1 Department of Nephrology, University Hospital Charité, Humboldt Universität zu Berlin and 2 Institute of Clinical Pharmacology and Toxicology, Benjamin Franklin Medical Center, Freie Universität Berlin, Berlin, Germany
The endothelin types
Endogenous peptidergic factors with vasoactive properties were first described by Hickey et al. in 1985 [1]. Yanagisawa and co-workers found that porcine endothelial cells generated a 21-aminoacid peptide, endothelin-1 in 1988 [2]. An i.v. injection of this new endothelium-derived peptide revealed that endothelin-1 (ET-1) is one of the most potent vasoconstrictors in vivo. Two further members of the ET family were described later and termed ET-2 and ET-3. Most studies on the ET system have focused on ET-1. Release of the active ET-1 peptide requires cleavage of a Trp21Val22 bond in the carboxy terminal of the precursor molecule, big ET-1. This reaction is catalysed by a membrane-bound metalloprotease, ET-converting enzyme (ECE-1). ET synthesis is mainly regulated on the level of gene transcription when vasoconstrictor peptides, inflammatory cytokines, and physical factors (e.g. hypoxia and shear stress) act on the endothelium.
Endothelin and the kidney
Although the normal human kidney contains mRNA for all three isoforms of ET, ET-1 appears to be the only peptide expressed at the protein level. Expression of ET-1, its precursor, big ET-1, and the ECE in the non-diseased kidney is largely confined to the glomerular and vascular endothelium (see [36] for reviews). However, when proteinuria is present, ET-1 expression can also occur in the tubules [7]. ET-1 released by the renal vasculature or nephron segments mainly acts in an autocrine and/or paracrine manner. ET-1 mediates its biological effects by interacting with two receptors, the ETA and ETB receptor, which belong to the family of rhodopsin-like, G-protein-coupled receptors. The ETA receptor is preferentially expressed in vascular smooth-muscle cells and mediates the potent constrictor actions of ET-1. The ETB receptor is primarily expressed in endothelial cells and binds all three ET isoforms. When this receptor is activated, nitric oxide (NO) and prostacyclin are released due to an activation of the calcium-dependent endothelial NO synthase (eNOS) [36]. The ETB receptor is widely expressed in renal epithelium, where, it probably promotes sodium excretion and regeneration of damaged tubular epithelium [8,9]. In some vascular beds the ETB receptor is also expressed on vascular smooth-muscle cells where it may also mediate constrictor actions [10]. In the human and rat kidney, however, selective stimulation of the ETB receptor increased renal blood flow rather than reducing it [11,12]. The above-described properties of the ET system clearly indicate that it acts mainly on the paracrine level.
The ET system has been implicated in the pathogenesis of several kidney diseases such as acute renal failure, lupus nephritis, diabetic nephropathy, and especially kidney diseases characterized by fibrosis (for reviews, see [36,13]). But we have to keep in mind that all these conditions are multifactorial and that the exact pathophysiological role of the ET systemas a part of a local paracrine networkin these conditions is difficult to analyse. One approach to study the primary pathophysiological effects of the ET system is the expression of its components in transgenic animals and the study of the resulting phenotypes.
The transgenic animal models
There are several approaches to the generation of transgenic animals for cardiovascular research that have been described elsewhere in detail [1416]. Most of these techniques have also been used to establish transgenic models to analyse the ET system.
Overexpression of the human ET-1 gene under the control of its natural promoter in mice is associated with a pathological renal phenotype characterized by an age-dependent development of interstitial fibrosis and glomerulosclerosis, leading to a progressive decrease in glomerular filtration rate without alterations of blood pressure. This blood-pressure-independent fibrotic remodelling of the kidney occurred despite a rather low overexpresion rate of ET-1. Tissue concentrations were only elevated by about 50%, indicating that ET-1 is a very potent profibrotic peptide hormone in vivo, at least in the kidney [17]. A blood-pressure-independent fibrotic remodelling of the kidney was also seen in transgenic rats overexpressing the human ET-2 gene under control of its promoter. However, the extent of kidney fibrosis was less pronounced and mainly restricted to the glomeruli [18,19]. This is most probably due to the preferential expression of the transgene within the glomeruli in ET-2 transgenic rats as shown by in situ hybridization [18], whereas the transgene is ubiquitously expressed within the entire kidney of ET-1 transgenic mice [17].
The absence of hypertension in these transgenic animal models of the ET system was unexpected, since an i.v. injection of ET-1 or ET-2 causes a very sustained vasoconstriction [26]. The lack of hypertension in all models with a chronically activated ET system studied so far is most probably the result of compensatory activation of vasodilator systems such as the NO system. The molecular mechanism leading to an enhanced NO synthesis in ET-1 transgenic mice and ET-2 transgenic rats are currently analysed. According to recent reports [19,20] a bolus injection of the NO synthesis inhibitor L-NAME induces an exaggerated hypertensive response in the ET-2 transgenic rats as well as in ET-1 transgenic mice. This observation apparently corroborates the hypothesis that the NO system counterregulates the ET overexpression in these models, leading to a normotensive phenotype.
The knock-out animal models
Other studies have chosen the loss of function approach to study the phenotypic relevance of the ET system. Homozygous -/- ET-1 knock-out mice died immediately after birth due to craniopharyngeal malformations resulting in an inability to breathe normally. Heterozygous +/- ET-1 knock-out mice with reduced ET-1 tissue concentrations showed an elevated blood pressure. Enhanced basal and hypercapnia-induced (pCO2-induced) sympathetic nerve activity in the heterozygous ET-1 +/- mice seem to be responsible, at least in part, for the unexpected finding of blood pressure elevation in animals with reduced endogenous tissue ET-1 [22]. Some other causes that might contribute to blood pressure elevation in ET -/- mice have been excluded, such as salt-sensitivity [23] or respiratory abnormalities, in analogy to the findings in homozygous knock-out mice [21]. An elevated blood pressure was also detected in ETB knock-out mice and also after pharmacological blockade of the ETB receptor using selective ETB receptor antagonists. This indicates that in vivo the non-stimulated endogenous ET system acts as a depressor rather than a pressor system [24,25]. The major phenotype in ETB deficient mice is the absence of enteric ganglionic cells, leading to a congenital megacolon with early postnatal death from Hirschsprung disease [26]. Therefore analysis of renal and cardiovascular consequences of ETB deficiency in these animals is rather difficult. The most elegant approach to resolve this problem was the technique recently adopted by Yanagisawas's group. They rescued ETB-deficient mice from the gastrointestinal consequences of ETB deficiency by selective expression of ETB receptors in intestinal ganglion cells. This was achieved by cross-breeding ETB knock-out mice with mice overexpressing the ETB gene under the control of the dopamine ß-hydroxylase gene promoter directing ETB expression to nerve cells (Yanagisawa et al., 6th International Conference on Endothelin 1999, Montreal, Canada). Hypertension was salt dependent in these mice, indicating that the regulation of salt excretion in the kidneys is partially ETB dependent. Earlier patch-clamp studies suggest that the ETB receptor regulates tubular sodium channel activity via an increase in the mean closure time in distal nephron cells [27].
Disruption of the ET-3 gene in mice [28] also causes the absence of enteric ganglion cells leading to Hirschsprung disease. These findings indicate that the interaction of ET-3 with the ETB receptor is essential in the development of neural crest-derived cell lines essential for enteric ganglion cells. Whether a disruption of the ET-3 gene also causes salt sensitive hypertension is unknown so far.
ETA knock-out mice, on the other hand, are characterized by severe craniofacial deformities and defects in the cardiovascular outflow tract, findings that resemble the human CATCH 22 or velocardiofacial syndrome [29].
What are the conclusions?
Taken together, the transgenic approach has successfully revealed that the ET system contributes essentially to normal embryonic development. There are obviously two ET system-dependent neural crest-driven developmental pathways: first the ET-1ETA receptor axismutations in this axis are associated with cranial and cardiac defects; second the ET-3ETB receptor axismutations in this axis are associated with epidermal melanocytes and enteric neurons. The findings in these animals led to the discovery of similar mutations in humans with Hirschsprung disease [30,31]. In adult life the ET system is most important in the renal and cardiovascular system.
With respect to the kidney, transgenic technologies revealed that a chronically activated renal ET system on its own causes kidney fibrosis without altering blood pressure. It was also shown that disruption of the renal ETB receptor causes salt-sensitive hypertension. The study of ET transgenic animals, therefore, has contributed significantly to our understanding of ET function in vivo.
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Notes
Correspondence and offprint requests to: Prof. Dr. Martin Paul, Institute of Clinical Pharmacology and Taxicology, Freie Universität Berlin, Benjamin Franklin Medical Center, Garystrasse 5, D-14195 Berlin Germany.
References