S. Cantaro

Cyclosporin‐induced endothelial dysfunction and hypertension: are nitric oxide system abnormality and oxidative stress involved?

Abstract Hypertension is a major side effect of cyclosporin (CsA). While the mechanism(s) responsible are unclear, CsA‐induced endothelial dysfunction and CsA‐induced hypertension have been attributed to the CsA effect on the endothelial‐derived factors controlling vasomotor tone. Endothelial nitric oxide (NO) is crucial in the maintenance of a state of basal vasodilation, and recent studies have suggested an NO‐mediated counterregulatory mechanism protective from CsA‐induced vasoconstriction. Our study evaluates endothelial nitric oxide synthase (ecNOS) gene status (PCR analysis) and plasma levels of NO metabolites (ELISA) in kidney and heart transplant patients under chronic CsA treatment with CsA‐induced hypertension. Since CsA increases superoxide production, which metabolises NO, plasma hydroperoxides from cholesterol esters and from triglycerides and peroxynitrite were also evaluated (HPLC) as an index of the presence of superoxides and of “oxidative stress”. Quantification of monocyte ecNOS mRNA and NO metabolites plasma levels from patients and controls (C) demonstrated NO system upregulation in patients notwithstanding the hypertension. The mean ecNOS to β‐actin ratio was 1.80 ± 0.85 in patients vs 0.40 ± 0.09 in C (P < 0.04). NO metabolites were 34.03 ± 14.32 μM in patients vs 11.53 ± 5.64 μM in C (P < 0.001). Hydroperoxides from cholesterol esters and from triglycerides were also increased in patients, 3.4 ± 1.4 vs 1.3 ± 0.6 integrated area units (i. a. u.), P < 0.007 and 10.6 ± 6.4 vs 1.3 ± 0.8 i.a.u., P < 0.008, respectively, as well as the peroxynitrite plasma level, 0.32 ± 0.11 μM/l vs undetectable in C. This study confirms a CsA‐induced NO system upregulation in transplanted patients. However, the NO‐mediated counterregulatory system to CsA‐induced vasoconstriction, present in normals, could be canceled in patients by CsA‐induced superoxide (O2−) and free radical production which, by increasing NO metabolism, could contribute to CsA‐induced vasoconstriction and hypertension and predispose to atherosclerosis.

Increased urinary NO2−/NO3− and cyclic guanosine monophosphate levels in patients with bartter's syndrome: Relationship to vascular reactivity

Nitric oxide (NO) is a potent endogenous vasodilator and plays a pivotal role in the control of vascular tone by the formation of cyclic guanosine monophosphate (GMP). Patients affected by Bartters syndrome have lower than normal vascular reactivity with normohypotension and decreased peripheral resistances in spite of biochemical and hormonal abnormalities typical of hypertension, and it is possible that increased production of NO may be involved in maintaining this reduced vascular response and vasodilatation. We have examined this possibility by studying NO2-/NO3- and cyclic GMP urinary excretions to assess NO production in vivo in seven patients affected by Bartters syndrome compared with seven healthy controls. A group of five patients with hypokalemia other than Bartter syndrome (pseudo-Bartters) was also included in the study to evaluate the effect of hypokalemia on NO production. NO2-/NO3- urinary excretion (0.45 +/- 0.14 v 0.25 +/- 0.04 micromol/micromol urinary creatinine [controls], P < 0.005, v 0.28 +/- 0.05 [pseudo-Bartters], P < 0.01) and cyclic GMP urinary excretion (0.057 +/- 0.028 v 0.022 +/- 0.01 micromol/micromol of urinary creatinine [controls], P < 0.009, v 0.024 +/- 0.004 [pseudo-Bartters], P < 0.02) were increased in patients with Bartters syndrome in comparison with controls and pseudo-Bartters, and a linear correlation between these two parameters was also present (P < 0.001). We conclude that in Bartters syndrome the increased NO2-/NO3- and cyclic GMP urinary excretions point to an increased NO synthesis, which could account for the reduced vascular response of the disease, therefore adding its role in determining the vascular hyporeactivity of Bartters syndrome.

Reduced susceptibility to oxidation of low-density lipoprotein in patients with overproduction of nitric oxide (Bartter's and Gitelman's syndrome)

Background The oxidation of low-density lipoprotein (LDL) might play an important role in the development of atherosclerosis. Objective To establish whether greater than normal production of nitric oxide (NO) in vivo protects LDL from oxidation. Patients and methods We studied nine subjects affected by Bartters and Gitelmans syndrome (both characterized by greater than normal production of NO), and 10 subjects matched for age, sex and lipid levels as controls. LDL particles were isolated from plasma by density gradient ultracentrifugation. Susceptibility of LDL to oxidation was evaluated after incubation with copper sulfate solution, by measuring the formation of conjugated dienes, the thiobarbituric acid-reactive substances, and the volatile peroxidation products of n-3 (propanal) and n-6 (pentanal and hexanal) polyunsaturated fatty acids. Phospholipid fatty acid composition of LDL was determined by gas chromatography. LDL α-tocopherol concentrations were measured. Results Patients with Bartters and Gitelmans syndrome had LDL particles smaller and/or denser than those of controls [Rf = 0.38 ± 0.03 versus 0.42 ± 0.02 (mean ± SD), P < 0.01], which hence were assumed to be more oxidizable. The phospholipid fatty acid composition of LDL and the α-tocopherol concentrations did not significantly differ between patients and controls. The duration of the lag phase, which is the time preceding formation of conjugated dienes, did not differ between groups, but the lag phase times were related to urinary excretion of nitrite/nitrate from patients (r = 0.66, P < 0.05). Moreover, patient LDL had produced less thiobarbituric acid-reactive substances after 5 h (P < 0.04), and less pentanal and hexanal after 5 and 6h (P < 0.04 and P < 0.02, respectively) than had that of controls. Conclusions Greater than normal production of NO in vivo is associated with lower than normal susceptibility of LDL to oxidation in vitro, suggesting that NO plays a protective role in the development of atherosclerosis.

Diagnosis of Iron Deficiency in Patients Undergoing Hemodialysis

To diagnose iron deficiency in patients undergoing hemodialysis, the percentage of hypochromic RBCs (with cellular hemoglobin concentration <280 g/L [HYPO%]) and mean reticulocyte hemoglobin content (CHret) provided by the Siemens ADVIA 120 and 2120 analyzers (Siemens Diagnostic Solutions, Tarrytown, NY) were proposed as alternatives to biochemical tests. Sysmex, with its XE-5000 analyzer (Sysmex, Kobe, Japan), also proposed the percentage of erythrocytes with cellular hemoglobin content lower than 17 pg (%Hypo-He) and equivalent of the mean reticulocyte hemoglobin content (Ret-He) with similar clinical applications. Our aim was to verify the clinical usefulness of the biochemical and cellular parameters as predictors of iron deficiency in patients undergoing long-term hemodialysis. We studied 69 patients undergoing hemodialysis 3 times weekly. The baseline values of serum ferritin and percentage of transferrin saturation were poor predictors of iron responsiveness. Better ability was demonstrated by reticulocyte indices (area under the curve [AUC], 0.74 for CHret and 0.72 for Ret-He; best cutoff values, 31.2 and 30.6 pg, respectively) and erythrocyte parameters (AUC, 0.72 for HYPO% and 0.68 for %Hypo-He; best cutoff values, 5.8 and 2.7, respectively). The newly proposed Ret-He and %Hypo-He can provide clinicians with information equivalent to CHret and HYPO%.

Intracellular calcium signalling and vascular reactivity in Bartter's syndrome

We investigated patients affected by Bartters syndrome in the attempt to localize the intracellular defect mediating the reduced intracellular Ca2+ mobilization that may be responsible for the decreased vascular reactivity characteristic of Bartters syndrome. Using the formylmethionyl-leucyl-phenylalanine (fMLP) receptor system, which causes, intracellular calcium release, we investigated fMLP-stimulated intracellular inositol 1,4,5-trisphosphate (IP3) production as well as the number and affinity of fMLP receptors in neutrophils from Bartters syndrome patients and healthy controls. Scatchard plot analysis of radioactive fMLP binding to neutrophils indicated that there were no differences in either cell receptor number and affinity for ligand between healthy controls (n = 5) and patients with Bartters syndrome (n = 5): 6,151 +/- 1,431 vs. 7,112 +/- 2,566 receptors/cell; K(D): 0.446 +/- 0.14 vs. 0.454 +/- 0.09 pM of fMLP. 5- and 10-second fMLP-stimulated intracellular IP3 production was instead reduced in patients affected by Bartters syndrome: 2.479 +/- 1.07 vs. 4.073 +/- 1.04 nmol/10(7) cells at 5 s (n = 8; p < 0.01); 1.673 +/- 0.741 vs. 3.766 +/- 1.348 nmol/10(7) cells at 10 s (n = 8; p < 0.005). The results of this study indicate that the anomaly of intracellular calcium mobilization in patients with Bartters syndrome arises from a defect at the postreceptor level. The anomalous calcium signalling that takes place in Bartters syndrome may provide a mechanism for the hyporesponsiveness to pressor stimuli characteristic of these patients.

Nifedipine Can Preserve Renal Function in Patients Undergoing Aortic Surgery with Infrarenal Crossclamping

Sixteen patients diagnosed with an aneurysm of abdominal aorta or Leriche disease underwent elective aortic surgery involving crossclamping of infrarenal aorta (ICC). These patients were randomized into two equal groups and 8 patients were infused with nifedipine starting from the isolation of aorta until the end of surgery (group A) while another 8 patients were infused with low-dose dopamine (group B) over the same surgical course. Plasma endothelin (ET) was measured before the induction of anesthesia, at the beginning and at the end of the clamp period and at the end of the operation. Intraoperatively, creatinine clearance and urinary excretion of PGE2, 6-keto PGF1 alpha and TxB2 were also determined before, during and after aortic crossclamping. Preoperative GFR as well as preinduction cardiac index (CI) and pulmonary capillary wedge pressure (PCWP) of the two groups did not differ. During cross-clamping plasma ET rose significantly in both groups. However, after clamp removal, plasma ET decreased in group A while it remained elevated in group B. Urinary excretion of TxB2, PGE2 and 6-keto PGF1 alpha increased during clamp in both groups, but the ratio of PGE2 + 6-keto PGF1 alpha/TxB2 during and after clamp was significantly higher in group A than in B. Postclamp creatinine clearance decreased in group B, and increased in group A; postoperative value of GFR was unchanged in group A and decreased significantly in group B. In conclusion, infusion of nifedipine, in contrast to dopamine, prevented the decrease of GFR in patients undergoing aortic surgery. This effect could be mediated by a nifedipine modulation of ET vascular synthesis and/or a preferential renal synthesis of vasodilating prostanoids.

Synthesis and catabolism of PGE2 by a nephroblastoma associated with hypercalcemia without bone metastases

PGE2 overproduction by a nephroblastoma associated with hypercalcemia was clearly demonstrated in a 2‐month‐old girl. Compared with normal tissue, tumor showed greater phospholipase A2 and PGE2 synthetase activities but metabolized PGE2 at a faster rate. Of the enzymes involved in PGE2 synthesis, those which transform arachidonic acid into PGE2 seem to be more active.

Full Pattern of Urinary Prostaglandins in Bartter’s Syndrome

Dr. Lorenzo Calò, Divisione di Nefrologia, Istituto di Medicina Interna, Università di Padova, Via Giustiniani 2, I-35128 Padova (Italy) Sir, males and 5 females, age range 19–56 years) and in 9 healthy controls cross-matched for sex and age. After Since Gill et al.’s [1] original description of increased urine extraction with organic solvents and silica gel col-urinary excretion of prostaglandin E2 (PGE2) in Bartter’s umn chromatography [3], PGE2 and PGF2α were assayedsyndrome, many controversies have arisen about the role as previously described [4], and 6-keto-PGF,α and TxB2played by prostaglandins in this disease, and it is still by the use of commercially available kits (Amersham, debated whether prostaglandin overproduction is a conAmersham, UK). stant feature and which prostanoid is more frequently As far as the mean urinary excretion is concerned, in involved. Bartter’s syndrome we found a significant increase in Different prostanoids, in fact, sometimes exert oppoPGE2 (627.5 ± 275.6 vs. 359.6 ± 126.6 ng/day; t = 2.62, site effects on renal handling of electrolytes, regulation of p < 0.02) and 6-keto-PGFlα (394.8 ± 190.1 vs. 194.7 ± 40.3renin release and renal action of antidiuretic hormone, ng/day; t = 3.09, p < O.Ol), a significant decrease in TxB2and can counteract the intrarenal action of angiotensin II (123.2 ± 57.3 vs. 184.1 ± 43.9 ng/day; t = 2.47, p < 0.05)in diffferent ways [2]. Therefore, it should be important to and no difference in PGF2α (1,588.6 ± 588.1 vs.know the full urinary pattern of the main arachidonic 1,500.4 ± 488.8 ng/day; t = 0.33, p = n.s.). However, con-acid metabolites in patients affected by Bartter’s synsidering the cutoff values corresponding to eitherdrome to clarify the role played by prostanoids in this mean + 1.64 SD or mean -1.64 SD (i.e. the one-sided 95thsyndrome, but it has never been done. or 5th percentile, respectively), the picture becomes more We have addressed this issue trying to answer two complicated. In fact, PGE2 values were above the upper main questions: which prostaglandin of the cyclooxygelimit in 4 patients, no PGF2α values were above the nornase pathway is more frequently involved in Bartter’s mal range, 6-keto-PGFια values were above the upper

Effect of doxazosin in mild to moderate hypertensive patients with insulin-dependent diabetes mellitus

Abstract Diabetic patients often develop hypertension, and the presence of both hypertension and diabetes doubles the risk of death from coronary heart disease (CHD). Moreover, the presence and importance of abnormalities such as high low-density lipoprotein (LDL) cholesterol and triglycerides levels as CHD risk factors in insulin-dependent diabetes mellitus type 1 have been downplayed, while increasing evidence suggests that the management of type 1 patients should include control of dyslipidemia and hyperglycemia and an effective antihypertensive treatment able also to reduce risk factors for coronary artery events. In this study we assessed the antihypertensive and metabolic effects of doxazosin in hypertensive patients with type 1 diabetes. We show that the drug normalizes blood pressure, and while no improvement in glucose control was observed, it reduced total cholesterol and increased HDL cholesterol as well as the HDL to total cholesterol ratio. The changes of the various parameters studied, including the calculated CHD risk score based on the Framingham equation, suggest that doxazosine can reduce the CHD risk for hypertensive type 1 patients.

Idiopathic hypercalciuria: O2−NO relationship and altered bone metabolism

The pathogenesis of idiopathic hypercalciuria (IH) has not been elucidated yet, but a correlation between IH and altered bone metabolism has been proposed. Since nitric oxide (NO) regulates osteoclasts’ bone resorption, a possible role for NO can be suggested. In this study we evaluated iNOS gene expression by reverse transcription of mRNA from monocytes, followed by polymerase chain reaction in patients with IH subdivided into fasting (FH) and absorptive (AH) hypercalciuria. Since superoxide (O2−), which metabolizes NO, is overproducted by osteoclasts during bone resorption, peroxynitrite plasma level was evaluated as index of O2−. Vertebral BMD in IH as a whole group was lower vs controls (C) (Z score=-1.78±0.2 vs 0.51±0.25, p<0.001), but only FH patients showed a reduced bone density (2.13±0.18 vs 0.51±0.25, p<0.0001). PTH and calcitriol were not different. FH showed an increase in b-ALP vs AH and C (41.1±2.6 vs 30.1±3.9 vs 26.6±3.6 U/l p<0.02), and higher uHP, either on NCD (17.7±1.6 vs 11.4±1.3 mg/g uCr, p<0.04) or after LCD (26.7±2.5 vs 16.7±1.9, p<0.01). Cells from FH patients, but not from both AH patients and C, expressed iNOS. Peroxynitrite plasma level was elevated in FH (0.30±0.07) μmol/l while not detectable in AH and C. This study confirms an altered bone metabolism only in FH which shows an abnormal NO system. The increased iNOS gene expression in FH, in fact, points toward an altered NO system’s activity downstream the generation of NO. A possible interaction of NO with O2−, which breaks down NO, and the role of this interaction in the pathophysiology of IH is discussed.