PANEL DE DISCUSION
Mario Negri Institute for Pharmacological Research.
Via Gavazzeni 11,24125 Bergamo,Italy
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Endothelins: synthesis, receptors and effects
Endothelins comprise a family of three isopeptides: endothelin-1, -2, and -3, containing 21-amino acids encoded by three distinct genes on different chromosomes (1,2). The 5 gene-flanking region of the human ET-1 precursor contains binding sites of fos/jun complex, activating protein 1 and nuclear factor 1, as well as sequences for the acute phase reactant regulatory elements involved in ET-1 induction under acute physical stress in vivo. Transcription of ET-1 gene leads to the production of preproendothelin-1, consisting of 212 amino acids and cleaved by a neutral endopeptidase to form the biologically inactive precursor big endothelin, ultimately converted to mature peptide by a phosphoramidon-sensitive membrane bound metalloprotease called endothelin converting enzyme (ECE). Three different ECE-1 isoforms differing in their N-terminal regions derive from a single gene through the use of alternative promoters. ECE isoforms are located either at the cell surface cleaving the bigET-1 supplied from outside the cell or in the trans-Golgi network converting endogenously produced ET-1. ET-1 is secreted from the cells either through a constitutive pathway assuring a continuosly release through secretory vesicles or a stimulated pathway in which ET-1, stored in Weiber-Palade bodies, is released to the cell surface after stimulation.
Beside endothelial and smooth muscle cells of blood vessels, also kidney, heart, brain, lung, pancreas and spleen cells synthetize ET-1 (1), whose secretion is predominantly abluminal (3,4).
Endothelins exert their biological activity after binding to specific cell surface receptors, identified as seven transmembrane domain G protein-coupled receptors (5,6). Endothelin type-A receptor (ETA) has higher affinity for ET-1 and ET-2 and less for ET-3; ETB, the type-B receptor, binds the three isopeptides with nearly identical affinity. The ETA receptors mediate vasoconstriction and cell proliferation, whereas ETB receptors can mediate vasodilation or vasoconstriction, are responsible for ET-1 clearance from the circulation and can modulate ET-1 own synthesis (7). Specific binding sites for endothelin have been identified in numerous fetal and adult organs including lung, heart, brain and kidney,relative abundance of ETA and ETB receptors in the kidney depending on the species (7).
When injected in the rat, ET-1 and ET-3 dose-dependently increase the systemic blood pressure after an initial depression or effect due to induction of prostacyclin and nitric oxide released from endothelial cells (8). The increase in systemic blood pressure (1,2) is prolonged, mostly dependent on renal, mesenteric, and muscular vessel constriction and sustained by an increased cardiac output. Systemic and renal vasoconstriction was prevented by a nonselective ETA and ETB receptor antagonist while ETA receptor antagonist only abolished systemic vasoconstriction, suggesting a role for ETB receptor in the renal response to ET-1 in the rat. In humans, ET-1 infusion causes profound renal vasoconstriction in healthy volunteers, in the face of unchanged systolic blood pressure (9), suggesting a unique susceptibility of renal vessels to ET-1 vasoconstriction in different species. ET-1 regulates volume homeostasis by increasing plasma renin activity and aldosterone secretion and also affects sodium and water balance (10).
ET-1 induces proliferation of cultured vascular smooth muscle cells and human umbilical vein endothelial cells. It is a strong mitogen for mesangial cells and fibroblasts, and by favoring cell proliferation also promotes matrix protein gene transcription (10). Endothelin-1 is chemotactic for blood monocytes (11), a property which in vivo could contribute to the tubulointerstitial damage that accompanies most progressive renal diseases (12).
Endothelin in progressive renal injury
In progressive renal diseases the early insult to the kidney depends on an hemodynamic adaptation of remaining units presumably triggered by focal obliteration of most nephrons affected by the original disease (13). Over time glomerulosclerosis and tubule atrophy develop further reducing renal mass in a self-perpetuating process of tissue injury which normally ends with interstitial fibrosis and progressive renal dysfunction. The renal community has discussed for years whether hemodynamic factors were the sole contributors to glomerular and tubulointerstitial injury or whether others mechanisms were also operating. Recent studies have suggested that proteinuria, previously considered just a marker of the severity of a given disease, may itself be pathogenic (12).
Of interest in this context are findings that the amount of protein recovered in the urine correlated better than any other clinical or laboratory parameters - including the nature of the underlying disease - with the tendency to progress to end stage kidney both in animals and humans. Enhanced protein traffic can cause disease progression through extensive endocytosis of proximal tubular cells that acquire an inflammatory phenotype ultimately responsible of progressive injury of the renal interstitium (Figure). On this line are data that exposure of proximal tubular cells in culture to increasing concentrations of albumin, IgG or transferrin induced a dose-dependent increase in ET-1 production, which was mainly released basolaterally (4). If the same condition would occur in vivo, ET-1 may accumulate in the interstitial compartment and, by binding to specific receptors on interstitial fibroblasts, may induce their proliferation and accumulation of matrix synthesis. A number of studies have convincingly indicated ET-1 as a profibrotic agent for the kidney.
Enhanced renal gene expression and synthesis of ET-1 occurs in vivo in proteinuric progressive renal disease models due to immune or nonimmune damage to the kidney. The remnant kidney model in rats is characterized by a time-dependent increase in renal ET-1 gene expression, paralleled by an increase in urinary excretion of the peptide; both correlating with the degree of interstitial fibrosis ang glomerulosclerosis (14). Gene expression of endothelin receptors in the kidneys of rats undergoing renal mass ablation was differentially modulated during the course of the disease. While ETA receptors were unchanged a selective upregulation of ETB receptors was found, possibly as a consequence of compensatory renal hypertrophy as recently demonstrated in ETB knock-out mice (15). Changes of ET-1 urinary excretion were also observed in rats with passive Heymann nephritis, an immune model of glomerular disease resembling human membranous nephropathy (16). Up-regulation of renal ET-1 and ETB receptor gene expression in NZB/WF1 mice with an immunological disease reminiscent of human lupus as well as in rats after puromycin aminonucleoside injection has been described (17,18). In experimental diabetes mRNA for ET-1 is overexpressed in the kidney in the face of unchanged ETA and ETB receptors (19).
Direct evidence for ET-1 to play a role in progressive renal damage derives from studies with transgenic animals. Transgenic mice for the human ET-1 promoter have increased renal synthesis of ET-1 and develop renal fibrosis and glomerulosclerosis with no changes in systemic blood pressure (20). Furthermore rats transgenic for the human ET-2 gene are normotensive but have renal lesions reminiscent of those of rats with remnant kidney (21).
Effectiveness of ET receptor antagonists to slow or even halt renal disease progression has been consistently reported. In the rat remnant kidney, a specific antagonist for the ETA receptor (22) ameliorates renal functional impairment and protects against glomerular and tubulointerstitial structural injury. An orally active compound with antagonizing properties for ETA and ETB receptors (23) even prolonged animal survival. Similar results have been obtained by other investigators (24). The renoprotective effect of ETA receptor antagonists also was evident in mice with experimental lupus nephritis. In rats with streptozotocin-induced experimental diabetes either if treated at the time of induction of the disease or when animals had already overt proteinuria (25,26).
Manipulation of the endothelin system in the rat by a selective ETA receptor antagonist ameliorated renal dysfunction, prevented renal damage (27), and even prolonged survival in chronic renal allograft nephropathy (28). Of note, ET receptor antagonists, that consistently reduce renal damage, did not invariably normalize proteinuria, strenghtening the hypothesis that excessive ET-1 is not a cause but rather a consequence of increased glomerular protein traffic. Theoretically, the combination of a drug that effectively reduces proteinuria with an endothelin receptor antagonist would have offered a superior renoprotection than single treatments alone. This is actually demonstrated by recent data obtained in a severe model of proteinuric progressive nephropathy not completely responsive to ACE inhibitors in term of reduction of proteinuria, in which combined administration of ACE inhibitor and a selective ET receptor antagonist ameliorated renal function impairment and prevented renal damage better than each drug alone (29).
Evidence for a potential role of ET-1 is also available in patients with chronic progressive nephropathies, based on plasma and urine immunoreactive ET-1 measurements (30). Plasma concentration of ET-1 in patients with end-stage renal failure undergoing hemodialysis are one- to twofold greater than normal (31), possibly as a consequence of an impairment of clearance. However, the fact that urinary excretion of ET-1 is also increased in patients with chronic glomerulonephritis (30) suggests that renal generation of ET-1 in these diseases is increased. The recent finding of a concomitant upregulation of ET-1 and ETB and ECE-1 a gene expression in patients with glomerulopathy and high proteinuria is highly suggestive of a activation of renal endothelin system in man predicted by animal studies in progressive renal diseases (23). Moreover, administration of an ETA/ETB receptor antagonist to patients with chronic renal failure reduced blood pressure and renal vascular resistance (32).
In conclusion, pre-clinical observations convincingly document a role of ET-1 in progressive renal disease and studies with endothelin receptor antagonists indicate that these compounds, while having a modest antiproteinuric effect, effectively prevent renal fibrosis.
Clinical studies are now emerging indicating a role for endothelin also in human progressive nephropathies. Due to the availability of endothelin receptor antagonists for human use, future clinical studies are now needed.
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