vascular calcifications: pathogenesis, management, and impact on clinical outcomes. J. Am. Soc. Nephrol. 17, S267–273. Capaldo, B., et al., 2001. Abnormal vascular reactivity in growth hormone deficiency. Circulation 103, 520–524. Cittadini, A., et al., 2002. Supraphysiological doses of GH induce rapid changes in cardiac morphology and function. J. Clin. Endocrinol. Metab. 87, 1654–1659. Cupisti, A., et al., 2000a. Responses of the skin microcirculation to acetylcholine and to sodium nitroprusside in chronic uremic patients. Int. J. Clin. Lab. Res. 30, 157–162. Cupisti, A., et al., 2000b. Responses of the skin microcirculation to acetylcholine in patients with essential hypertension and in normotensive patients with chronic renal failure. Nephron 85, 114–119. Farkas, K., et al., 2005. Impairment of skin microvascular reactivity in hypertension and uraemia. Nephrol. Dial. Transplant. 20, 1821–1827. Feldt-Rasmussen, B., et al., 2007. Growth hormone treatment during hemodialysis in a randomized trial improves nutrition, quality of life, and cardiovascular risk. J. Am. Soc. Nephrol. 18, 2161–2171. Ganau, A., et al., 1992. Patterns of left ventricular hypertrophy and geometric remodeling in essential hypertension. J. Am. Coll. Cardiol. 19, 1550–1558. Gasser, P., Bühler, F.R., 1992. Nailfold microcirculation in normotensive and essential hypertensive subjects, as assessed by video-microscopy. J. Hypertens. 10, 83–86. Hana, V., et al., 2002. Reduced microvascular perfusion and reactivity in adult GH deficient patients is restored by GH replacement. Eur. J. Endocrinol. 147, 333–337. Holdt, B., et al., 2005. Comparative evaluation of two newly developed devices for capillary viscometry. Clin. Hemorheol. Microcirc. 33, 379–387. Irving, R.J., et al., 2002. Microvascular correlates of blood pressure, plasma glucose, and insulin resistance in health. Cardiovasc. Res. 53, 271–276. Ito, H., et al., 1993. Time course of functional improvement in stunned myocardium in risk area in patients with reperfused anterior infarction. Circulation 87, 355–362. Jung, F., et al., 2001. Primary cutaneous microangiopathy in heart recipients. Microvasc. Res. 62, 154–163. Köhler, R., et al., 2005. Impaired EDHF-mediated vasodilation and function of endothelial Ca-activated K channels in uremic rats. Kidney Int. 67, 2280–2287. Kruger, A., et al., 2006. Laser Doppler flowmetry detection of endothelial dysfunction in end-stage renal disease patients: correlation with cardiovascular risk. Kidney Int. 70, 157–164. Lanes, R., et al., 2005. Endothelial function, carotid artery intima-media thickness, epicardial adipose tissue, and left ventricular mass and function in growth hormone-deficient adolescents: apparent effects of growth hormone treatment on these parameters. J. Clin. Endocrinol. Metab. 90, 3978–3982.
Levey, A.S., et al., 1999. A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease Study Group. Ann. Intern. Med. 130, 461–470. Li, G., et al., 2008. Growth hormone exerts acute vascular effects independent of systemic or muscle insulin-like growth factor I. J. Clin. Endocrinol. Metab. 93, 1379–1385. London, G.M., et al., 2002. Impairment of arterial function in chronic renal disease: prognostic impact and therapeutic approach. Nephrol. Dial. Transplant. 17 (Suppl. 11), 13–15. London, G.M., et al., 2004. Forearm reactive hyperemia and mortality in end-stage renal disease. Kidney Int. 65, 700–704. Mahesh, S., Kaskel, F., 2008. Growth hormone axis in chronic kidney disease. Pediatr. Nephrol. 23, 41–48. Møller, N., et al., 1989. Effects of growth hormone on insulin sensitivity and forearm metabolism in normal man. Diabetologia 32, 105–110. Morimoto, L.M., et al., 2005. Variation in plasma insulin-like growth factor-1 and insulin-like growth factor binding protein-3: genetic factors. Cancer Epidemiol. Biomark. Prev. 14, 1394–1401. Napoli, R., et al., 2003. Acute effects of growth hormone on vascular function in human subjects. J. Clin. Endocrinol. Metab. 88, 2817–2820. Oltman, C.L., et al., 2000. Mechanism of coronary vasodilation to insulin and insulin-like growth factor I is dependent on vessel size. Am. J. Physiol. Endocrinol. Metab. 279, E176–181. Oomen, P.H., et al., 2002. Reduced capillary permeability and capillary density in the skin of GH-deficient adults: improvement after 12 months GH replacement. Clin. Endocrinol. (Oxf) 56, 519–524. Passauer, J., et al., 2000. Evidence in vivo showing increase of baseline nitric oxide generation and impairment of endothelium-dependent vasodilation in normotensive patients on chronic hemodialysis. J. Am. Soc. Nephrol. 11, 1726–1734. Passauer, J., et al., 2005a. Nitric oxide in chronic renal failure. Kidney Int. 67, 1665–1667. Passauer, J., et al., 2005b. Reduced agonist-induced endothelium-dependent vasodilation in uremia is attributable to an impairment of vascular nitric oxide. J. Am. Soc. Nephrol. 16, 959–965. Pignoli, P., et al., 1986. Intimal plus medial thickness of the arterial wall: a direct measurement with ultrasound imaging. Circulation 74, 1399–1406. Rabkin, R., et al., 2005. Growth hormone resistance in uremia, a role for impaired JAK/ STAT signaling. Pediatr. Nephrol. 20, 313–318. Rossi, M., et al., 2008. Blunted post-ischemic increase of the endothelial skin blood flowmotion component as early sign of endothelial dysfunction in chronic kidney disease patients. Microvasc. Res. 75, 315–322. Sahn, D.J., et al., 1978. Recommendations regarding quantitation in M-mode echocardiography: results of a survey of echocardiographic measurements. Circulation 58, 1072–1083. Schmidt, R.J., Baylis, C., 2000. Total nitric oxide production is low in patients with chronic renal disease. Kidney Int. 58, 1261–1266. Schumann, L., et al., 1996. Microcirculation of the fingernail fold in CAPD patients: preliminary observations. Perit. Dial. Int. 16, 412–416. Shamim-Uzzaman, Q.A., et al., 2002. Altered cutaneous microvascular responses to reactive hyperaemia in coronary artery disease: a comparative study with conduit vessel responses. Clin. Sci. (Lond) 103, 267–273. Sigrist, M.K., McIntyre, C.W., 2008. Vascular calcification is associated with impaired microcirculatory function in chronic haemodialysis patients. Nephron, Clin. Pract. 108, c121–c126. Thum, T., Bauersachs, J., 2006. Growth hormone regulates vascular function—what we know from bench and bedside. Eur. J. Clin. Pharmacol. 62 (Suppl. 13), 29–32. Thum, T., et al., 2007. Growth hormone treatment improves markers of systemic nitric oxide bioavailability via insulin-like growth factor-I. J. Clin. Endocrinol. Metab. 92, 4172–4179. Turner, J.D., et al., 1988. Induction of mRNA for IGF-I and -II during growth hormonestimulated muscle hypertrophy. Am. J. Physiol. 255, E513–517. Twickler, T.B., et al., 2000. Growth hormone (GH) treatment decreases postprandial remnant-like particle cholesterol concentration and improves endothelial function in adult-onset GH deficiency. J. Clin. Endocrinol. Metab. 85, 4683–4689. van Guldener, C., et al., 2007. Homocysteine and asymmetric dimethylarginine (ADMA): biochemically linked but differently related to vascular disease in chronic kidney disease. Clin. Chem. Lab. Med. 45, 1683–1687. Wu, H.Y., et al., 1994. Endothelial-dependent vascular effects of insulin and insulin-like growth factor I in the perfused rat mesenteric artery and aortic ring. Diabetes 43, 1027–1032. Zheng, Z., et al., 2005. Cardiac resistance to growth hormone in uremia. Kidney Int. 67, 858–866. Zoccali, C., et al., 2002. Left ventricular hypertrophy, cardiac remodeling and asymmetric dimethylarginine (ADMA) in hemodialysis patients. Kidney Int. 62, 339–345.
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Microvascular Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y m v r e
Regular Article
Short-term growth hormone treatment and microcirculation: Effects in patients with chronic kidney disease Richard Nissel a,1, Dagmar-Christiane Fischer a,⁎,1, Andreas Puhlmann a, Birgit Holdt-Lehmann b, Andrea Mitzner c, Michael Petzsch d, Thomas Körber d, Michael Tieβ e, Reinhard Schmidt c, Dieter Haffner a a
Department of Pediatrics, University Children's Hospital, Rembrandtstrasse 16/17, 18057 Rostock, Germany Institute of Clinical Chemistry and Laboratory Medicine, University of Rostock, Germany Department of Nephrology, University of Rostock, Rostock, Germany d Department of Cardiology, University of Rostock, Rostock, Germany e Health Care Center, Dialysis Association North, Rostock, Germany b c
a r t i c l e
i n f o
Article history: Received 27 January 2009 Revised 21 April 2009 Accepted 26 May 2009 Available online 3 June 2009 Keywords: Endothelial dysfunction Chronic kidney disease Growth hormone treatment Nailfold capillaroscopy Microcirculation Venous occlusion plethysmography Cardiac index
a b s t r a c t Endothelial dysfunction is common in patients with chronic kidney disease (CKD) and contributes significantly to the high long-term cardiovascular morbidity and mortality. The short-term cardiovascular effects of recombinant human growth hormone (rhGH) in CKD patients (stages III–V) and healthy controls (n = 15 each) were explored in a single-center, non-randomized pilot study. Subjects were investigated before, after a 7 day treatment with rhGH, and after a 7 day wash-out period. Microcirculation was assessed by nailfold capillaroscopy and leg strain gauge plethysmography. Echocardiography was performed and serum concentrations of IGF-I and IGF-binding protein-3 (IGFBP-3) were determined. Before the start of rhGH therapy, mean post-ischemic maximum flow velocity of erythrocytes (VRBC) and leg blood flow (LBF) in CKD patients were significantly reduced to 68% and 75% of that seen in controls, whereas VRBC and LBF under resting conditions were comparable. Treatment with rhGH significantly increased VRBC and LBF under resting conditions. Whereas maximum post-ischemic VRBC was improved by rhGH in patients and controls, maximum post-ischemic LBF increased in controls only. This was paralleled by a non-significant reduction of total vascular resistance, and increased heart rate and cardiac index. In conclusion, CKD patients respond to short-term rhGH treatment with significantly improved capillary blood flow, whereas only minor effects on total peripheral resistance and cardiac output were noted. © 2009 Elsevier Inc. All rights reserved.
Introduction Endothelial dysfunction is an early manifestation of the atherogenic process in patients with chronic kidney disease (CKD) and appears to be a strong predictor of the overall increased cardiovascular mortality in these patients (Briese et al., 2006; Brown et al., 1994; Kruger et al., 2006; London et al., 2002; London et al., 2004). The mechanisms by which uremia promotes cardiovascular complications are poorly understood. Apart from conventional risk factors and metabolic aberrations typically associated with chronic renal failure (e.g. oxidative stress, chronic (micro-) inflammation, and disturbed mineral metabolism), chronic uremia negatively affects endothelial signaling and the physiological activity of endogenous growth hormone (GH) (Briese et al., 2006; Cannata-Andia et al., 2006; Köhler et al., 2005). GH is of major importance for maintenance of cardiovascular and endothelial function in humans and GH deficiency has been associated with increased carotid intima-media thickness (cIMT) and endothelial dysfunction (Lanes
⁎ Corresponding author. Fax: +49 381 494 7044. E-mail address: dagmar-christiane.fi[email protected] (D.-C. Fischer). 1 These authors contributed equally to this study and should both be considered as first authors. 0026-2862/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.mvr.2009.05.006
et al., 2005; Thum and Bauersachs, 2006; Thum et al., 2007). In fact, resistance to GH due to a diminished expression of the GH receptor together with an impairment of the respective downstream signal transduction cascades is a hallmark of uremia (Rabkin et al., 2005). As a consequence, secretion and thus physiological activity of insulin-like growth factor I (IGF-I) decreases. This is further aggravated by the concomitant increase in circulating IGF-binding proteins competing with the IGF-I receptor (Mahesh and Kaskel, 2008; Rabkin et al., 2005; Zheng et al., 2005). Furthermore, endothelial cells are endowed with high affinity receptors for IGF-I and IGF-I exerts a vasodilatory action mediated by endothelial NO-synthase/cycloxygenase (eNOS/COX) pathways in conduit arteries and most likely by endothelium-derived hyperpolarizing factors (EDHFs) in microvessels (Bar et al., 1988; Oltman et al., 2000; Thum et al., 2007; Wu et al., 1994). Reduced availability of and/or reduced response to NO/prostacyclin (PGI2) and EDHF of conduit arteries and resistant vessels have been accused as major contributors to the uremia-associated endothelial dysfunction (Köhler et al., 2005; Passauer et al., 2005a,b). Supplementation of GH in GH-deficient patients has been shown to decrease cIMT, to improve endothelial function and the cardiovascular risk profile (Lanes et al., 2005; Twickler et al., 2000). Furthermore, a recent study on 6-months rhGH therapy in CKD patients on maintenance hemodialysis revealed
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beneficial effects with respect to nutritional state, quality of life and the serum profile of cardiovascular risk markers (Feldt-Rasmussen et al., 2007). We performed a non-randomized pilot study to investigate the effects of short-term rhGH treatment (1 week) with respect to endothelial and cardiac function in non-malnourished CKD patients in comparison to age- and sex-matched controls. Materials and methods Study protocol The study (registered at http://eudract.emea.europa.eu; EudraCT, No.: 2006-000016-25) was designed as a single-center, nonrandomized pilot study (patients and healthy controls) and gained approval from the Ethics Committee of the University of Rostock. All patients and controls gave written informed consent.
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Clinical examinations Patients and controls were examined before onset of rhGH treatment (t0), after 7 days of rhGH treatment (t1), and after a 7 day wash-out period (t2). At these time points, investigation of serum marker indicative for GH activity was paralleled by assessment of endothelial and cardiovascular function. In particular, nailfold video-microscopy, venous occlusion plethysmography, measurement of carotid intimamedia thickness (cIMT), and echocardiography were performed. At each time point, study participants were examined after an overnight fasting period (at least 8 h) in the morning and an interval of at least 24 h after the last dialysis session. Patients took their regular morning medication regardless of clinical examinations but were not allowed to smoke before finalization of the individual investigations. At t1, examinations were done within 12 to 15 h after the last rhGH dosing. Vascular properties
Patients and controls Inclusion criteria: adult CKD patients on conservative treatment with a glomerular filtration rate (GFR) b 60 ml/min/1.73m2 (CKD stage III/IV), or undergoing hemodialysis treatment (CKD stage V) were eligible for this study. Exclusion criteria: a history of diabetes mellitus, familial hyperlipidemia, malignant tumors or alcohol abuse as well as pregnancy or signs of lower extremity varicose veins were exclusion criteria. A total of 15 patients (CKD stage III/IV: n = 6; CKD stage V: n = 9; mean time on dialysis: 3.42 ± 2.54 years; 95% CI: 1.46–5.37; mean GFR in patients on conservative treatment: 16.2 ± 6.0 ml/min/1.73m2, 95% CI: 10.4–22.2) were enrolled and the baseline clinical characteristics are given in Table 1. The primary renal diseases were chronic glomerulonephritis (n = 4), autosomal-dominant polycystic kidney disease (n = 3), interstitial nephritis (n = 2), and juvenile nephronophthisis (n = 2). In the remaining four patients renal disease was due to IgA nephropathy, IgM nephropathy, renal artery stenosis and obstructive uropathy, respectively. To reduce variability within the population, 15 healthy control persons were selected from over 80 volunteers by matching them pairwise for sex, age, smoking habits and usage of oral contraceptives in female patients (Table 1). Study medication Patients and healthy controls received rhGH (Genotropin(, Pfizer, Berlin, Germany) for 7 days. The drug was administered every evening in a dosage of 1.33 mg/m2 body surface area (approximately 30 μg/kg) by subcutaneous injection. Concomitant medications are summarized in Table 1 and were not changed during the study period.
Intravital video capillary microscopy was used to examine blood flow in nailfold capillaries. A light microscope (reflectance mode; total magnification 80×) equipped with a green cold light source and a video imaging unit was employed essentially as described (Jung et al., 2001; Schumann et al., 1996). In brief, erythrocyte velocity was measured after the patient has had adapted to room temperature (22 °C) and the skin temperature was at least 27.4 °C. The arm selected for investigations was positioned at heart level and gently immobilized using a vacuum pillow. The fourth finger of the right hand (left hand, when right arm was the one with the hemodialysis fistula) was fixated with the nailfold area under the microscope. For analysis of the registered images (video camera: Philips CCP VCM 6250; recorder: Panasonic AG7350) the CapImage software package (Version 6.01; Zeintl, Heidelberg, Germany) was used. Per capillary, erythrocyte velocity was measured at least 10 times per minute to compensate for the vasomotion related rhythmic fluctuations and the average of these measurements represents the erythrocyte velocity (VRBC) under resting conditions. A pneumatic occlusion cuff was placed around the upper arm and arterial occlusion was achieved by inflating the cuff to 300 mm Hg for a period of 3 min. During post-ischemic reactive hyperemia erythrocyte velocity was recorded until deceleration to pre-ischemic velocity occurred and the maximum erythrocyte velocity after cessation of ischemia was determined. Representative examples for VRBC as a function of time under resting conditions and after ischemia are given in Fig. 1. Capillary density was determined essentially as described (Gasser and Bühler, 1992). Capillary microscopy was performed by the same trained investigator (BHL) and within the same temperaturecontrolled room throughout the study. The intra-individual coefficient of variation was ≤20% for determination of VRBC.
Table 1 Baseline clinical characteristics of CKD patients and healthy controls.
Gender Age (range, 95% CI) Body mass index Serum albumin Smoking Blood pressure systolic diastolic Medication Contraceptives Antihypertensive drugsa Lipid lowering drugsb Folic acid 25-hydroxyvitamin D Calcitriol
CKD patients
Controls
(Male/Female) [years] [kg/m2] [g/l] (yes/no) [pack years] (mm Hg) (mm Hg)
9/6 43.3 ± 17.6 (range: 18–69; 95% CI: 33.6–53.1) 24.6 ± 4.3 (CI: 22.2–27.0) 36.8 ± 4.5 (CI: 34.3–39.3) 4/11 1.8 ± 3.6 (CI: 0.1–4.1) 128 ± 17 (CI: 118–137) 76 ± 14 (CI: 69–84)
9/6 40.2 ± 12.7 (range: 21–69; 95% CI: 33.2–47.2) 24.5 ± 3.2 (CI: 22.7–26.3) 39.0 ± 3.5 (CI: 37.0–40.9) 4/11 2.7 ± 6.7 (CI: 0.1–6.9) 121 ± 13 (CI: 114–128) 75 ± 9 (CI: 70–80)
(yes/no) (yes/no) (yes/no) (yes/no) (yes/no) (yes/no)
1/5 11/4 6/9 3/12 0/15 13/2
2/4 0/15 0/15 0/15 0/15 0/15
Data are presented as mean ± SD, 95% CI are given in brackets. a Calcium channel antagonists, β-adrenoreceptor antagonists, α-adrenoreceptor antagonists, ACE inhibitors, and diuretics. b Fluvastatin in three, simvastatin in two and lovastatin in one patient.
p-value 0.5795 0.9456 0.1512 1.0000 0.6720 0.2383 0.7807 1.0000 b 0.0001 0.0084 0.2235 1.0000 b 0.0001
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Serum concentrations of asymmetric dimethyl-arginine (ADMA) were determined with an enzyme immunoassay (DLD, Hamburg, Germany). Determination of nitrate and nitrite as surrogate markers for NO was done with a modified Griess procedure (Nitric oxide assay kit, Assay Designs, Ann Arbor, USA) after removal of proteins by means of ultrafiltration through a 10,000 MWCO filter (VectaSpin Micro, Whatman, Dassel, Germany). Assays were performed essentially as described by the manufacturer and all samples were assayed in duplicate. Dynamic blood viscosity was assessed by use of a Reverse Flow Viscosimeter (Holdt et al., 2005). The coefficients of variation were below 10% for determination of blood viscosity, ADMA and NO, respectively. Statistical analysis Fig. 1. Recording of post-ischemic reactive hyperemia by nailfold capillaroscopy. Erythrocyte velocity measured in a healthy control (●\●) and in a CKD patient (○- -○) under resting conditions and after induction of ischemia is shown. The maximal erythrocyte velocity (Vmax) is indicated.
Venous occlusion plethysmography was performed on both legs with the Compactus device (Gutmann-Medizinelektronik, Germany) as described previously (Briese et al., 2006). Subjects were laying with legs and ankles supported above heart level. A pneumatic occlusion cuff was placed around each upper leg and connected with an automatic inflator (Compactus; Gutmann-Medizinelektronik, Germany). Mercury strain gauges were placed on the largest part of the lower legs distal to the knee. After calibration, venous occlusion was achieved by cuff inflation to 60 mm Hg for the measurement of baseline leg blood flow (LBF, expressed in ml/100 ml/min). After inflating the occlusion cuffs for a period of 5 min to at least 50 mm Hg above the systolic pressure (or at maximum 180 mm Hg), reactive hyperemic blood flow was measured every 10 s for 100 s. The post-ischemic peak flow (PIPF) was defined as the highest flow measured during this period expressed in absolute values (ml/100 ml/min). The total post-ischemic flow during this period (TPIF) was defined as the area under the plethysmography curve (AUC) (Briese et al., 2006). All examinations were done after an acclimatization period (ca. 20 min) within the same temperature-controlled room and by the same trained observer (RN). The intra-individual coefficient of variation was ≤15%. Carotid intima-media-thickness (cIMT) was assessed by highresolution B-mode ultrasound using a EUB-525 Duplex Scanner (Hitachi, Tokyo/Japan) equipped with a 12-MHz linear array transducer and echo-tracking system (Pignoli et al., 1986). Four measurements in each of the two carotid arteries were performed. The mean was calculated and used for further analysis. Blood pressure and heart rate were measured by the standard sphygmomanometric method after the patients has been in the supine position for at least 10 min preceding standard and pulsed Doppler-echocardiographic examinations at time of enrollment and after 7 days of rhGH treatment. Left ventricular end-diastolic diameter, thickness of interventricular septum and left ventricular posterior wall as well as stroke volume and cardiac output were measured (Lanes et al., 2005; Sahn et al., 1978). Left ventricular mass, relative wall thickness, left ventricular mass index for body surface area and total peripheral resistance were calculated according to standard procedures (Ganau et al., 1992).
Results are given as mean± standard deviation if not indicated otherwise. When appropriate, range and/or 95% confidence interval (CI) are shown. Normal distribution of the data was assessed by the Shapiro– Wilks test. The longitudinal changes in cardiovascular and biochemical data were evaluated by repeated measure ANOVA including a withinsubject factor (t0; t1; t2) and, a between-subject factor (patients and controls). Pairwise comparisons between time points were performed using the CONTRAST option of the general linear models procedure in the SAS-software (v 8.2). A Bonferoni–Holm adjustment was made to account for multiple testing. Differences in the distribution of specific attributes (e.g. smoking/non-smoking, use of drugs) in the treatment groups were analyzed by Chi-square Test. A p-valueb 0.05 was considered as statistically significant. Results Baseline characteristics of CKD patients and controls are presented in Table 1. At time of enrollment (t0), patients showed well controlled hypertension, and no signs of malnutrition, i.e. normal BMI and serum albumin levels.
Laboratory methods Blood was obtained prior to plethysmography and was stored on ice until delivered to the laboratory, where preparation of serum and plasma took place. Samples were stored at −20 °C until analysis. GFR was calculated according to the “Modification of Diet in Renal Disease Study Prediction Equation” (MDRD) (Levey et al., 1999). Homocysteine, IGF-I and IGFBP-3 were determined by immunoassay-procedures combined with either fluorescence-induced polarization or chemiluminescence (FPIA, IMx, Abbott, Wiesbaden Germany and Immulite 1000, Siemens, Bad Nauheim, Germany). The molar ratio of IGF-I and IGFBP-3 was calculated essentially as described by Morimoto (Morimoto et al., 2005).
Fig. 2. Effect of rhGH treatment on cutaneous microcirculation in CKD patients and controls. CKD patients and healthy controls were investigated before (t0), after 7 days of rhGH treatment (t1), and after a 7-day wash-out period (t2). At these time points, erythrocyte velocity (VRBC) under resting conditions (A), and post-ischemic maximum erythrocyte velocity (Vmax; B) were measured and data are given as mean ± SEM. Significant differences between patients and controls (⁎) as well as significant changes compared to baseline (#) are indicated.
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6.24); controls, 7.5 ± 0.85 loops/mm (95% CI: 7.00–7.99); p b 0.001). Whereas VRBC under resting conditions was rather similar in patients and controls, the maximum post-ischemic VRBC was only about 68% of that seen in controls (Fig. 2). After 1 week of rhGH therapy (t1) VRBC under resting conditions and post-ischemia was significantly enhanced in CKD patients and controls (each p b 0.05 versus baseline; Fig. 2). At the end of the seven day wash-out period VRBC determined at either condition was still significantly higher in controls compared to pretreatment values (each p b 0.05 t2 versus t0; Fig. 2). By contrast, this was not seen in CKD patients. Capillaroscopy revealed no significant differences between CKD patients on conservative treatment and on hemodialysis (data not shown). Venous occlusion plethysmography At baseline, LBF at rest was comparable in both groups (p = 0.7431; Fig. 3A). Mean post-ischemic peak flow (PIPF) and total post-ischemic flow (TPIF) in CKD patients were approximately 75% of the values determined in controls (patients versus controls, each p b 0.05 Figs. 3B and C). After 1 week of rhGH therapy (t1) patients and controls presented with a significantly enhanced LBF at rest (+34% and +55% in CKD patients and controls, respectively; each p b 0.01 versus baseline). The mean PIPF remained rather constant throughout the study period (Fig. 3B) and only healthy subjects responded to rhGH therapy with a slight, but significant increase in TPIF (Fig. 3C). At the end of the wash-out period (t2), LBF at rest was still higher compared to the pretreatment values, but was already lower than at the end of the treatment period. However, this effect reached significance in controls only. At the same time (t2) CKD patients presented with slightly enhanced values for PIPF and TPIF (Figs. 3B and C). There were no significant differences between CKD patients on conservative treatment compared to those on hemodialysis with respect to LBF during the study period (data not shown). Cardiac function
Fig. 3. Data obtained by venous occlusion plethysmography in CKD patients and controls. CKD patients and healthy controls were investigated before (t0), after 7 days of rhGH treatment (t1), and after a 7-day wash-out period (t2). At these time points, leg blood flow at rest (LBF; A), post-ischemic peak flow (PIPF; B) and total post-ischemic flow (TPIF; calculated as the area under the curve of LBF; C) were measured. Data are given as mean ± SEM. Significant differences between patients and controls (⁎) as well as significant changes compared to baseline (#) are indicated.
Intravital video capillary microscopy Nailfold capillary density was significantly reduced in CKD patients compared to controls (patients, 5.57 ± 1.16 loops/mm (95% CI: 4.90–
CKD patients showed significantly increased interventricular septum diameter, LV wall thickness, LV mass index, systolic blood pressure, and cIMT (each p b 0.05; Table 2). RhGH significantly increased heart rates by a mean of 15% and 13% in patients and controls, respectively (each p b 0.01; Fig. 4A). This resulted in a concomitant increase in cardiac output and cardiac index which reached statistical significance in controls but not in CKD patients (Fig. 4B). The increased cardiac index, together with an unchanged blood pressure, resulted in a slight decrease of systemic vascular resistance (patients, −9%, p = 0.09; controls, −14%, p b 0.05; Fig. 4C). There were no significant differences between CKD patients on conservative treatment compared to those on hemodialysis with respect to cardiac function (data not shown).
Table 2 Doppler-echocardiographic data, blood pressure, and carotid intima-media-thickness (cIMT) in CKD patients and controls at baseline and after 7 days of rhGH treatment. CKD patients LV end-diastolic diameter Interventricular septum LV posterior wall LV mass index Relative wall thickness Single stroke volume Cardiac output Systolic BP Diastolic BP cIMT
[cm] [cm] [cm] [g/m2] [ml] [l/min] [mm Hg] [mm Hg] [mm]
Controls
Baseline
rhGH
Baseline
RhGH
5.07 ± 0.60 (4.74–5.40) 1.18 ± 0.28 (1.02–1.34)⁎ 1.12 ± 0.23 (0.99–1.25)⁎ 128 ± 50 (100–156)⁎ 0.44 ± 0.08 (0.40–0.49) 75.0 ± 13.6 (67.0–82.5) 4.81 ± 0.97 (4.34–5.24) 128 ± 17 (118–137) 76 ± 14 (69–84) 0.80⁎ ± 0.21 (0.68–0.91)
5.01 ± 0.55 (4.66–5.36) 1.23 ± 0.27 (1.06–1.39)⁎ 1.13 ± 0.18 (1.01–1.24)⁎ 117 ± 51 (86–148) 0.45 ± 0.07 (0.41–0.50)⁎
4.97 ± 0.42 (4.74–5.20) 1.00 ± 0.08 (0.95–1.05) 0.98 ± 0.09 (0.93–1.03) 93 ± 13 (86–100) 0.40 ± 0.05 (0.37–0.42) 68.9 ± 16.1 (58.3–76.6) 4.23 ± 0.86 (3.69–4.64) 121 ± 13 (114–128) 75 ± 9 (70–80) 0.67 ± 0.09 (0.63–0.72)
4.96 ± 0.44 (4.72–5.20) 0.99 ± 0.10 (0.94–1.05) 0.97 ± 0.10 (0.91–1.02) 91 ± 15 (83–100) 0.39 ± 0.05 (0.36–0.42) 69.6 ± 13.8 (60.2–76.2) 4.88 ± 1.16 (4.21–5.55)⁎⁎
71.0 ± 13.1 (61.8–83.8) 5.33 ± 1.25 (4.56–6.28) 132 ± 13 (125–139)⁎ 77 ± 10 (71–83) n.d.
CKD = chronic kidney disease; LV = left ventricular; BP = blood pressure; cIMT = carotid intima-media-thickness; n.d. = not done. ⁎ = significant difference patients vs. controls (p b 0.05). ⁎⁎ = significant difference vs. baseline (p b 0.05); data are presented as mean ± SD, 95% CI are given in brackets.
122 ± 11 (115–128) 75 ± 8 (71–80) n.d.
498.9 ± 177.9⁎⁎(400.3–597.4) 7.61 ± 3.78⁎⁎(5.52–9.71) 0.26 ± 0.10⁎⁎(0.21–0.32) 35.5 ± 17.2(27.0–48.5) 0.76 ± 0.25(0.63–0.89) 9.06 ± 2.07(7.9–10.2)
Washout (t2) rhGH (t1)
602.9 ± 254.6⁎⁎(461.9–744.0) 8.46 ± 2.47⁎⁎(7.09–9.83) 0.27 ± 0.16⁎⁎(0.18–0.36) 90.8 ± 44.8⁎(66.0–115.6) 0.98 ± 0.22⁎(0.86–1.10) 20.1 ± 6.2⁎,⁎⁎(16.6–23.7) 195.5 ± 84.1⁎(148.9–242.0) 6.53 ± 1.98⁎(5.43–7.63) 0.11 ± 0.05(0.08–0.14) 107.9 ± 42.5⁎(84.4–129.8) 0.93 ± 0.23(0.80–1.06) 24.9 ± 9.0⁎(19.7–30.2) [μmol/l] [μmol/l] [μmol/l]
[ng/ml] [mg/l]
221.5 ± 110.6(160.3–282.8) 7.47 ± 2.68⁎(5.98–8.95) 0.11 ± 0.04(0.09–0.13) 93.6 ± 47.8⁎(67.1–120.0) 1.05 ± 0.27⁎(0.90–1.20) 25.6 ± 8.9⁎(20.7–30.5)
Controls
Baseline (t0) Baseline (t0)
Washout (t2) rhGH (t1) CKD patients
IGF = Insulin-like growth factor; BP = binding protein; NO = nitric oxide; ADMA = asymmetric dimethyl-arginine. Data are presented as mean ± SD and 95% CI are given in brackets. ⁎ = significant difference patients vs. controls (p b 0.05). ⁎⁎ = significant difference vs. baseline (p b 0.05).
Fig. 4. Cardiac function in CKD patients and controls. Assessment of cardiac function was performed before (t0), and after 7 days of rhGH treatment (t1). Heart rate (A), cardiac index (B) and total peripheral resistance (TPR) (C) are given as mean ± SEM. Significant differences between patients and controls (⁎) as well as significant changes compared to baseline (#) are indicated.
Table 3 Biochemical markers of GH activity and endothelial function in CKD patients and healthy controls before, after 7 days of rhGH treatment, and after a seven day wash-out period.
Before onset of treatment mean serum level of IGF-I was slightly and mean serum level of IGFBP-3 was markedly higher in CKD patients compared to controls (each p b 0.01), whereas the molar ratio of IGF-I/IGFBP-3 was similar in both groups (p = 0.8593; Table 3). At the end of the 7-day treatment period, mean serum concentrations of IGF-I were about three times higher than at time of enrollment. This was accompanied by a less pronounced increase of the IGFBP-3 concentration and approximately doubling of the molar IGF-I/IGFBP-3 ratio (each p b 0.01). During the 7-day treatment period, serum concentration of urea decreased from 18.9 ± 4.3 mmol/l (95% CI: 16.5–21.3) to 14.3 ± 5.6 mmol/l (95% CI: 11.2–17.4) in CKD patients and from 4.2 ± 1.3 mmol/l (95% CI: 3.4–4.9) to 3.1 ± 1.0 mmol/l (95% CI: 2.6–3.7) in controls (each p b 0.05). Serum concentrations of NO, ADMA, and homocysteine were significantly higher in CKD patients compared to controls (each p b 0.02; Table 3). Whereas NO and ADMA levels remained constant throughout the study, homocysteine levels were significantly lowered after rhGH in CKD patients. No significant changes in hematocrit, dynamic blood viscosity, and body weight, or side effects (e.g. fluid retention) were noted in both treatment groups during the study period (data not shown).
130.2 ± 54.7(99.9–160.5) 4.09 ± 0.92(3.58–4.60) 0.12 ± 0.04(0.09–0.14) 41.1 ± 25.3(25.8–52.6) 0.80 ± 0.25(0.67–0.93) 10.4 ± 3.4(8.5–12.2)
Systemic effects of rhGH
170.6 ± 65.2⁎⁎(134.5–206.7) 4.81 ± 1.53⁎⁎(3.97–5.66) 0.13 ± 0.05(0.11–0.16) 41.8 ± 20.7(28.3–52.2) 0.82 ± 0.18(0.68–0.97) 9.84 ± 3.16(8.1–11.6)
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IGF-I IGFBP-3 IGF-I/IGFBP-3 molar ratio NO ADMA Homocysteine
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Discussion Growth hormone has long been known to maintain and to mediate cardiovascular function at the level of blood vessel reactivity and cardiac structure (Cittadini et al., 2002; Farkas et al., 2005; Hana et al., 2002; Irving et al., 2002). A relative GH deficiency is a common feature of chronic uremia and endothelial dysfunction is an early manifestation of the atherogenic process in CKD patients. Furthermore, endothelial dysfunction turned out to be a strong predictor of the overall increased cardiovascular mortality in these patients (Briese et al., 2006; Brown et al., 1994; Cupisti et al., 2000a; Cupisti et al., 2000b; Farkas et al., 2005; Jung et al., 2001; Kruger et al., 2006; London et al., 2002; London et al., 2004; Rossi et al., 2008; ShamimUzzaman et al., 2002; Sigrist and McIntyre, 2008). In the current study, nailfold capillaroscopy and venous occlusion plethysmography were used to evaluate rhGH effects on microcirculation in non-malnourished CKD patients with well controlled hypertension. RhGH increased capillary blood flow under resting conditions as well as during reactive hyperemia in CKD patients and healthy subjects. In addition, a significantly increased leg blood flow was noted in all study participants under resting conditions. PIPF and TPIF showed only small and non-significant changes in CKD patients, whereas in healthy controls, TPIF was significantly increased after 1 week of rhGH treatment. This discrepancy with respect to the results obtained by capillaroscopy and plethysmography is most likely due to the fact that the latter allows no discrimination between skin microcirculation and skeletal blood flow, whereas capillaroscopy measures skin microcirculation only. Interestingly, in both groups an improved endothelial function was noted even 1 week after cessation (i.e. at t2) of treatment, albeit this effect reached statistical significance in healthy subjects only (Figs. 2 and 3). At the same time, the systemic IGF-I levels as well as the IGFBP-3 concentration were still significantly elevated in controls (Table 3). Thus, we have to consider that the rhGH induced stimulation of IGF-I and the related binding proteins lasted longer in controls compared to CKD patients. It remains to be elucidated, whether this is due to an attenuated release of IGF-I from cellular storages after withdrawal of exogenous rhGH or reflects sensibilization of the GH–IGF-I axis secondary to the administration of rhGH. This hypothesis is indirectly supported by the known GH-resistance in CKD patients, which presented 1 week after withdrawal of rhGH with only slightly and non-significantly elevated IGF-I and IGFBP-3 levels (Rabkin et al., 2005). It is still a matter of discussion, whether rhGH directly or via IGF-I mediates endothelial function, but either of both mediators preferentially activate the eNOS/COX pathways leading to release of NO and prostanoids, respectively. Whereas the vasodilatory effect of NO and prostanoids is at maximum in conduit arteries, EDHFs came into play with decreases in vessel diameter being almost solely responsible for vasodilation in capillaries (Bar et al., 1988; Li et al., 2008; Oltman et al., 2000; Thum et al., 2007; Wu et al., 1994). Therefore, our results suggest that rhGH may affect the release of EDHFs and that this effect is predominant under the condition of chronic uremia. In both study groups the seven-day treatment with rhGH had beneficial effects on the cardiac index and total peripheral resistance. All of these findings are most likely related to the rhGH induced enhancement of microcirculation, rather than to functionally relevant cardiac growth and/or change in cardiac structure (Ito et al., 1993; Turner et al., 1988). This point of view is supported by a recent study showing, that a four-week rhGH treatment in healthy subjects was associated with functional but not with structural adaptation of the cardiovascular system (Cittadini et al., 2002). The overall lower impact of rhGH on cardiovascular and endothelial function in CKD patients in the present study is most probably due to the global GH insensitivity in uremia (Mahesh and Kaskel, 2008; Rabkin et al., 2005). Data on short-term cardiovascular effects of rhGH in CKD patients are grossly lacking. Instead, the effects of rhGH therapy on endothelial and/or cardiovascular function have been investigated in healthy
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subjects, in patients with GH deficiency and in patients with congestive heart failure (Capaldo et al., 2001; Farkas et al., 2005; Hana et al., 2002; Irving et al., 2002; Lanes et al., 2005; Møller et al., 1989; Napoli et al., 2003; Oomen et al., 2002; Twickler et al., 2000). Results of these studies indicate that the beneficial effects of rhGH therapy on vascular function are likely to be, at least in part, due to an improved availability of NO. This concept is especially appealing for two reasons. Firstly, chronic uremia has been associated with an impaired generation of NO due to inhibition of synthesis as well as activation of trapping reactions, leading to overall diminished systemic concentrations of NO (Passauer et al., 2005a; Schmidt and Baylis, 2000). Homocysteine and ADMA are thought to impair NOdependent vasodilation, to increase smooth muscle cell proliferation, and to enhance platelet dysfunction (van Guldener et al., 2007). Furthermore, elevated ADMA and homocysteine levels have been associated with atherosclerotic disease, left ventricular dysfunction, and cardiovascular mortality in uremic patients (van Guldener et al., 2007; Zoccali et al., 2002). Secondly, elegant in-vivo and in-vitro experiments point to acute and direct NO-mediated vascular effects of rhGH in healthy subjects (Li et al., 2008; Napoli et al., 2003). Obviously, our data on the serum concentrations of NO and ADMA do not fit into these concepts. Our CKD patients presented with systemic NO concentrations about twice of those seen in healthy subjects, whereas quite similar concentrations of ADMA, a potent inhibitor of eNOS, were determined in both groups. Therefore, we had to consider, that in our patients the NO pathway was already fully activated at time of enrollment. This is in line with a previous report on functional investigation of the NO pathway in uremic patients (Passauer et al., 2000). In this study, evidence was obtained that rather the availability of NO under baseline conditions but its generation on demand is impaired. Since our functional assessment of vascular function indicated parallel (and positive) effects in both cohorts, we expected to see also an increase of the circulating NO concentrations. To our surprise, this was not the case. Whereas for CKD patients the fairly constant levels of NO fit well to the hypothesis of an already fully activated NOgenerating system, the situation is less clear in healthy controls. Beside the technical problems related to the direct quantitative determination of NO the design of our study was quite different from those described recently (Baylis and Vallance, 1998; Li et al., 2008; Napoli et al., 2003). Whereas patients and controls received rhGH as a bolus immediately before functional assessment, we administered rhGH over a period of 7 days and used apparently non-invasive assessment of the endothelial function timely apart from rhGH administration. Thus, it could well be, that we missed the acute and transient effects of rhGH on NObioavailability but detected the overall improvement of capillary endothelial vasodilation. One may speculate, whether the induction of NO is in fact a very rapid and transient effect, probably mediated by direct interaction between rhGH and the GH receptor, whereas the induction of EDHFs occurs later and their biological activity may last longer. Furthermore, it remains to be elucidated whether rhGH mediates EDHFs directly or via IGF-I. Our study has several limitations. The design of the study was open and not randomized and the number of patients was rather low, leaving a possibility for bias. Considering the relatively short time period, the fact that all patients were kept on constant medication during the study, and the complete remission of the vasodilatory effects in CKD patients after the wash-out period, a time-dependent bias seems unlikely. However, whether the positive effects on microcirculation in CKD patients will last during longer treatment periods remains to be elucidated. In conclusion, treatment with rhGH significantly increased microcirculation under resting conditions in CKD patients and healthy subjects. This was paralleled by a significantly increased heart rate, reduction of total vascular resistance, and an increased cardiac index in controls, whereas the latter two findings did not reach statistical significance in CKD patients. If rhGH treatment is able to modulate
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