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

Abstract

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.

Figure 1

Tissue expression of renalase. (A) Northern blot analysis of human tissues using the MGC12474 clone as a probe. The upper band in skeletal muscle and lower bands in kidney and liver may represent alternatively spliced forms of renalase. (B) Western blot analysis of rat tissues using a renalase polyclonal antibody. (C–F) Renalase expression in kidney. (C) In situ hybridization analysis of human kidney. Left: antisense probe. Open arrow indicates the glomerulus; filled arrow indicates proximal tubules. Scale bar: 40 μm. Right: sense probe control. Magnification, ×200. (D) Immunolocalization in human kidney. Left: anti-renalase antibody. Filled arrow indicates proximal tubules. Scale bar: 40 μm. Right: preimmune serum. Magnification, ×630. (E) Immunofluorescence in human kidney. Left panels: anti-renalase antibody, thick arrow denotes the glomerulus; thin arrows indicate proximal tubules. Right panels: preimmune serum. Scale bars: 40 μm. (F and G) Renalase expression in heart. (F) In situ hybridization analysis of human heart. Left: antisense probe. Open arrow indicates blood vessels; filled arrow indicates ventricular myocytes. Scale bar: 40 μm. Right: sense probe control. Magnification, ×200. (G) Immunolocalization in human heart. Left: anti-renalase antibody. Open arrow indicates blood vessels; filled arrows indicate ventricular myocytes. Scale bar: 40 μm. Right: preimmune serum. Magnification, ×630.

Figure 2

Renalase is a secreted amine oxidase. (A) Detection of renalase in culture medium of HEK293 cells transiently transfected with renalase cDNA. Control, secondary antibody alone. (B) Western blot analysis of human plasma using an anti-renalase antibody. Normal, plasma from individuals with normal renal function; Control protein, human recombinant renalase protein; ESRD, plasma from patients with ESRD receiving hemodialysis. (C) Renalase metabolizes catecholamines. Ten micrograms of GST-renalase fusion protein was used for each assay; amine oxidase is expressed as H₂O₂ production (nmol/mg/min). (D) Renalase activity is insensitive to known monoamine oxidase inhibitors. The renalase activity was assessed as described in C, with dopamine, epinephrine, and norepinephrine as substrates. The renalase activity was examined in the absence of inhibitor (Alone), in the presence of 1 μM clorgyline, or in the presence of 1 μM pargyline. Renalase activity is expressed as arbitrary fluorescence units/10 μg protein. In control studies, clorgyline (1 μM) and pargyline (1 μM) inhibited MAO-A activity by 83.9% ± 2.3% (n = 3) and 82.4% ± 1.9% (n = 3), respectively. (E) Affinity purification of human renalase. The anti-renalase polyclonal antibody was used to isolate protein from human urine. Lane 1: renalase from human urine; lane 2: control with secondary antibody alone.

Figure 3

Effect on renalase of hemodynamic parameters. (A) Cardiac response before and after an i.v. bolus injection of 4 μg/g body wt. The arrow denotes the timing of renalase injection. VP, left ventricular pressure; VP max, maximal left ventricular pressure; HR, heart rate; dP/dt, rate of change in left ventricular pressure, a measure of cardiac contractility. (B) Pressure-volume changes before and after renalase injection. (C) Renalase dose-response curve: cardiac contractility. (D) Renalase dose-response curve: mean arterial pressure (MAP).