Nitrosative stress by cyclosporin A in the endothelium: studies with the NO-sensitive probe diaminofluorescein-2/diacetate using flow cytometry.

BACKGROUND: We have reported previously that exposure of endothelial cells to cyclosporin A (CsA) may result in the regulation of specific genes, such as the endothelial nitric oxide synthase. In the context of endothelial toxicity, whether this represents an adaptive response to injury at the physiological level or contributes further to the generation of nitrosative stress remains to be investigated. The precise reactive species potentially produced by CsA and their capacity to modify proteins functionally are not well known. METHODS: We assessed the possibility of detecting intracellular nitric oxide (NO) by flow cytometry with the cell-permeable probe diaminofluorescein-2 diacetate (DAF-2/DA). RESULTS: CsA increased the intracellular 2 h accumulation of DAF-2T, the oxidized form of DAF-2/DA, in an L-NAME-sensitive process. When CsA was re-added for 2 h to bovine aortic endothelial cells previously pre-incubated with CsA for 16 h, an additional increase in the accumulation of DAF-2T was achieved. CONCLUSIONS: In this work, we provide data showing that intracellular NO can be detected by flow cytometry with DAF-2/DA and suggesting two potential mechanisms (transcriptional and post-translational) for the intracellular accumulation of NO in vascular endothelial cells exposed to the immunosuppressant CSA:

Transcriptional induction of endothelial nitric oxide gene by cyclosporine A. A role for activator protein-1.

We have previously shown that the immunosuppressant cyclosporine A (CsA) increases the activity, the protein level, and the steady-state levels of the mRNA of the endothelial nitric-oxide synthase (eNOS) gene in bovine aortic endothelial cells (BAEC). We have now investigated the mechanisms responsible for these effects. Preincubation with an inhibitor of RNA polymerase II abolished CsA-induced eNOS up-regulation. Nuclear run-on experiments demonstrated a 1.6-fold increase in the induction of eNOS gene by CsA. In agreement with these results, transient transfections showed that CsA augmented the transactivation of the eNOS promoter. Electrophoretic mobility shift assays showed an increase in the activator protein-1 (AP-1) DNA binding activity in BAEC treated with CsA. An increase in the level of c-fos mRNA and in the nuclear content of c-Fos protein was detected in BAEC treated with CsA. Site-directed mutagenesis of the AP-1 cis-regulatory element in the context of the human eNOS promoter resulted in the abrogation of the induction mediated by CsA. Hence, up-regulation of eNOS mRNA by CsA is a transcriptional phenomenon involving the proximal AP-1 site in the 5'-regulatory region of the human eNOS gene. Furthermore, our data exemplify how immunosuppressive drugs may result in the regulation of specific genes involved in the homeostasis of endothelial function, such as eNOS.

La ciclosporina A produce anión superóxido intracelular en el endotelio / Cyclosporine A produces intracellular superoxide anion in the endothelium

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CsA and FK506 up-regulate eNOS expression: role of reactive oxygen species and AP-1.

Cyclosporine A (CsA) and FK506 increase endothelial nitric oxide synthase (eNOS) mRNA expression in cultured bovine aortic endothelial cells (BAEC). CsA appears to increase eNOS mRNA levels mainly by increasing the rate of transcription, although a small contribution of mRNA stabilization could not be ruled out. CsA and FK506 induced an increase of ROS synthesis with the fluorescent probe used, DHR123. The ROS generating system glucose oxidase (GO) increased the expression of eNOS mRNA in BAEC. This upregulation of eNOS mRNA by CsA or GO was abrogated by catalase. As AP-1 is a redox-sensitive transcription factor and the bovine eNOS promoter has an AP-1 consensus sequence, a role of this factor in the up-regulation of eNOS mRNA was studied. Electrophoretic mobility shift assays were consistent with an increase in AP-1 DNA-binding activity in BAEC treated with CsA or glucose oxidase. The potential participation of ROS and the transcription factor AP-1 in the regulation of eNOS gene expression is suggested.

Role of reactive oxygen species in the signalling cascade of cyclosporine A-mediated up-regulation of eNOS in vascular endothelial cells.

1. Cyclosporine A (CsA) increases eNOS mRNA expression in bovine cultured aortic endothelial cells (BAEC). As some effects of CsA may be mediated by reactive oxygen species (ROS), present experiments were devoted to test the hypothesis that the CsA-induced eNOS up-regulation could be dependent on an increased synthesis of ROS. 2. CsA induced a dose-dependent increase of ROS synthesis, with the two fluorescent probes used, DHR123 (CsA 1 microM: 305+/-7% over control) and H2DCFDA (CsA 1 microM: 178+/-6% over control). 3. Two ROS generating systems, xanthine plus xanthine oxidase (XXO) and glucose oxidase (GO), increased the expression of eNOS mRNA in BAEC, an effect which was maximal after 8 h of incubation (XXO: 168+/-21% of control values. GO: 208+/-18% of control values). The ROS-dependent increased eNOS mRNA expression was followed by an increase in eNOS activity. 4. The effect of CsA on eNOS mRNA expression was abrogated by catalase, and superoxide dismutase (SOD). In contrast, the antioxidant PDTC augmented eNOS mRNA expression, both in basal conditions and in the presence of CsA. 5. The potential participation of the transcription factor AP-1 was explored. Electrophoretic mobility shift assays were consistent with an increase in AP-1 DNA-binding activity in BAEC treated with CsA or glucose oxidase. 6. The present results support a role for ROS, particularly superoxide anion and hydrogen peroxide, as mediators of the CsA-induced eNOS mRNA up-regulation. Furthermore, they situate ROS as potential regulators of gene expression in endothelial cells, both in physiological and pathophysiological situations.

Effects of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, atorvastatin and simvastatin, on the expression of endothelin-1 and endothelial nitric oxide synthase in vascular endothelial cells.

Endothelial dysfunction associated with atherosclerosis has been attributed to alterations in the L-arginine-nitric oxide (NO)-cGMP pathway or to an excess of endothelin-1 (ET-1). The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been shown to ameliorate endothelial function. However, the physiological basis of this observation is largely unknown. We investigated the effects of Atorvastatin and Simvastatin on the pre-proET-1 mRNA expression and ET-1 synthesis and on the endothelial NO synthase (eNOS) transcript and protein levels in bovine aortic endothelial cells. These agents inhibited pre-proET-1 mRNA expression in a concentration- and time-dependent fashion (60-70% maximum inhibition) and reduced immunoreactive ET-1 levels (25-50%). This inhibitory effect was maintained in the presence of oxidized LDL (1-50 microg/ml). No significant modification of pre-proET-1 mRNA half-life was observed. In addition, mevalonate, but not cholesterol, reversed the statin-mediated decrease of pre-proET-1 mRNA levels. eNOS mRNA expression was reduced by oxidized LDL in a dose-dependent fashion (up to 57% inhibition), whereas native LDL had no effect. Statins were able to prevent the inhibitory action exerted by oxidized LDL on eNOS mRNA and protein levels. Hence, these drugs might influence vascular tone by modulating the expression of endothelial vasoactive factors.

Lipid composition of platelets in patients with uremia.

AIMS: The lipid composition of platelets and the function of these cells in patients with uremia were studied. METHODS: Fourteen patients and 14 normal volunteers were studied. Platelet lipids including phospholipids and cholesterol, as well as the platelet aggregation response to agonists, were studied. RESULTS: The amount of platelet phospholipids was decreased in patients compared to controls (338.0 +/- 79 vs. 511.6 +/- 125 nmol/10(9) cells; p < 0.001), while the percentage of the five main specimens of these compounds was normal. The content of platelet cholesterol in patients (97.8 +/- 17.0 microg/10(9) cells) was similar to that in controls (91.7 +/- 26.0 microg/10(9) cells). Consequently, the cholesterol:phospholipid ratio in uremic platelets was increased (0.75 +/- 0.1 vs. 0.46 +/- 0.1; p < 0.01). Although this feature is associated with hyperreactive platelets, the aggregation tests were defective for adenosin diphosphate (p < 0.01), arachidonic acid (p < 0.01), epinephrine (p < 0.01) and collagen (p < 0.001). This behavior is probably due to the multifactorial platelet defect described in uremia.

Androgen therapy for anaemia of chronic renal failure. Indications in the erythropoietin era.

The high cost of recombinant human erythropoietin has led us to consider the existing indications for androgen treatment of anaemia in patients with chronic renal failure. In the present work, we have tried to identify those patients on haemodialysis for whom androgens could constitute a therapeutic alternative. The evolution of haemoglobin concentration was analysed in 84 patients (67 males and 17 females) treated with a cycle of nandrolone decanoate (200 mg per week given intramuscularly, for six months). In the total group of patients, haemoglobin rose from 69 g/L to 87 g/L (p < 0.01). The increment in haemoglobin was not related to sex, basal haemoglobin, primary renal disease, or dose of nandrolone decanoate corrected by body weight. However, we observed a relationship between this increment in haemoglobin and patient age. Haemoglobin increased by 8 g/L in patients younger than 46 years (n = 29), by 18 g/L in patients aged between 46 and 55 years (n = 28), and by 27 g/L in patients older than 55 years (n = 27) (p < 0.01 between groups). In the last group, haemoglobin concentration at the end of androgen treatment was 101 +/- 16 g/L. The haemoglobin level reached during androgen treatment was maintained for over a year after androgen withdrawal in 55% of the responder patients. A reversible rise in the serum concentration of triglycerides was the main side-effect observed. Nandrolone decanoate therapy was not associated with hepatotoxicity or an increase in blood pressure. Voice change and mild hirsutism were observed in most of the women receiving nandrolone decanoate, and these secondary effects constitute a real disadvantage to its use in females. In conclusion, our results showed that androgens are a useful alternative in the treatment of anaemia in male haemodialysis patients older than 55 years. Furthermore, the response obtained was similar to that observed with erythropoietin, but at a lower cost.

[Endocrine changes and sexual dysfunction in kidney transplantation and hemodialysis: comparative study]. / Alteraciones endocrinas y disfunción sexual en trasplante renal y hemodialisis: estudio comparativo.

OBJECTIVE: The objective of this paper is to compare the hormonal changes and sexual activity between transplanted patients and patients in regular haemodialysis (HD). MATERIAL AND METHODS: 130 patients, 98 RT carriers and 32 with CRF were evaluated with regard to the sexual function. The etiology of CRF is similar in both groups. All patients underwent hormonal determinations (FSH, LH, Prolactin, Testosterone, Oestradiol and PTH), complete serum testing and other diagnostic studies done selectively, 38 of 130 patients (14 in HD and 24 transplants) answered a personal questionnaire on sexual activity. RESULTS: Oestradiol and prolactin levels are higher in the HD group compared to transplanted patients (p < 0.05). 70% of RT patients maintain their libido versus 35% of those in dialysis (p < 0.01). The former group refers good erection in 55% cases versus 21% of dialysis patients (p < 0.01). Intercourse frequency and degree of overall satisfaction is higher in the RT group (N.S.). CONCLUSIONS: No significant differences were found in FSH, LH and testosterone levels. The normality of FSH levels may be a reflection of the integrity of the germinal line. 65% HD patients refer decreased or absent libido, to which the higher levels of prolactin and oestradiol found could contribute. No relationship was found between sexual function with the type of immunosuppression or the graft function.

Urinary calcium excretion in treated and untreated essential hypertension.

A high prevalence of hypercalciuria has been reported in patients with essential hypertension. Nevertheless, the clinical and therapeutic implications of this finding have scarcely been studied. This study was designed to determine the prevalence of hypercalciuria in an unselected population with essential hypertension and to analyze the relationship between the urinary calcium and the clinical and therapeutic status of these patients. This article presents a prospective study of 112 patients with essential hypertension and 49 healthy normotensive control subjects. Urinary excretion rates of calcium, sodium, chloride, potassium, urinary calcium/creatinine index, the fractional excretion of sodium, potassium and uric acid, the creatinine clearance and serum values of creatinine, urea, uric acid, electrolytes, total proteins, parathormone (intact molecule), plasma renin activity, aldosterone, glucose, and insulin (fasting and after an oral glucose load) were performed in every patient and control subject. Untreated hypertensive patients had a higher prevalence of hypercalciuria (35% had a urinary calcium/creatinine ratio > 0.20 versus 20% of treated hypertensives and 2% of control subjects; P < 0.001). Patients on thiazide or beta-blocker monotherapy had lower urinary excretion rates of calcium and urate than patients on calcium-antagonist monotherapy or untreated patients. Urinary calcium, sodium, and urate correlated positively both in treated and untreated essential hypertension patients. Patients with the higher urinary calcium levels also had higher excretion rates of sodium and urate, higher creatinine clearance rates, and lower serum creatinine and serum uric acid levels. It was concluded that hypercalciuria is a frequent finding of untreated essential hypertension. The association of high urinary calcium levels with high urinary urate excretion rates in the same patient may predispose to development of lithiasis in patients with essential hypertension. Antihypertensive drugs have a variable effect on calciuria-uricosuria, which may constitute an additional criterion in the selection and individualization of therapy. Thiazides and beta-blockers can decrease calciuria and uricosuria and, therefore, the lithogenic risk in these patients.

Membranous nephropathy: recurrence after kidney transplantation.

BACKGROUND: It is supposed that about 5% of dialysis patients had membranous nephropathy as a cause for their renal failure. Despite of this prevalence, only 33 cases of recurrent membranous nephropathy after kidney transplantation have been reported in the English literature. METHODS: Among 509 recipients of renal allografts, membranous glomerulonephritis was the cause of renal failure in five patients, who received six transplants. RESULTS: Recurrence of the disease was observed in three allografts (50%) in three patients, all of them were on treatment with cyclosporin and low-dose prednisone. Proteinuria appeared at 2, 5 and 19 months after grafting. One patient experienced a spontaneous remission after 12 months and he is free from proteinuria and with good renal function after 5 years. The remaining two patients presented progressive renal function deterioration and returned to haemodialysis 24 and 17 months after th appearance of proteinuria. In these patients increasing the immunosuppression did not produce any beneficial effect. One of those patients underwent a second transplant; recurrence of the membranous nephropathy has not been observed after 3 years of follow-up. CONCLUSIONS: In this study three new cases of recurrence of membranous nephropathy are reported. One patient experienced a spontaneous remission of proteinuria. Recurrence of membranous nephropathy in renal allograft was very high in our series. Its appearance was associated with poor prognosis of the graft in most patients, although spontaneous remission of proteinuria is possible.

Androgen versus erythropoietin for the treatment of anemia in hemodialyzed patients: a prospective study.

According to this facility's protocol for the treatment of anemia in hemodialyzed patients, androgens were administered to male patients aged over 50 yr and recombinant human erythropoietin was administered to male patients below 50 yr of age and to female patients. In the study presented here, both therapeutic approaches have been prospectively analyzed. Patients were divided into two groups. Group A was composed of 18 patients, aged 62 +/- 12 yr, treated with nandrolone decanoate (200 mg/wk im) for 6 months; Group B was composed of 22 patients (6 men, 16 women) aged 47 +/- 15 yr, treated with subcutaneous recombinant human erythropoietin (initial dose, 6000 IU/wk) for 6 months. The increases of hemoglobin were similar in both groups; Group A, from 7.3 +/- 0.8 to 10.8 +/- 1.7 g/dL (P < 0.001), and Group B, from 7 +/- 0.6 to 10.4 +/- 1 g/dL (P < 0.001). In Group A, increases of triglycerides (159 +/- 71 versus 267 +/- 153 mg/dL, P < 0.001), serum albumin (3.9 +/- 0.3 versus 4.2 +/- 0.3 g/dL, P < 0.05), and dry weight (62.1 +/- 9.8 versus 64.9 +/- 10.1 kg, P < 0.001) were observed, which remained unmodified in Group B. Blood pressure control worsened in one patient (6%) from Group A, and in ten patients (45%) from Group B (P < 0.05). In conclusion, androgens produced an improvement in anemia in selected patients, similar to that achieved by use of recombinant human erythropoietin but at a lower cost. Androgens also have an appreciable anabolic effect and did not increase the blood pressure.