Update on endothelins - biology and clinical implications.

In the decade since the initial discovery and characterization of endothelin, its biology as a powerful vasoconstrictor has been dramatically demonstrated. Studies have clarified the existence of endothelin isoforms, complex mechanisms of biosynthesis, interaction with specific receptors, and pathogenic implications. We are on the brink of using endothelin antagonism as a clinical treatment for disease processes where endothelin plays an important role, including congestive heart failure and hypertension. Novel observations have been made about the unexpectedly profound contribution endothelins make to normal fetal maturation, especially in cardiac and enteric development.

The angiotensin type II receptor tonically inhibits angiotensin- converting enzyme in AT2 null mutant mice.

BACKGROUND: Pharmacologic inhibition of the angiotensin-converting enzyme (ACE) limits angiotensin II (Ang II)-induced vasoconstriction and cellular proliferation. There is emerging evidence that some of the beneficial effects of ACE inhibitors may be endogenously available through the angiotensin receptor type 2 (AT2). METHODS: To evaluate whether AT2 modulates ACE activity, we used an high-performance liquid chromatography (HPLC)-based enzymatic assay in tissues from AT2 knockout mice (Agtr2-/y) and cultured cells. These studies were complimented by physiologic studies of pharmacologic inhibition of AT2. RESULTS: Circulating (C) and tissue ACE activities in heart (H), lung (L), and kidney (K) were doubled in Agtr2-/y mice compared with wild-type mice [162.9 +/- 17.6 mU/mL (C), 97.7 +/- 20.7 (H), 6282.1 +/- 508.3 (L), and 2295.0 +/- 87.0 (K) mU/g tissue for Agtr2-/y vs. 65.3 +/- 35.4 mU/mL (C), 44.5 +/- 8.7 (H), 3392.4 +/- 495.2 (L), and 1146.1 +/- 217.3 (K) mU/g tissue for wild-type mice, P < or = 0.05, 0.025, 0.002, and 0.0001, respectively]. Acute pharmacologic inhibition of AT2 [PD123319 (PD), 50 microg/kg/min, i. v.] significantly increased ACE activity in kidneys of wild-type mice (1591.2 +/- 104.4 vs. 1233.6 +/- 88.0 mU/g tissue in saline-infused mice, P < 0.05; P < 0.01 vs. uninfused, wild-type mice). Moreover, ACE activity increased in A10 cells exposed to PD (10-6 mol/L) together with Ang II (10-7 mol/L), but not with an AT1 antagonist (losartan, 10-6 mol/L). This heightened ACE activity appears functionally relevant because infusion of angiotensin I caused more prompt hypertension in Agtr2-/y mice than in wild-type littermates. Likewise, infusion of bradykinin, also a substrate for ACE, caused significantly less hypotension in Agtr2-/y mice than controls. CONCLUSIONS: These studies indicate that AT2 functions to decrease ACE activity tonically, which may, in part, underlie AT2's increasingly recognized attenuation of AT1-mediated actions.

ACE I/D gene polymorphism predicts renal damage in congenital uropathies.

We investigated angiotensin converting enzyme gene (ACE I/D) polymorphism as a risk for progressive renal damage in congenital uropathies. The ACE I/D genotype was determined in 196 Caucasian patients with congenital uropathies and 163 individuals with no clinical or sonographic evidence of any urological malformations. The study group included patients with ureteropelvic junction obstruction (n=49), primary obstructive megaureter (n=19), primary vesicoureteral reflux (VUR) (n=67), and posterior urethral valves (n=27). Thirty-four patients were excluded because of additional diseases or insufficient follow-up. There was no difference in the ACE I/D distribution between children with uropathies and normal controls (II 16%, ID 56%. DD 28% vs. II 26%, ID 50%, DD 24%). Renal lesions were found in 99 of 162 children by ultrasonography, intravenous pyelography, and nuclear scans. In these children there was significant over-representation of the DD genotype (II 11%, ID 53%, DD 36%) compared with normals (P<0.005, X2=14.9) or with patients with uropathies but no renal lesions (II 23%, ID 62%, DD 15%, P<0.005, X2=14.9). Because ACE I/D has been linked with progressive deterioration of renal function, we evaluated a subset of patients with initially normal kidneys who developed radiographic renal lesions (n=28). Among these patients there was an even greater over-representation of the DD genotype (II 0%, ID 43%, DD 57%, P<0.001, X2=22.6) compared with patients with uropathies but no radiographic lesions. Multivariate analysis revealed that the DD genotype is a risk factor for parenchymal destruction, which was independent of time of diagnosis, surgical intervention, or urinary tract infection. This finding was particularly relevant in patients with VUR who constituted the majority with initially normal kidneys who developed radiographic damage (22/28). Indeed, the odds ratio of developing parenchymal damage with VUR was significantly increased if the individual had the DD genotype (4.2, 95% confidence interval 1.4-13.0). In conclusion the ACE I/D gene polymorphism is a risk factor for renal parenchymal damage in patients with congenital urological abnormalities and appears particularly relevant in children with VUR, where it is an independent predisposing factor.

Potent antihypertrophic effect of the bradykinin B2 receptor system on the renal vasculature.

BACKGROUND: Angiotensin type 1 (AT1) receptor-deficient mice (Agtr1-/-), which selectively lack both AT1A and AT1B receptor genes, are characterized by marked intrarenal vascular thickening. In the present study, we explored the possible involvement of the kinin-kallikrein system in the development of this renal vascular hypertrophy. METHODS: Wild-type and Agtr1-/- mice were examined for the developmental regulation pattern of the kinin-kallikrein system and treated with aprotinin (a kallikrein inhibitor), AcLys [D-b Nal7, Ile8] des-Arg9-bradykinin (a bradykinin B1 receptor antagonist), or Hoe-140 (a bradykinin B2 receptor antagonist) from 3 to 14 days of age. RESULTS: The normal postnatal up-regulation of kininase II was organ-specifically suppressed in Agtr1-/- kidneys at 2 and 3 weeks of age. Immunohistochemical staining in Agtr1-/- mice revealed tissue kallikrein staining along the nephron from connecting tubules to cortical collecting tubules in proximity to the hypertrophic vasculature, whereas tissue kallikrein staining was confined to connecting tubules in wild-type mice. Aprotinin and Hoe-140 accelerated the vascular hypertrophy significantly as determined by wall thickness ratio, whereas B1 receptor antagonism had no effect. CONCLUSION: The kinin-kallikrein system in the Agtr1-/- mouse kidney is functionally activated by local suppression of kininase II and extensive redistribution of kallikrein to perivascular areas. This activation, specific to the kidney, serves to dampen a development of the marked vascular hypertrophy. These results demonstrate, to our knowledge for the first time, the antihypertrophic effect of the bradykinin B2 receptor system on the renal vasculature in vivo.

Angiotensin type 2 receptor is important in the normal development of the ureter.

In humans, the actions of angiotensin II are transduced through the AT1 and AT2 receptors which have recently been implicated in renal organogenesis. Polymorphisms in the human angiotensin II receptor genes have been linked to cardiovascular and nephrological disorders. In this study we evaluated 35 patients with either primary obstructive megaureter or posterior urethral valves. Each was genotyped for the A1166 AT1 polymorphism and the recently described A-1332G AT2 transition. The incidence of these genetic variants was also evaluated in normal controls without any ultrasonographic urological abnormalities. Similar to our previous findings in congenital urological abnormalities, the AT1 receptor genotype distribution did not differ between patients with either primary obstructive megaureter or posterior urethral valves versus controls. In contrast, compared with normal controls, there was a dramatic increase in the occurrence of the AT2 A-1332G transition in patients with primary obstructive megaureter (75.0% vs. 41.9% in controls, P<0.025). In patients with posterior urethral valves, there was no difference in the occurrence of the transition versus controls (36.9%, P=NS). Thus, there is no correlation between the AT1 receptor gene polymorphism and urological abnormalities. However there is an increased incidence in the AT2 genetic variant in patients with primary obstructive megaureter.

Angiotensin II, type 2 receptor in the development of vesico-ureteric reflux.

OBJECTIVE: To investigate if mutation of the angiotensin II (Ang II) receptors AT2 is involved in primary vesico-ureteric reflux (VUR) in humans. PATIENTS AND METHODS: Genetic polymorphisms in the AT1 and AT2 receptors was evaluated in 23 patients having the most common congenital urological abnormality, namely primary congenital VUR. The occurrence of the A1166C transition in the AT1 receptor gene and the A-1332G transition in the AT2 receptor gene were evaluated and compared with the incidence in normal controls with no urological abnormalities. RESULT: The distribution of the AT1 receptor genotypes was no different between patients with VUR and healthy controls. Furthermore, 10 of 23 (44%) patients with VUR and seven of 19 (42%) controls carried the AT2 receptor gene variation. These results contrast with our previous finding of an association between the A-1332G transition in the AT2 receptor gene and primary obstructive megaureter, and pelvi-ureteric junction obstruction. CONCLUSIONS: We propose that while the AT2 receptor is crucial for the normal development of the ureter, it does not contribute to the processes which culminate in VUR, which is primarily an abnormality in the bladder trigone.

IgA nephropathy.

Immunoglobulin A nephropathy results from the abnormal deposition of IgA immunoglobulin in the glomerulus, which leads to the characteristic presentation of painless hematuria. It is the most common glomerulonephritis worldwide. Originally described 30 years ago, it was thought to follow a benign course. We now know that IgA nephropathy leads to progressive renal destruction in about one third of affected patients. Alteration in glycosylation of circulating IgA may be an important pathophysiologic mechanism that predisposes to IgA deposition, although how this leads to parenchymal damage remains unclear. Hypertension, high-grade proteinuria, and elevated serum creatinine levels are known risk factors for progressive renal destruction. In addition to these well-recognized risk factors, there appear to be genetic variants, particularly within the angiotensin-converting enzyme gene, that portend a worse outcome.

Role of the angiotensin type 2 receptor gene in congenital anomalies of the kidney and urinary tract, CAKUT, of mice and men.

Angiotensin type 2 receptor gene null mutant mice display congenital anomalies of the kidney and urinary tract (CAKUT). Various features of mouse CAKUT impressively mimic human CAKUT. Studies of the human type 2 receptor (AGTR2) gene in two independent cohorts found that a significant association exists between CAKUT and a nucleotide transition within the lariat branchpoint motif of intron 1, which perturbs AGTR2 mRNA splicing efficiency. AGTR2, therefore, has a significant ontogenic role for the kidney and urinary tract system. Studies revealed that the establishment of CAKUT is preceded by delayed apoptosis of undifferentiated mesenchymal cells surrounding the urinary tract during key ontogenic events, from the ureteral budding to the expansive growth of the kidney and ureter.

Role of the renin-angiotensin system in disorders of the urinary tract.

PURPOSE: The role of the renin-angiotensin system in the homeostasis of fluid, electrolyte and blood pressure has been known for quite some time. Recent developments indicate that angiotensin has a profound role, not only in the developing urinary tract but also in the response of the urinary tract to injury. In this review we outline these characteristics. MATERIALS AND METHODS: We summarize the clinical approach to congenital abnormalities of the kidney and urinary tract, and report new data obtained in genetically engineered mice. Furthermore, we present the connection between the mutant mice observations and human congenital abnormalities. RESULTS: Genetically engineered mutants clearly indicate that the renin-angiotensin system is important for normal renal and urological development. As in glomerular disease, the renin-angiotensin system is involved in progressive damage due to urological disease. CONCLUSIONS: While the renin-angiotensin system is important for blood pressure regulation, it also affects the embryogenesis of the urinary tract and modulates renal injury due to specific disease processes. The importance of angiotensin and its blockade provides an exciting avenue for possible early treatment in children with congenital anomalies of the kidney and urinary tract.

Renal angiotensin converting enzyme promotes renal damage during ureteral obstruction.

PURPOSE: We and others have shown that angiotensin II has a pivotal role in renal damage in various renal injuries. Although most angiotensin II actions are associated with the angiotensin type 1 receptor, there is increasing evidence that the angiotensin type 2 receptor also transduces some important effects of angiotensin II. In this regard we recently observed that mice with genetically engineered disruption of the angiotensin type 2 receptor, termed Agtr2 mutants, are more susceptible to structural renal damage after ureteral obstruction. Recent evidence suggests that a genetically determined increase in angiotensin converting enzyme activity in humans promotes end organ damage. Therefore, we determined whether renal damage in Agtr2 mutants is associated with heightened angiotensin converting enzyme activity. MATERIALS AND METHODS: We studied 28 wild type and 19 Agtr2 mutant mice with unilateral ureteral obstruction. Seven days after obstruction was created serum samples were obtained to evaluate angiotensin converting enzyme activity. The obstructed and contralateral kidneys were harvested for histological analysis and determination of renal angiotensin converting enzyme activity by high pressure liquid chromatography. RESULTS: Renal angiotensin converting enzyme was uniformly higher than serum angiotensin converting enzyme in normal wild type and Agtr2 mutant mice. However, even at baseline Agtr2 mutant mice had strikingly higher renal angiotensin converting enzyme activity than normal wild type mice (mean plus or minus standard error 1,492+/-83 versus 450+/-60 milliunits per gm. tissue weight, p <0.0005). Histological analysis revealed more extensive parenchymal damage in the obstructed kidneys of mutant mice than in identically treated controls. Notably while unilateral ureteral obstruction decreased renal angiotensin converting enzyme activity in each group, activity remained persistently higher in the Agtr2 mutants than in normal mice (mean 742+/-146 versus 310+/-43 milliunits per gm. tissue weight, p <0.005). CONCLUSIONS: We propose that elevated renal angiotensin converting enzyme activity contributes to more severe renal parenchymal damage in ureteral obstruction by promoting the availability of growth factors, such as angiotensin II, or depleting antiproliferation factors, such as bradykinin or nitric oxide. These findings complement previous observations that angiotensin converting enzyme inhibition preserves the renal parenchyma after injury, including obstruction.

Angiotensin II type I receptor polymorphism in African Americans lower frequency of the C1166 variant.

The C1166 variant, an A to C substitution polymorphism at the 1166 position of the angiotensin II type I (AT1) receptor, has been previously associated with hypertension in Caucasians. This study determines the frequency of the C1166 variant in an African American population. Normotensive African American (n = 99) and Caucasian (n = 100) subjects were genotyped to determine the frequency of the C1166 variant. This study establishes the frequency of the C1166 variant in African Americans (0.05 +/- 0.01) and demonstrates a significantly lower frequency in African Americans compared with Caucasians (0.05 vs. 0.25, respectively, chi 2 = 30.7, p < < 0.001, 1 df).

Endothelin in ischemic acute renal failure.

The endothelin peptides comprise a family of potent and long-lasting vascoconstrictors, to which the renal microcirculation is particularly susceptible. Increased renal endothelin expression is observed after a variety of injurious stimuli, including ischemia, and persists for days after resolution of the initial injury. Autoinduction of its own production is likely to be a central mechanism underlying endothelin's prolonged effects. Furthermore, antagonizing endothelin reveals its role in maintaining the postischemic glomerular dysfunction that typifies ischemic acute renal failure.

Angiotensin converting enzyme gene polymorphism: potential silencer motif and impact on progression in IgA nephropathy.

Since the renin angiotensin system (RAS) is established as an important factor in renal disease progression, we determined whether RAS alleles that have been linked to variability in outcome in several cardiovascular diseases also affect progression of IgA nephropathy. These genetic variants include: (1) angiotensin I converting enzyme deletion polymorphism in intron 16 (ACE I/D), reported to be associated with increased risk of myocardial infarction as well as left ventricular hypertrophy; (2) a point mutation in the angiotensinogen (Agt) gene resulting in a methionine to threonine substitution at residue 235 (M235T), reported to be associated with hypertension in Caucasians; and (3) an angiotensin receptor type I (ATR) A to C transition at bp 1166 (A1166C) which shows synergy with the deleterious effects of the ACE DD genotype in myocardial infarction. We examined these polymorphisms by PCR amplification of genomic DNA samples from 64 Caucasian patients in the USA (age 6 to 83 years) with biopsy-proven IgA nephropathy whose renal status was followed for an average of almost seven years. Patients who presented with and maintained normal serum creatinine (Cr, < 1.5 mg/dl), had ACE genotype frequencies of II:35%, ID:61%, DD:4%. By contrast, in patients with progression (initially normal Cr increased to a mean of 4.5 +/- 0.86 mg/dl), ACE genotype frequencies were II:22%, ID:44%, DD:33% (P = 0.057 by Fishers's exact test, vs. non-progressors). The association of the DD genotype with progression was even more striking when patients with other risk factors (hypertension and/or heavy proteinuria) were excluded. In this subgroup, the genotype frequencies in patients with stable creatinine versus those with deterioration in renal function was 53%, 47%, and 0% versus 0%, 40%, and 60%, respectively, for II, ID, and DD genotypes (P = 0.009 by Fisher's exact test, progressors vs. non-progressors). Further, sequence analysis of the I gene polymorphism revealed a potential 13 bp silence motif. Neither the Agt 235T nor the ATR A 1166C gene variants, however, was associated with deterioration of renal function. Taken together, these results indicate that, although polymorphism in each of the three genes in the RAS system has been linked to cardiovascular diseases, only the ACE I/D polymorphism is associated with progressive deterioration in renal function in IgA nephropathy. Since previous observations link ACE polymorphism with ACE activity, these findings imply a widespread importance of ACE in modulating destructive processes in different organs.

Combined antagonism of endothelin A/B receptors links endothelin to vasoconstriction whereas angiotensin II effects fibrosis. Studies in chronic cyclosporine nephrotoxicity in rats.

Both functional and structural damage characterize nephrotoxicity due to cyclosporine (CsA) with accumulating evidence for dissociation of mechanisms that lead to each of these processes. We studied the role of endothelin (Et) and angiotensin II (AII), since each of these peptides can modulate vasoconstriction as well as parenchymal destruction. Salt-depleted rats were treated with daily CsA (15 mg/kg s.c.) for 5 weeks (group 1, CsA, n = 13). Separate groups of CsA-treated rats received either a combined antagonist of both EtA/EtB receptors (group 2, CsA+aEtA/B, 100 mg/kg/day p.o., n = 6) or angiotensin I-converting enzyme inhibitor (group 3, CsA+ACEI, enalapril 200 mg/L drinking water, n = 8). Glomerular filtration rate (GFR) was assessed by creatinine clearance (Ccr) in conscious rats at 3 and 5 weeks. At 3 weeks, serum creatinine was 1.5 +/- 0.1 mg/dl in group 1 rats, 1.2 +/- 0.2 mg/dl in group 2 rats (P < 0.05 vs. CsA), and 2.3 +/- 0.8 mg/dl in group 3 rats. Ccr was 0.87 +/- 0.08 ml/min in group 1. In group 2, GFR was remarkably preserved (1.14 +/- 0.11 ml/min, P < 0.05 vs. group 1). By contrast, GFR in group 3 rats was lower (0.31 +/- 0.08 ml/min) than either aEtA/B-treated or even CsA-treated rats (P < 0.0005 vs. group 1, P < 0.0005 vs. group 2). At 5 weeks, the same pattern emerged; serum creatinine was 2.5 +/- 0.2 mg/dl in group 1, 1.2 +/- 0.1 in group 2 (P < 0.0005 vs. CsA), and 3.4 +/- 0.9 in group 3 (P < 0.025 vs. CsA+aEtA/B). Ccr had decreased dramatically in CsA-treated rats to 0.18 +/- 0.03 ml/min. GFR was preserved in CsA+aEtA/B rats (0.51 +/- 0.03 ml/min, P < 0.0005 vs. group 1), while profound hypofiltration was apparent in CsA+ACEI rats (0.12 +/- 0.03 ml/min, P < 0.0005 vs. group 2). In salt-depleted control animals, GFR was 0.62 +/- 0.02 ml/min. Despite striking functional preservation in response to antagonism of Et receptors, tubular vacuolization/dilatation, as well as arteriolopathy, was not different among the CsA-treated groups. Tubulointerstitial fibrosis was also not different between CsA and CsA+aEtA/B rats (on a 0-4 scale, 1.05 +/- 0.14 vs. 0.87 +/- 0.14, P = NS). In contrast, both tubular vacuolization/dilatation and interstitial fibrosis were significantly greater in all CsA-treated groups compared with salt-depleted controls. However, in the CsA+ACEI group that had the most severe hypofiltration at each time point, tubulointerstitial fibrosis was 0.69 +/- 0.06 (P < 0.05 vs. CsA).(ABSTRACT TRUNCATED AT 400 WORDS)

Nitric oxide and endothelin in pathophysiological settings.

The role of the endothelium is now known to encompass the generation of many potent cytokines which impact endothelial cells, adjacent tissue such as smooth muscle cells, and distant sites in an autocrine, paracrine, and endocrine manner, respectively. This review addresses two of these cytokines, nitric oxide and endothelin, and describes how each effects the functions of endothelial cells, including regulation of platelet aggregation and coagulation, regulation of vasomotor tone, modulation of inflammation, and the regulation of cellular proliferation. The emphasis is on the increasingly recognized importance of the autocrine and paracrine mechanisms by which nitric oxide and endothelin act. In particular, autoinduction of endothelin is proposed as a central mechanism underlying endothelin's renowned effects. Additionally, specific nitric oxide/endothelin interactions are discussed by which each cytokine modulates the production and actions of the other. The net effect observed in a variety of physiological and pathophysiological settings, therefore, reflects a balance of these opposing functions.

Endothelin A receptor mediates functional but not structural damage in chronic cyclosporine nephrotoxicity.

Chronic treatment with cyclosporine (CsA) is limited not only by glomerular hypofiltration but also by structural damage. The pathogenesis of these nephrotoxicities was studied in a model of chronic CsA-induced renal damage. Salt-depleted rats were treated with daily CsA (15 mg/kg sc) for approximately 3 weeks, at which time renal function was measured and kidneys were harvested for morphologic assessment. A separate group of rats (CsA + BQ123) were identically treated with CsA but in addition also received simultaneous treatment with a specific endothelin A (EtA) receptor antagonist, BQ123, which was continuously delivered via sc osmotic pump (1 mg/kg per hour) and maintained throughout the study. Chronic CsA treatment caused profound functional and structural damage, although blood pressure was normal (102 +/- 6 mm Hg); GFR was 0.05 +/- 0.02 mL/min per 100 g body wt, and RPF was 0.15 +/- 0.06/100 g body wt. Renal injury was scored on a scale of 0 to 4 and showed dilation/vacuolization of 1.07 +/- 0.29 and tubulointerstitial fibrosis of 0.78 +/- 0.17. Arteriolopathy was present in 78 +/- 4% of arterioles. Chronic antagonism of the EtA receptor preserved renal function: GFR was 0.15 +/- 0.03 mL/min per 100 g body wt, and RPF was 0.32 +/- 0.08/100 g body wt (P < 0.05 for GFR versus CsA). Blood pressure was not affected: 104 +/- 8 mm Hg.(ABSTRACT TRUNCATED AT 250 WORDS)