Heino Velazquez

Renalase is a novel, soluble monoamine oxidase that regulates cardiac function and blood pressure

The kidney not only regulates fluid and electrolyte balance but also functions as an endocrine organ. For instance, it is the major source of circulating erythropoietin and renin. Despite currently available therapies, there is a marked increase in cardiovascular morbidity and mortality among patients suffering from end-stage renal disease. We hypothesized that the current understanding of the endocrine function of the kidney was incomplete and that the organ might secrete additional proteins with important biological roles. Here we report the identification of a novel flavin adenine dinucleotide-dependent amine oxidase (renalase) that is secreted into the blood by the kidney and metabolizes catecholamines in vitro (renalase metabolizes dopamine most efficiently, followed by epinephrine, and then norepinephrine). In humans, renalase gene expression is highest in the kidney but is also detectable in the heart, skeletal muscle, and the small intestine. The plasma concentration of renalase is markedly reduced in patients with end-stage renal disease, as compared with healthy subjects. Renalase infusion in rats caused a decrease in cardiac contractility, heart rate, and blood pressure and prevented a compensatory increase in peripheral vascular tone. These results identify renalase as what we believe to be a novel amine oxidase that is secreted by the kidney, circulates in blood, and modulates cardiac function and systemic blood pressure.

Adaptation of the distal convoluted tubule of the rat. Structural and functional effects of dietary salt intake and chronic diuretic infusion.

We studied the effects of dietary NaCl intake on the renal distal tubule by feeding rats high or low NaCl chow or by chronically infusing furosemide. Furosemide-treated animals were offered saline as drinking fluid to replace urinary losses. Effects of naCl intake were evaluated using free-flow micropuncture, in vivo microperfusion, and morphometric techniques. Dietary NaCl restriction did not affect NaCl delivery to the early distal tubule but markedly increased the capacity of the distal convoluted tubule to transport Na and Cl. Chronic furosemide infusion increased NaCl delivery to the early distal tubule and also increased the rates of Na and Cl transport above the rates observed in low NaCl diet rats. When compared with high NaCl intake alone, chronic furosemide infusion with saline ingestion increased the fractional volume of distal convoluted tubule cells by nearly 100%, whereas dietary NaCl restriction had no effect. The results are consistent with the hypotheses that (a) chronic NaCl restriction increases the transport ability of the distal convoluted tubule independent of changes in tubule structure, (b) high rates of ion delivery to the distal nephron cause tubule hypertrophy, and (c) tubule hypertrophy is associated with increases in ion transport capacity. They indicate that the distal tubule adapts functionally and structurally to perturbations in dietary Na and Cl intake.

Cyst formation and activation of the extracellular regulated kinase pathway after kidney specific inactivation of Pkd1

Polycystic kidney disease (ADPKD) results from failure of the kidney to properly maintain three-dimensional structure after loss of either polycystin-1 or -2. Mice with kidney selective inactivation of Pkd1 during embryogenesis develop profound renal cystic disease and die from renal failure within 3 weeks of birth. In this model, cysts form exclusively from cells in which Cre recombinase is active, but the apparent pace of cyst expansion varies by segment and cell type. Intercalated cells do not participate in cyst expansion despite the presence of cilia up to at least postnatal day 21. Cystic segments show a persistent increase in proliferation as determined by bromodeoxyuridine (BrdU) incorporation; however, the absolute proliferative index is dependent on the underlying proliferative potential of kidney tubule cells. Components of the extracellular regulated kinase (MAPK/ERK) pathway from Ras through MEK1/2 and ERK1/2 to the effector P90(RSK) are activated in both perinatal Pkd1 and adult Pkd2 ortholgous gene disease models. The pattern of MAPK/ERK activation is focal and does not correlate with the pattern of active proliferation identified by BrdU uptake. The possibility of a causal relationship between ERK1/2 activation and cyst cell proliferation was assessed in vivo in the acute perinatal Pkd1 model of ADPKD using MEK1/2 inhibitor U0126. U0126 treatment had no effect on progression of cyst formation in this model at doses sufficient to reduce phospho-ERK1/2 in cystic kidneys. Cysts in ADPKD exhibit both increased proliferation and activation of MAPK/ERK, but cyst growth is not prevented by inhibition of ERK1/2 activation.

Renal Mechanism of Trimethoprim-induced Hyperkalemia

Table. SI Units Hyperkalemia is increasingly being recognized in patients with human immunodeficiency virus (HIV) infection and the acquired immunodeficiency syndrome (AIDS) [1-7]. In this setting, it is likely that hyperkalemia is the result of inadequate renal potassium excretion. Three mechanisms could be responsible for renal potassium retention: adrenal insufficiency with inadequate production of aldosterone; acute renal failure with reduced glomerular filtration and damage to tubule cells; and inhibition of potassium secretion. Most attention has focused on the first two mechanisms [1-8]. We are aware, however, of three reports of reversible hyperkalemia [4, 9, 10] that suggest that a therapeutic agent may have a direct action on renal tubules to suppress potassium transport. A common factor in the three reports was the administration of trimethoprim for treatment of Pneumocystis carinii pneumonia. The purpose of our study was to test the hypothesis that trimethoprim causes hyperkalemia by a direct action on the distal nephron cells responsible for secreting potassium. Methods Study in Humans All patients receiving high-dose trimethoprim (20 mg/kg per day), with either sulfamethoxazole or dapsone, at the Yale-New Haven Hospital during a 4-month period, were included. As a part of the clinical management of these patients, serum measurements of sodium, potassium, and creatinine levels were recorded before, during, and after trimethoprim treatment. In some patients, increased serum potassium levels (>5.0 mmol/L) were identified while patients were receiving trimethoprim. In a group of these patients, we reviewed the clinical course and recommended further evaluation to search for causes of hyperkalemia. This evaluation included measurements of serum glucose, renin, aldosterone, and cortisol levels as well as osmolality; measurements of urinary sodium, potassium, chloride, glucose, and protein levels as well as osmolality; and examination of the urinary sediment. The tubule fluid/plasma concentration ratio for potassium in the cortical collecting duct (transtubular potassium gradient) [11, 12] was calculated from urine and serum values for potassium and osmolality as (K)urine/[K]serum)/([Osm]urine/[Osm]serum). The ability of cosyntropin to stimulate cortisol secretion was determined in patients whose cortisol level was < 552 nmol/L (<20 g/dL). Studies in Rats Male Sprague-Dawley rats, allowed free access to standard rat chow and tap water up to the time of the experiment, were anesthetized before surgical exposure of one kidney, as previously described [13]. The ureter was cannulated. Intravenous Infusion of Trimethoprim A salt solution (140 mmol/L sodium chloride, 4 mmol/L potassium chloride) was infused at 15 mL/h per kg body weight, and 45 minutes was allowed for equilibration after surgical preparation was completed. Subsequently, six 30-minute urine collections were obtained. The first 90 minutes was period I (collections 1 to 3), and the subsequent 90 minutes was period II (collections 4 to 6). The control and experimental groups of animals differed only in that at 90 minutes (after period I), 0.64 g/L of trimethoprim was added to the intravenous infusate (the trimethoprim infusion rate was 9.6 mg/h per kg body weight) in the experimental group and was maintained throughout period II. Sodium, potassium, and chloride concentrations were measured in urine [13]. The urine flow rate was measured, and the excretion rates for fluid, sodium, potassium, and chloride were calculated. Microperfusion of Distal Tubules Microperfusion experiments were done as described previously [13, 14]. Distal tubules were perfused with an artificial tubule fluid with or without trimethoprim (the composition of the perfusion solutions is given in Table 1. A perfusion pipette was positioned at the upstream end of the tubule, and a collection pipette was positioned at the downstream end. The perfusion pump was set to deliver 15 nL/min. A paired design was used as follows: After the first tubule fluid sample was collected, both the collection pipette and the perfusion pipette were removed: Then a second perfusion pipette containing a different solution [the order of solutions was alternated] and a second collection pipette were used to collect a second tubule fluid sample. The volume of collected samples was measured. Sodium, potassium, chloride, and inulin concentrations in perfused and collected fluids were measured, as described previously [14]. Osmolality and pH of bulk solutions were measured [14]. The perfusion rate was calculated from the collection rate and the inulin concentrations. Net fluid transport was calculated as the difference between perfusion and collection rates. Net transport rates for sodium, potassium, and chloride levels by each distal tubule were determined. Transepithelial voltage across the wall of the late distal tubule was measured, as previously described [15]. In experiments designed to test the effect of different concentrations of trimethoprim on transepithelial voltage, a higher perfusion rate (30 compared with 15 nL/min) was used to minimize changes in luminal ion composition along the length of the perfused tubule. Table 1. Distal Tubule Flow Rates, Collected Ion Concentrations, and Transport Rates with Control and Trimethoprim Solutions* Statistical Analysis Results were analyzed using the t-statistic. A P value of less than 0.05 (95% CI) was statistically different. Results Human Studies Records from 30 consecutive inpatients who were treated with high-dose trimethoprim at Yale-New Haven Hospital between 10 July and 18 November 1992 were reviewed. No patients were excluded from this study. All patients were HIV positive and were treated for presumed or confirmed P. carinii pneumonia. Twenty-three of the patients were treated with trimethoprim-sulfamethoxazole, and seven were treated with trimethoprim-dapsone. On average, the length of the trimethoprim treatment period was 5.3 2.79 days (mean SD; range, 1 to 13 days). Figure 1 shows that the serum potassium concentration increased by 0.6 mmol/L (CI, 0.29 to 0.95 mmol/L) during treatment with trimethoprim. When the drug was discontinued, the potassium concentration decreased to pretreatment values. In 15 of 30 patients (50%), the serum potassium concentration was more than 5.0 mmol/L on at least 1 day during trimethoprim treatment. Severe and potentially life-threatening hyperkalemia (potassium > 6.0 mmol/L) occurred in three patients (10%). The serum creatinine concentration was slightly higher during trimethoprim treatment than during recovery (mean difference, 15.9 mmol/L; CI, 5.3 to 26.5 mmol/L [0.18 mg/dL; CI, 0.06 to 0.30 mg/dL]). None of the patients were taking nonsteroidal anti-inflammatory drugs, converting-enzyme inhibitors, or potassium-sparing diuretics. Figure 1. Effect of trimethoprim on serum potassium concentration in patients with AIDS. Renal and adrenal function were evaluated in seven patients during hyperkalemia (potassium > 5 mmol/L). The mean serum potassium concentration was 5.9 0.9 mmol/L, and the urinary potassium concentration was 11.3 5.8 mmol/L (mean SD). Oliguria was not present in any of the patients, and the serum creatinine concentration was not increased above baseline (mean difference, 17.7 mmol/L; CI, 9.72 to 43.3 mmol/L [0.2 mg/dL; CI, 0.11 to 0.49 mg/dL]). The transtubular potassium gradient calculated for the cortical collecting duct [11, 12] was 1.9 1.1 (mean SD). This value was low (expected range was 6 to 11) for a plasma potassium concentration of >5 mmol/L. In three patients, the transtubular potassium gradient was calculated both during and after treatment with trimethoprim. After discontinuation of trimethoprim, the transtubular potassium gradient increased to normal values in all three patients (mean difference, 4.5; CI, 1.4 to 7.5). The urinary sodium concentration was 103 65.7 mmol/L, and there was mild hyponatremia (132 2.8 mmol/L). The plasma cortisol (497 152 nmol/L, supine [18.0 5.5 g/dL, supine]); renin (0.667 0.25 ng/[L x s], supine [2.4 0.9 ng/mL per hour, supine]); and aldosterone (535 264 pmol/L, supine [19.3 9.5 ng/dL, supine]) levels were all high normal or increased during hyperkalemia. In two patients with borderline serum cortisol levels (221 and 469 nmol/L [8 and 17 g/dL]), cosyntropin stimulation test results were normal. Glucose levels were all within the normal range. Rat Studies The effect of trimethoprim on potassium and sodium excretion rates in the whole kidney is shown in Figure 2. Compared with the control group, trimethoprim decreased potassium excretion by 572 nmol/min (CI, 299 to 845 nmol/min). The reduction in potassium excretion during trimethoprim infusion was 40% (CI, 21% to 60%) of the control rate measured in period II. Although sodium excretion in the control group increased with time, trimethoprim caused a twofold larger increase in sodium excretion. The difference between these changes, 1192 nmol/min (CI, 240 to 2142), was statistically significant. The increase in sodium excretion during trimethoprim infusion was 46% (CI, 9% to 83%) of the control rate measured in period II. There was no effect of trimethoprim on urine flow or chloride excretion rate (Appendix 1). Appendix Table 1. Figure 2. The scales depict the change () in ion excretion rate with time (period II minus period I) in control and experimental (Trimethoprim) animals. Panel A Panel B Table 1 gives flow rates, lumen ion concentrations, and ion transport rates from in vivo microperfusion of distal tubules of rats. Figure 3 shows that 1 mmol/L of trimethoprim inhibited net potassium secretion by 59% (CI, 26% to 92%). The rate of net sodium absorption did not decrease. Rates of net chloride and water absorption were also not affected. Figure 3. Net potassium transport during perfusion of 14 distal tubules with control and trimethoprim (TMP) solutions. Figure 4 shows the effect of trimethoprim concent

Catecholamines Regulate the Activity, Secretion, and Synthesis of Renalase

Background— We previously identified renalase, a secreted novel amine oxidase that specifically degrades circulating catecholamines. Parenteral administration of either native or recombinant renalase lowers blood pressure, heart rate, and cardiac contractility by metabolizing circulating catecholamines. Renalase plasma levels are markedly reduced in patients with chronic kidney disease. It is not known whether endogenous renalase contributes to the regulation of catecholamines. Methods and Results— We show here that circulating renalase lacks significant amine oxidase activity under basal conditions (prorenalase) but that a brief surge of epinephrine lasting <2 minutes causes renalase activity to increase from 48±18 to 2246±98 arbitrary units (n=3; P<0.002). Enzyme activation is detectable within 30 seconds and sustained for at least 60 minutes. Analysis of epinephrine-mediated hemodynamic changes in normotensive rats indicates that prorenalase becomes maximally activated when systolic pressure increases by >5 mm Hg. The catecholamine surge also leads to a 2.8-fold increase in plasma renalase concentration. Cultured cells exposed to dopamine upregulate steady-state renalase gene expression by >10-fold. The time course of prorenalase activation is abnormal in rats with chronic kidney disease. Conclusions— These data identify a novel mechanism for the regulation of circulating catecholamines. In the renalase pathway, excess catecholamine facilitates the conversion of prorenalase, an inactive plasma amine oxidase, to renalase, which can degrade catecholamines. Excess catecholamines not only regulate the activation of prorenalase but also promote its secretion and synthesis. Because chronic kidney disease is associated with a number of systemic abnormalities, including activation of the sympathetic nervous system, increased catecholamines levels, cardiac hypertrophy, and hypertension, renalase replacement is an attractive therapeutic modality owing to its role in catecholamine metabolism.

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.)

Expression of the thiazide-sensitive Na-Cl cotransporter by rabbit distal convoluted tubule cells.

A thiazide-sensitive Na-Cl cotransporter contributes importantly to mammalian salt homeostasis by mediating Na-Cl transport along the renal distal tubule. Although it has been accepted that thiazide-sensitive Na-Cl cotransport occurs predominantly along the distal convoluted tubule in rats and mice, sites of expression in the rabbit have been controversial. A commonly accepted model of rabbit distal nephron transport pathways identifies the connecting tubule, not the distal convoluted tubule, as the predominant site of thiazide-sensitive Na-Cl cotransport. The thiazide-sensitive Na-Cl cotransporter has been cloned recently. The present experiments were designed to localize sites of thiazide-sensitive Na-Cl cotransporter mRNA expression along the rabbit distal nephron. Nonradioactive in situ hybridization with a thiazide-sensitive Na-Cl cotransporter probe was combined with immunocytochemistry with an antibody that recognizes distal convoluted tubule cells and with a Na+/Ca2+ exchanger antibody that recognizes only connecting tubule cells. The results indicate that thiazide-sensitive Na-Cl cotransporter mRNA is highly expressed by cells of the distal convoluted tubule and not by connecting tubule cells. Segments that stain with the Na+/Ca2+ exchanger antibody (connecting tubules) do not demonstrate thiazide-sensitive Na-Cl cotransporter mRNA expression. We conclude that the predominant site of thiazide-sensitive Na-Cl cotransporter mRNA expression in rabbit distal nephron is the distal convoluted tubule and that sites of mRNA expression of electroneutral Na and Cl transport are similar in rabbits, rats, and mice.

Endothelial glucocorticoid receptor is required for protection against sepsis

The glucocorticoid receptor (GR) is ubiquitously expressed on nearly all cell types, but tissue-specific deletion of this receptor can produce dramatic whole organism phenotypes. In this study we investigated the role of the endothelial GR in sepsis in vivo and in vitro. Mice with an endothelial-specific GR deletion and controls were treated with 12.5 mg/kg LPS and phenotyped. Mice lacking GR showed significantly increased mortality, more hemodynamic instability, higher nitric oxide levels, and higher levels of the inflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) compared with controls. There were no differences in rates of apoptosis or macrophage recruitment between the two groups. Both endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) expression were increased after LPS challenge in mice with endothelial GR deficiency, and aminoguanidine, a specific iNOS inhibitor in mice was able to rescue hemodynamic collapse in these animals. In vitro, human umbilical vein cells (HUVECs) subjected to GR knockdown by siRNA showed increased expression of eNOS at baseline that persisted after treatment with LPS. Both eNOS and iNOS mRNA was increased by qPCR. In HUVECs lacking GR, NF-κB levels and NF-κB–dependent genes tissue factor and IL-6 were increased compared with controls. Thus, endothelial GR is a critical regulator of NF-κB activation and nitric oxide synthesis in sepsis.

Cloning and localization of a double-pore K channel, KCNK1: exclusive expression in distal nephron segments

The K-selective channel, TOK1, recently identified in yeast, displays the unusual structural feature of having two putative pore regions, in contrast to all previously cloned K channels. Using the TOK1 pore regions as probes, we identified a human kidney cDNA encoding a 337-amino acid protein (hKCNK1) with four transmembrane segments and two pore regions containing the signature sequence of K channels. Amino acid identity to TOK1 is only 15% overall but 40% at the pores. Northern analysis indicates high expression of a 1.9-kb message in brain > kidney >> heart. Nephron segment localization, carried out in rabbit by reverse transcription-polymerase chain reaction, reveals that KCNK1 is expressed in cortical thick ascending limb, connecting tubule, and cortical collecting duct. It was not detected in the proximal tubule, medullary thick ascending limb, distal convoluted tubule, and glomerulus. We conclude that KCNK1 is a unique, double-pore, mammalian K channel, distantly related to the yeast channel TOK1, that is expressed in distal tubule and is a candidate to participate in renal K homeostasis.