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Oxytocin is known to have an antidiuretic effect, but the mechanisms underlying this effect are not completely understood. We infused oxytocin by osmotic minipump into vasopressin-deficient Brattleboro rats for five days and observed marked antidiuresis, increased urine osmolality, and increased solute-free water reabsorption. Administration of oxytocin also significantly increased the protein levels of aquaporin-2 (AQP2), phosphorylated AQP2 (p-AQP2), and AQP3 in the inner medulla and in the outer medulla plus cortex. Immunohistochemistry demonstrated increased AQP2 and p-AQP2 expression and trafficking to the apical plasma membrane of principal cells in the collecting duct, and increased AQP3 expression in the basolateral membrane. These oxytocin-induced effects were blocked by treatment with the vasopressin V2 receptor antagonist SR121463B, but not by treatment with the oxytocin receptor antagonist GW796679X. We conclude that vasopressin V2 receptors mediate the antidiuretic effects of oxytocin, including increased expression and apical trafficking of AQP2, p-AQP2, and increased AQP3 protein expression. 19: 225-232, 200819: 225-232, . doi: 10.1681 In recent years, the understanding of the molecular basis for the antidiuretic effect of vasopressin has been substantially advanced. The vasopressin V2 receptor 1 and renal aquaporin (AQP) water channels 2 have been cloned. Vasopressin has been shown to mediate both long-and short-term effects on AQP2 in the principal cells of the collecting duct. The long-term effect of vasopressin leads to increased expression of AQP2, whereas the shortterm effects involve trafficking of AQP2 to the apical membrane of the principal cells. 3 There is also evidence that vasopressin increases AQP3 protein expression on the basolateral membrane of the principal cells 4 and up-regulates the Na-K-2Cl cotransporter, 5 the initiator of the countercurrent concentrating mechanism. J Am Soc NephrolOxytocin (OT) is also known to possess antidiuretic properties. 6 In this regard, use of OT to induce labor in pregnancy has been associated with water retention and hyponatremia. 7 OT has been shown in vitro to increase osmotic water transport in microdissected renal inner medullary collecting ducts (IMCD) 8 and in vivo to cause an antidiuresis in vasopressin-deficient Brattleboro rats. 9,10 These effects were reversed by a vasopressin V2 receptor antagonist, suggesting that OT stimulation of vasopressin receptors mediates the antidiuresis. These in vitro observations were not altered by two different OT receptor antagonists. 8 The effect of OT on the long-and short-term regulation of AQP2 or the expression of AQP3 and Na-K-2Cl cotransporter has not been studied. The present study was therefore undertaken to advance the knowledge at the

ANG II plays a major role in renal water and sodium regulation. In the immortalized mouse renal collecting duct principal cells (mpkCCD(cl4)) cell line, we treated cells with ANG II and examined aquaporin-2 (AQP2) protein expression, trafficking, and mRNA levels, by immunoblotting, immunofluorescence, and RT-PCR. After 24-h incubation, ANG II-induced AQP2 protein expression was observed at the concentration of 10(-10) M and increased in a dose-dependent manner. ANG II (10(-7) M) increased AQP2 protein expression and mRNA levels at 0.5, 1, 2, 6, and 24 h. Immunofluorescence studies showed that ANG II increased the apical membrane targeting of AQP2 from 30 min to 6 h. Next, the signaling pathways underlying the ANG II-induced AQP2 expression were investigated. The PKC inhibitor Ro 31-8220 (5 × 10(-6) M) and the PKA inhibitor H89 (10(-5) M) blocked ANG II-induced AQP2 expression, respectively. Calmodulin inhibitor W-7 markedly reduced ANG II- and/or dDAVP-stimulated AQP2 expression. ANG II (10(-9) M) and/or dDAVP (10(-10) M) stimulated AQP2 protein levels and cAMP accumulation, which was completely blocked by pretreatment with the vasopressin V2 receptor (V2R) antagonist SR121463B (10(-8) M). Pretreatment with the angiotensin AT(1) receptor (AT1R) antagonist losartan (3 × 10(-6) M) blocked ANG II (10(-9) M)-stimulated AQP2 protein expression and cAMP accumulation, and partially blocked dDAVP (10(-10) M)- and dDAVP+ANG II-induced AQP2 protein expression and cAMP accumulation. In conclusion, ANG II regulates AQP2 protein, trafficking, and gene expression in renal collecting duct principal cells. ANG II-induced AQP2 expression involves cAMP, PKC, PKA, and calmodulin signaling pathways via V2 and AT(1) receptors.

The effect of endotoxemia (lipopolysaccharide, 2.5 mg/kg ip) was investigated in aquaporin (AQP) 1 knockout (KO) compared with wild-type (WT) mice. At baseline, KO mice exhibited higher water intake (WI) and urine output (UO). After endotoxemia, WI and UO remained higher in the KO than WT mice, and urine osmolality was lower. The higher serum osmolality in AQP1-KO mice during endotoxemia was associated with higher AQP2 (133 +/- 8 vs. 100 +/- 3%, P < 0.01), AQP3 (140 +/- 8 vs. 100 +/- 4%, P < 0.001) and Na(+)-K(+)-2Cl(-) cotransporter type 2 (NKCC2; 152 +/- 14 vs. 100 +/- 15%, P < 0.05) expression than that in WT mice. These responses during endotoxemia in the AQP1-KO mice compared with WT were associated with lower glomerular filtration rate (GFR) (69 +/- 8 vs. 96 +/- 8 ml/min, P < 0.05) and renal blood flow (0.77 +/- 0.1 vs. 1.01 +/- 0.1 ml/min, P < 0.01). Urinary sodium excretion and fractional sodium excretion were higher in KO compared with WT mice in endotoxemia and were accompanied by more severe tubular injury. With water repletion and comparable serum osmolalities, GFR was still lower in KO (57 +/- 13 vs. 120 +/- 6 ml/min, P < 0.01) compared with WT during endotoxemia. The abundance of AQP2 and AQP3 protein in KO mice was not different from WT mice; however, NKCC2, Na(+)/H(+) exchanger type 3, and fractional sodium excretion remained higher in KO compared with WT. Thus the polyuria in AQP1-KO mice does not protect against endotoxemia-induced acute kidney injury but rather absence of AQP1 predisposed to enhanced endotoximic renal injury.

Vascular heme oxygenase (HO) regulates vascular tone in normal conditions and in some pathologic circumstances (e.g., sepsis). However, its possible role in the pathogenesis of arterial vasodilation in cirrhosis is unknown. To address this question, the expression and activity of HO in arterial vessels was studied in rats at 1, 2, and 4 weeks after bile duct ligation (BDL) or sham operation. A progressively increased expression of HO-1 was found in aorta and mesenteric arteries of BDL rats in a close chronologic relationship with the progression from acute cholestatic liver injury (1 week) to the fully developed cirrhosis with intense systemic arterial vasodilation (4 weeks). No changes were found in the expression of the constitutive isoform HO-2. HO-1 was mainly located in vascular smooth muscle cells of the arterial wall. Aortic HO activity increased in parallel with the expression of HO-1 (up to 600% in rats with cirrhosis compared with sham rats) and correlated with hemodynamic parameters. Increased expression of HO-1 and HO activity were also found in other organs, such as liver and spleen, though to a lesser extent compared with vascular tissue. The acute administration of an inhibitor of HO to cirrhotic rats, at a dose that normalized aortic HO activity, was associated with significantly greater effects on arterial pressure, total peripheral vascular resistance, and cardiac index, compared with effects in sham rats. In conclusion, these findings are consistent with a role for HO in the pathogenesis of arterial vasodilation in cirrhosis.