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Hormonal regulation of water and sodium handling in the collecting duct

 

Ulla Seehusen Kruchov van Deurs

Summary

This PhD thesis consists of two different studies on pharmacological interventions during congestive heart failure (CHF). These pharmacological interventions were directed toward the kidney with the goal of diminishing the development of edema and hyponatremia. In part one, the focus is on the renal mechanisms during the course of CHF, which lead to excessive water
retention. More specifically, the aim was to elucidate whether an interaction exists between the V2 and the AT1 receptor in the renal collecting ducts (CD). Part two examines the renal site of action of a potential new aquaretic, ZP120. ZP120 induces aquaresis, antinatriuresis and antikaliuresis, and is currently in a phase II clinical trial as an aquaretic during the acute phase of water retaining disease states such as CHF.
PART 1
During the late stages of CHF retention of free water is seen as the result of baroreceptor mediated vasopressin (AVP) release. AVP induces an upregulation of aquaporin 2 (AQP2) expression and apical membrane localization in renal CDs. In rats with an early stage of this disease there seems to be an increase in sensitivity to vasopressin, since increased AQP2 expression and apical localization has been described in presence of normal plasma AVP levels (Hadrup et al., 2004). AVP binding to the V2 receptor in the CDs induces activation of adenylate cyclase (AC) and therefore results in an increase in intracellular cyclic Adenosine Mono Phosphate (cAMP) levels. In vitro studies have shown that Angiotensin II (ANGII) potentiates AVP-mediated cAMP accumulation (Klingler et al.,1998), suggesting the existence of an interaction between the signaling pathways downstream from the V2 and AT1 receptors. Recent studies from our laboratory have shown that long term treatment with the ANGII type 1 (AT1) receptor antagonist, losartan, decreases the expression of AQP2, and reduces AVP-mediated CD water reabsorption in rats with CHF (Staahltoft et al., 2002). Therefore the aim of part 1 was to further study a potential interaction between the V2 and AT1 receptors in CHF rats.
To examine the long term effect of the AT1 receptor antagonist, losartan, in CHF, CHF was induced in rats by left anterior descending coronary artery ligation (LCAL). Sham operated rats were used as controls. 3 weeks after LCAL, left ventricular end diastolic pressure (LVEDP) was determined by introducing a 3F microtip catheter into the left ventricle. CHF rats were characterized by LVEDP above 10 mmHg. Rats were then randomly divided to receive 4 weeks infusion with losartan (10 mg/kg/day) or vehicle. In CHF rats receiving vehicle infusion a correlation existed between the level of CHF and renal expression of AQP2 in cortex and inner medulla (IM). This correlation was abolished by four weeks AT1 receptor blockade.
It has previously been shown that ANGII potentiates AVP mediated cAMP accumulation. This was tested in freshly isolated inner medullary CDs (IMCDs). IMCDs were isolated from untreated Wistar male rats and incubated with increasing concentrations of vasopressin (AVP 10-12-10-6 M) with or without the presence of ANGII (10-10-10-7 M) at 30 °C for 10 minutes. 3-isobutyl-1-methylxanthine (IBMX, 5x10-4 M) was added to inhibit phosphodiesterase activity. An ANGII concentration of 10-7 M potentiated AVP-mediated cAMP accumulation. This might reflect a relevant physiological and/or pathophysiological interaction between the V2 and AT1 receptors, since local intratubular ANGII concentration have been reported of 10-8 M, which is significantly higher than circulating ANGII levels (~10-10 M) (Cervenka et al., 1999; Seikaly et al., 1990). The effect of losartan in the CHF rats could be mediated by a central or a peripheral mechanism of action. To examine this further, renal excretory responses to co-administration of AVP and ANGII were to be examined in Brattleboro (BB) rats, which do not produce AVP. First, BB rats were placed in metabolic cages and divided into groups receiving osmotic minipumps delivering either AVP (840 ng/day) or vehicle. Furthermore, the effect of the synthetic AVP analogue, dDAVP (240 ng/day) was evaluated. Here it was found that infusion of dDAVP, but not AVP, significantly and consistently decreased urine volume and increased urine osmolality throughout a 10 day period reflected in an increase in renal AQP2 and pAQP2 expression levels. The same pattern was seen during a five day infusion study. Following 2 days AVP infusion urine volume was significantly decreased and urine osmolality was significantly increased. This effect of AVP was reflected in increased protein expression levels of AQP2 and phosphorylated AQP2 (pAQP2). These results suggest that in the polyuric BB rat compensatory mechanisms sets in between 2 and 5 days after AVP infusion start that reestablish pre-treatment conditions. The model was never systematically tested with the co-administration of AVP and ANGII, since it was concluded that the model is not good for reliable answers on this matter.