Apoptosis plays a role in the regulation of heart mass and architecture, and might contribute to the cardiac remodelling seen in renovascular hypertension. It is not known whether the beneficial effects of angiotensin-converting enzyme (ACE) inhibition or calcium channel blockade on cardiac remodelling are linked to the modulation of apoptosis. To test this hypothesis, we established four groups of rats: (i) sham-operated controls, (ii) a group that underwent the two-kidney/one-clip (2K1C) procedure, (iii) a group with 2K1C treated for 12 weeks with quinapril (6mg·day -1 ·kg -1 ), and (iv) a group with 2K1C treated for 12 weeks with diltiazem (24mg·day -1 ·kg -1 ). Treatment started 2 weeks after clipping. Systolic blood pressure was reduced to a similar extent by quinapril and diltiazem (2K1C, 223±19mmHg; 2K1C+quinapril, 149±15mmHg; 2K1C+diltiazem, 160±40mmHg; both P <0.01 compared with 2K1C alone). Left ventricular weight, interstitial fibrosis and perivascular fibrosis were reduced significantly by both drugs. The apoptotic index (apoptotic cells/total cell number) was increased 21.6-fold ( P <0.01) after quinapril treatment as compared with the 2K1C group, but was not affected by calcium channel blockade. In conclusion, our study demonstrates that ACE inhibition, in contrast with calcium channel blockade, may cause regression of cardiac hypertrophy/remodelling in 2K1C renovascular hypertensive rats through enhanced apoptosis.

Plasma endothelin (ET)-1 concentrations have been shown to be elevated in patients receiving calcineurin-inhibitors (CI). We investigated urinary and plasma ET-1 (uET-1, pET-1), BigET-1 (uBigET-1, pBigET-1) concentrations, and plasma soluble endothelin-converting enzyme (ECE) concentrations in 381 adult caucasian kidney allograft recipients with graft survival of more than 2 years from the outpatients department of our clinic. Blood and urine probes were always drawn immediately before morning dosage of immunosuppressants. Mean of urinary protein excretion (meanProt) and mean of serum creatinine (meanCrea) were calculated from all available measurements in the most recent year. uET-1 and uBigET-1 were adjusted for urinary protein excretion by calculating uET-1/meanProt and uBigET-1/meanProt. Patients ( n = 310) were on a cyclosporine A or FK506 (CI-group) based immunosuppression protocol, and 71 patients were immunosuppressed without use of CI (nonCI group). Time since transplantation was similar in both groups (mean±S.D.; CI-group: 7.55±2.50; nonCI-group: 7.74±3.06 years, P = 0.240) as well as meanCrea (mean±S.D.; CI-group: 1.97±1.34; nonCI-group: 1.77±1.29mg/dl, P = 0.326). pET-1 was significantly higher in the CI-group, compared with nonCI (mean±S.D.; 0.87±1.4 versus 0.56±0.76fmol/ml, P = 0.011). pBigET-1 was similar (mean±S.D.; 0.85±1.41 versus 0.70±1.21fmol/ml, P = 0.33). ECE concentrations were higher in the CI group (mean±S.D.; 14.30±18.02 versus 9.23±7.42ng/ml, P = 0.001). uET-1/meanProt and uBigET-1/meanProt concentration were similar in the CI-group compared with the nonCI-group (mean±S.D.; uET-1/meanProt: 15±24 versus 21±40pmol/g, P = 0.139; uBigET-1/meanProt: 34±55 versus 19±23pmol/g, P = 0.248). pET-1 elevation in patients receiving CI might be more likely to be due to elevated conversion of pBig-ET-1 by more ECE, and not to higher concentrations of pBigET-1.

Endothelin-1 (ET-1) is a potent pro-fibrotic growth factor. However, little is known about its specific effects on the synthesis of matrix proteins in vivo . We used male 12-month-old ET-1 transgenic mice characterized by transgene expression in the kidney and (to a lesser extent) in the heart. Global cardiac and renal matrix protein synthesis was analysed after Sirius Red and periodate–Schiff staining. Specific expression of collagen types I, III and IV, laminin and fibronectin was examined using immunohistochemistry followed by computer-aided image analysis. Analysis of blood pressure revealed that mean arterial blood pressure was similar in ET-1 transgenic mice and controls. The total cardiac matrix protein content was increased in the myocardium of ET-1 transgenic mice. Analysis of specific cardiac matrix proteins showed increased cardiac expression of collagen type III (+211%; P <0.001) and laminin (+128%; P <0.01) in transgenic mice. The expression of collagen types I and IV and fibronectin was not altered. Global analysis of renal matrix proteins confirmed earlier studies showing pronounced interstitital fibrosis and glomerulosclerosis. Laminin expression was markedly increased in the glomerula (+152%; P <0.01) and even more so in the interstitium (+211%; P <0.001), whereas expression of collagen type III was reduced in glomerula (-48%; P <0.01) and interstitial tissue (-55%; P <0.01) of ET-1 transgenic mice. In conclusion, a primary overexpression of ET-1 does not cause uniformly enhanced synthesis of matrix proteins. In contrast, the effects of ET-1 on the matrix protein pattern is tissue-specific. The major renal and cardiac alterations in matrix proteins induced by ET-1 is a marked enhancement of laminin expression.

Transgenic overexpression of erythropoietin (Epo) in mice increases haematocrit to a mean of 80% in adult mice, leading to an increase in blood viscosity and volume. As a consequence, renal tissue endothelin-1 (ET-1) concentrations are significantly elevated in erythropoietin-overexpressing (Epo+) mice (mean±S.E.M; Epo+, 798±71; Epo-, 400±25pg/g tissue; P <0.01). To investigate the pattern of expression of the primary translation product of the ET-1 gene, prepro-ET-1, in kidneys of (Epo+) mice, we generated crossbred mice overexpressing the human EPO gene with mice carrying a reporter gene construct expressing the LacZ gene under the control of the human prepro-ET-1 promotor sequence (LacZ+/Epo+). For comparison, we generated (LacZ+/Epo-) mice from the same strains. After Bluo-Gal staining of frozen kidney sections ( n = 10 in each group), intracellular blue precipitates as indicators of prepro-ET-1 promotor activity were detectable in tubular and vascular endothelium and glomerular cells in (LacZ+/Epo-) as well as (LacZ+/Epo+) mice. Comparison of the amount of blue precipitates by semiquantitative scoring showed a significant increase in reporter gene activity in tubular epithelium of (LacZ+/Epo+) mice (mean±S.E.M.; LacZ+/Epo+, 1.64±0.18; LacZ+/Epo-, 1.00±0.19; P <0.05). Reporter gene activity was not significantly elevated in epithelium of small intrarenal arteries of (LacZ+/Epo+) mice (mean±S.E.M.; LacZ+/Epo+, 0.86±0.14; LacZ+/Epo-, 0.38±0.21; P = 0.08) and was similar in glomerular cells (mean±S.E.M.; LacZ+/Epo+, 1.28±0.16; LacZ+/Epo-, 1.14±0.21; P = 0.6). The main source of elevated ET-1 tissue concentration in kidneys of (Epo+) mice therefore seems more likely to be tubular than vascular endothelium or glomerular cells.

The renal endothelin (ET) system is involved in the pathogenesis of kidney fibrosis as well as blood pressure control by regulating tubular sodium excretion. Long-term effects of ETA receptor blockade on blood pressure and kidney function in spontaneously hypertensive rats (SHRs) on a high-salt diet are unknown. We treated SHRs on a 6% (w/v) NaCl sodium diet (SHR-S) for 48 weeks with the ETA antagonist LU 135252 (whose selectivity for ETA is 150 times greater than for ETB) with 10, 30 and 100mg·kg -1 ·day -1 or placebo. The ETA antagonist had at no time-point any effect on blood pressure. Glomerular filtration rate was normal in SHR-S and not altered by LU 135252. However, urinary albumin excretion was markedly reduced by the ETA antagonist (SHR-S, 145±50mg/day; SHR-S+10mg·kg -1 ·day -1 LU 135252, 33±11mg/day, P <0.05 versus SHR-S; SHR-S+30mg·kg -1 ·day -1 LU 135252, 55±16mg/day and SHR-S+100mg·kg -1 ·day -1 LU 135252, 32±11mg/day, P <0.05 versus SHR-S at both concentrations). Total urinary protein excretion was likewise significantly reduced by treatment with 10mg·kg -1 ·day -1 LU 135252 (SHR-S, 0.25±0.06g/day; SHR-S+10mg·kg -1 ·day -1 LU 135252, 0.089±0.01g/day, P <0.05 versus SHR-S). The higher dosages of LU 135252 showed only a trend towards reduction of total urinary protein excretion. Computer-aided image analysis after haematoxylin/eosin and periodic acid-Schiff staining revealed that treatment with 10mg·kg -1 ·day -1 LU 135252 significantly reduces the media/lumen ratio of intrarenal arteries. Higher dosages of LU 135252 were less effective. Renal matrix protein synthesis in SHR-S was not altered by LU 135252. In conclusion, the renal ET system contributes in a blood-pressure-independent manner to the regulation of urinary protein excretion and renal vascular hypertrophy in SHR-S. Lower doses of the ETA antagonist were more effective, indicating that a potential additional blockade of the ETB receptor using higher doses of LU 135252 seems to oppose the beneficial effects of a sole ETA blockade. Urinary protein excretion is an independent risk factor of chronic renal failure, thus ETA antagonists might be a therapeutic tool to prevent proteinuria-induced chronic renal failure.

Endothelin (ET) A receptor antagonists have been shown to be beneficial in rat models of chronic kidney allograft dysfunction. We investigated urinary and plasma ET-1 (uET-1, pET-1) and BigET-1 (uBigET-1, pBigET-1) concentrations, and plasma soluble ET-converting enzyme (ECE) concentration in 310 adult Caucasian kidney allograft recipients with graft survival of more than 2 years from the outpatients department of our clinic. All patients were on cyclosporine A- or FK506-based immunosuppression protocols. From all available measurements since transplantation, we calculated the slope of serum creatinine -1 /year (slopeCrea) as a parameter for progression of chronic graft dysfunction, as well as the mean of serum creatinine (meanCrea) from most recent year before measurements as a parameter for actual graft function. The slope of urinary protein excretion/year (slopeProt) and mean of urinary protein concentration (meanProt) from most recent year was calculated analogue. uET-1 and uBigET-1 were adjusted for protein excretion by calculating uET-1/meanProt and uBigET-1/meanProt. Blood and urine probes for measurements were always drawn immediately before morning dosage of immunosuppressants. There was no significant correlation of any measured component of the ET system with slopeCrea or slopeProt. MeanCrea (mg/dl) was significantly correlated with pBigET-1 (fmol/ml) and pET-1 (fmol/ml) (pBigET-1: r = 0.179, P = 0.001; pET-1: r = 0.161, P = 0.009). The other measured components of the ET systems were not significant correlated with meanCrea. In conclusion, the actual graft function is associated with elevated pET-1 and BigET-1 concentrations as it is well known from other forms of impaired kidney function. However, the actual concentration of ET-1, soluble ECE, and BigET-1 in urine and plasma in our study is not associated with parameters for progression of chronic graft dysfunction.

Pre-eclampsia complicates approximately 5–7% of pregnancies and it may be deleterious to both maternal and fetal health. In a prospective study, we investigated plasma endothelin (ET)-1 concentration within the 24th and 36th gestational week in non-smoking pregnant women. Thirty women fulfilled the criteria for the diagnosis of pre-eclampsia according to the American College of Obstetricians and Gynaecologists: de novo arterial hypertension after the 20th gestational week in at least two separate measurements and proteinuria of more than 300mg/l in a random specimen. For comparison, we analysed blood samples from 125 non-pre-eclamptic pregnant women. ET-1 concentrations were higher in pre-eclamptic pregnancies at both time points (mean±S.D.: 1.07±2.00 versus 0.54±0.56pg/ml, P = 0.045 at 24th week; 0.75±1.20 versus 0.44±0.45pg/ml, P = 0.023 at 36th week). Receiver operating characteristic (ROC) curves revealed a significant interaction between ET-1 plasma concentrations at the 36th week and the diagnosis of pre-eclampsia [area under the curve (AUC)±S.E.M.: 0.657±0.049, P = 0.008] and a cut-off value at 0.30pg/ml. Multivariate analysis showed a 4.6-fold higher chance (95% confidence interval: 1.7–12.1, P = 0.002) for the diagnosis of pre-eclampsia in pregnant women with ET-1 plasma concentration higher than 0.30pg/ml at the 36th week. Interaction between ET-1 plasma concentration at the 24th week and diagnosis of pre-eclampsia was not significant in ROC curve analysis (AUC±S.E.M.: 0.594±0.071, P = 0.278). Interestingly, we found a strong positive correlation between ET-1 concentration in the 24th and 36th week in linear regression analysis in pre-eclamptic ( r = 0.99, P <0.001) and non-pre-eclamptic pregnancies ( r = 0.61, P <0.001) with a slightly, non-significant decrease from the 24th to 36th week (for group means see above), indicating individual plasma ET-1 levels even in non-pre-eclamptic pregnancies. Linear regression analysis showed no correlation between blood pressure or urine protein excretion and ET-1 plasma concentration in non-pre-eclamptic pregnant women. In conclusion, our prospective study indicates that the ET system is, in contrast to most other forms of human hypertension, activated in pre-eclamptic pregnant women.