Alterations in vasomotor control of coronary resistance vessels in remodelled myocardium of swine with a recent myocardial infarction

The mechanism underlying the progressive deterioration of left ventricular (LV) dysfunction after myocardial infarction (MI) towards overt heart failure remains incompletely understood, but may involve impairments in coronary blood flow regulation within remodelled myocardium leading to intermittent myocardial ischemia. Blood flow to the remodelled myocardium is hampered as the coronary vasculature does not grow commensurate with the increase in LV mass and because extravascular compression of the coronary vasculature is increased. In addition to these factors, an increase in coronary vasomotor tone, secondary to neurohumoral activation and endothelial dysfunction, could also contribute to the impaired myocardial oxygen supply. Consequently, we explored, in a series of studies, the alterations in regulation of coronary resistance vessel tone in remodelled myocardium of swine with a 2 to 3-week-old MI. These studies indicate that myocardial oxygen balance is perturbed in remodelled myocardium, thereby forcing the myocardium to increase its oxygen extraction. These perturbations do not appear to be the result of blunted β-adrenergic or endothelial NO-mediated coronary vasodilator influences, and are opposed by an increased vasodilator influence through opening of KATP channels. Unexpectedly, we observed that despite increased circulating levels of noradrenaline, angiotensin II and endothelin-1, α-adrenergic tone remained negligible, while the coronary vasoconstrictor influences of endogenous endothelin and angiotensin II were virtually abolished. We conclude that, early after MI, perturbations in myocardial oxygen balance are observed in remodelled myocardium. However, adaptive alterations in coronary resistance vessel control, consisting of increased vasodilator influences in conjunction with blunted vasoconstrictor influences, act to minimize the impairments of myocardial oxygen balance.
Introduction
Heart failure constitutes a major cardiovascular disorder of which the incidence and prevalence are increasing, principally due to an increased survival of acute myocardial infarction (MI) in conjunction with an ageing population. The mechanism underlying the progressive deterioration of left ventricular (LV) dysfunction towards overt heart failure remains incompletely understood, but may involve (1) loss of cardiomyocytes through apoptosis [75], (2) a primary reduction in contractile function of the surviving myocardium [97], and/or (3) alterations in extracellular matrix leading to progressive LV dilation [87]. In addition, myocardial blood flow (MBF) abnormalities, resulting in impaired myocardial O2 delivery to the non-infarcted regions (leading to secondary contractile dysfunction and/or enhanced apoptosis), have been suggested to contribute to the progression of LV dysfunction after MI [99]. For example, in vivo studies in rats [54, 55] and swine [108] indicate a reduction in MBF reserve of up to 35% in the surviving remodelled LV myocardium, 3–8 weeks after infarction. Furthermore, in patients with overt heart failure [94], but also in patients with only asymptomatic LV dysfunction [95], flow reserve is reduced in the non-stenotic myocardial regions. In line with these clinical observations, we observed in a porcine model of post-infarct remodelling that during increased O2-demand induced by exercise, the increase in coronary blood flow (CBF) is impaired resulting in perturbations in oxygen delivery [43, 70]. The reduction in flow reserve and the perturbed oxygen delivery during exercise are caused, at least in part, by insufficient growth of the coronary vasculature to maintain flow capacity commensurate with myocardial hypertrophy, in conjunction with a decrease in diastolic pressure time index resulting from the elevated heart rate and particularly elevated LV diastolic pressures [43]. In addition, coronary vasomotor tone may also be increased secondary to neurohumoral activation and endothelial dysfunction, further adding to the perturbations in CBF. However, little is known about the alterations in vasomotor control in coronary resistance vessels within remodelled myocardium (Fig. 1). For this reason we undertook a series of studies to determine whether neurohumoral (autonomic nervous system and renin-angiotensin system), local metabolic, and endothelial control mechanisms of coronary resistance vessel tone are altered in swine with remodelled myocardium produced by a recent MI.
Fig. 1Alterations in determinants of oxygen supply and demand in remodelled myocardium in swine with a 3-week-old myocardial infarction (MI). The net effect of these alterations is a decrease in oxygen supply/demand ratio
Characteristics of LV remodelling and dysfunction after MI in swine
Left ventricular remodelling was produced by permanent ligation of the left circumflex coronary artery. This ligation results in a circumscribed transmural infarction of the lateral LV wall, comprising 20–25% of the total LV [82, 96]. LV dysfunction in awake resting MI swine is characterised by 20–30% decreases in cardiac output, stroke volume and LVdP/dt
max and a tripling of LV filling pressure. This difference between LVdP/dt
max, cardiac output and stroke volume in normal and MI swine remained constant between ∼10 and ∼32 days, indicating that the degree of LV dysfunction and the circulatory adaptations in MI were stable during this observation period [43]. Similarly, we observed that already during the first week after infarction significant LV remodelling occurs, consisting of LV dilation and hypertrophy that remain fairly stable between 1 and 6 weeks after infarction [98]. During exercise, cardiac output, LV systolic pressure and LVdP/dt
max increased almost in parallel in MI and normal animals up to 3 km/h, after which curves diverged (Fig. 2); 4 km/h was also the maximally attainable exercise level for most MI swine.
Fig. 2Cardiovascular responses to exercise in normal swine and swine with a ∼3-week-old MI. 0L = lying, 0S = standing. Data are mean ± SEM; *P < 0.05 versus 0L, †P < 0.05 MI versus normal a corresponding exercise level. Data are from Haitsma et al. [43]
Left ventricular dysfunction produced by MI results in neurohumoral activation, characterized by a trend towards elevated plasma levels of catecholamines, but normal circulating levels of renin, angiotensin II and aldosterone at rest. The latter may have been due to the increments in atrial natriuretic peptide (ANP) and endothelin, which can suppress renin and aldosterone release [83]. In resting MI swine, ANP doubled within 24 h, recovered to 50% above normal values within 2 weeks and remained stable between 2 and 6 weeks after infarction, while renin and norepinephrine levels remained normal under resting conditions [98]. In contrast to the discrete neurohumoral activation in resting swine with MI, exercise resulted in exaggerated increases in catecholamines and ANP and increases in endothelin, angiotensin II, and aldosterone (Fig. 3). While resting circulating levels of norepinephrine were still normal and the relative sympathetic drive in response to exercise was preserved in MI, the cardiac responsiveness to exercise (both heart rate and LVdP/dt
max) was already blunted 3 weeks after infarction [43], likely due to β-adrenoceptor desensitization and/or downregulation [100].
Fig. 3Neurohumoral responses in exercising swine with a ∼3-week-old MI. In the norepinephrine, epinephrine and dopamine panels, data shown for N represent 0L, 1, 2, 3, 4 and 5 km/h and data for MI represent 0L, 1, 2, 3 and 4 km/h. In all other panels, data shown for N represent 0L, 1, 3 and 5 km/h and data for MI represent 0L, 1, 3 and 4 km/h. Mean data points were fitted with second order curves. VO2 = O2-consumption. Data are mean ± SEM; *P < 0.05 vs 0L, †
P < 0.05 MI versus Normal. Data are from Haitsma et al [43]
Myocardial O2 balance in remodelled myocardium
Marked decreases in myocardial perfusion occur in pacing-induced severe heart failure in swine [88] and dogs [85, 90], especially in the more vulnerable subendocardial layers. Although one study in dogs indicated that the lower MBF is principally the result of a lower myocardial O2 consumption (MVO2) [90], studies in swine suggest that the impaired perfusion is, at least in part, responsible for the deterioration of LV function because the interstitial edema and disruption of collagen fibers in the subendocardium resemble the ultrastuctural changes that occur with recurrent ischemia [49, 89]. In animal models of severe pressure-overload induced LV hypertrophy, selective underperfusion of the subendocardium can produce myocardial ischemia during exercise and result in post-exercise myocardial stunning [2, 102]. The contribution of perfusion abnormalities in the remote surviving myocardium to LV dysfunction after a MI remains unclear. Studies in rats demonstrated a 25–40% reduction in coronary flow reserve in the surviving myocardium at four [55] and eight [54] weeks after MI. Similarly, maximum subendocardial blood flow was blunted by 40% in anesthetized swine with heart failure 3 weeks after a MI [108]. We hypothesized that the decreased flow reserve could limit the increase in MBF to the hypertrophied myocardium during exercise when hemodynamic abnormalities and neurohumoral activation are exacerbated, thereby impairing myocardial O2-supply.
Three weeks after infarction, blood flow per gram of myocardium in the (remote) LV anterior wall of resting swine with MI was similar to that in normal animals (Fig. 4), confirming previous studies in rats and swine [54, 55, 108]. Interestingly, we observed a trend towards slightly higher blood flows in the outer two layers (P = 0.09), suggesting that despite hypertrophy of the surviving myocardium, metabolic demand in the outer, but not the inner, layers was still slightly elevated, 3 weeks after infarction [43]. During exercise, MBF increased but was redistributed in favor of the subepicardium in MI compared to normal swine. These perturbations were most likely due to increased extravascular compressive forces, resulting from a reduction in diastolic time fraction (secondary to impaired relaxation and increased heart rate) and elevated LV filling pressures [43, 97], that impede MBF, particularly in the subendocardial layers. The decreased MBF necessitated a small increase in O2 extraction that resulted in a slightly lower coronary venous O2 tension (Fig. 4), which actually may have been underestimated as the lower myocardial capillary density in swine with MI possibly prevented a greater increase in O2 extraction [43, 70]. The observation that O2 extraction was forced to increase indicates that increases in extravascular forces are not fully compensated by a concomitant lowering of coronary vasomotor tone in remodelled myocardium during exercise. These observations prompted us to further investigate the control of coronary vasomotor tone in remodelled myocardium during exercise.
Fig. 4Myocardial blood flow and O2-balance in the left ventricular anterior wall of N and MI ∼3 weeks after myocardial infarction. Epi subepicardial, OM outer mid; IM inner mid; Endo subendocardial. MVO2 = myocardial O2-consumption; MEO2 = myocardial O2 extraction; CVPO2 = coronary venous O2 tension In the top left panel data myocardial blood flow data are shown for resting (Rest, lying) conditions, and during maximum exercise (Ex, 5 km/h in N and 4 km/h in MI). Data are mean ± SEM; *P < 0.05 versus 0L, †
P < 0.05 MI versus Normal. Data are from Haitsma et al [43]
Vasomotor control of the coronary microcirculation in remodelled myocardium
Neurohumoral control
Cardiac dysfunction is accompanied by a hemodynamic defense reaction consisting of salt and water retention, peripheral vasoconstriction and cardiac stimulation, which serves to partially restore cardiac output and to increase systemic vascular resistance in order to maintain arterial pressure [56]. An integral part of this defense reaction involves alterations in autonomic balance, consisting of an increase in sympathetic activity and a decrease in parasympathetic activity [56].
Sympathetic control
In patients with advanced heart failure plasma noradrenaline levels are already increased under resting conditions [11, 36]. These increased levels result principally from increased sympathetic nerve activity although impaired reuptake may also contribute [56]. Prolonged exposure to elevated noradrenaline levels results in desensitization and downregulation of the β-adrenergic receptors [8, 32, 101]. During exercise, the increases in catecholamine levels are exaggerated in patients with heart failure as compared to healthy controls [35], which is aimed at maintaining chronotropic and inotropic responses to exercise [34].
Also in swine with LV dysfunction produced by a 2–3-week-old MI, we observed exaggerated increases in arterial and coronary venous catecholamine levels during treadmill exercise [23, 43], at a time when resting catecholamine levels were still in the normal range [43, 98]. The exaggerated exercise-induced increases in catecholamine levels reflect increased sympathetic activity, which acts to maintain the chronotropic and inotropic responses to acute exercise. This concept is supported by the observation that β-adrenoceptor blockade produced slightly greater decreases in the chronotropic response during exercise in MI as compared to normal swine. In contrast, β-adrenoceptor blockade in MI swine resulted in a smaller decrease in global LV contractility compared to normal swine, in particular during exercise. The latter findings are consistent with a reduced left ventricular myocardial β-adrenoceptor responsiveness [100].
In normal swine and dogs, β-adrenergic receptor activation contributes to coronary vasodilatation during exercise [27, 39]. The β-adrenergic coronary vasodilatation results in an increase in myocardial oxygen delivery that is commensurate with the increase in oxygen consumption, so that myocardial oxygen extraction and hence coronary venous O2 tension remain constant (Fig. 5). In MI swine, the net β-adrenergic vasodilator influence on the coronary circulation is maintained. However, in view of the exaggerated increases in catecholamine levels during exercise, these findings suggest a diminished β-adrenergic responsiveness of the coronary resistance vessels after MI.
Fig. 5Effect of saline, α-adrenoceptor blockade (phentolamine, 1 mg/kg iv) and/or β-adrenoceptor blockade (propranolol, 0.5 mg/kg iv) and of muscarinic receptor blockade (atropine, 30 μg/kg/min, iv) and/or β-adrenoceptor blockade (propranolol, 0.5 mg/kg iv) on the response of coronary venous O2 tension (CVPO2) plotted as a function of myocardial O2 consumption (MVO2) in seven normal and 7 MI swine. *P < 0.05 versus corresponding Control; †
P < 0.05 Atropine + Propranolol versus corresponding Atropine or Phentolamine + Propranolol versus corresponding Phentolamine; Data are mean ± SEM. Data are from Duncker et al. [23]
In dogs, α-adrenoceptor activation limits the exercise-induced increase in CBF, thereby necessitating an increase in myocardial O2 extraction, which leads to a decrease in coronary venous O2 tension [39, 51]. In contrast, α-adrenoceptors do not contribute to regulation of coronary blood flow in normal swine during exercise [27]. We found that, in accordance with our findings in normal swine, administration of the α-adrenoceptor blocker phentolamine had also no effect on coronary venous O2 tension of MI swine (Fig. 5). These findings indicate that even in the presence of exaggerated increments in catecholamine levels in MI swine during exercise, α-adrenoceptors do not contribute to regulation of tone in porcine coronary resistance vessels [23].
Parasympathetic control
The shift in the sympathovagal balance, with an increased sympathetic activity [12, 33] and a blunted parasympathetic activity is reflected in reduced heart rate variability and reduced baroreceptor reflex sensitivity [6, 12, 30, 76] in patients with advanced heart failure. We observed that a maximal dose of the muscarinic receptor blocker atropine produced a similar increase in resting heart rate in swine with MI as compared to normal swine, suggesting preserved parasympathetic activity under resting conditions [23]. In contrast, the atropine-induced increase in heart rate during exercise (particularly at higher exercise levels) was blunted in MI swine, while the increase in LVdP/dt
max was abolished [23]. These results are consistent with the concept that gradual inhibition of parasympathetic influence on the heart during exercise was more pronounced in swine with MI compared to normal swine. Importantly, these findings suggest that after MI, at a time when parasympathetic tone under basal resting conditions is normal, a more pronounced inhibition of parasympathetic tone occurs with increasing exercise intensity. Since parasympathetic activity can presynaptically modulate sympathetic activity [1], it is likely that the greater degree of withdrawal of parasympathetic tone during exercise contributed to the exaggerated increase in sympathetic activity during exercise. This is also supported by the observation that in the presence of propranolol, the effects of atropine were no longer different between MI and normal swine [23].
In resting dogs, parasympathetic activity exerts a direct vasodilator influence on coronary resistance vessels that is mediated via nitric oxide [109]. In contrast, in resting swine parasympathetic activity exerts an indirect vasoconstrictor effect on the coronary resistance vessels (which wanes at increasing exercise intensity) that is mediated via inhibition of β-adrenergic vasodilatation [27]. In contrast to the loss of the inhibitory influence of the parasympathetic system on β-adrenoceptor mediated cardiostimulation, we observed that its effects on the coronary circulation were maintained in MI compared to normal swine (Fig. 5) [23]. These findings indicate that at this stage of LV dysfunction, parasympathetic control of β-adrenoceptor-mediated coronary vasodilatation is unimpaired.
Angiotensin II
The renin-angiotensin system plays an important role in cardiovascular homeostasis by contributing to the regulation of blood volume, blood pressure and vascular tone. Angiotensin II (ANG-II) exerts its effects on vascular tone through binding to the AT1-receptor, resulting in vasoconstriction, as well as binding to the AT2-receptor, evoking vasodilation [14]. Both receptor subtypes have been identified in the coronary microcirculation [4, 107], suggesting that endogenous ANG-II may contribute to the regulation of coronary vascular tone and to the regulation of myocardial perfusion. Under pathological circumstances, i.e. after MI, the renin-angiotensin system is activated, resulting in increased plasma levels of ANG-II, particularly during exercise [34, 43]. Moreover, there is evidence that AT1-receptor density in the viable region of the myocardium is increased early after MI [62, 98], suggesting that its vasoconstrictor influence on the coronary vasculature could be increased, which may limit myocardial perfusion thereby exacerbating LV dysfunction.
Contrary to our hypothesis, we observed a loss of ANG-II induced vasoconstrictor influence, reflected by the lack of increase in coronary venous O2 tension (Fig. 6), despite increased plasma ANG-II levels and maintained AT1 receptor densities in coronary arterioles isolated from remodelled myocardium of MI swine [68]. It is unlikely that a generalized loss of vasodilator capacity in the remote myocardium contributed to the blunted vasodilator response to the AT1 receptor blocker irbesartan, as we have previously shown that vasodilation produced by nitroprusside is unperturbed [44]. Although an increased AT2-receptor expression could have acted to limit ANG-II induced vasoconstriction [84] this is unlikely as AT2-mRNA was not altered in coronary arteries from patients with ischemic heart disease [103]. Moreover, the dramatic increases in ANG-II levels that we observed after irbesartan did not result in enhanced, but rather blunted, coronary vasodilation [68]. Therefore, the observation of a reduced vasoconstrictor influence of endogenous ANG-II is best explained by AT1-receptor-desensitization, which is in accordance with studies in dogs with pacing-induced heart failure [78], in rats with pressure-overload LV hypertrophy [63], and in rats with LV remodeling after MI [84], that demonstrated blunted vasoconstrictor responses to exogenous ANG-II.
Fig. 6Effect of AT1-receptor blockade with irbesartan (1 mg/kg iv) on the relation between myocardial O2 consumption (MVO2) and coronary venous O2 tension (CVPO2) in normal swine and swine with a 2–3-week-old myocardial infarction. Data are means ± SE; *P < 0.05 versus corresponding control relation, †
P < 0.05 effect of irbesartan different in MI versus Normal animals. Data are from Merkus et al. [68]
Local metabolic control
Adenosine
Adenosine has been proposed to be a metabolic messenger that regulates coronary resistance vessel tone in response to changes in metabolic needs of the myocardium [5]. However, adenosine receptor blockade with 8PT and/or augmenting adenosine catabolism with intracoronary adenosine deaminase had either no effect [3] or produced a small decrease [28, 67, 93] in basal coronary venous O2 tension (reflecting vasoconstriction), but did not interfere with the normal exercise-induced increase in CBF and O2 delivery [3, 28, 67, 93], indicating that adenosine is not critical for the exercise-induced coronary vasodilation or that loss of adenosine-mediated vasodilation can be compensated for by increased contribution of other vasodilator pathways to maintain adequate metabolic vasodilation.
In contrast to the lack of evidence for an essential role of adenosine in regulation of CBF under physiological conditions, endogenously released adenosine does contribute to coronary vasodilation when there is insufficient supply of O2 [61]. Similarly, adenosine production could be increased in the remodelled myocardium after MI as a result of the perturbations in the myocardial O2 balance [43, 70, 108]. However, we found no evidence for an increased contribution of adenosine to regulation of coronary resistance vessel tone in remodelled myocardium of swine with a recent MI [71], as adenosine receptor blockade with 8PT caused a similar decrease in coronary venous O2 tension in post-infarct remodelled hearts and normal hearts both at rest and during exercise (Fig. 7). These findings are in agreement with observations in dogs with pressure-overload LV hypertrophy, in which adenosine receptor blockade did not affect CBF either at rest or during exercise [65].
Fig. 7Effect of the adenosine receptor antagonist 8-phenyltheophylline (8PT, 5 mg/kg iv), the KATP channel blocker glibenclamide (Glib, 3 mg/kg iv) or their combination on myocardial O2 balance in the LV anterior free wall of normal swine and swine with a recent MI. MVO2 = myocardial O2 consumption; CVPO2 = coronary venous O2 tension; Data are mean ± SEM; Data are mean ± SEM; *P ≤ 0.05 versus corresponding control; †
P ≤ 0.05 effect of Glib was blunted at higher MVO2 levels (Glib × MVO2); ‡
P ≤ 0.05 effect of Glib different after MI (Glib × MVO2 × MI). Data are from Merkus et al. [71]
Several reasons could be forwarded for the failure to observe a larger contribution of adenosine to regulation of coronary resistance vessel tone. First, it is possible that the perturbations in the myocardial O2 balance were too mild to increase adenosine production. Second, the activity of enzymes that regulate tissue adenosine levels may have been altered [16, 17]. For example, the activity of adenosine deaminase, the enzyme responsible for breakdown of adenosine to inosine, was found to be elevated in LV hypertrophy [10, 13, 15]. Furthermore, there is evidence that the activity of cytosolic 5′ nucleotidase, which converts 5′AMP to adenosine, is lower in certain models of pressure-overload [13] and volume-overload [10] induced LV hypertrophy, which could be related to intermittent hypoperfusion of the hypertrophic myocardium [41]. Together these enzymatic alterations, which act to decrease myocardial levels of adenosine may have prevented a significant increase in myocardial adenosine levels in the post MI remodelled hearts.
Finally, it is possible that an increased role of adenosine in hypertrophied myocardium is masked by an increased contribution of other vasodilator systems during adenosine receptor blockade, as the process of metabolic vasodilation is thought to be mediated through multiple parallel or redundant pathways. Thus, KATP channel activity may have increased in response to adenosine receptor blockade to compensate for the loss of adenosine-mediated vasodilation. Hence, we evaluated the interactions between these vasodilator pathways.
KATP channels
In addition to the role of KATP channels in the regulation of CBF under physiological conditions [24, 25, 31], there is evidence for an increased KATP channel activity in the coronary circulation of remodelled hearts. For example, in anesthetized dogs with pacing-induced severe heart failure [106], the KATP channel blocker glibenclamide resulted in an exaggerated vasoconstrictor response as compared to normal dogs. Interestingly, in dogs subjected to only 1 week of pacing (when LV function was still normal), KATP channel activity was not different from normal dogs [106], suggesting that KATP channel activity in the basal resting state was only enhanced in the presence of overt heart failure. Similarly, in awake dogs with compensated pressure-overload induced LV hypertrophy, the reduction in CBF produced by glibenclamide was similar to that in normal dogs [65]. During exercise, however, glibenclamide produced a greater reduction in CBF in hypertrophied hearts, indicating increased KATP-channel contribution to coronary vasodilation when O2 requirements of the hypertrophied heart were augmented [65].
In swine with MI-induced moderate LV remodeling and dysfunction, glibenclamide caused a marked decrease in coronary venous O2 tension in remodelled left ventricle under resting conditions, that was similar to the decrease in coronary venous O2 tension in normal hearts (Fig. 7; [71]). Although the vasoconstriction under resting conditions in response to KATP channel blockade was similar in normal and post-MI remodelled hearts, the responses to exercise were different. Thus in normal swine, the effects of KATP channel blockade waned during exercise, suggesting that other vasodilator systems compensated for the loss of KATP channels during exercise. In contrast, in the post-MI remodelled hearts the effects of KATP channel blockade were maintained during exercise. Our findings, which are consistent with the observations in dogs [65], support the hypothesis that KATP channel opening is of greater importance in resistance vessel dilation during exercise in hypertrophied than in normal hearts. It is likely that with the progression from LV dysfunction to overt heart failure, increased KATP channel activity may also become important under resting conditions [106].
Interaction between KATP channels and adenosine
In contrast to the canine heart in which adenosine can act as a back-up system [29, 81], adenosine and KATP channels appear to exert additive vasodilator influences on coronary vasomotor tone in the normal porcine heart [67]. Thus, the coronary vasoconstriction that occurs in response to combined adenosine receptor blockade and KATP channel blockade equalled the sum of the vasoconstriction induced by blockade of the individual pathways. Adenosine mediates its vasodilator effect on porcine coronary resistance vessels via KATP, KCa and Kv channels [9, 46–48] It is therefore possible that following KATP channel blockade adenosine maintained its vasodilator influence via KCa and/or Kv channels.
In contrast to the normal porcine heart, the magnitude of the constriction induced in remodelled hearts by combined blockade of adenosine receptors and KATP channels was virtually identical to that produced by blockade of KATP channels alone [71]. These findings could be interpreted to suggest that in remodelled myocardium the vasodilator influence of endogenous adenosine was entirely mediated through opening of KATP channels, observations that are corroborated by findings in pressure-overload hypertrophied canine hearts [65]. Taken together these observations in the porcine and canine coronary circulations suggest that, although the magnitude of the vasodilator influence exerted by endogenous adenosine was similar in normal and remodelled hearts, its effector pathway was different.
Endothelial control
Several studies have indicated that endothelial dysfunction, in particular a decreased production of NO and an increased production of endothelin, could aggravate LV dysfunction due to the peripheral vasoconstriction-induced increase in LV afterload, coronary vasoconstriction, and increased myocardial O2 consumption [26, 59, 80, 105].
Nitric oxide
Clinical studies indicate that chronic heart failure is accompanied by blunted vasodilator responses to endothelium-dependent receptor mediated vasodilators (particularly acetylcholine) in the microcirculation of the LV myocardium [92], leg [45, 57], and forearm [19, 52, 57, 64]. In the canine model of pacing-induced end-stage congestive heart failure, attenuated vasodilator responses of resistance vessels to acetylcholine in vivo have also been observed in the microvasculature of the hindleg circulation [22, 64] and the coronary circulation [105]. In swine with a 2–3-week-old MI, we observed reduced vasodilator responses in the systemic and coronary microvasculature to ATP, in doses which we have previously shown to be completely abolished by pretreatment with the eNOS-inhibitor NLA [26]. The findings of a blunted ATP-induced vasodilation are in agreement with the hypothesis that agonist-induced eNOS-mediated NO production is blunted 2–3 weeks after MI.
A loss of NO-mediated vasodilation could enhance the progression of LV dysfunction to heart failure. This is supported by studies in dogs with pacing-induced dilated cardiomyopathy, in which the loss of basal NO production in the LV myocardium coincides with the progression from LV dysfunction to overt heart failure [80, 105]. However, in swine with a 2–3-week-old MI, we did not find any evidence of a reduced coronary vasodilator influence of endogenous NO as decreases in coronary venous O2 tension produced by NLA in resting and exercising MI swine were similar to those of normal swine (Fig. 8). In heart failure patients, studies on the contribution of NO to basal microvascular tone in the forearm, leg, or total systemic bed have yielded equivocal results with responses varying from blunted [45, 58, 64], to maintained [60], and even enhanced [20, 42] increases in vascular tone following NO synthase inhibition. It is possible that a maintained or increased NO production as observed in some studies, was the result of increased iNOS expression [21, 79], as part of a generalized inflammatory response in end-stage heart failure, that occurred in the presence of either a decreased [21, 86] or increased [37, 50] eNOS expression. Since NLA can block all three isoforms of NOS [7], we performed additional experiments, in which we blocked iNOS with aminoguanidine, to determine whether an upregulation of iNOS-mediated NO production masked a reduction in eNOS activity. iNOS blockade had no effect on coronary vasomotor tone, demonstrating that NO production via iNOS does not contribute significantly to vascular tone in MI swine, and, consequently, that basal and exercise-induced endothelial NO production is maintained early after MI [44].
Fig. 8Effect of inhibition of NO synthase by NLA (20 mg/kg iv) on myocardial O2 extraction and coronary venous PO2 at rest (lying) and during treadmill exercise in MI and N. Data are mean ± SEM. *P < 0.05 NLA versus corresponding Control; there were no significant differences in the responses to NLA between MI and N either at rest or during exercise. Data are from Haitsma et al. [44]
The reason for the maintained basal and exercise-induced NO production in the presence of a blunted vasodilator response to ATP is unclear. However, Traverse et al. [91] have shown that the amount of NO, produced after stimulation with an agonist is larger than the amount of NO produced during moderate exercise (60% increase in heart rate). Hence, the maximal capacity of NO production may be reduced, whereas the capacity of eNOS is sufficient to maintain basal and exercise-induced NO production. This explanation is unlikely since agonist-induced dilation is already affected at the lowest dose of administered ATP (which probably releases less NO than strenuous exercise). Moreover, higher doses of ATP still produce more dilation. Another explanation for the divergent results between ATP and exercise-induced increases in NO-production may be that ATP activates eNOS through a different mechanism than shear stress. ATP-induced activation of eNOS is mediated through a calcium-calmodulin dependent pathway [74] whereas shear stress activates eNOS through Akt-mediated phosphorylation [18], resulting in calcium-independent activation of eNOS. Hence, it is possible that perturbations in the calcium homeostasis of endothelial cells contributed to the selective impairment of ATP-induced vasodilation in swine with MI.
Endothelin
Despite the increased plasma levels of endogenous ET in swine with a 2–3 week old MI, its vasoconstrictor influence on the coronary circulation was reduced (Fig. 9 [69, 70]). To determine whether this was the result of blunted receptor responsiveness or reduced local ET-production, we studied the vasoconstriction induced by exogenous ET. The coronary vasoconstrictor influence to exogenous ET-1 in vivo was reduced after MI, indicating a reduced coronary vascular responsiveness to ET. Paradoxically, a recent study showed that ischemic heart disease results in upregulation of ETA and ETB receptor mRNA in human coronary arteries [104]. This is in accordance with our measurements in isolated coronary arterioles obtained from sham-operated swine and swine with a MI, which showed that the ET responsiveness in vessels from animals with a MI was actually increased [70]. The discrepancy between the in vivo and the in vitro findings suggests that ET receptor sensitivity is modulated in vivo, and that this modulation is apparently lost in vitro. Possible modulators of ET-receptor sensitivity are adenosine and NO, which have been shown to desensitize ET-receptors on the coronary vasculature [72, 73], and which may have been lost in the in vitro set-up due to lack of surrounding myocardium and intravascular blood flow. However, since we observed similar vasoconstrictor responses to blockade of adenosine receptors and NO production in MI and normal swine, these vasodilators would seem unlikely explanations for the observed reduced ET receptor sensitivity in MI swine in vivo.
Fig. 9Effect of ETA receptor blockade with EMD (3 mg/kg iv) on myocardial oxygen balance in normal swine and swine with a recent MI. Data are means ± SE; *P < 0.05 versus corresponding Control relation, †
P < 0.05 effect of EMD waned during exercise. Data are from Merkus et al. [70]
In conclusion, our observations suggest that when additional coronary vasodilation is required in the hypertrophied myocardium after MI, withdrawal of the ET-mediated vasoconstrictor influence contributes to a shift in vasomotor tone towards vasodilation. These findings may also explain in part why clinical trials of ET-receptor antagonists in heart failure have failed to show therapeutic value of these compounds [40, 77].
Conclusions and physiological relevance
Under physiological circumstances, the heart matches its blood supply to the demand of the myocardium by altering the balance of vasodilator and vasoconstrictor influences, i.e. an increase in myocardial O2 demand results in an increased influence of vasodilators (opening of KATP channels, NO, adenosine [53], β-adrenergic vasodilation [27]) and a decreased influence of the potent vasoconstrictor endothelin-1 [66, 69]. Studies from our laboratory performed during the past 10 years have shown that following MI myocardial O2 balance in the remodelled LV is perturbed, necessitating vasodilation and recruitment of coronary flow reserve, as evidenced by increased opening of KATP channels [71]. Importantly, however, not only an increased KATP channel vasodilator influence, but also blunting of the coronary vasoconstrictor influences of endogenous endothelin [70] and angiotensin II [68], aid in preventing a more significant impairment of oxygen supply to the remodelled myocardium (Figs. 10, 11). Our studies suggest that generalized blunting of vasoconstrictor influences is one of the first adaptive mechanisms to reduce coronary resistance vessel tone and increase myocardial blood supply. This adaptation, which occurs in the healthy porcine heart during acute exercise, also appears operative in remodelled myocardium. Blunting of vasoconstrictor influences may be a physiologically favorable strategy, since it is more energy-efficient to blunt vasoconstrictor influences than to synthesize vasodilators.
Fig. 10Myocardial oxygen balance in normal and MI swine. Shown are the actual relations between MVO2 and CVPO2 in 30 normal swine (open circles) and 20 MI swine (open triangles) under control conditions. In addition, we have depicted the computed relations in MI swine if the ET (solid diamonds) and ANG II (solid squares) vasoconstrictor influences (which were both attenuated in MI swine) and the KATP (solid triangles) vasodilator influences (which were enhanced in MI swine) would have been identical to those in normal swine. The graph clearly illustrates that the adaptations in coronary vasomotor control act to blunt perturbations in oxygen balance in remodelled myocardium of swine with a recent MI
Fig. 11Alterations in vasomotor balance in the coronary resistance vessels within remodelled myocardium in swine with a 2–3-week-old myocardial infarction
Although the shift in coronary tone towards vasodilatation acts to blunt the flow perturbations in the remodelled heart, it does not appear to fully restore myocardial oxygen balance. This is also supported by the observation that KATP channel activation is increased, consistent with the presence of metabolic distress. Whether these perturbations in myocardial oxygen delivery blood are sufficiently severe to contribute to the progressive deterioration of contractile function of the remodelled left ventricle remains to be established, but it is of interest to note that we previously found that troponin I proteolysis occurred in remodelled myocardium, which was associated with a loss of myofilament force development [97]. Intermittent myocardial ischemia, as may occur in remodelled hearts during exercise or excitement, has been shown to be able to promote troponin I proteolysis [38] and could thereby mediate the flow perturbation-induced progressive deterioration of LV function after MI. Definitive proof of our hypothesis must await future studies demonstrating that prevention of such flow perturbations will indeed prevent progressive loss of contractile function of the remodelled porcine left ventricle.

swine
myocardial infarction
coronary blood flow
myocardial oxygen balance
exercise


Simultaneous prescribing of atypical antipsychotics, conventional antipsychotics and anticholinergics—a European study

Objective The aim of this study was to investigate to what extent atypical antipsychotics, conventional antipsychotics and anticholinergics are prescribed simultaneously in daily clinical practice in Europe.
Introduction
All antipsychotics are solely registered for use as monotherapy. The prescription of two or more antipsychotics simultaneously, also called antipsychotic polypharmacy, has no empirical foundation [1]. However, in the past antipsychotic polypharmacy with two conventional (‘early generation’) antipsychotics was frequently practised with reported frequencies ranging from approximately 10–69% [2]. In the last years the conventional antipsychotics are increasingly replaced by the atypical (‘second generation’) antipsychotics. Few studies from the UK and the US have focused on antipsychotic polypharmacy with atypical antipsychotics and reported frequencies ranging from 13% to 68% [3–7].
We carried out a pharmaco-epidemiological study to atypical antipsychotic polypharmacy and included data of patients of several psychiatric hospitals in various European countries. Additionally, we also focused on the co-prescription of anticholinergic drugs. There is considerable evidence that atypical antipsychotics cause fewer extrapyramidal side effects than conventional antipsychotics [8]. Therefore, there should be less need for anticholinergic co-medication to be prescribed with the atypicals.
This European study was conducted to investigate to what extent atypical antipsychotics, conventional antipsychotics and anticholinergics are being prescribed simultaneously in daily clinical practice.
Method
The study was carried out with the co-operation of members of the ‘European Pharmacists for Psychiatry and Neurology’ (EPPN), an organisation of hospital pharmacists working in psychiatry and neurology. All participating hospital pharmacists were working in psychiatric hospitals. Data were collected between November 1998 and October 1999. In that period the hospital pharmacists screened their hospital pharmacy database on one single day for patients who had been using an atypical antipsychotic for a minimum of 6 weeks. The 6-week period was chosen so that titration to the atypical antipsychotic should have been completed. No restrictions were imposed with regard to age and diagnosis.
The following data were collected for each patient included: age, sex, type and dose of the atypical(s) and, if prescribed, type and dose of conventional antipsychotics (maximum of four) and the type of anticholinergics (maximum of four). “As needed” (PRN) medication was considered as “used” medication. The doses of antipsychotic depot-medications were recalculated to daily oral doses [9]. All data were checked for improbable doses, duplication of patients and other errors.
Median doses of the antipsychotics are given, because of positively skewed distributions. The frequencies with which antipsychotic polypharmacy was practised and anticholinergics were prescribed are calculated for the total group of patients and per individual atypical antipsychotic.
Conventional antipsychotics with a chlorpromazine equivalent dose of more than 40 mg (compared to 100 mg chlorpromazine) are considered to be low potency drugs [10, 11]. The frequencies with which antipsychotic polypharmacy was practised are calculated with and without the low-potent antipsychotics.
Results
Hospital pharmacists from six West European countries participated: Belgium, Denmark, France, Germany, The Netherlands and Scotland. In total, data were received for 2,765 patients. Of these, 40 patients (1.5%) were excluded for the following reasons: patients mentioned twice (n = 10), no atypical antipsychotic prescribed (n = 8), improbably low clozapine doses (n = 5) and other errors (n = 17).
Table 1 shows details of the participating countries and patients. Most patients came from Belgium (40.9%), followed by The Netherlands (26.2%) and France (16.5%).
Table 1Number of hospitals and patients included in the participating countries with the mean age of patients and the percentages of male patientsCountryNumber of hospitals (%)Number of patients (%)Mean age (sd)% of malesBelgium19 (42.2%)1115 (40.9%)47.9 (15.6)59%The Netherlands5 (11.1%)715 (26.2%) 46.1 (17.9)54%France10 (22.2%)449 (16.5%)42.3 (14.5)61%Denmark4 (8.9%)217 (8.0%)48.1 (16.4)52%Scotland6 (13.3%)183 (6.7%)45.8 (17.7)51%Germany1 (2.2%)46 (1.7%)38.5 (13.9)60%Total45 (100%)2725 (100%)46.2 (16.4)57%
Table 2 shows the frequencies with which the atypical antipsychotics were prescribed, the median dose, the frequencies with which of antipsychotic polypharmacy (with and without the low-potent conventional antipsychotics) was practised, the frequencies with which anticholinergics were administrated and the frequencies of no co-prescription of antipsychotics or anticholinergics.
Table 2.Details of the atypical antipsychotics prescribed: their frequencies; median dose; the frequencies of patients prescribed antipsychotic polypharmacy (with and without low-potent antipsychotics) and anticholinergics; the frequencies of no co-prescription of antipsychotics and anticholinergics (n = 2,725)Atypical antipsychoticNumber of patients (%)Median dose (mg) Antipsychotic polypharmacy (%) (including low-potent antipsychotics)Antipsychotic polypharmacy (%) (excluding low-potent antipsychotics)*Anticholinergic prescription (%)No co-prescription of antipsychotics or anticholinergics (%)Risperidone1095 (40.2%)4.045.124.736.542.0Clozapine683 (25.1%)400.031.218.924.557.7Olanzapine562 (20.6%)15.037.022.120.356.2Sulpiride133 (4.9%)300.049.225.831.141.7Amisulpride132 (4.8%)600.054.123.345.928.6Sertindole37 (1.4%)16.062.224.327.029.7Quetiapine16 (0.6%)550.050.012.512.543.8Zotepine2 (0.1%)225.0000100Two atypicals65 (2.4%)********40.0****Total2725 (100%)–42.1%24.4%30.1%47.1%* See Table 3 for the classification of the low-potent antipsychotics** Total median dose not calculated*** By definition 100%**** By definition 0%
Risperidone was prescribed most often (40.2%), followed by clozapine (25.1%), olanzapine (20.6%) and others (in total 14.1%). For 65 patients (2.4%) 2 atypical antipsychotics were prescribed; the combinations used most were clozapine/risperidone (n = 18), olanzapine/risperidone (n = 12) and sulpiride/risperidone (n = 11). In our population of patients using an atypical, antipsychotic polypharmacy was prescribed in 42.1% of cases. Of patients receiving antipsychotic polypharmacy 31.7%, 8.5%, 1.3% and .6% were prescribed two, three, four, and five or more antipsychotics respectively.
Table 3 shows the antipsychotics prescribed concurrently with the atypicals. The conventional antipsychotics prescribed most often were levomepromazine, haloperidol, prothipendyl, cyamemazine and zuclopentixol. When the low-potent conventional antipsychotics are not included in the analysis, 24.4% of the patients was prescribed antipsychotic polypharmacy.
Table 3The frequencies and median doses antipsychotics prescribed concurrently for patients using atypical antipsychoticsAntipsychoticAntipsychotic prescribed concurrently (%) Median dose (mg)Levomepromazine*16.6%50.0 Haloperidol11.2%10.0 Prothipendyl*9.5%80.0 Cyamemazine*7.1%100.0 Zuclopenthixol7.6%28.5 Pipamperon*5.6%80.0 Dehydrobenzperidol4.7%10.0 Thioridazine*4.4%100.0 Clotiapine4.4%40.0 Chlorpromazine*3.1%200.0 Flupenthixol2.7%5.9 Fluphenazine2.4%9.0 Bromperidol2.4%7.5 Chlorprothixeen*1.8%100.0 Perphenazine1.5%16.0 Pimozide1.4%4.0 Other conventional antipsychotics9.2%–Second atypical4.2%–Total100.0%–* Considered as low-potent antipsychotic
In total 30.1% of the patients were prescribed an anticholinergic drug. The anticholinergics prescribed most often were biperiden (28.3%), procyclidine (19.9%), trihexyphenidyl (16.7%) and tropatepine (13.4%).
In total 47.1% of the patients are prescribed the atypical antipsychotic without any other antipsychotic or anticholinergic, 22.8% are prescribed an atypical plus one or more other antipsychotics, 10.8% are given an atypical plus an anticholinergic and 19.3% an atypical plus an anticholinergic and one or more other antipsychotics.
Discussion
This European study clearly shows that it is common clinical practice to prescribe an atypical antipsychotic and one or more other antipsychotics simultaneously. Low-potent antipsychotics may be added to an atypical antipsychotic mainly because of their sedative, anxiolytic and anticholinergic properties, rather than because of their antipsychotic properties. Even when the low-potent conventional antipsychotics are not included in the analysis, we found that antipsychotic polypharmacy was prescribed for almost a quarter of the patients. Additionally, we did not expect to find that almost a third of the patients were prescribed an anticholinergic in addition to the atypical drug. The results of this study imply that more than half (52.9%) of the patients are not being treated in the way according to the psychiatric handbooks and guidelines: The atypical is frequently combined with another antipsychotic and/or an anticholinergic.
Several studies have shown that antipsychotic polypharmacy with two conventional antipsychotics was in frequent use in clinical practice [2]. Others have shown that fairly large numbers of patients (ranging from 18.5% to 56%) are being prescribed a therapy that combines clozapine with a conventional antipsychotic [12–14]. Recently, a few studies have focused on antipsychotic polypharmacy with the atypical antipsychotics, with reported frequencies ranging from 13% to 68% [3–7]. There could be several reasons to explain this large variation in co-prescribing such as the definition of antipsychotic polypharmacy, whether inpatients or outpatients were included, stage of the disease, the differences in prescription patterns between countries and hospitals and different use of guidelines.
We consider several hypotheses for the relatively high rate of antipsychotic polypharmacy involving atypicals. First of all, it could be that the patients with an inadequate treatment response to one antipsychotic are treated with combinations of antipsychotics, including the atypical antipsychotics. However, there are hardly any studies that show that antipsychotic polypharmacy is effective in patients who do not respond to one antipsychotic [1].
Secondly, it is also possible that antipsychotic polypharmacy is continued although the patient shows no improvement because psychiatrists are hesitant to discontinue any medication in patients with persistent psychotic symptoms.
Thirdly, it could be that in some patients the recommended doses of the atypical antipsychotics are too low to be effective and that adding a second antipsychotic is in fact a dose-increase strategy. In such cases, however, a higher dose of one particular atypical might be just as effective.
Fourthly, when switching between two antipsychotics it is common to titrate the first drug downwards while simultaneously titrating the second drug upwards. If the patient responds halfway through the titrating process the clinician might decide to continue both antipsychotics—a situation referred to as “the cross-titration trap” [15]. Our study included only patients who had been using an atypical antipsychotic for more than 6 weeks, so the titrating process would normally have been completed.
It is remarkable that the potential risks of antipsychotic polypharmacy have received limited research attention [16]. However, Waddington et al. [17] found that antipsychotic polypharmacy is associated with reduced survival among patients with schizophrenia. Centorrino et al. [16] reports that the risk of adverse effects is 56% higher with antipsychotic polypharmacy.
There have been previous reports of anticholinergics being added to atypical antipsychotics. Of patients using clozapine 19% [12] and 37% [14] were also prescribed anticholinergics. Anticholinergics were added to 54.9% of patients using atypical antipsychotics [3]. These numbers are in line with our finding (30.1%). The prescription of this combined therapy could be attributed to the use of a second, almost always conventional, antipsychotic (19.3%), which might induce extrapyramidal symptoms. But nevertheless, 10.8% of patients are prescribed an atypical antipsychotic in combination with an anticholinergic without any other (conventional) antipsychotics. Minzenberg et al. [18] studied the effects of anticholinergic properties of psychiatric medications and found clinically significant effects on memory and complex attention.
The frequencies of atypical polypharmacy in the separate countries range between 26.1% (Germany) and 49.1% (Belgium), without the low-potent antipsychotics it varies between 17.1% (Denmark) and 31.6% (Belgium). However, these numbers should be appraised cautiously because this study was not primary set up to detect differences between the various countries (some countries are under represented).
Furthermore, the study does not allow to draw inferences about differences between the various atypicals in antipsychotic polypharmacy and anticholinergic use. These differences could easily be biased as a result of the different introduction data of the atypicals in the diverse countries. For example, at the moment of data-collection olanzapine and sertindole had been introduced very recently in most countries, quetiapine was available only in Scotland and zotepine was used only in a clinical trial.
Nevertheless, the similarity between the frequencies with which antipsychotic polypharmacy is prescribed for the various atypicals (excluding quetiapine and zotepine, on account of their low frequencies) is remarkable: Excluding low-potent antipsychotics the frequencies range from 18.9% to 25.8%.
The study however has its limitations.
First of all, the population included is not a random sample from Western European countries that participated. Therefore, hypothetically it could be argued that the results were biased because only hospitals practising extremely high rates of antipsychotic polypharmacy were included. However, it is unlikely that only such hospitals were selected.
Secondly, we estimate, on the basis of the information obtained from the participating hospitals, that 90% of the data are from inpatients. The symptomatology of inpatients may be more severe than that of outpatients, and such patients may be prescribed antipsychotic polypharmacy more frequently than outpatients [3].
Thirdly, we did regard PRN-medication as “used” medication. This may have heightened our results somewhat but cannot explain the frequent prescription of antipsychotic polypharmacy. A major advantage of this study is the large number of patients included and the differentiation made between high- and low-potent antipsychotics. This makes extrapolation more feasible.
Conclusion
It is common practice to prescribe a combination of atypical and conventional antipsychotics. Furthermore, the use of atypical antipsychotics does rule out the use of anticholinergics. On the contrary, atypical antipsychotics are often prescribed in combination with anticholinergics. Apparently, monotherapy involving the atypicals is not considered to be an effective therapy for a substantial number of patients in clinical practice.
Possible conflicts of interest
P.N. van Harten gives lectures on symposia and congresses that are sponsored by pharmaceutical companies.

anticholinergics
europe
polypharmacy
pharmaco-epidemiology
antipsychotic agents
drug therapy combination
drug use study


Prevention and treatment of renal osteodystrophy in children on chronic renal failure: European guidelines

Childhood renal osteodystrophy (ROD) is the consequence of disturbances of the calcium-regulating hormones vitamin D and parathyroid hormone (PTH) as well as of the somatotroph hormone axis associated with local modulation of bone and growth cartilage function. The resulting growth retardation and the potentially rapid onset of ROD in children are different from ROD in adults. The biochemical changes of ROD as well as its prevention and treatment affect calcium and phosphorus homeostasis and are directly associated with the development of cardiovascular disease in pediatric renal patients. The aims of the clinical and biochemical surveillance of pediatric patients with CRF or on dialysis are prevention of hyperphosphatemia, avoidance of hypercalcemia and keeping the calcium phosphorus product below 5 mmol2/l2. The PTH levels should be within the normal range in chronic renal failure (CRF) and up to 2–3 times the upper limit of normal levels in dialysed children. Prevention of ROD is expected to result in improved growth and less vascular calcification.
Introduction
Chronic renal failure is associated with specific abnormalities of skeletal homeostasis, commonly called renal osteodystrophy (ROD), which if not treated appropriately during the critical phases of skeletal growth can result in bone deformities and a disturbed growth pattern. The main factors for the development of ROD are disturbances in the calcium phosphate homeostasis, in vitamin D and parathyroid hormone metabolism as well as alterations in the somatotroph axis, i.e., on the endocrine and paracrine levels. In recent years it has been recognized that the spectrum of renal bone disease covers ‘high-’ as well as ‘low-turnover’ conditions. As a consequence of chronic renal failure itself and of the treatment of renal bone disease, high plasma phosphate levels and an elevated calcium phosphorus product are common. These are important risk factors for the development of vascular calcification and cardiovascular morbidity and mortality in young adults who have been on renal replacement therapy since childhood [1, 2]. Because aluminium-containing phosphate binders are no longer indicated in children, aluminium-related osteopathy is not considered in these recommendations.
The European Pediatric Peritoneal Working Group (EPPWG) was established in 1999 by pediatric nephrologists with a major interest in peritoneal dialysis and has, among others, published guidelines on chronic and acute peritoneal dialysis [3, 4, 5, 6]. The group incorporates pediatric nephrologists from 12 European countries. One of the functions of the group is to establish expert guidance in important clinical areas associated with chronic renal failure and dialysis [now the European Pediatric Dialysis Working Group (EPDWG)] in conjunction with other members of the multidisciplinary team. These guidelines were initiated and discussed at meetings of the group and developed by e-mail discussion to develop consensus of opinion based upon cumulative clinical experience and reported studies.
Recommendations
Recommendation 1
Clinical, biochemical and radiological markers of renal bone disease should be monitored regularly. The clinical markers of renal bone disease to be prevented are signs of overt rickets, slipped femoral epiphysis and disturbances of growth. The biochemical markers are plasma phosphate, calcium, alkaline phosphatase, bicarbonate and intact parathyroid hormone (PTH). The minimal frequency of measurements (and target ranges) for biochemical markers in a stable phase vary according to renal function (Table 1). If the patient has active ROD, additional blood samples may be required. Table 1 Frequency of measurements for biochemical and radiological markers of renal osteodystrophyMarker1Frequency of measurement (every x month)TargetGFRGFRGFR <15, dialysis59–3029–15(Evidence)Calcium or631Normal range (corrected calcium)2Ionized calciumNormal range2Phosphate631Normal range for age bandCalcium phosphorus product631≤5.0 mmol2/l2Target range 3.3–4.4 mmol2/l2, 3Alkaline phosphatase631Normal range for age bandSerum bicarbonate/base excess631Normal range, at least: bicarbonate >22 mmol/lBase excess >−5 mmol/lIntact PTH/whole PTH631Normal range in moderate CRF (GFR>29 ml/min/1.73m2)Up to 2–3 times upper limit of normal in advanced CRF or on dialysis25-(OH) vitamin D3As indicated4As indicated4As indicated4>20 ng/lLeft hand and wrist X-ray6–12No radiological signs of hyperparathyroidismNo Looser zones or osteopeniaCalcium: mmol/l in mg/dl: ×4, phosphate mmol/l in mg/dl: ×3.0969; calcium phosphorus product: mmol2/l2 in mg2/l2: ×12.387. 2Corrected calcium (mg/dl) = measured calcium concentration (mg/dl) + 0.8 × [4-measured albumin concentration (g/dl)]; corrected calcium (mmol/l) = measured calcium concentration (mmol/l) +0.2 × [4-measured albumin concentration (g/dl)]. 3Depending on age. 4Only in patients with suspected vitamin D deficiency
Plasma calcium must be adjusted for albumin levels (= corrected calcium) or by measuring ionized (free) calcium, in patients with hypoalbuminemia or acid-base disorders. Ionized calcium values are often available on blood gas analysis systems with calcium-sensitive electrodes, but correct values are only obtained in validated conditions. Alkaline phosphatase as a marker for osteoblast activity has particular importance in both low and high-turnover bone disease with elevated or low alkaline phosphatase serum levels, respectively.
PTH should be monitored monthly in advanced CRF (GFR <15 ml/min/1.73m2) because of its rapid changes. Different assays are available for the measurement of PTH. In principle, intact PTH(1–84) (iPTH) should be measured because it is believed to represent the active hormone. For this purpose, two-site assays were developed at the end of the 1980s [7]. However, PTH fragments accumulating in end-stage renal disease may also show biological activity. Furthermore, recent research using plasma samples from healthy and renal patients analyzed by HPLC and the iPTH assay demonstrated cross-reactivity of the intact PTH-assay with a PTH-fragment (PTH7–84). Therefore, new assays were developed that detect only PTH molecules with a complete N-terminal end, called “whole-PTH” assays [8]. In adult and pediatric dialysis patients, these “whole PTH” assays yielded 30–60% lower PTH levels than the intact PTH assay. Despite theoretical considerations, no significant improvement in the distinction between the different forms of renal bone disease using the “whole PTH” assays has yet been demonstrated in clinical practice [9, 10, 11, 12].
Radiological signs of renal bone disease, which alone are not sensitive enough to indicate therapy-adaptations, include signs of hyperparathyroidism and growth zone lesions. Periosteal resorption zones and metaphyseal changes are the most obvious signs. In late adolescent and young adult patients, Looser zones as specific signs of osteomalacia may be found. The sites and expression of bone lesions are age-dependent according to the age-dependent growth and remodeling at different sites of the skeleton. In pre-school children, the metaphysis of the distal radius and ulna may present only minor abnormalities, whereas more severe lesions may be seen at the upper and lower femoral epiphysis. Adynamic bone disease shows no specific radiological changes. However, extraosseous calcifications, fractures or osteopenia may indicate adynamic bone disease.
Histological evaluation of bone biopsy specimens remain the “gold standard” in assessment of renal osteodystrophy. However, due to its invasive nature, bone biopsies are not performed in clinical practice, but they remain an important tool for research [12].
Recommendation 2
Metabolic acidosis should be corrected (evidence). Chronic metabolic acidosis leads to increased bone resorption and inhibits endochondral bone formation in animal experiments [13, 14]. Infants with isolated chronic metabolic acidosis show growth retardation [14]. In adult hemodialysis patients, negative aspects of chronic metabolic acidosis on metabolism have been reported [15]. Therefore, metabolic acidosis should be corrected by a stepwise approach: The first step is optimization of the dialysis regimen both for HD- and PD-patients [4, 15]. Since the introduction of HCO3-based PD solutions [16], metabolic acidosis is a less frequent problem. In combination with a daytime-dwell, patients may even be at risk for alkalosis [17]. However, if these adaptations do not result in correction of the metabolic acidosis, sodium bicarbonate should be administered orally. Whenever possible, formulations that dissolve in the small intestine should be used. The use of sodium citrate increases the risk of aluminium absorption [18, 19]. Acidosis should be corrected to the normal range of the local laboratory.
Recommendation 3
The plasma-phosphate level should be kept within the normal age-specific range (evidence). In children with chronic renal failure, hyperphosphatemia is observed at GFR levels below 40 ml/min/1.73m2 [20] and almost always in children on dialysis. Hyperhosphatemia has several deleterious effects on PTH secretion [21, 22], parathyroid cell proliferation [23] and soft tissue (vascular) calcification [1, 2].
Increased phosphate levels stimulate PTH secretion in vitro [24] and in vivo [21, 25] independent of calcium and 1,25(OH)2 vitamin D3 levels. This effect of phosphate on PTH secretion occurs via post-transcriptional processes by regulating pre-pro-PTH mRNA stability [26]. Furthermore, a reduction of serum-phosphate results in a parallel reduction of PTH levels without change in serum calcium [27]. Hyperphosphatemia accelerates parathyroid cell proliferation [23], which can result in nodular hyperplasia and severe hyperparathyroid bone disease necessitating parathyroidectomy (see below).
Increased plasma phosphate levels have a profound effect on soft tissue and vascular calcification, which are often observed in young patients on or even after dialysis [1, 2, 28], increasing the risk of cardiovascular morbidity and mortality. Block et al. [29] found an increased risk of death for serum phosphate exceeding 2.09 mmol/l in adult patients; other studies described a much lower phosphate level (~1.4–1.6 mmol/l) at which a higher incidence of calcifications was found [1, 30, 31]. The histology of the calcification pattern is quite distinct from the process of atherosclerosis. In renal patients, extensive calcification of the tunica media occurs even after short periods of dialysis, as shown by investigations of the epigastric artery in adult patients [32]. Experimental work gave evidence for an active role of phosphate in this process as phosphate induces osteoblastic marker followed by calcification in cultured smooth muscle cells [33]. Dialysis patients are at risk of heart failure. Although this is a multifactorial process, it was demonstrated in animal experiments that high plasma phosphate levels per se accelerated cardiac fibrosis [34].
Recommendation 4
If plasma phosphate is elevated, phosphate intake should be limited to the recommended levels. Dietary intake of protein and as a consequence of phosphate in western countries usually exceeds the recommended intake at least in adolescent dialysis patients. Therefore, dietary counseling should be performed by a trained dietician [35]. If the dietary records show a protein intake above the recommended level for pediatric dialysis patients, the patients and their parents should be trained to reduce their phosphate intake. This may start with the reduction of dairy products and/or substitution with special low-phosphorus products and a reduction of meat intake.
Recommendation 5
In case of hyperphosphatemia, the dialysis efficacy should be optimized (evidence). Phosphate is slowly transported via the peritoneal membrane into the dialysate [36]. The dialysate to plasma ratio (D/P) for phosphate is volume and time related; after 4 h in standard peritoneal equilibration tests, this ratio reaches the value of 0.5–0.6, which is a much lower ratio than for urea, which is usually nearly 1. Therefore, an increase in phosphate removal should consider not only an increase in dwell volume to 1,000–1,400 ml/m2 BSA, but also dwell time optimization, avoiding a too short dwell time. A daytime dwell should be added. The details of a dialysis regimen optimization has been described elsewhere [5]. However, due to the low clearance of phosphate achievable even with an optimized PD regimen, dietary phosphate restriction and the use of oral phosphate binders are almost always necessary.
The dialytic phosphate removal kinetic during a hemodialysis session differs from urea kinetic: after an early initial drop the phosphatemia decrease only slowly [37, 38]. Therefore, it is admitted that the hemodialysis phosphate purification capacity is directly impacted by the duration of the dialysis session, because the phosphate shift from the intracellular to the vascular compartment is time-dependent. Therefore, longer dialysis sessions [39] or daily dialysis sessions [40, 41, 42] offer a unique, powerful dialysis method for optimal phosphate purification. The hemodialysis modality, HD, HF or HDF, and the type of the synthetic membrane used have only a limited impact on dialytic phosphate removal [38], whereas the dialysate composition in terms of glucose and bicarbonate concentration are presumed to be of importance [42, 43, 44], modulating a shift of intracellular phosphate trapped in the cells, where it is not available for dialysis exchanges.
Recommendation 6
For control of hyperphosphatemia, aluminium-free phosphate binders should be administered (evidence). Phosphate binders are necessary to reduce phosphate absorption from the gut. Ca-containing phosphate binders, i.e., calcium carbonate (CaCO3, elemental calcium content 40%) or calcium acetate (CaAc, elemental calcium content 25%) should be used as the first line. No data are published on the efficacy and safety of calcium acetate/magnesium carbonate compound phosphate binders in pediatric patients. The upper intake level of elemental calcium is suggested to be 2,500 mg/day for healthy children above 4 years of age [19]. Whereas for adult dialysis patients the DOQI guidelines suggest to limit the elemental calcium intake to 2,000 mg/day, no safe upper level can be given for the pediatric age band. However, a positive calcium balance in the range of +200–+300 mg in the growing skeleton should be maintained. In contrast, a too high calcium load should be avoided, because one of the identified risk factors for soft tissue calcification in pediatric CRI patients was cumulative calcium intake [2, 24].
Calcium-containing phosphate binders are started at approximately 500 mg per 200 mg phosphate content of the diet (0–1 years, 1–2×500 mg; 1–4 years, 2–3×500 mg/day; 5–8 years, 3–4×500 mg/day; 9–18 years, 5×500 mg). An alternative approach is a start dose of approximately 50 mg/kg/day, which is well below the doses given in clinical studies. Phosphate binders are then adjusted to normalize serum-phosphate and calcium. CaCO3 can be crushed to fine powder, or a 10% solution can be used for administration in infants, often via a feeding tube. The phosphate binders should be taken with meals, because fecal excretion of phosphate was higher when calcium acetate was given with meals instead of in between meals [45]. Biochemically, CaAc has a higher phosphate binding capacity, which is independent of the pH. The higher efficacy of CaAc, calculated on a weight basis compared to CaCO3, for phosphate control was also shown in clinical practice in adult and pediatric patients [46, 47]. Unfortunately, compliance of the patients with the intake of phosphate binders is often poor and should be checked regularly. It may be helpful if the patient is involved in selecting the flavor and size of the phosphate binder and has regular contact with the dietician at clinic appointments. Snacks during the day are often a source of additional phosphate and require additional phosphate binders.
Patients treated with Ca-containing phosphate binders and active vitamin D metabolites are particularly at risk for the development of hypercalcemia. Therefore, in case of hypercalcemia, active vitamin D metabolites should be stopped (see recommendation 12). If hypercalcemia, the most common side-effect of Ca-containing phosphate binder therapy, persists or the calcium phosphorus product exceeds 5.0 mmol2/l2 with the use of Ca-containing phosphate binders, the calcium content in the dialysis fluid should be reduced [5]. Furthermore, the dose of the calcium-containing phosphate binders should be reduced whenever possible or they should be replaced by Ca- and aluminium-free phosphate binders, because epidemiological studies have shown a direct relationship between serum phosphate and calcium phosphorus product on mortality [29, 48]. The only commercially available aluminium- and calcium-free phosphate binder is Sevelamer. This compound is usually taken orally as capsules, but anecdotally can be delivered via enteral tubes by dissolving the capsule in 5 ml water (instruction Fa genzyme). Aluminium-containing phosphate binders or calcium citrate increase intestinal aluminium absorption and should not be used in pediatric dialysis patients. An alternative phosphate binder that is available is lanthanum carbonate, which has a high affinity for phosphate, and is minimally absorbed in the intestine. In a randomized study in adult patients, lanthanum carbonate controlled plasma phosphate levels well and induced less adynamic bone disease than CaCO3 [49]. However, no long-term data on the effect of lanthanum on bone, on which surface lanthanum can accumulate [50], and its safety profile for use in children are available yet.
Recommendation 7
Vitamin D deficiency should be avoided (evidence). In early renal failure, an increase of PTH correlates positively with 25(OH)-vitamin D3 levels [51]. Renal synthesis of 1,25(OH)2 D3 is impaired in chronic renal disease. However, extrarenal cells, i.e., macrophages and osteoblasts, are also capable of 1,25(OH)2 D3 production [52]. In contrast to the kidney, the extra-renal synthesis is strictly substrate dependent. It has been shown that supplementation with vitamin D3 in elderly CRF patients with 25(OH)-vitamin D3 levels between 20 and 50 pg/ml decreased PTH serum concentrations [53]. Furthermore, 25-OH-vitamin D3, but not 1,25(OH)2 D3, improved muscular function and phosphate content [54]. Therefore, vitamin D deficiency should be prevented.
Recommendation 8
Marked hyperparathyroidism should be prevented in children with CRF prior to dialysis (evidence). Due to the risk of persisting bone disease and the development of parathyroid adenoma, an increase in PTH above normal to slightly elevated levels should be prevented in children with CRF. Therefore, low doses of active vitamin D metabolites should be given in time. In adult patients this treatment regimen has resulted in controlled iPTH without negative aspects [55]. In children with moderate renal failure (GFR >30 ml/min/1.73 m2), normal levels of PTH in association with strictly controlled phosphate levels are associated with a normal ratio of intact PTH to “whole” PTH and normal levels of alkaline phosphatase, indicating a physiological PTH secretion and unremarkable bone turnover [10]. In such patients, slight catch-up growth with PTH levels at the upper limit of normal was reported [56]. In a sub-group analysis, improved growth was restricted to patients with enteral feeding tubes.
Recommendation 9
PTH levels should be kept at two to three times the upper limit of the normal range in end-stage renal disease (evidence). Grossly elevated PTH concentrations (>four times the upper normal range) in the presence of normal or high serum calcium and high alkaline phosphatase are almost always associated with high-turnover bone disease [56, 57]. The exception is intermittent therapy with high-dose active vitamin D metabolites, because 1,25(OH)2 vitamin D3 directly inhibits osteoblastic function and proliferation. Hyperphosphatemia can occur in hyperparathyroid bone disease because of excessive bone resorption.
However, in pediatric dialysis patients low and even normal levels of PTH are reported to be associated with low turnover bone disease [57]. A PTH concentration higher than in healthy subjects is needed to stimulate bone turnover due to resistance of the skeleton to PTH in advanced renal failure. If PTH falls into the (low) normal range, the risk of hypercalcemia increases [58]. Hyperphosphatemia and hypercalcemia can develop in low turnover bone disease because the skeleton is unable to take up enough phosphate and calcium. Low bone turnover or adynamic bone disease is increasingly observed in adult [59] as well as in pediatric PD patients [56]. Risk factors for adynamic bone disease are high calcium intake (Ca-containing phosphate binders), therapy with high doses of active vitamin D metabolites, peritoneal dialysis using dialysis fluid with high calcium content and age (adolescents after the growth spurt). Biochemically, hypercalcemia and/or PTH levels within or below the normal range are indicative of low turnover bone disease [56, 58], which may adversely affect growth in dialysed children [60]. Therefore, active vitamin D therapy should be reduced or stopped if PTH falls below the low normal range [57, 59, 61]. If PTH remains below normal despite normal calcium and phosphate levels, the dialysate should be changed to low calcium solutions (1.0 or 1.25 mmol/l) to stimulate PTH secretion [61], if this is not already used as the standard solution [5].
Recommendation 10
If PTH is elevated in children with CRF or if PTH is elevated more than two to three times normal in the presence of Pi <2 mmol/l in dialyzed children, active vitamin D metabolites should be administered orally (evidence). Treatment with active vitamin D metabolites results in biochemical and/or histological improvement in patients with high-turnover bone disease. Doses of 1,25(OH)2 vitamin D3 or 1α-(OH) vitamin D3 usually range from 0.1 µg/day to 0.75 µg/day, with a starting dose of 20–40 ng/kg. High doses often induce hypercalcemia, necessitating the reduction or discontinuation of vitamin D therapy (see hypercalcemia).
As to the mode of administration of the active vitamin D metabolites, intravenous or oral therapy, have the same efficacy [63]. Therefore, the oral route should be preferred in children on dialysis. Doses should be given in the evening, because fewer episodes of hypercalcemia have been reported compared to taking the vitamin D metabolites in the morning [64]. Intermittent therapy was shown to be effective to suppress elevated PTH serum levels [59]. However, recent data indicate that large intermittent 1,25(OH)2 vitamin D3 doses adversely affect bone turnover [57, 61, 65] and chondrocyte activity [66] resulting in low turnover bone disease and in reduced growth [65, 66, 67, 68]. In addition, episodes of hypercalcemia occur with similar frequency with the intermittent or oral administration of vitamin D metabolites [63, 69]; furthermore, the intermittent mode of administration was not shown to be more effective in suppressing PTH levels in adult [70] and pediatric [63] patients in prospective, randomized trials. For the above mentioned reasons, intermittent high-dose vitamin D therapy should be avoided in pediatric patients.
Three newer vitamin D analogs, so called “non-hypercalcemic” vitamin D analogs (doxercalciferol, paricalcitol and 22-oxa-calciferol), have been introduced in clinical use in adult patients with secondary hyperparathyroidism due to CRF. Despite their ability to induce less hypercalcemia in animal experiments, only one clinical study with a head-to-head comparison of the new vitamin D analogue (paricalcitol) is available demonstrating a borderline reduction of hypercalcemia or a decrease in calcium phosphorus product [71]. No data are available for their use in children.
Agents that specifically enhance the sensitivity of the calcium-sensing receptor, called calcimimetics, used in combination with active vitamin D metabolites in adult dialysis patients resulted in a persistent decrease of PTH levels without elevated calcium phosphorus product [72]. If plasma Ca or Pi is high in the presence of elevated PTH, the calcimimetics may become first choice. However, no data for pediatric patients are available.
Recommendation 11
Treatment with growth hormone should not be started in the presence of severe hyperparathyroid bone disease. It is well established that the uremic growth retardation is at least in part due to disturbed pulsatile secretion of growth hormone as well as a peripheral growth hormone resistance [73]. After correction of metabolic acidosis and normalization of caloric intake, administration of recombinant human growth hormone increases growth velocity resulting in catch-up growth and in improved final height [74]. Growth hormone directly stimulates growth cartilage proliferation and metabolism and osteoblast activity [75, 76]. This growth promoting effect of GH was suspected to increase the metaphyseal instability induced by severe hyperparathyroidism. In hyperparathyroid bone disease, i.e., osteitis fibrosa, a fibrous layer is formed at the metaphyseal junction of the growth plate with the bone tissue. This can lead to epiphyseal slipping resulting in gross deformities of the affected long bones [77]. Therefore, parathyroid hormone, as well as calcium and phosphate levels, should be treated towards the recommended normal ranges prior to administration of GH. However, in a recent prospective study, no increased frequency of epiphyseal slipping associated with the use of growth hormone was reported [78]. After the start of GH administration, an increase of PTH levels was observed [79, 80].
Recommendation 12
In case of hypercalcemia, active vitamin D metabolites and calcium-containing phosphate binders should be stopped and dialysate changed to low calcium solutions (evidence). Hypercalcemia and elevated calcium phosphorus product (>5.0 mmol2/l2) should be corrected promptly (Fig. 1). Vitamin D metabolites should be discontinued and in case of persisting hypercalcemia the dialysate calcium decreased to low calcium dialysate. Laboratory values should be controlled weekly until improvement is seen. One exception is hypercalcemia due to severe hyperparathyroidism and high-turnover bone disease. In addition, in patients with residual renal function, loop diuretics, i.e., furosemide may increase calcium excretion. If hypercalcemia is not corrected despite the discontinuation of active vitamin D metabolites, calcium-containing phosphate binders should be replaced by calcium-free phosphate binders to reduce the calcium load. Hypercalcemia is reported in up to 25% of patients taking calcium-containing phosphate binders ([1, 29], see recommendation 6). Fig. 1 Clinical algorithm for treatment of elevated calcium phosphorus product in children with CRF. Ca-cont calcium containing; Ca-free calcium-free; phos-binder phosphate binder; vit D vitamin D; ADBD adynamic bone disease
Recommendation 13
The calcium phosphorus product should be kept within the normal range, at least below 5.0 mmol2/l2 (60 mg2/dl2) (evidence). One of the risk factors for cardiovascular morbidity was shown to be an elevated calcium phosphorus product above 5.0 mmol2/l2 (see also recommendation 2). If the calcium phosphorus product is elevated above this level, phosphate and/or calcium levels should be reduced. This may be achieved by an increase in phosphate binders, reduction/stopping of active vitamin D metabolites and use of low calcium (Ca++ 1.0–1.25 mmol/l) dialysate.
Recommendation 14
Parathyroidectomy has to be considered in case of severe, therapy-refractory hyperparathyroidism with radiological signs in combination with hypercalcemia and/or elevated calcium phosphorus product (evidence). Severe hyperparathyroidism may no longer react to even high doses of 1,25(OH)2 vitamin D3 and reduction of phosphate levels. The clinical observations in adults show that patients with at least one parathyroid gland larger than 0.5 cm3 or 1.0 cm in diameter usually do not respond to active vitamin D metabolites [81, 82]. Therefore, persisting grossly elevated PTH levels in the presence of high-dose active vitamin D metabolites, radiological signs of hyperparathyroidism on wrist X-ray and/or high serum calcium and normal phosphate and/or elevated calcium phosphorus product indicate the need for surgical parathyroidectomy. Medical parathyroidectomy by alcohol injection was described in adult patients [83], but to our knowledge not in pediatric patients. Parathyroid hormone stimulates bone turnover and regulates calcium homeostasis, both of which are of utmost importance in the growing skeleton. Therefore, subtotal parathyroidectomy or autotransplantation of parathyroid tissue is recommended in children or adolescents, avoiding hypoparathyroidism ([84], review). However, these procedures carry the risk of recurrence of severe parathyroid tissue hyperplasia. The use of the new calcimimetic agents [85] may help to prevent the most severe hyperparathyroidism and therefore modify the approach to that condition.

chronic renal failure
vitamin d
parathyroidectomy
secondary hyperparathyroidism
calcium-phosphate product