Niveles de células progenitoras endoteliales circulantes en pacientes hipertensos tratados / Circulating endothelial progenitor cell levels in treated hypertensive patients

Introducción: Muchos pacientes hipertensos óptimamente tratados de sus factores de riesgo cardiovascular (FRCV) siguen presentando complicaciones cardiovasculares. Las células progenitoras endoteliales (CPE) han demostrado ser fundamentales para la reparación del daño endotelial en tejidos isquémicos. Por ello, hemos estudiado los niveles de CPE y del factor de crecimiento del endotelio vascular (VEGF) en pacientes hipertensos en tratamiento con buen control de la presión arterial (PA). Material y métodos: Se recogió una muestra de sangre de pacientes hipertensos tratados que presentaban unas cifras de PA adecuadas para los objetivos individuales. Los niveles plásticos de CPE CD34+/KDR+ y CD34+/VE-cadherina+ se midieron mediante citometría de flujo. La concentración de VEGF se cuantificó mediante ELISA. Como controles se incluyó un grupo de sujetos sin FRCV tradicionales. Resultados: Hemos incluido 108 pacientes (61 ± 12 años, 47,2% hombres), de los cuales un 82,4% presentaba PA < 140/90 mmHg, un 91,7% control de la diabetes (HbA1c < 7%), el 81,5% cLDL < 130 o 100 mg/dl y el 85,2% no fumaba, aunque el 45,4% presentaba obesidad (IMC ≥ 30 kg/m2). A pesar de que, en conjunto, sus parámetros bioquímicos no diferían de los del grupo control, los pacientes hipertensos presentaban una disminución significativa de los niveles plasmáticos de células CD34+/KDR+ y CD34+/VE-cadherina+, aunque la concentración plasmática de VEGF era significativamente mayor en los pacientes hipertensos que en los sujetos control. Conclusiones: Los pacientes hipertensos tratados muestran una disminución significativa de los niveles plasmáticos de CPE que podría ser responsable, al menos en parte, del riesgo residual que presentan estos pacientes, sugiriéndose que las CPE podrían ser una importante diana terapéutica

Introduction: Most optimally treated hypertensive patients still have an around 50% increased risk of any cardiovascular event, suggesting the possible existence of unidentified risk factors. In the last years there has been evidence of the essential role of circulating endothelial progenitor cells (EPCs) in the maintenance of endothelial integrity and function, increasing the interest in their involvement in cardiovascular disease. In this study, the circulating levels of EPCs and vascular endothelial growth factor (VEGF) are investigated in treated hypertensive patients with adequate control of blood pressure (BP). Material and methods: Blood samples were collected from treated hypertensive patients with controlled BP. Plasma levels of EPCs CD34+/KDR+ and CD34+/VE-cadherin+ were quantified by flow cytometry. Plasma concentration of VEGF was determined by ELISA. A group of healthy subjects without cardiovascular risk factors was included as controls. Results: A total of 108 hypertensive patients were included (61±12 years, 47.2% men) of which 82.4% showed BP < 140/90 mmHg, 91.7% and 81.5% controlled diabetes (HbA1c <7%) and cLDL (<130 or 100 mg/dL), respectively, and 85.2% were non-smokers. Around 45% of them were obese. Although patients had cardiovascular parameters within normal ranges, they showed significantly lower levels of CD34+/KDR+ and CD34+/VE-cadherin+ compared with healthy control group, although plasma VEGF concentration was higher in patients than in controls. Conclusions: Despite an optimal treatment, hypertensive patients show a decreased number of circulating EPCs that could be, at least in part, responsible for their residual cardiovascular risk, suggesting that these cells could be a therapeutic target

[Circulating endothelial progenitor cell levels in treated hypertensive patients]. / Niveles de células progenitoras endoteliales circulantes en pacientes hipertensos tratados.

INTRODUCTION: Most optimally treated hypertensive patients still have an around 50% increased risk of any cardiovascular event, suggesting the possible existence of unidentified risk factors. In the last years there has been evidence of the essential role of circulating endothelial progenitor cells (EPCs) in the maintenance of endothelial integrity and function, increasing the interest in their involvement in cardiovascular disease. In this study, the circulating levels of EPCs and vascular endothelial growth factor (VEGF) are investigated in treated hypertensive patients with adequate control of blood pressure (BP). MATERIAL AND METHODS: Blood samples were collected from treated hypertensive patients with controlled BP. Plasma levels of EPCs CD34+/KDR+ and CD34+/VE-cadherin+ were quantified by flow cytometry. Plasma concentration of VEGF was determined by ELISA. A group of healthy subjects without cardiovascular risk factors was included as controls. RESULTS: A total of 108 hypertensive patients were included (61±12 years, 47.2% men) of which 82.4% showed BP<140/90 mmHg, 91.7% and 81.5% controlled diabetes (HbA1c <7%) and cLDL (<130 or 100 mg/dL), respectively, and 85.2% were non-smokers. Around 45% of them were obese. Although patients had cardiovascular parameters within normal ranges, they showed significantly lower levels of CD34+/KDR+ and CD34+/VE-cadherin+ compared with healthy control group, although plasma VEGF concentration was higher in patients than in controls. CONCLUSIONS: Despite an optimal treatment, hypertensive patients show a decreased number of circulating EPCs that could be, at least in part, responsible for their residual cardiovascular risk, suggesting that these cells could be a therapeutic target.

Ezetimibe reduces plaque inflammation in a rabbit model of atherosclerosis and inhibits monocyte migration in addition to its lipid-lowering effect.

BACKGROUND AND PURPOSE: Ezetimibe, a selective inhibitor of intestinal cholesterol absorption, might also suppress inflammatory components of atherogenesis. We have studied the effects of ezetimibe on two characteristics of atherosclerotic plaques (infiltrate and fibrosis) and on expression of inflammatory genes in a rabbit model of accelerated atherosclerosis. EXPERIMENTAL APPROACH: Femoral atherosclerosis was induced by a combination of endothelial desiccation and atherogenic diet. Animals were randomized to ezetimibe (0.6 mg x kg(-1) x day(-1)), simvastatin (5 mg x kg(-1) x day(-1)), ezetimibe plus simvastatin or no treatment, still on atherogenic diet. A control group of rabbits received normolipidemic diet. KEY RESULTS: Rabbits fed the normolipidemic diet showed normal plasma lipid levels. Either the normolipidemic diet or drug treatment reduced the intima/media ratio (normolipidemic diet: 22%, ezetimibe: 13%, simvastatin: 27%, ezetimibe + simvastatin: 28%), compared with rabbits with atherosclerosis. Ezetimibe also decreased macrophage content and monocyte chemoattractant protein-1 expression in atherosclerotic lesions. Furthermore, ezetimibe reduced the increased activity of nuclear factor kappaB in peripheral blood leucocytes and plasma C-reactive protein levels in rabbits with atherosclerosis. In THP-1 cells, ezetimibe decreased monocyte chemoattractant protein-1-induced monocyte migration. Importantly, the combination of ezetimibe with simvastatin was associated with a more significant reduction in plaque monocyte/macrophage content and some proinflammatory markers than observed with each drug alone. CONCLUSIONS AND IMPLICATIONS: Ezetimibe had beneficial effects both on atherosclerosis progression and plaque stabilization and showed additional anti-atherogenic benefits when combined with simvastatin. Its effect on monocyte migration provides a potentially beneficial action, in addition to its effects on lipids.

Losartan improves resistance artery lesions and prevents CTGF and TGF-beta production in mild hypertensive patients.

Although structural and functional changes of resistance arteries have been proposed to participate in arterial hypertension (HTA) outcome, not all therapies may correct these alterations, even if they normalize the blood pressure (BP). The aim of this study was to investigate the mechanisms of the protection afforded by the angiotensin receptor antagonist losartan in resistance arteries from patients with essential HTA. In all, 22 untreated hypertensive patients were randomized to receive losartan or amlodipine for 1 year and the morphological characteristics of resistance vessels from subcutaneous biopsies were evaluated. Protein expression of connective tissue growth factor (CTGF), transforming growth factor beta (TGF-beta), and collagens III and IV was detected by immunohistochemistry. In comparison with normotensive subjects, resistance arteries from hypertensive patients showed a significant media:lumen (M/L) ratio increment and a higher protein expression of CTGF, TGF-beta, and collagens. After 1 year of treatment, both losartan and amlodipine similarly controlled BP. However, M/L only decreased in patients under losartan treatment, whereas in the amlodipine-treated group this ratio continued to increase significantly. The administration of losartan prevented significant increments in CTGF, TGF-beta, and collagens in resistance arteries. By contrast, amlodipine-treated patients showed a higher vascular CTGF, TGF-beta, and collagen IV staining than before treatment. Our results show that the administration of losartan, but not amlodipine, to hypertensive patients improves structural abnormalities and prevents the production of CTGF and TGF-beta in small arteries, despite similar BP lowering. These data may explain the molecular mechanisms of the better vascular protection afforded by drugs interfering with the renin-angiotensin system.

Renal tubulointerstitial damage caused by persistent proteinuria is attenuated in AT1-deficient mice: role of endothelin-1.

Using angiotensin II (AngII) type 1A receptor-deficient mice [AT1(-/-)], in which we induced protein overload nephropathy, we explored the potential implication of AngII and endothelin-1 (ET-1) in the tubulointerstitial damage because of persistent proteinuria. At day 7, AT1(-/-) showed marked proteinuria to a similar extent to that of wild-type mice (WT). However, at day14, AT1(-/-) had significantly less proteinuria, renal damage, transforming growth factor-beta, and matrix mRNA expression and mortality. AT1(-/-) also showed a significant diminution in the activation of the transcriptional factors nuclear factor-kappaB and AP-1. Unexpectedly, AT1(-/-) had a higher interstitial infiltration than WT. The administration of the angiotensin-converting enzyme inhibitor quinapril to WT caused a marked improvement in proteinuria and renal lesions, resembling that seen in untreated AT1(-/-). However, the interstitial infiltration persisted in AT1(-/-) when treated with quinapril. Because ET-1 may participate in the recruitment of mononuclear cells, we also studied the implication of this peptide. AT1(-/-) had a significantly higher ET-1 expression in tubular epithelial cells than WT. The administration of the dual ETA/ETB antagonist bosentan to AT1(-/-) considerably reduced the interstitial infiltrates. Bosentan also exerted a beneficial effect on proteinuria, renal lesions, and mortality in WT. These data show that in overload nephropathy, proteinuria and renal lesions are, to a large extent, AngII-dependent. The up-regulation of ET-1 in tubular epithelial cells in AT1(-/-), associated with interstitial infiltrates, suggests that the combination of drugs interfering with both vasopeptides may be of therapeutic interest in renal diseases with severe proteinuria and tubulointerstitial damage.

Mycophenolate mofetil prevents salt-sensitive hypertension resulting from nitric oxide synthesis inhibition.

Recent studies have suggested that subtle microvascular and tubulointerstitial injury in the kidney can cause salt-sensitive hypertension. To test this hypothesis, we determined whether the mild renal disease induced by transient blockade of nitric oxide (NO) synthesis would result in salt-sensitive hypertension and whether prevention of the renal injury by coadministration of the immunosuppressive agent mycophenolate mofetil (MMF) would block the development of salt sensitivity. N(omega)-nitro-L-arginine-methyl ester (L-NAME; 70 mg/100 ml in the drinking water) was administered for 3 wk to rats with or without MMF (30 mg x kg(-1) x day(-1) by gastric gavage), followed by a 1-wk "washout" period in which the MMF was continued, which was followed in turn by placement on a high-salt (4% NaCl) diet for an additional 4 wk. Renal histology was examined at 3 and 8 wk, and blood pressure was measured serially. L-NAME treatment resulted in acute hypertension and the development of mild renal injury. During the washout period, blood pressure returned to normal, only to return to the hypertensive range on exposure of the animals to a high-salt diet. MMF treatment prevented the development of hypertension in response to a high-salt diet. This correlated with the ability of MMF to inhibit specific aspects of the renal injury, including the development of segmental glomerulosclerosis, the infiltration of T cells and ANG II-positive cells, and the thickening of afferent arterioles.

Mycophenolate mofetil prevents salt-sensitive hypertension resulting from angiotensin II exposure.

BACKGROUND: Interstitial mononuclear cell infiltration is a feature of experimental models of salt-sensitive hypertension (SSHTN). Since several products of these cells are capable of modifying local vascular reactivity and sodium reabsorption, we investigated whether mycophenolate mofetil (MMF), a drug known to inhibit infiltration and proliferation of immune cells, would modify the SSHTN induced by angiotensin II (Ang II) infusion. METHODS: Sprague-Dawley rats received Ang II for two weeks using subcutaneous minipumps. A high-sodium (4% NaCl) diet was started on the third week and was maintained until the eighth week. MMF (30 mg/kg, N = 15), an immunosuppressive drug, or vehicle (N = 15) was given daily by gastric gavage during the initial three weeks. Sham-operated rats (N = 9) were used as controls. Body weight, blood pressure (tail-cuff plethysmography), and serum creatinine were determined weekly. Urinary malondialdehyde (MDA) excretion, renal histology, and immunohistology, including the presence of Ang II and superoxide-producing cells, were analyzed at the end of Ang II infusion and at eight weeks. RESULTS: MMF treatment did not modify hypertension induced during exogenous Ang II infusion, but prevented the subsequent SSHTN. Tubulointerstitial injury resulting from Ang II infusion was significantly reduced by MMF treatment, as were proliferative activity, T-cell infiltration and activation (interleukin-2 receptor expression), superoxide-producing cells, and urinary MDA excretion. Ang II-producing cells were present in the renal tubulointerstitium of rats with SSHTN (60 +/- 30 Ang II-positive cells/mm(2) at 8 weeks) and were reduced by two thirds in the MMF-treated group. Forty percent of lymphocytes infiltrating the tubulointerstitium stained positive for Ang II. The expression of Ang II receptors in the kidney was unmodified. CONCLUSIONS: SSHTN resulting from Ang II infusion is associated with infiltration and activation of immune cells that produce Ang II. MMF treatment reduces these features and prevents the development of SSHTN.

Activation of NF-kappaB in tubular epithelial cells of rats with intense proteinuria: role of angiotensin II and endothelin-1.

The mechanisms by which persistent proteinuria induces interstitial inflammation and fibrosis are not well known, although nuclear factor-kappaB (NF-kappaB), which regulates the transcription of many genes involved in renal injury, could be implicated. In rats with intense proteinuria, we studied the renal activation of NF-kappaB as well as the potential involvement of the vasoactive hormones angiotensin II (Ang II) and endothelin-1 (ET-1). Uninephrectomized Wistar-Kyoto rats receiving 1 g/d of BSA had proteinuria but no renal morphological lesions at day 1. By contrast, tubular atrophy and/or dilation and mononuclear cell infiltration were observed after 8 or 28 days of BSA administration, coinciding with maximal proteinuria. In relation to control uninephrectomized rats, the renal cortex of nephritic rats showed an increment in the activation of NF-kappaB at all time periods studied. By in situ Southwestern histochemistry, NF-kappaB activity was mainly localized in proximal tubules, interstitial mononuclear cells, and, to a lesser extent, the glomeruli. The administration of the ACE inhibitor quinapril plus the ET(A)/ET(B) receptor antagonist bosentan during 28 days to BSA-overloaded animals diminished proteinuria, renal lesions, and NF-kappaB activity more markedly than single drugs. Cultured tubular epithelial cells exposed to BSA revealed an intense NF-kappaB activation in a time- and dose-dependent manner. Incubation of cells with receptor antagonists of Ang II (AT(1): losartan and AT(2): PD-123,319) or ET-1 (ET(A): BQ123 and ET(B): IRL1038) inhibited significantly the BSA-induced NF-kappaB activity (90%, 75%, 90%, and 60% of inhibition versus basal, respectively). Our results show that overload proteinuria causes NF-kappaB activation in tubular epithelial cells both in vivo and in vitro. The vasoactive peptides Ang II and ET-1 appear to be implicated in this effect. The results reveal a novel mechanism of perpetuation of renal damage induced by persistent proteinuria.

Angiotensin-converting enzyme is upregulated in the proximal tubules of rats with intense proteinuria.

Persistent proteinuria is considered a deleterious prognostic factor in most progressive renal diseases. However, the mechanisms by which proteinuria induces renal damage remain undetermined. Since proximal tubular cells possess all the machinery to generate angiotensin II (Ang II), we approached the hypothesis that proteinuria could elicit the renal activation of the renin-angiotensin system in a model of intense proteinuria and interstitial nephritis induced by protein overload. After uninephrectomy (UNX), Wistar-Kyoto rats received daily injections of 1 g BSA or saline for 8 days. The mean peak of proteinuria was observed at the fourth day (538+/-89 versus 3+/-1 mg/24 h in UNX controls; n=12; P<0.05) and was increased during the whole study period (at the eighth day: 438+/-49 mg/24 h; n=12; P=NS). Morphological examination of the kidneys at the end of the study showed marked tubular lesions (atrophy, vacuolization, dilation, and casts), interstitial infiltration of mononuclear cells, and mesangial expansion. In relation to UNX control rats, renal cortex of BSA-overloaded rats showed an increment in the gene expression of angiotensinogen (2.4-fold) and angiotensin-converting enzyme (ACE) (2.1-fold), as well as a diminution in renin gene expression. No changes were observed in angiotensin type 1 (AT1) receptor mRNA expression in both groups of rats. By in situ reverse transcription-polymerase chain reaction and immunohistochemistry, ACE expression (gene and protein) was mainly localized in proximal and distal tubules and in the glomeruli. By immunohistochemistry, angiotensinogen was localized only in proximal tubules, and AT1 receptor was localized mainly in proximal and distal tubules. In the tubular brush border, an increase in ACE activity was also seen (5. 5+/-0.5 versus 3.1+/-0.7 U/mg protein x10(-4) in UNX control; n=7; P<0.05). Our results show that in the kidney of rats with intense proteinuria, ACE and angiotensinogen were upregulated, while gene expression of renin was inhibited and AT1 was unmodified. On the whole, these data suggest an increase in Ang II intrarenal generation. Since Ang II can elicit renal cell growth and matrix production through the activation of AT1 receptor, this peptide may be responsible for the tubulointerstitial lesions occurring in this model. These results suggest a novel mechanism by which proteinuria may participate in the progression of renal diseases.

Renal expression of parathyroid hormone-related protein (PTHrP) and PTH/PTHrP receptor in a rat model of tubulointerstitial damage.

BACKGROUND: PTHrP, which appears to act as a growth/differentiation factor in a variety of tissues, is present in the kidney; however, its role is unclear. METHODS: The expression of PTHrP and the PTH/PTHrP receptor were investigated by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry in the remnant kidney of uninephrectomized (UNX) rats after protein overloading [1 g/day of bovine serum albumin (BSA)]. RESULTS: Compared with UNX-control rats, proteinuria in BSA-overloaded animals was detected within the first 24 hours and increased during the entire study period (28 days). Kidney examination by light microscopy showed no significant renal lesions at day 1 of BSA treatment, whereas at days 8 and 28, tubular lesions, infiltration of mononuclear cells, and mesangial expansion were observed. PTHrP mRNA expression in the renal cortex was already increased at day 1 (fourfold) and plateaued between days 8 and 28 (12- and 15-fold, respectively) in BSA-overloaded animals compared with UNX-control rats. At day 8, immunohistochemical analysis with two different anti-PTHrP antibodies showed a dramatic increase of PTHrP staining in the damaged proximal and distal tubules from BSA-overloaded rats with respect to UNX-control rats. Moreover, intense PTHrP immunostaining was also observed in glomerular mesangial and endothelial cells in BSA-overloaded rats, but not in the UNX-control rats. A reciprocal decrease of PTH/PTHrP receptor mRNA and immunostaining, without significant changes in the cellular localization (proximal and distal tubule, and glomerular mesangial and epithelial cells) of the PTH/PTHrP receptor positivity was found to occur in the renal cortex of BSA-overloaded rats. At day 8, coinciding with the up-regulation of PTHrP, an increase in the angiotensin converting enzyme and preproendothelin-1 gene expression was observed in the renal cortex of BSA-overloaded rats compared with UNX-control rats. CONCLUSIONS: These results indicate that PTHrP can be added to the group of genes that are up-regulated in proximal tubular cells in response to intense proteinuria. Our results, together with previous findings, suggest that the vasoactive hormones angiotensin II and endothelin-1 could participate in the PTHrP production in the renal cortex of BSA-overloaded rats. Further experiments are required to clarify the mechanisms of PTHrP up-regulation and its possible role in the response to renal damage in this animal model.

Platelet-activating factor stimulates gene expression and synthesis of matrix proteins in cultured rat and human mesangial cells: role of TGF-beta.

Platelet-activating factor (PAF) is a potent inflammatory mediator that participates in the pathogenesis of proteinuria and glomerular damage. However, the role of this lipid in glomerular sclerosis remains unknown. This study examines the effect of PAF on the regulation of extracellular matrix proteins by rat and human mesangial cells. PAF increased in a dose-dependent manner the gene expression of fibronectin and type IV collagen, but not type I collagen. Moreover, an increase in cell-associated and soluble fibronectin synthesis was also seen. These effects were abolished by BN52021 and WEB2086, two different PAF receptor antagonists. Because transforming growth factor (TGF)-beta has been considered a profibrogenic cytokine, this study also evaluated whether PAF effects might be mediated by the production of endogenous TGF-beta. PAF caused an increase in TGF-beta1 mRNA expression (by a protein kinase C-dependent pathway) and TGF-beta activity. Moreover, PAF-induced fibronectin synthesis was totally abolished when an anti-TGF-beta-neutralizing antibody was added to the culture medium, suggesting that PAF stimulates fibronectin synthesis, at least in part, through the induction of TGF-beta. Addition of cycloheximide, a protein synthesis inhibitor, upregulated PAF-induced fibronectin mRNA expression but downregulated PAF-induced TGF-beta1 gene expression, suggesting the existence of different regulatory transcriptional factors of the two proteins. These results suggest that PAF may be implicated in matrix accumulation during renal injury and therefore contribute to the pathogenesis of glomerulosclerosis.

Endothelin-1 upregulation in the kidney of uninephrectomized spontaneously hypertensive rats and its modification by the angiotensin-converting enzyme inhibitor quinapril.

Endothelin (ET-1) is a potent vasoconstrictor that plays an important role in the control of renal circulation and tubular function. The contribution of this peptide to the pathogenesis of systemic hypertension and renal failure remains largely undefined. In spontaneously hypertensive rats (SHR) uninephrectomized at 20 weeks of age (UNX-SHR) and followed until 45 weeks of age, we determined ET-1 gene expression in renal tissue by reverse transcription-polymerase chain reaction and its localization by in situ hybridization in paraffin-embedded kidney sections. Age-matched SHR and normotensive Wistar-Kyoto (WKY) rats were chosen as controls. At the end of the follow-up, UNX-SHR had high systolic blood pressure, intense proteinuria, mesangial expansion, focal and segmental glomerular sclerosis, and tubulointerstitial lesions. In relation to WKY and SHR, UNX-SHR exhibited an increase in ET-1 gene expression in renal cortex and medulla. By in situ hybridization and immunoperoxidase staining, an overexpression of ET-1 gene and protein were seen in mesangial and glomerular epithelial cells and in some proximal tubules and vessels. Angiotensin-converting enzyme (ACE) activity was significantly increased in the renal brush border. Since in mesangial cells, angiotensin II induces ET-1 synthesis, a group of UNX-SHR received the ACE inhibitor quinapril from the time of UNX. These animals had a decrease in blood pressure, proteinuria, and serum and brush border ACE activity and in the expression and synthesis of ET-1 in all renal areas. On the whole, these data show that UNX-SHR have an upregulation of ET-1 gene and protein in several structures of the kidney compared with SHR and WKY rats. Quinapril diminished ACE activity and ET-1 expression and synthesis coincidentally with an improvement in proteinuria and morphological lesions. The beneficial effects of ACE inhibitors may be due to the diminution of both angiotensin II and ET-1 generation.

Quinapril decreases renal endothelin-1 expression and synthesis in a normotensive model of immune-complex nephritis.

Angiotensin-converting enzyme (ACE) inhibitors diminish proteinuria and the progression to renal failure in several experimental models of renal injury. Endothelin-1 (ET-1) possesses potent biological actions on renal vessels and has been considered as a potential mediator of renal damage. Because angiotensin II (Ang II) induces ET-1 synthesis in endothelial and mesangial cells, we hypothesized that some of the beneficial effects of the ACE inhibitors could result from the blockade of ET-1 synthesis. In a normotensive model of immune-complex nephritis, in which there exists an increase in renal ACE activity, the effect of the ACE inhibitor quinapril on preproET-1 and ETA receptor mRNA expression, as well as on ET-1 protein levels, was examined in this study. In relation to controls, nephritic rats showed an increase in preproET-1 and ETA receptor gene expression in renal cortex and medulla, coinciding with the maximal renal ACE activity. PreproET-1 mRNA (in situ hybridization) and ET-1 protein (immunohistochemistry) were localized in glomerular capillary walls, mesangial and glomerular epithelial cells, as well as in the brush border of some proximal tubules, and in small vessels. In nephritic rats, there was an increase in preproET-1 mRNA levels and ET-1 protein in all of these areas, without modification of their distribution. The administration of the ACE inhibitor quinapril decreased proteinuria and morphological lesions, preproET-1 gene transcription, and ET-1 protein levels, as well as the ETA receptor mRNA. The results from this study show that in a normotensive model of immune-complex nephritis, there was an overexpression of ET-1 in several structures of the kidney that was downregulated by quinapril administration. The beneficial effect of ACE inhibitors could be a result of the modulation of local production of Ang II and ET-1.

An orally active ETA/ETB receptor antagonist ameliorates proteinuria and glomerular lesions in rats with proliferative nephritis.

The proliferation of mesangial cells and the extracellular matrix expansion constitute the most outstanding morphological aspects of the majority of progressive glomerular diseases. In vitro, endothelin-1 (ET-1) is mitogenic for mesangial cells and induces matrix protein synthesis. We studied the possible participation of ET-1 in the pathogenesis of renal damage in a normotensive model of proliferative nephritis. Coincidentally with maximal proteinuria and glomerular lesions, an increase was found in the glomerular mRNA expression of preproET-1 and the ETA receptor (10 and 6 times compared to controls, respectively), but not of the ETB receptor, and in ET-1 urinary excretion (217 +/- 33 vs. 84 +/- 4 pg ET-1/24 hr, N = 4 to 5, P < 0.05). By in situ hybridization, an increase in preproET-1 mRNA expression in glomerular endothelial, epithelial and mesangial cells, and in come tubular cells was observed. The administration of bosentan, an ETA/ETB receptor antagonist, had a beneficial effect on the evolution of nephritis preventing the appearance of intense proteinuria (76 +/- 35 vs. 380 +/- 77 mg/24 hr, N = 4 to 5, P < 0.05), the morphological lesions and the renal function impairment (creatinine clearance 367 +/- 46 vs. 268 +/- 33 microliters/min/100 g, N = 4 to 5). Simultaneously, there was a decrease in ET-1 urinary excretion (88 +/- 14 vs. 217 +/- 33 pgET-1/24 hr, N = 4,5, P < 0.05) and in the renal preproET-1 mRNA expression. The mean systolic blood pressures remained in the normal range in all animals. These data indicate that ET-1 participates in the pathogenesis of proteinuria and glomerular injury in a model of proliferative nephritis. The nonpeptidic orally active ETA/ETB receptor antagonists could be useful in the treatment of some human nephritis.

Endothelin-1 induces loss of proteoglycans and enhances fibronectin and collagen production in cultured rabbit synovial cells.

Endothelin-1 exerts a wide range of biological actions besides its characteristic vasoconstrictor function. The potential participation of endothelin-1 in rheumatic diseases has hardly been explored. We have studied the possible role of endothelin-1 as a modulator of extracellular matrix turnover in cultured rabbit synoviocytes. In relation to basal levels, endothelin-1 increased the mRNA levels of collagen I and fibronectin at 24 h (130 +/- 9% and 132 +/- 18%, respectively), but did not modify the expression of decorin core proteoglycan. Endothelin-1 also decreased proteoglycan metabolism (about 50% of proteoglycan synthesis inhibition and 270 +/- 32% of degradation rate vs. basal, P < 0.05 in both cases) and enhanced total collagen (1.5 +/- 0.5 vs. 0.8 +/- 0.2 microgram hydroxyproline/microgram DNA in basal, P < 0.05) and fibronectin protein synthesis (157 +/- 14% of [35S] methionine incorporation vs. basal, P < 0.05). The endothelin ETA receptor antagonist BQ-123 (Cyclo D-trp-D-asp-pro-D-val-leu) displaced [125I]endothelin-1 binding and inhibited endothelin-1 effects on extracellular matrix components. The cell incubation with indomethacin totally reversed the endothelin-1 effect. These data suggest that endothelin-1 may be an important mediator of the pathogenesis of joint damage, disturbing the extracellular synovial matrix turnover through the endothelin ETA receptors.

Effects and interactions of endothelin-1 and angiotensin II on matrix protein expression and synthesis and mesangial cell growth.

Mesangial cell growth and accumulation of extracellular matrix proteins constitute key features of progressive glomerular injury. Endothelin-1 (ET-1) and angiotensin II (Ang II), two potent vasoconstrictor agents, evoke a number of similar responses in mesangial cells. In rat mesangial cells, we compared ET-1 and Ang II effects on matrix protein production and cell proliferation as well as the potential interaction between the two hormones. When cells in 0.5% fetal calf serum were incubated for 24 hours with various concentrations of ET-1 or Ang II, both peptides stimulated, in a dose-dependent manner, fibronectin and type IV collagen mRNA expression, fibronectin synthesis, and mitogenesis. Incubation with specific receptor antagonists of both hormones demonstrated that endothelin subtype A (ETA) and angiotensin type 1 (AT1) receptors were involved. Preincubation of cells with two different protein kinase C inhibitors or with a neutralizing anti-transforming growth factor-beta antibody, but not an unrelated IgG, diminished the peptide-induced fibronectin synthesis. A dual interrelation seems to exist between ET-1 and Ang II. Thus, the AT1 receptor antagonist losartan and the angiotensin-converting enzyme inhibitors quinaprilat and captopril diminished the ET-1-mediated effects, whereas, the ETA receptor antagonist BQ-123 diminished the Ang II-induced fibronectin synthesis and mesangial cell proliferation. Our results suggest that ET-1 and Ang II stimulate matrix protein synthesis and mesangial cell mitogenesis through ETA and AT1 receptors, respectively, by complicated mechanisms, implicating protein kinase C activation, synthesis of transforming growth factor-beta, and release of one peptide by the other. These data could be important for a better understanding of the participation of vasoactive substances in the pathogenesis of glomerulosclerosis.

Comparative effects of dopexamine and dopamine on glycerol-induced acute renal failure in rats.

Acute renal failure (ARF) was induced in rats by intramuscular injection of 50% glycerol, 10 mL/kg body weight. Rats were given isotonic saline (1.5 mL/h) dopexamine hydrochloride (dopexamine, 100 microg/h) or dopamine (100 microg/h), commencing either immediately after glycerol administration and maintained during all the observation time (90 min, acute studies) or 20 min before administration of glycerol and during 60 min (chronic studies). Renal function was assessed during 90 min after induction of ARF in anesthetized rats and during 3 days following ARF induction in conscious animals. In anesthetized rats treated with dopexamine or dopamine, the reduction in insulin and para-aminohippuric acid clearance was markedly lower than that observed in untreated animals. In conscious animals, urinary flow and creatinine clearance were higher in rats treated with dopamine or dopexamine than in the non-treated group. Rats treated with dopexamine had higher renal Na+ and K+ excretion than dopamine-treated rats. Survival was higher in the dopexamine group than in either of the other two groups. These results demonstrate that pretreatment with dopexamine or dopamine significantly improves the course of ARF, with better survival after treatment with dopexamine.

Involvement of angiotensin II and endothelin in matrix protein production and renal sclerosis.

PURPOSE: To review the evidence that links angiotensin II and endothelin as growth factors and as modifiers of extracellular matrix synthesis in renal cells, with particular reference to the effects of angiotensin converting enzyme (ACE) inhibition in models of renal injury. IN VITRO STUDIES: In cultured mesangial cells, both angiotensin II and endothelin promote contraction, proliferation/hypertrophy, signal transduction pathways, the activation of early growth genes, and the generation of inflammatory mediators, cytokines and growth factors. Both hormones have been shown to promote the synthesis of fibronectin and collagen in a dose-dependent manner. ACE inhibition attenuates the effect of endothelin-1, one of three isoforms of endothelin. ANIMAL STUDIES: In experimental models of renal injury, chiefly in those characterized by increased intraglomerular pressure, ACE inhibition has reduced proteinuria and glomerular and interstitial sclerosis. HUMAN STUDIES: ACE inhibition has been shown to have major beneficial effects in patients with diabetic nephropathy, even in those with normal blood pressure. CONCLUSIONS: Although the renal-protective effects of ACE inhibitors in experimental and human renal injury may reflect systemic and/or local hemodynamic effects of these drugs, their modulatory actions on extracellular matrix synthesis and proteinuria may contribute to the benefit of ACE-inhibitor therapy.

The potential role of inflammatory and fibrogenic cytokines in the glomerular diseases.

In recent years increasing evidence has been accumulated on the role of cytokines in mediating glomerular and renal damage. Many such cytokines are released in the inflamed glomeruli by leukocytes and intrinsic glomerular cells. Cytokines not only recruit inflammatory cells into the injured glomeruli, but also induce a variety of responses on glomerular cells that range from a direct toxic effect to shape changes, proliferation, and induction of the release of inflammatory mediators and extracellular matrix, and could promote further glomerular damage. Moreover, exogenous administration of cytokines has induced glomerular injury in healthy animals and has enhanced renal damage in animals with glomerulonephritis. Anti-cytokine strategies have proved to be effective therapeutical alternatives in experimental models of glomerular diseases and may provide a more specific approach to the management of human glomerulonephritis.