Manuel Pestana

Mycophenolate Mofetil versus Cyclophosphamide for Induction Treatment of Lupus Nephritis

Recent studies have suggested that mycophenolate mofetil (MMF) may offer advantages over intravenous cyclophosphamide (IVC) for the treatment of lupus nephritis, but these therapies have not been compared in an international randomized, controlled trial. Here, we report the comparison of MMF and IVC as induction treatment for active lupus nephritis in a multinational, two-phase (induction and maintenance) study. We randomly assigned 370 patients with classes III through V lupus nephritis to open-label MMF (target dosage 3 g/d) or IVC (0.5 to 1.0 g/m(2) in monthly pulses) in a 24-wk induction study. Both groups received prednisone, tapered from a maximum starting dosage of 60 mg/d. The primary end point was a prespecified decrease in urine protein/creatinine ratio and stabilization or improvement in serum creatinine. Secondary end points included complete renal remission, systemic disease activity and damage, and safety. Overall, we did not detect a significantly different response rate between the two groups: 104 (56.2%) of 185 patients responded to MMF compared with 98 (53.0%) of 185 to IVC. Secondary end points were also similar between treatment groups. There were nine deaths in the MMF group and five in the IVC group. We did not detect significant differences between the MMF and IVC groups with regard to rates of adverse events, serious adverse events, or infections. Although most patients in both treatment groups experienced clinical improvement, the study did not meet its primary objective of showing that MMF was superior to IVC as induction treatment for lupus nephritis.

Renalase deficiency aggravates ischemic myocardial damage

Chronic kidney disease (CKD) leads to an 18-fold increase in cardiovascular complications not fully explained by traditional risk factors. Levels of renalase, a recently discovered oxidase that metabolizes catecholamines, are decreased in CKD. Here we show that renalase deficiency in a mouse knockout model causes increased plasma catecholamine levels and hypertension. Plasma blood urea nitrogen, creatinine, and aldosterone were unaffected. However, knockout mice had normal systolic function and mild ventricular hypertrophy but tolerated cardiac ischemia poorly and developed myocardial necrosis threefold more severe than that found in wild-type mice. Treatment with recombinant renalase completely rescued the cardiac phenotype. To gain insight into the mechanisms mediating this cardioprotective effect, we tested if gene deletion affected nitrate and glutathione metabolism, but found no differences between hearts of knockout and wild-type mice. The ratio of oxidized (NAD) to reduced (NADH) nicotinamide adenine dinucleotide in cardiac tissue, however, was significantly decreased in the hearts of renalase knockout mice, as was plasma NADH oxidase activity. In vitro studies confirmed that renalase metabolizes NADH and catecholamines. Thus, renalase plays an important role in cardiovascular pathology and its replacement may reduce cardiac complications in renalase-deficient states such as CKD.

Renalase Lowers Ambulatory Blood Pressure by Metabolizing Circulating Adrenaline

Background Blood pressure is acutely regulated by the sympathetic nervous system through the action of vasoactive hormones such as epinephrine, norepinephrine, and dopamine. Renalase, a recently described, secreted flavoprotein, acutely decreases systemic pressure when administered in vivo. Single‐nucleotide polymorphisms present in the gene are associated with hypertension, cardiac disease, and diabetes. Although renalases crystal structure was recently solved, its natural substrate(s) remains undefined. Methods and Results Using in vitro enzymatic assays and in vivo administration of recombinant renalase, we show that the protein functions as a flavin adenine dinucleotide– and nicotinamide adenine dinucleotide–dependent oxidase that lowers blood pressure by degrading plasma epinephrine. The enzyme also metabolizes the dopamine precursor l‐3,4‐dihydroxyphenylalanine but has low activity against dopamine and does not metabolize norepinephrine. To test if epinephrine and l‐3,4‐dihydroxyphenylalanine were renalases only substrates, 17 246 unique small molecules were screened. Although the search revealed no additional, naturally occurring compounds, it identified dobutamine, isoproterenol, and α‐methyldopa as substrates of renalase. Mutational analysis was used to test if renalases hypotensive effect correlated with its enzymatic activity. Single–amino acid mutations that decrease its enzymatic activity to varying degrees comparably reduce its hypotensive effect. Conclusions Renalase metabolizes circulating epinephrine and l‐3,4‐dihydroxyphenylalanine, and its capacity to decrease blood pressure is directly correlated to its enzymatic activity. These findings highlight a previously unrecognized mechanism for epinephrine metabolism and blood pressure regulation, expand our understanding of the sympathetic nervous system, and could lead to the development of novel therapeutic modalities for the treatment of hypertension. (J Am Heart Assoc. 2012;1:e002634 doi: 10.1161/JAHA.112.002634.)

The effect of dietary sodium restriction on neurohumoral activity and renal dopaminergic response in patients with heart failure

This work evaluates the effect of a low‐sodium diet on clinical and neurohumoral parameters and on renal dopaminergic system activity in heart failure (HF) patients.

Assessment of renal dopaminergic system activity in the nitric oxide‐deprived hypertensive rat model

1 The present paper reports changes in the urinary excretion of dopamine, 5‐hydroxytryptamine and amine metabolites in nitric oxide deprived hypertensive rats during long‐term administration of NGnitro‐L‐arginine methyl ester (l‐NAME). Aromatic L‐amino acid decarboxylase (AAAD) activity in renal tissues and the ability of newly‐formed dopamine to leave the cellular compartment where the synthesis of the amine has occurred were also determined.

Attenuation of the cardiovascular and metabolic complications of obesity in CD14 knockout mice

AIMS Although toll-like receptors (TLR) are known to mediate the metabolic complications of obesity, the mechanisms underlying its activation remain largely unknown. The present study analyzed a model of diet-induced obesity in mice lacking the TLR4/TLR2 co-receptor CD14. MAIN METHODS Six-week-old male mice lacking CD14 (n = 16) were allocated to either a control diet or a high-fat high-simple carbohydrate diet (5.4 kcal/g; 35% fat; 35% sucrose), and compared with C57BL/6 (WT; n = 15) controls. After 12 weeks, body composition, basal sympathetic activity, non-invasive blood pressure and glucose tolerance were evaluated. Hepatic and adipose tissues were collected for mRNA quantification, histology and LPS incubation. KEY FINDINGS In both WT and CD14 knockout mice, obesity was accompanied by TLR2 and TLR4 upregulation. However, obese mice lacking CD14 presented decreased lipid and macrophage content in hepatic and adipose tissues, lower urinary levels of noradrenaline, decreased systolic blood pressure, reduced fasting plasma glucose and blunted glucose intolerance, compared with obese WT group. In the presence of exogenous sCD14, adipose tissue incubation with LPS-induced TLR2 and TNF-alpha upregulation in both WT and CD14 knockout obese mice. SIGNIFICANCE In our model of diet-induced obesity, mice lacking CD14 showed lower adiposity and hepatic steatosis, improved glucose homeostasis, blunted sympathetic overactivity and reduced blood pressure elevation. This was observed in the presence of preserved TLR4 and TLR2 gene expression, and intact TLR4 signaling pathways. These results suggest that CD14-mediated TLR activation might contribute to the cardiovascular and metabolic complications of obesity.

Aging, High Salt Intake, and Renal Dopaminergic Activity in Fischer 344 Rats

The present study examined renal dopaminergic activity and its response to high salt (HS) intake in adult (6-month-old) and old (24-month-old) Fischer 344 rats. Daily urinary excretion of L-3, 4-dihydroxyphenylalanine (L-DOPA), dopamine, and its metabolites 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid was similar in adult and old rats; by contrast, daily urinary excretion of norepinephrine in old rats was almost twice that in adult animals. HS intake (1% NaCl) over a period of 24 hours resulted in a 2-fold increase in the urinary excretion of dopamine, DOPAC, and norepinephrine in adult animals but not in old animals. Norepinephrine and L-DOPA plasma levels did not change during HS intake and were similar in both groups of rats. The natriuretic response to an HS intake in old rats (from 4.7+/-0.4 to 10.7+/-2.0 nmol. kg(-1). d(-1); Delta=6.0+/-0.9 nmol. kg(-1). d(-1)) was less than in adult rats (from 5.2+/-0.4 to 13.5+/-2.5 nmol. kg(-1). d(-1); Delta=8.3+/-0.8 nmol. kg(-1). d(-1)). A diuretic response to HS intake was observed in adult rats (from 20.9+/-2.3 to 37.6+/-2.8 mL. kg(-1). d(-1)) but not in old rats (from 37.7+/-5.7 to 42.3+/-6. 0 mL. kg(-1). d(-1)). Dopamine levels and dopamine/L-DOPA ratios in the renal cortex of old rats were greater than in adult rats. HS intake increased both dopamine levels and dopamine/L-DOPA ratios in the renal cortex of adult rats but not in old rats. Aromatic L-amino acid decarboxylase activity was higher in old rats than in adult rats; HS intake increased L-amino acid decarboxylase activity (nmol. mg protein(-1). l5 min(-1)) in adult rats (from 67+/-1 to 93+/-1) but not in old rats (from 86+/-2 to 87+/-2). Dopamine inhibited Na(+),K(+)-ATPase activity in proximal tubules obtained from adult rats, but it failed to exert such an inhibitory effect in old rats. It is concluded that renal dopaminergic tonus in old rats is higher than in adult rats but fails to respond to HS intake as observed in adult rats. This may be due in part to the inability of dopamine to inhibit Na(+),K(+)-ATPase activity in old rats.

Renal Dopaminergic System Activity in the Rat Remnant Kidney

Background: Renal dopamine exerts natriuretic and diuretic effects by activating D1-like receptors. Uninephrectomy results in increased renal dopaminergic activity and dopamine-sensitive enhanced natriuresis. Methods: The present study evaluated renal adaptations in sodium handling and the role of dopamine in rats submitted to ¾ nephrectomy: right nephrectomy and excision of both poles of the left kidney (¾nx rats). Results: Two weeks after surgery the absolute urinary levels of dopamine were markedly reduced in ¾nx rats whereas the urinary dopamine excretion per % of residual nephrons was significantly increased in the remnant kidney of ¾nx rats. The Vmax values for renal aromatic L-amino acid decarboxylase, the enzyme responsible for the synthesis of renal dopamine, were decreased in ¾nx rats. Renal catechol-O-methyltransferase activity, the enzyme responsible for the methylation of dopamine, was increased in ¾nx rats whereas the renal activities of monoamine oxidases A and B did not differ between ¾nx and Sham animals. Volume expansion (5% body weight) resulted in similar natriuretic responses in ¾nx and Sham rats. During D1 antagonist administration (Sch-23390, 30 µg·h–1·kg–1) the natriuretic response to volume expansion was reduced in ¾nx rats more pronouncedly than in Sham animals. Conclusion: The decrease in absolute renal dopamine output in ¾nx rats is related with reduced renal synthesis and enhanced O-methylation of the amine. However, this is accompanied in ¾nx rats by increased renal dopamine excretion per residual nephrons and dopamine-sensitive enhanced natriuresis.

Assessment of renal dopaminergic system activity during cyclosporine A administration in the rat.

1 Administration of cyclosporine A (CsA; 50 mg kg−1 day−1, s.c.) for 14 days produced an increase in both systolic (SBP) and diastolic (DBP) blood pressure by 60 and 25 mmHg, respectively. The urinary excretion of dopamine, DOPAC and HVA was reduced from day 5–6 of CsA administration onwards (dopamine from 19 to 46%, DOPAC from 16 to 48%; HVA from 18 to 42%). In vehicle‐treated rats, the urinary excretion of dopamine and DOPAC increased (from 7 to 60%) from day 5 onwards; by contrast, the urinary excretion of HVA was reduced (from 27 to 60%) during the second week. 2 No significant difference was observed between the Vmax and Km values of renal aromatic L‐amino acid decarboxylase (AAAD) in rats treated with CsA for 7 and 14 days or with vehicle. 3 Km and Vmax of monoamine oxidase types A and B did not differ significantly between rats treated with CsA for 7 and 14 days or with vehicle. 4 Maximal catechol‐O‐methyltransferase activity (Vmax) in homogenates of renal tissues obtained from rats treated with CsA for 7 or 14 days was significantly higher than that in vehicle‐treated rats; Km(22.3 ± 1.5 μm) values for COMT did not differ between the three groups of rats. 5 The accumulation of newly‐formed dopamine and DOPAC in cortical tissues of rats treated with CsA for 14 days was three to four times higher than in controls. The outflow of both dopamine and DOPAC declined progressively with time and reflected the amine and amine metabolite tissue contents. No significant difference was observed between the DOPAC/dopamine ratios in the perifusate of renal tissues obtained from CsA‐ and vehicle‐treated rats. In addition, no significant differences were observed in k values or in the slope of decline of both DA and DOPAC between experiments performed with CsA and vehicle‐treated animals. 6 The Vmax for the saturable component of L‐3, 4‐dihydroxyphenylalanine (L‐DOPA) uptake in renal tubules from rats treated with CsA was twice that of vehicle‐treated animals. Km in CsA‐ and vehicle‐treated rats did not differ. 7 The decrease in the urinary excretion of sodium and an increase in blood pressure during CsA treatment was accompanied by a reduction in daily urinary excretion of dopamine. This appears to result from a reduction in the amount of L‐DOPA made available to the kidney and does not involve changes in tubular AAAD, the availability of dopamine to leave the renal cells and dopamine metabolism. The enhanced ability of the renal tissues of CsA‐treated animals to synthesize dopamine, when exogenous L‐DOPA is provided, results from an enhanced activity of the uptake process of L‐DOPA in renal tubular cells.

Deamination of newly‐formed dopamine in rat renal tissues

1 The present study has examined the formation of dopamine and 3,4‐dihydroxyphenylacetic acid (DOPAC) in slices of the rat renal cortex and the renal medulla loaded with exogenous l‐β‐3,4‐dihydroxyphenylalanine (l‐DOPA). The effects of pargyline and of two selective inhibitors of monoamine oxidase (MAO) types A and B, respectively Ro 41–1049 and Ro 19–6327, on the deamination of newly‐synthesized dopamine in kidney slices incubated with exogenous l‐DOPA were also tested. The assay of l‐DOPA, dopamine, noradrenaline and DOPAC was performed by means of h.p.l.c. with electrochemical detection. 2 Incubation of renal slices with exogenous l‐DOPA resulted in a concentration‐dependent accumulation of dopamine and DOPAC; the tissue levels of newly‐formed dopamine and DOPAC in slices of the renal medulla were 6–8% of those in cortical slices. 3 Pargyline (0.1 mm) produced a marked decrease (84% reduction) in the formation of DOPAC in kidney slices loaded with 1.0 mm l‐DOPA; this effect was accompanied by a 17% increase in the accumulation of dopamine. Similar effects were obtained at higher concentrations of pargyline (0.5 and 1.0 mm). At 5.0 and 10.0 mm pargyline, a marked decrease (46 and 76% reduction) in the accumulation of newly‐formed dopamine was observed. 4 The accumulation of dopamine and DOPAC was found to be time‐dependent in experiments in which tissues were incubated with 5 and 10 μm l‐DOPA for 5, 10, 20 and 30 min. Pargyline (0.1 mm) produced an increase in the accumulation of dopamine at all incubation periods and decreased the formation of DOPAC. 5 Ro 41–1049 (50, 100 and 250 nm) was found to produce a concentration‐dependent increase of newly‐formed dopamine (16–66% increase) and reduced DOPAC formation (44–89% reduction). Ro 19–6327 (50, 100 and 250 nm), was found not to affect the accumulation of newly‐formed dopamine, but significantly reduced the formation of DOPAC. 6 It is concluded that deamination of newly‐formed dopamine in kidney slices loaded with l‐DOPA constitutes an important mechanism of amine inactivation. The results presented also suggest that most of the MAO, located inside the compartment where the synthesis of dopamine occurs, is of the A type.