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Occasional survey
Renal excretion of endothelin in children
Istva´nMa´ttyus1, L. B. Zimmerhackl2, A. Schwarz2, M. Brandis2, M. Milte´nyi1, and T. Tulassay1
1First Department of Pediatrics, Semmelweis Medical University, Budapest, Hungary
2Albert-Ludwigs University, Freiburg, Germany
Received May 7, 1996; received in revised form January 14, 1997; accepted January 17, 1997
Abstract. Endothelin (ET) is a peptide with profound va-
soconstrictive potential. First isolated from porcine endo-
thelial cell supernatant, it is produced also by smooth
muscle, epithelial and circulating cells. Besides vasocon-
striction, a wide spectrum of biological activities of ET (via
activation of membrane receptors) has been described.
These include regulation of other hormones and neuro-
transmitters, cellular growth and proliferation, broncho-
constriction, and, in the kidney, natriuresis and water
diuresis. ET exerts its effects mainly in an autocrine and
paracrine fashion. A high concentration of ET is found in
urine, compared with plasma originating mainly from the
kidney itself. In this review we focus on the role of urinary
excretion of ET in children. ET excretion was determined
under different physiological and pathological conditions.
In premature infants and newborns, the daily excretion of
ET (corrected for body surface) was higher than in older
children; it was constant, and comparable to the values in
healthy adults after the age of 2 years. Renal ET excretion
correlated positively with urine flow in both healthy and
sick children. Conditions with tubular and/or collecting
duct cell damage, such as severe hypoxia, hemolytic-ure-
mic syndrome, renal transplantation, diabetes mellitus,
chronic renal failure, and contrast media cytotoxicity were
characterized by elevated urinary excretion of ET. In con-
clusion, the renal excretion of ET is influenced by several
factors, probably reflecting the intrarenal ET production.
ET has a low specificity with regard to renal injury.
Key words: Endothelin – Urine – Receptors – Synthesis
Introduction
In the mid 1980s Hickey et al. [1] found a potent vasoactive
substance in cultured endothelial cells. In 1988 Yanagisawa
et al. [2] described a new peptide isolated and purified from
cultured porcine endothelial cells, called endothelin (ET). It
is a 21-amino acid peptide with two disulfide bonds, whose
structure is similar to that of venom of the Israeli burrowing
asp. Two other isopeptides of ET have been characterized:
the initially isolated human/porcine ET is now termed ET-1
and the others are called ET-2 and ET-3. The latter two
differ from ET-1 in the 2 and 6 amino acid positions, re-
spectively [2]. Structure and activity studies have shown
that the two intramolecular disulfide bonds, hydrophobic
carboxyl terminal, and the hairpin loop configuration are
essential for their bioactivity [3]. They are encoded by three
separate genes found in the genome of all investigated
mammalian species, including humans. ET is not only
synthesized in endothelial cells, but also in smooth muscle
cells, epithelial cells, and circulating cells. Besides its
profound vasoconstrictive potential, ET modulates inotropy
and chronotropy, neurotransmission, and bronchoconstric-
tion, and increases vascular permeability, inhibits sodium
reabsorption in the renal collecting ducts, induces mito-
genesis, and regulates other hormones and cytokines [4, 5].
These effects are mediated through the activation of spe-
cific receptors identified in various target organs [6, 7].
Two subtypes of ET receptors are found: ETA and ETB.
The ETA receptor specifically binds ET-1, while ETB has
equal affinity for all three isoforms [6, 7]. The synthesis of
ET is regulated by a variety of stimuli, such as endotoxin,
anoxia, shear stress, arginine vasopressin, angiotensin II,
transforming growth factors, phorbol esters, and thrombin
over the last few years immunoreactive ET (IR-ET) has
been detected in different body fluids, including plasma,
milk, saliva, amniotic fluid, and urine [8]. The production,
secretion, and physiological effects of ET have been
studied extensively, but there are few studies regarding the
pathophysiological role of ET in the urine of children.
Thus, this review will focus on the recent advances in the
knowledge of ET in the urine of children and its relation-
ship to kidney function under physiological and patho-
physiological conditions.
Correspondence to: L. B. Zimmerhackl, Universita¨ts-Kinderklinik,
Mathildenstrasse 1, D-79106 Freiburg, Germany
Pediatr Nephrol (1997) 11: 513–521
IPNA 1997
Synthesis of ET
Although ET was initially described as a peptide released
from large vessel endothelial cells [2], other sites of
synthesis have subsequently been discovered. ET is also
expressed in brain, lung, heart, and kidney. Within the
kidney endothelial cells, glomerular, tubular epithelial, and
mesangial cells can all synthesize ET [4, 9]. The mature ET
peptide is generated through several steps. The best char-
acterized is the synthesis of ET-1. The preproET-1 gene,
which is located on chromosome 6, encodes a 212-amino
acid peptide. The expression of preproET-1 mRNA is
regulated by a mechanism involving receptor-mediated
mobilization of intracellular calcium (CA2+) and activation
of protein kinase C, which results in the synthesis of c-jun
protein. The binding activity of c-jun protein to the tran-
scription factor ap-1-responsive element of the ET-1 gene
increases, and this causes the induction of preproET-1
mRNA [10]. PreproET-1 is cleaved by an endopeptidase
forming a 38-amino acid intermediary structure called big
ET. The microtubular system is important in transferring
the synthesized ET-1 to the cell surface. In the next step big
ET is cleaved between the Tryp21-Val22 into the mature ET
form by a specific endopeptidase called ET converting
enzyme, which is a membrane-bound metalloprotease, de-
scribed by Xu et al. [11], which contains a 21-amino acid
transmembrane domain and a 9-amino acid zinc-binding
part. The distribution of the enzyme in the different tissues
is heterogenous. In the kidney, ET converting enzyme was
found in tubular epithelial cells and in vascular endothelial
cells, including the glomerulus.
The secretion of ET is regulated by a Ca2+-calmodulin
complex and by intracellular Ca2+ released from the Ca2+
store. ET release from endothelial cells is luminal and
contraluminal [12], but the concentration of ET in local
tissue may greatly exceed that of the blood. This suggests
that the relative release may differ and that local release of
ET may be primarily directed to the effector loci. Wagner et
al. [12] have shown that, both in basal and stimulated se-
cretion, about 80% of ET-1 is secreted towards the baso-
lateral direction of the cultured endothelial cell, which is
similar to the report of Uchida et al. [13] in MDCK cells.
Secreted basolaterally it can interact with receptors on the
underlying vascular smooth muscle or other cells with ET
receptors in a paracrine fashion, and the small distance
between basolaterally secreted ET and its effector may also
diminish the amount needed for effective concentration.
Receptors and intracellular signalling
It was observed that all three ETs have a diversity of
pharmacological activities, with different potencies in
various tissues (Table 1). Affinity crosslinking experiments
and radioligand binding studies have also supported the
existence of multiple subtypes of ET receptors [14, 15].
Two subtypes of ET receptors, termed ETA and ETB, have
been identified [7, 16], and there are reports of the possi-
bility of a third receptor called non-ETA/non-ETB [17, 18],
which may be a subtype of ETB [19]. ETA has a high af-
finity only for ET-1, while ETB binds to all three isoforms.
Both are in the cellular membrane, have seven transmem-
brane domains, and function by mediating intracellular
signal transduction through G-proteins. The mRNAs of
both ETA and ETB receptors can be detected in a number
of tissues, and the differences in expression of these re-
ceptors modulate the activity of ET [20]. ETA receptors are
found in smooth muscle cells of blood vessels and bronchi
[20], in myocardial and mesangial cells [21]. ETB receptors
have been detected in the endothelial cells of smaller blood
vessels in a number of organs, in glia and plexus cells of the
brain, in renal tubular epithelial and in smooth muscle and
myocardial cells. In the kidney, expression of ETA mRNA
was found in renal arteries and in the mesangial cells, with
the greatest expression in the afferent and efferent arterioles
(Table 1). Signals of ETB were also observed in the inner
stripe of the outer medulla, probably corresponding to the
vasa recta, and in the renal cortex, probably corresponding
to glomeruli [6, 20].
At least two intracellular signalling pathways for ET
action have been identified. ET increases membrane dia-
cyglycerol, thus activating protein kinase C, it also induces
phosphatidylinositol breakdown, which results in release of
Ca2+ from intracellular stores [22]. The increase in inositol
triphosphate and/or Ca2+ mobilizes extracellular Ca2+ by
activating voltage-dependent Ca2+ channels. The activation
of ETA receptors results in cellular contraction, via vas-
cular smooth muscle and mesangial cells [22]. ETB re-
ceptors were first found on the surface of endothelial cells;
ETB receptor activation leads to vascular dilatation. The
514
Table 1. Endothelin (ET) binding sites and ET mRNA in different parts of the nephron
ETA ETB Effect mRNA ET Ref.
Glomerulus arteriole afferent+efferent
smooth muscle + Muscle cell contraction,
vasoconstriction [25, 28]
Glomerulus mesangium + ET synthesis + NO synthesis (+) [40]
Glomerulus endothelium + ET synthesis + NO synthesis + [40]
Glomerulus epithelium + [34]
Proximal tubulus non-A- non-B? Inhibition of Na reabsorption [48]
Loop Henle + ? [66]
Distal tubulus (MDCK, LLCPK) + AVP antagonism via inhibition
cyclicAMP + [6, 48]
Collecting duct + AVP antagonism via inhibition
cyclicAMP 2+ [25, 28]
Vasa recta + + Vasoconstriction + ? + [24, 88]
NO, Nitric oxide; Na, sodium; AVP, arginine vasopressin
cellular mechanism for this effect is the same as for the
ETA receptor, i.e., activation of the constitutive Ca2+/cal-
modulin-dependent nitric oxide (NO) synthetase and
phospholipase A2[22, 23]. The released and newly syn-
thesized NO and prostacyclin mediate the depressor re-
sponse. ETB receptors are involved in the persistent in-
crease in preproET-1 expression, autoinduction of ET
synthesis, diuretic response, ET-induced cellular prolifera-
tion, and probably in the clearance of circulating ET-1 via
uptake in the lung [5, 20].
Regulating factors
ET is synthesized by many cell types and has many im-
portant effects in addition to vasoconstriction. Its release, or
the expression of the ET gene in various cell types, is af-
fected by several factors under normal and pathophysiolo-
gical conditions. Transforming growth factor-βand
thrombin increase ET mRNA levels and ET release from
endothelial cells and microdissected IMCD cells [24]. In
endothelial cell culture, ET-1 transcription is activated by
epinephrine, arginine vasopressin, bradykinin,
cyclosporin A (CsA), ethanol, and phorbol esters (activa-
tors of protein kinase C) [4, 20]. Ferri et al. [25] demon-
strated that insulin stimulated ET-1 release from cultured
endothelial cells in a dose-dependent fashion. ET-1 release
persisted for 24 h, and was also observed at physiological
insulin concentrations. This may effect de novo protein
synthesis, rather than ET-1 release from intracellular stores.
It was also demonstrated that acute moderate hypoxia was
significantly associated with elevated ET secretion in en-
dothelial and probably renal epithelial cells [26]. Morise et
al. [27] showed that, after injection of endotoxin, the signs
of renal dysfunction could be antagonized by anti-ET-1
antibody administration. Shear stress [28] enhanced ET
secretion by endothelial cells. Angiotensin II was found to
be the most potent stimulator of ET production and release
by endothelial cells [29, 30], and induced ET secretion in a
time- and dose-dependent manner. Most of the stimulators
of ET production and release are protein kinase C activa-
tors, suggesting that this enzyme plays an important role in
the regulation of ET synthesis.
Less is known regarding the factors suppressing ET
synthesis. NO modulates the actions of ET at several steps,
possibly by blunting the increase in intracellular Ca2+ via
interference with a postreceptor pathway [5]. It was also
shown that NO reduced the affinity of the ET receptor for
its ligand [31], possibly by reacting with the thiol group of
the receptor. ET-2 suppresses the synthesis of ET-1 via ETB
receptors through induction and release of NO and pros-
tacyclin [5]. ET-1 has an affinity for ETB receptors which
are activated during elevated synthesis and release of NO
and thus constitute an important mechanism for termination
of ET activity. Kohno et al. [32] demonstrated that natriu-
retic peptides inhibit the production of ET. The actions of
ET may also be affected by antagonizing ET converting
enzyme [5, 11].
Besides these regulating factors, ET has an unusual
means of regulating its own production. Hunley et al. [5]
described a rapid and long-lasting increase in preproET-1
gene expression after stimulation with ET-1. This en-
hancement of mRNA expression is mediated by ETB re-
ceptors, and can be blocked by an ETB antagonist but not a
selective ETA antagonist. These data show that the ETB
receptor also stimulates the synthesis of its own ligand,
which suggests that cells with ETB receptors, once stimu-
lated, need no further external source of ET-1 for persistent
ET-1 action. In different diseases activated endothelial and
mesangial cells may serve both as sources and as effector
sites for ET. Further investigations are needed to clarify
which factors are involved in the regulation of these com-
plex reactions; e.g., endothelial cell activation of the ETB
receptor may result in upregulation or, via NO production,
antagonism of ET synthesis.
ET in plasma
After the discovery of ET, several studies reported serum or
plasma ET levels in different pathophysiological circum-
stances, with conflicting interpretation [1, 4, 33]. The cir-
culating half-life of ET is short (2–3 min), and it is almost
completely metabolized in the pulmonary capillaries by the
enzyme neutral endopeptidase [34], probably by ETB-
mediated uptake [19]. Little circulating ET is cleared in the
urine. ET is synthesized by the endothelial cells, but as
most is released on the basolateral and not the luminal side,
local levels are different from circulating levels [12, 13].
Depending on the nature and magnitude of the stimulus,
circulating ET may be elevated in different pathophysio-
logical or experimental conditions. However, it is not clear
whether an elevated level of circulating ET is necessary for
its actions. There are reports of lower plasma ET levels
[35]. The effects of ET can be detected long after the
normalization of the plasma level. These data suggest that
the concentration of ET in the plasma does not correlate
with its activity in different cells and tissues, and that the
circulating levels depend on the balance of the luminal
(mostly) endothelial synthesis and metabolism.
Elevated plasma levels were observed in patients with
chronic renal failure [36, 37] and nephrological diseases
[33, 38], hemolytic-uremic syndrome (HUS) [39], early
infancy [40], preterm infants [40], during pregnancy [41],
and after renal transplantation [42].
ET in urine
After the discovery of ET, IR-ET was detected in human
urine [33, 43, 44] at levels greater than in plasma, although
it seemed to be unstable [8, 43]. More recently, Worgall et
al. [45] reported that IR-ET was stable in urine for more
than 24 h. Using specific radioimmunoassays, competitive
enzyme immunoassays, and high-pressure liquid chroma-
tography of urine extracts, the molecular form of urinary
ET was investigated by several researches. Recent data
show that most of the urinary IR-ET is big ET. Matsumoto
et al. [46] demonstrated that IR-big ET-3 and IR-big ET-2
were the major species in urine from healthy humans, and
IR-big ET 1 and IR-ET-1 were found in relatively low
concentrations. Large amounts of ET converting enzyme
515
were found in the kidney, suggesting that in the urine big
ET can be converted to ETand different pathophysiological
circumstances may facilitate this conversion without
changing the total amount of IR-ET. These data indicate
that the ET family peptides should be investigated with
specific assay methods, with particular attention to big ET
[46].
Source of urinary ET
Despite the higher concentration in urine than plasma, the
urinary excretion of ET does not correlate with the glo-
merular filtration rate (GFR), filtered load or plasma levels
[33]. Several investigators have found that the clearance of
125iodine-labelled ET-1 from the blood into the urine is very
low [34, 47]: less than 1% of the total radioactivity injected
was recovered in the urine. It was suggested that the filtered
ET-1 is subject to proteolytic degradation by neutral en-
dopeptidase along the brush border of the proximal tubuli
[34].
ET mRNA has been detected in endothelial cells of
different vessels, including glomerular capillaries, me-
sangial cells, and in different renal tubular cells [9, 48].
After stimulation of ET production by fetal calf serum and
transforming growth factor-β, the highest production was
found in the epithelial cells of collecting ducts of the inner
medulla [49]. Ujiie et al. [9] and Kohan [50] demonstrated
that inner medullary tubules produce significantly larger
amounts of ET than other nephron segments. Polymerase
chain reaction products of ET-1 mRNA were also detected
in this area. Renal epithelial cell lines (LLCPK and
MDCK) and glomerular epithelial cells synthesize and re-
lease ET-1 [49, 51]. Taken together these data suggest that
urinary ET is produced mainly in the kidney by several
different cell types, including mesangial, epithelial, and
endothelial cells.
An additional source of urinary ET may be the urinary
tract itself, anywhere from the calices to the bladder. The
preproET-encoding gene was found in the epithelial, en-
dothelial, and smooth muscle cells of the urinary bladder
[52]. The amount of ET secreted by the urinary tract is
small, but it may nevertheless contribute to the total urinary
ET.
Normal values in children
In adults there are several studies indicating that the normal
range of urinary ET excretion is between 20 and 100 ng/24 h
[33, 44, 53, 54] (Table 2). The reason for this wide range is
probably the difference in commercial ET kits used and in
the methods of urine extraction. Little information is
available on urinary ET excretion in children, either under
pathological or normal conditions. In our investigations
[55], the range of urinary ET excretion in healthy children
between 2.9 and 17 years of age was 59.3+3.68 ng/24 h
×1.73 m2body surface area (mean+SEM), which is within
the normal range measured in adults and by others [45].
Factors influencing urinary ET excretion in children
Influence of age – ET in newborns
Daily excretion of urinary ET was lower in younger than in
older children (Table 3). It correlated significantly with age
and the values corrected for body surface area remained
constant [55, 56]. These data suggest that there is a dif-
ference in secretion of ET between the various age groups,
which is predominantly due to body size and thus to dif-
516
Table 2. Normal values for urinary excretion of immunoreactive ET
(ng/24 h) (ng/24 h ×1.73 m2) Reference
Adults 77+5 [33]
41.5+17 [44]
27.3+3 [53]
91+16 [54]
Children
4–8 years 56.2+4.8 [40]
9–12 years 62.3+3.9 [40]
13–18 years 61.4+9.1 [40]
40.3 57.1 [45]
41.8+2.6 59.3+3.7 [55]
Newborns
Day 1 of life 28.1+4.3 [40]
Day 5 of life 53.6+3.0 [40]
Day 1 of life 20.5+6.8 Own observation
Day 5 of life 43.5+8.4 Own observation
Preterms
28 weeks of gestation 70.4+15.6 [56]
Days 1–3 of life
35 weeks of gestation 47.5+10.2 [55]
days 1–3 of life
ferences in kidney size. Thus we can compare urinary ET
excretion of different ages if we use values corrected for
body surface area [55] (Table 3).
Sanchez et al. [57] observed that the urinary ET-1
concentration was independent of the mode of delivery,
maturity, cardiorespiratory function, and intrauterine
growth of the neonate. In premature neonates we found
elevated urinary excretion [55], with a lower concentration
of urinary ET. The very low-birth-weight neonates of less
than 28 weeks’ gestational age were on mechanical venti-
lation. Therefore the influence of the respirator or increased
physical stress on ET excretion cannot be ruled out com-
pletely. In full-term healthy newborns, the urinary ET
concentration was higher on the 1st day than later, but the
ET excretion at 24 h corrected for body surface area was
lower than at 1 week ([40]; Mattyus et al., own observa-
tions). The elevation in daily excretion may reflect the
higher diuresis. The ET excretion of newborns was higher
(than of older children).
This difference can be explained by several factors. It is
known that ET has a growth-promoting and mitogenic ac-
tivity [4, 58, 59]. Such proliferative effects have been
shown in vascular smooth muscle and glomerular me-
sangial cells. 3H-thymidine uptake and cell number in-
dicated that the mitogenic effect of ET was greater than of
any other vasoconstrictor tested [60]. In different cell lines
of cardiac myocytes, fibroblasts, and smooth muscle cells,
ET stimulated DNA and protein synthesis [61]. Fant et al.
[58] found that ET stimulated the growth of the placenta,
suggesting that ET probably has an important role in the
regulation of fetal cellular development. Gro¨ne et al. [62]
reported that in human fetal renal tissue the total number of
ET binding sites was higher than in adult kidneys. These
data suggest that ET might have a growth-promoting effect
in developing kidneys and may mediate some of the
changes in glomerular and tubular functions observed in the
first days of extrauterine life. ET excreted in the urine is
mostly synthesized by renal cells [4, 55, 63]. The elevated
urinary excretion may indicate a higher synthesis in de-
veloping renal tissue, mainly in tubular epithelial cells.
This would also explain the elevated levels in preterm in-
fants.
Influence of diuresis
The total amount of ET excreted in the urine is greatly
influenced by diuresis (Table 3). We demonstrated that
forced diuresis significantly enhanced urinary excretion of
ET, while the amount of creatinine excreted remained un-
changed [55]. Urinary ET not only correlated with forced
but normal diuresis. This correlation was seen in newborns,
diabetic children, and in different nephrological diseases.
Sulyok et al. [40] and Worgall et al. [45] also found a
positive correlation between ET excretion and diuresis in
children with renal disease and in healthy controls.
However, the clinical interpretation of the flow depen-
dence of urinary ET excretion needs further clarification.
The elevated urine flow rate may be either the cause or the
result of enhanced ET-1 excretion. ET-1 synthesized mostly
by the epithelial cells of the inner medullary collecting
ducts, has an inhibitory effect on vasopressin-stimulated
cyclic AMP generation and water permeability in the same
cells [22, 64, 65]. In experiments with the ETB receptor
agonist sarafotoxin 6-c [17], a dose-dependent diuresis,
increase in free water excretion, and decrease in urine os-
molality, despite modest renal vasoconstriction, was ob-
served. These data suggest that ET-1-induced diuresis is
mediated by ETB receptors. However, the higher diuresis
might induce elevated ET synthesis in tubular cells of
collecting ducts, resulting in enhanced urinary excretion of
ET. Thus further investigations of the mechanism by which
diuresis influences renal ET excretion are needed.
Renal ET excretion in diabetes mellitus
According to more recent data on the role of ET in vascular
disturbances and cellular proliferation [5, 18], it may be
involved in the pathophysiological process of diabetic an-
giopathy and nephropathy. Ferri et al. [25] showed that
insulin stimulated ET-1 secretion from human endothelial
cells; this interaction could play a significant role in the
development of atherosclerotic lesions and nephropathy
under hyperinsulinemic conditions. There are some studies
on the urinary excretion of ET in adult patients with dia-
betes mellitus. Totsune et al. [53] did not find any change in
urinary ET, whereas others reported elevated ET excretion
[66]. In diabetic rats the urinary excretion of ET was ele-
vated [67]. We measured the renal ET-1 excretion in dia-
betic children under stable metabolic conditions and found
that both the daily excretion and the ratio of ET/creatinine
were significantly higher in diabetic children than in nor-
mal control subjects [68]. There were significant differ-
ences between the healthy and diabetic children with re-
spect to diuresis, with the diabetic children having higher
values.
The high diuresis in diabetic children may be due to
hyperfiltration and tubulopathy. The elevated ET excretion
in diabetes mellitus may also reflect greater ET synthesis
within the kidney. There is strong evidence that local ET
inhibits sodium and water reabsorption in the inner renal
medulla [69, 70], which suggests that one reason for the
elevated urinary output is the enhanced local ET synthesis.
A variety of renal disorders are characterized by alterations
517
Table 3. Physiological conditions and diseases with elevated urinary
ET in children
Conditions with increased ET-1 in urine Reference
Premature infants [18, 26]
Mature newborns, 1st day of life [26]
Water diuresis [18, 42]
Perinatal hypoxia [26]
Diabetes mellitus [52]
Vesicoureteral reflux [59, 60]
HUS [58]
Chronic renal failure [42]
Idiopathic hypercalciuria [42]
After kidney transplantation [42]
Cyclosporin A treatment [42]
Contrast media toxicity [68]
HUS, Hemolytic-uremic syndrome
in the level of urinary ET, which correlated with the se-
verity of the disease [4, 33, 36]. It is possible that the
elevated ET excretion is due to the renal tubular damage
observed in diabetes mellitus [71, 72].
ET excretion in tubular disturbances
(diabetic, ketoacidosis, vesicoureteral reflux)
During diabetic ketoacidosis, osmotic diuresis with loss of
salt and water causes severe volume depletion. Blood
pressure homeostasis is maintained by a significant rise in
peripheral vascular resistance [73, 74]. With further volume
depletion, kidney function deteriorates and GFR and renal
plasma flow decrease. Electrolyte disturbances and acidosis
enhance the alteration in kidney function and signs of renal
tubular dysfunction can be detected [75, 76]. The elevated
peripheral vascular resistance mediated by the renin-an-
giotensin-aldosterone system, the sympathetic nervous
system, vasopressin, and prostaglandins counterbalances
the reduced intravascular volume and compensates for so-
dium loss. In our investigations [63, 77], we detected a
decreased GFR associated with highly significant changes
in the excretion of tubular markers. Urinary α1-micro-
globulin was greatly elevated, signalling proximal tubular
damage, and decreased during recovery. Tomm-Horsfall
protein excretion was low during ketoacidosis, but in-
creased with a return to normal pH and normal blood
glucose levels. This indicates a reversible disturbance of
cell function of the ascending loop of Henle and the initial
distal tubule during ketoacidosis. The absolute amount of
ET-1 excreted remained constant despite the marked
changes in GFR and in proximal and distal tubular func-
tions, but the ET/creatinine ratio was significantly elevated
[63]. Based on these data, we can draw the following
conclusions: (1) there is no significant correlation between
GFR and renal ET excretion, as reported [33]; (2) urinary
ET excretion is independent of proximal and distal tubular
function; (3) the activated renin-angiotensin-aldosterone
system and sympathic nervous system have little influence
on the urinary ET, at least during ketoacidosis; (4) the in-
creased ET excretion relative to creatinine or GFR suggests
that ET plays a local role in the kidney under pathophy-
siological conditions such as diabetic ketoacidosis. To de-
termine the precise mechanism and physiological impor-
tance of this process further investigations are needed.
There are few studies of urinary ET excretion in ur-
ological diseases. This is particularly relevant to injury to
the renal medulla, where the majority of ET is synthesized.
In patients with vesicoureteral reflux (VUR) high levels of
urinary β2-microglobulin, N-acetyl-β-D-glucosaminidase,
and microalbumin have been reported [78]. Komeyama et
al. [79] found no correlation between any of these factors
and the renal ET-1/creatinine ratio in spot urine samples,
both in adults and in children, in agreement with our results
[63]; however ET-1/creatinine increased in proportion to
the grade of reflux, except for grade 5 reflux where it was
decreased. Thus ET-1 may be an indicator of renal tubular
injury in patients with primary VUR.
Urinary ET in nephrological diseases
The urinary excretion of ET in different nephrological
diseases in adults has been investigated in several studies
[33, 36]. Ohta et al. [33] found significantly elevated levels
of 24-h urinary ET excretion in patients with focal glo-
merulosclerosis, lupus nephritis, end-stage renal disease,
membranous proliferative glomerulonephritis, and IgA
nephropathy. Similar results were reported by Roccatello et
al. [38]. We have found an elevated urinary ET/creatinine
ratio in pediatric patients with minimal change glomerular
disease, mesangial proliferative glomerulonephritis, focal
glomerulosclerosis, and after kidney transplantation during
(CsA) therapy (unpublished data). In contrast, Worgall et
al. [45] did not find increased ET excretion in children with
nephrotic syndrome and stable glomerulonephritis, but re-
ported higher urinary ET excretion in patients with chronic
renal failure, idiopathic hypercalciuria, and after renal
transplantation. In children with HUS the elevation of daily
ET excretion was not significant, probably because of the
small number of patients studied [45]. However Siegler et
al. [39] detected elevated excretion of urinary ET in chil-
dren with HUS and related the severity of the disease to the
24-h excretion. Worgall et al. [45] demonstrated the urine
flow dependence of ET excretion described by others [40,
55], both in healthy controls and in renal diseases.
Urinary ET excretion in other diseases
ET may also have a role in the pathophysiology of a series
of illnesses besides nephrological diseases. Sofia et al. [80]
found elevated 24-h urinary ET excretion in adult patients
with chronic obstructive pulmonary disease, which in-
creased during acute exacerbations. A negative correlation
was found between arterial oxygen pressure and ET-1 ex-
cretion.
In patients with essential hypertension, Hoffmann et al.
[81] detected a lower daily excretion of ET-1 than in nor-
mal controls, which was not affected by the dietary sodium
content. Saito et al. [82] found a positive correlation be-
tween ET and sodium excretion in hypertonic patients.
In full-term and premature infants, many diseases affect
several organs at once, such as the cardiorespiratory sys-
tem, the central nervous system, and the kidney. There are
disturbances in the oxygenation, volume, electrolyte and
blood pH regulation. The changes, reversible at first, be-
come irreversible with severe pathophysiological con-
sequences. ET release is stimulated by hypoxia [26], en-
dotoxin [27], and several other factors, and has a role in the
regulation of platelet aggregation, coagulation, vasomotor
tone [4], cellular growth, and proliferation, and probably
regeneration [20]. On this basis, newborns treated in in-
tensive care units may also have changes in ET excretion.
Sulyok et al. [40] found that newborns with severe perinatal
asphyxia/infection had elevated urine concentrations and
daily excretion of ET-1. In dopamine-treated neonates there
was a significant flow-dependent increase in daily ET-1
excretion, but dopamine may also have stimulated the
synthesis of ET.
518
In animal experiments and in vitro studies [5, 18, 20, 83]
ET plays a role in the regulation of vasomotor tone, re-
generation of injured blood vessels, and probably in the
development of atherosclerotic plaques. Atherosclerosis is
a disease mainly of adults, but it is genetically determined,
and early laboratory signs are probably detectable in
childhood. There are several research groups investigating
the role of ET metabolism in its pathogenesis, and new
results will hopefully help in the prevention and therapy of
atherosclerosis.
ET and nephrotoxicity
ET and CsA
ET is involved in the nephrotoxicity observed with onco-
logical chemotherapy [84] and in the tubular toxicity of
CsA. This immunosuppressive agents given to patients
after transplantation of the heart, lungs, liver, and kidneys,
as well as in the nephrotic syndrome or chronic glomeru-
lonephritis. Among its recognized adverse side effects are:
hypertension, central nervous system toxicity, hepatotoxi-
city, and nephrotoxicity [85]. The latter ranges from a slight
decrease in GFR to progressive renal scarring. The early
renal involvement after renal transplantation intense re-
versible renal vasoconstriction, that may reflect altered
vascular reactivity due to endothelial cell damage. In-
travenous administration of CsA to rats results in an ap-
proximate tenfold elevation in plasma ET level and higher
systemic blood pressures [86]. One hour following CsA
administration, rabbit anti-porcine ET serum was infused
continuously into a branch of the main renal artery. Mi-
cropuncture investigations showed a large difference be-
tween the two parts of the kidney, that infused with ET
antiserum and that without antiserum [86]. In the glomeruli
exposed to ET antiserum the fall in single nephron GFR
was much less. In another study [87], 60 min after ad-
ministration of CsA the plasma level of ET was unchanged,
but the low glomerular plasma flow persisted. Kivlighn et
al. [88] found that a selective ETA receptor antagonist (BQ-
123) protected the kidney from CsA-induced hypoperfusion
and hypofiltration. These results implicate ET as an im-
portant component in CsA-induced glomerular dysfunction.
Worgall et al. [45] found elevated urinary excretion of
ET-1 in children after renal transplantation during CsA
therapy. However, the higher urinary ET excretion may be
the result of CsA therapy or of pathophysiological pro-
cesses affecting the kidney during transplantation. Other
reports of Yamakado et al. [42] and Watschinger et al. [89]
indicate that ET may participate in the pathogenesis of
acute rejection in the transplanted kidney.
ET and contrast media
ET-1 release from renal tubular cells is stimulated by
contrast media [90]; after contrast media application a short
vasodilatation is followed by a sustained vasoconstriction
in renal arterial vessels, similar to the effect of an infusion
of ET-1 [91]. We found that a significant elevation in ex-
cretion and concentration of urinary ET-1 could be detected
in all children after angiography [92]. The urinary levels of
tubular markers α1-microglobulin, N-acetyl-β-D-glucosa-
minidase, and villin were also higher, suggesting that in
contrast media nephrotoxicity the proximal tubules and
probably the inner medullary collecting ducts are affected.
The changes in these markers correlated with the changes
in ET excretion. In this study irreversible tubular damage
was not noted, but it is proposed that renal function should
be monitored during contrast media investigations.
Conclusion
In summary, in the kidney ET is involved in many physi-
ological and pathological processes, and the amount ex-
creted in the urine is the result of intrarenal synthesis in-
fluenced by several factors. ET could be a marker of renal
injury in different pathological processes both in children
and in adults, but its specificity is low. Thus ET should be
used with caution as a marker of distal tubular or collecting
duct epithelial function. Elevation of urinary ET may in-
dicate damage of distal tubular and/or inner medullary
collecting duct cells, due to hypoxia, inflammation, or ex-
posure to toxic agents, such as CsA. Further investigations
are needed to clarify the importance of ET in autocrine and
paracrine regulation of renal function and in the patho-
physiology of various renal diseases.
Acknowledgements. Dr. Istva´n Ma´ttyus was a recipient of a DAAD
stipendiary from the Deutscher Akademischer Austauschdienst. We
thank Dr. Phil. A. Brecht for secreterial assistance. We wish to thank
Professor Dr. F. Kaskel, Head, Division of Pediatric Nephrology,
SUNY, Stony Brook, New York, for his thorough criticism and help in
the revision of the manuscript. Work cited by the authors was sup-
ported by the German Research Foundation (DFG Zi 314).
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