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).

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.

REFERENCES

1. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T: A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332:411-415, 1988

2. Inoue A, Yanagisawa M, Kimura S, Kasuya Y, Miyauchi T, Goto K, Masaki T: The human endothelin family: three structurall y and pharmacologically distinct isopeptides predicted by three separate genes. Proc Natl Acad Sci U S A 86:2863-2867, 1989

3. Wagner OF, Christ G, Wojta J, Vierhapper J, Parzer S, Nowotney PJ, Schneider B, Waldhausl W, Binder B: Polar secretion of endothelin-1 by cultured endothelial cells. J Biol Chem 267:16066-16068, 1992

4. Zoja C, Morigi M, Figliuzzi M, Bruzzi I, Oldroyd S, Benigni A, Ronco PM, Remuzzi G: Proximal tubular cell synthesis and secretion of endothelin-1 on challenge with albumin and other proteins. Am J Kidney Dis 26:934-941, 1995

5. Arai H, Hori S, Aramori I, Ohkubo H, Nakanishi S: Cloning and expression of a cDNA encoding an endothelin receptor. Nature 348:730-732, 1990

6. Sakurai T, Yanagisawa M, Inoue A, Ryan US, Kimura S, Mitsui Y, Goto K, Masaki T: cDNA cloning, sequence analysis and tissue distribution of rat preproendothelin-1 mRNA. Biochem Biophys Res Commun 175:44-47, 1991

7. Benigni A, Remuzzi G: Endothelin antagonists. Lancet 353:133-138, 1999

8. De Nucci G, Thomas R, D'Orleans-Juste P, Antunes E, Walder C, Warner TD, Vane JR: Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc Natl Acad Sci USA 85:9797-9800, 1988

9. Sorensen SS, Madsen JK, Pedersen EB: Systemic and renal effect of intravenous infusion of endothelin-1 in healthy human volunteers. Am J Physiol 266:F411-F418, 1994

10. Simonson MS, Dunn MJ: Renal actions of endothelin peptides. Curr Opin Nephrol Hyperten 2:51-60, 1993

11. Achmad T, Rao G: Chemotaxis of human blood monocytes towards endothelin-1 and the influence of calcium channel blockers. Biochem Biophys Res Commun 189:994-1000, 1992

12. Remuzzi G, Ruggenenti P, Benigni A: Understanding the nature of renal disease progression. Kidney Int 51:2-15, 1997

13. Brenner BM, Meyer TW, Hostetter TH: Dietary protein intake and the progressive nature of kidney disease: The role of hemodynamically mediated glomerular injury in the pathogenesis of progressive glomerular sclerosis in aging, renal ablation, and intrinsic renal disease. N Engl J Med 307:652-659, 1982

14. Orisio S, Benigni A, Bruzzi I, Corna D, Perico N, Zoja C, Benatti L, Remuzzi G: Renal endothelin gene expression is increased in remnant kidney and correlates with disease progression. Kidney Int 43:354-358, 1993

15. Liu B, Yanagisawa M, Preisig P: The endothelin B (ETB) receptor mediates compensatory renal hypertrophy. J Am Soc Nephrol 10:496A, 1999

16. Zoja C, Liu X-H, Abbate M, Corna D, Schiffrin EL, Remuzzi G, Benigni A: Angiotensin II blockade limits tubular protein overreabsorption and the consequent up-regulation of endothelin-1 gene in experimental membranous nephropathy. Exp Nephrol 6:121-131, 1998

17. Nakamura T, Ebihara I, Fukui M, Osada S, Tomino Y, Masaki T, Goto K, Furuichi Y, Koide H: Renal expression of mRNAs for endothelin-1, endothelin-3 and endothelin receptors in NZB/W F1 mice. Renal Physiol Biochem 16:233-243, 1993

18. Nakamura T, Ebihara I, Fukui M, Osada S, Tomino Y, Masaki T, Goto K, Furuichi Y, Koide H: Modulation of glomerular endothelin and endothelin receptor gene expression in aminonucleoside-induced nephrosis. J Am Soc Nephrol 5:1585-1590, 1995

19. Fukui M, Nakamura T, Ebihara I, Osada S, Tomino Y, Masaki TM, Goto K, Furuichi Y, Koide H: Gene expression for endothelins and their receptors in glomeruli of diabetic rats. J Lab Clin Med 122:149-156, 1993

20. Hocher B, Thone-Reineke C, Rohmeiss P, Schmager F, Slowinski T, Burst V, Siegmund F, Quertermous T, Bauer C, Neumayer H-H, S chleuning W-D, Theuring F: Endothelin-1 transgenic mice develop glomerulosclerosis, interstitial fibrosis, and renal cysts but not hypertension. J Clin Invest 99:1380-1389, 1997

21. Liefeldt L, Bocker W, Schonfelder G, Zintz M, Paul M: Regulation of the endothelin system in transgenic rats expressing the human endothelin-2 gene. J Cardiovasc Pharmacol 26:S32-S35, 1995

22. Benigni A, Zoja C, Corna D, Orisio S, Longaretti L, Bertani T, Remuzzi G: A specific endothelin subtype A receptor antagonist protects against injury in renal disease progression. Kidney Int 44:440-444, 1993

23. Benigni A, Zoja C, Corna D, Orisio S, Facchinetti D, Benatti L, Remuzzi G: Blocking both type A and B endothelin receptors in the kidney attenuates renal injury and prolongs survival in rats with remnant kidney. Am J Kidney Dis 27:416-423, 1996

24. Nabokov A, Amann K, Wagner J, Gehlen F, Munter K, Ritz E: Influence of specific and non-specific endothelin receptor antagonists on renal morphology in rats with surgical renal ablation. Nephrol Dial Transplant 11:514-520, 1996

25. Nakamura T, Ebihara I, Tomino Y, Koide H: Effect of a specific endothelin A receptor antagonist on murine lupus nephritis. Kidney Int 47:481-489, 1995

26. Benigni A, Colosio V, Brena C, Bruzzi I, Bertani T, Remuzzi G: Unselective inhibition of endothelin receptors reduces renal dysfunction in experimental diabetes. Diabetes 47:450-456, 1998

27. Orth SR, Odoni G, Amann K, Strzelczyk P, Raschack M, Ritz E: The ETA receptor blocker LU 135252 prevents chronic transplant nephropathy in the "Fisher to Lewis" model. J Am Soc Nephrol 10:387-391, 1999

28. Rohmeiss P, Braun C, Conzelmann T, Vetter S, Schaub M, Back WE, Kirchengast M, van der Woude FJ: Improvement of chronic renal allograft rejection in rats with the oral endothelin A-receptor antagonist LU135252. J Am Soc Nephrol 9:656A, 1998 (abstract)

29. Benigni A, Corna D, Maffi R, Benedetti G, Zoja C, Remuzzi G: Renoprotective effect of contemporary blocking of angiotensin II and endothelin-1 in rats with membranous nephropathy. Kidney Int 54:353-359, 1998

30. Ohta K, Hirata Y, Shichiri M, Kanno K, Emori T, Tomita K, Marumo F: Urinary excretion of endothelin-1 in normal subjects and patients with renal disease. Kidney Int 39:307-311, 1991

31. Koyama H, Tabata T, Nishzawa Y, Inoue T, Morii H, Yamaji T: Plasma endothelin levels in patients with uraemia. Lancet 333:991-992, 1989

32. Webb DJ, Monge JC, Rabelink TJ, Yanagisawa M: Endothelin: new discoveries and rapid progress in the clinic. Trends Pharmacol Sci 19:5, 1998

DISCUSSION BOARD PANEL DE DISCUSION