Extracellular osmolarity is known as an important factor for the regulation of natriuretic peptide receptors (NPRs). We investigated the intra-renal osmoregulation of NPRs using renal medullectomized rats with bromoethylamine hydrobromide (BEA, 200 mg/kg). The administration of BEA caused the decreased food intake and body weight. Water intake was decreased on the first day and then increased from the second day. Urine volume was persistently increased from the first day and free water clearance was also increased from the second day. Urinary excretions of sodium and potassium were decreased on the second day and then recovered to control level. Plasma levels of atrial natriuretic peptide (ANP) and Dendroaspis natriuretic peptide (DNP) in BEA-treated rats were not different from control rats. The inactive renin was increased. The maximum binding capacities of 125 I-ANP as well as 125 I-DNP decreased in glomeruli and medulla of BEA-treated rat kidneys but the binding affinity was not changed. In renal cortex, the gene expressions of ANP, NPR-A, and NPR-B were not changed but that of NPR-C decreased. In renal medulla, the gene expressions of NPR-A, -B, and -C decreased without change in ANP mRNA. Both renal medullary osmolarity and sodium concentration by BEA treatment were lower than those in control kidney. The cGMP concentrations in renal medulla and urine in BEA-treated rats were higher than those in control rats. These results suggest that the increased cGMP production may be partly involved in the decrease in NPRs mRNA expression and their binding capacities by BEA-induced medullectomy.
The natriuretic peptide family consists of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and Dendroaspis natriuretic peptide (DNP) [20], each of which is derived from a separate gene [22]. ANP and BNP are secreted from cardiac myocytes in response to stretch [10], [27] and CNP is secreted from endothelium in response to shear stress [21]. However, the main sites of synthesis and the stimuli of secretion for DNP are still unknown. All of these natriuretic peptides (NPs) show diuretic, natriuretic, vasorelaxant, and antiproliferative activities by binding to their specific cell surface receptors [20]. Three different subtypes of natriuretic peptide receptors (NPR) have been characterized, purified, and cloned, i.e. NPR-A, -B, and -C [11], [24]. All NPs specifically bind to NPR-A except CNP which binds to NPR-B. These receptors are biological receptors, which contain particulate guanylyl cyclase (GC) catalytic activity and produces cGMP from GTP by binding its extracellular ligand. NPR-C receptor lacks the GC domain and hence acts as the clearance receptor for all of these NPs [11]. The biological functions of ANP depend on plasma ANP level and NPRs density.
The NPR-A gene has been shown to be regulated by NPs [2], endothelin [28], glucocorticoids, angiotensin II [12], transforming growth factor-β, chorionic gonadotrophin, growth hormone, and changes in extracellular osmolality [11]. The tonicity of the renal medulla is highly variable by virtue of changes in body fluid balance, such as water loading, dehydration, and high salt intake. Changes in tonicity in renal medulla regulate many genes [9]. It has been shown that hypertonicity induced by NaCl or sucrose but not urea, increased NPR-A activity, gene expression and promoter activity in inner medullary collecting duct (IMCD) cells [4]. Increased extracellular tonicity causes the reduction of endothelial NO synthase (eNOS) protein level, mRNA and gene promoter [3], [4]. From several series of experiments, Chen et al. [5] concluded that the reduction in eNOS expression with the concomitant decline in cGMP levels accounted for an osmotic stimulation of NPR-A gene. However, most of the studies have been done in in vitro conditions because change in body fluid in in vivo condition is always accompanied with change in plasma ANP level, which may autoregulate NPR-A expression. Therefore, we tested whether hypoosmolarity caused by renal medullectomy decreases NPRs in in vivo condition.
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Male Sprague–Dawley rats, weighing 200–250 g, obtained from the Orientbio Inc. (Seoungnam, Korea), were housed in individual cages in a temperature-controlled environment with 12-h light/12-h dark cycle and were maintained on standard laboratory chow (5L79 Purina rat and mouse 18% chow, Charles River Laboratories Inc., Wilmington, MA) ad libitum. All animal experimentation described in this study was conducted in accord with the Guiding Principles in the Care and Use of Animals by the American
The administration of BEA markedly decreased food intake on the first day and then recovered gradually (Fig. 1B). Body weight was dramatically decreased as compared to control rats (Fig. 1A). Water intake abruptly decreased on the first day and increased from the second day after BEA injection (Fig. 1C), and urine volume (Fig. 1D) increased from the first day. Urine osmolality was markedly decreased (Fig. 1E) and free water clearance increased from the second day after BEA injection (Fig. 1F).
The present study was carried out to define the osmoregulation of NPRs in chemical medullectomized rat kidney. BEA treatment caused the decreased Na+ concentration and osmalarity, and the increased basal cGMP level in renal medulla compared to control group. Urinary concentration ability markedly decreased and urine volume increased. Plasma ANP and DNP levels were not changed. The _B_ max of ANP and DNP binding sites as well as NPRs mRNA were decreased in renal medulla of BEA-treated rats.
This work was supported by the Korean Science and Engineering Foundation (R13-2008-005-00000-0, R01-2006-000-10554-0).
These authors contributed equally.
