Am J Physiol Renal Physiol. 2015 Jun 15;308(12):F1358-68.

Defective renal dopamine function and sodium-sensitive hypertension in adult ovariectomized Wistar rats: role of the cytochrome P-450 pathway.

 

Di Ciano LA1, Azurmendi PJ1, Colombero C2, Levin G2, Oddo EM1, Arrizurieta EE3, Nowicki S2, Ibarra FR4.
  • 1Instituto de Investigaciones Médicas A Lanari, Buenos Aires University, Buenos Aires, Argentina;
  • 2Centro de Investigaciones Endocrinológicas “Dr. César Bergadá” (CEDIE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)-FEI-División de Endocrinología, Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina;
  • 3Instituto de Investigaciones Médicas A Lanari, Buenos Aires University, Buenos Aires, Argentina; CONICET, Buenos Aires, Argentina.
  • 4Instituto de Investigaciones Médicas A Lanari, Buenos Aires University, Buenos Aires, Argentina; Departamento de Fisiología, Facultad de Medicina, Buenos Aires University, Buenos Aires, Argentina; and ilanari@pinos.com ibarraf@hotmail.com.

 

Abstract

We have previously shown that ovariectomy in adult Wistar rats under normal sodium (NS) intake results in an overexpression of the total Na(+)-K(+)-ATPase (NKA) α1-subunit (Di Ciano LA, Azurmendi PJ, Toledo JE, Oddo EM, Zotta E, Ochoa F, Arrizurieta EE, Ibarra FR. Clin Exp Hypertens 35: 475-483, 2013). Upon high sodium (HS) intake, ovariectomized (oVx) rats developed defective NKA phosphorylation, a decrease in sodium excretion, and an increment in mean blood pressure (MBP). Since NKA phosphorylation is modulated by dopamine (DA), the aim of this study was to compare the intracellular response of the renal DA system leading to NKA phosphorylation upon sodium challenge in intact female (IF) and oVx rats. In IF rats, HS caused an increase in urinary DA and sodium, in NKA phosphorylation state, in cytochrome P-4504A (CYP4A) expression, and in 20-HETE production, while MBP kept normal. Blockade of the D1 receptor (D1R) with the D1-like receptor antagonist SCH 23390 in IFHS rats shifted NKA into a more dephosphorylated state, decreased sodium excretion by 50%, and increased MBP. In oVxNS rats, D1R expression was reduced and D3R expression was increased, and under HS intake sodium excretion was lower and MBP higher than in IFHS rats (both P < 0.05), NKA was more dephosphorylated than in IFHS, and CYP4A expression or 20-HETE production did not change. Blockade of D1R in oVxHS rats changed neither NKA phosphorylation state nor sodium excretion or MBP. D2R and PKCα expression did not vary among groups. The alteration of the renal DA system produced by ovariectomy could account for the defective NKA phosphorylation, the inefficient excretion of sodium load, and the development of salt-sensitive hypertension.

KEYWORDS: CYP4A-20-HETE; Na+-K+-ATPase phosphorylation; dopamine D1 receptor; female hormones; ovariectomy; salt-sensitive hypertension

PMID: 25925257

 

Supplemental Material

Ovariectomy as a model of salt-sensitive hypertension in adult Wistar rats:

Under different conditions and causes, when ovary function declines, cardiovascular, metabolic, renal and bone disorders appear in the course of time. The absence of gonadal hormones secondary to ovariectomy (oVx) performed in normotensive adult rats causes several changes in the regulation of extracellular fluid volume, among which salt sensitive hypertension is the most obvious. Ovariectomy was performed at 60 days of life and animals were studied when they reached 150 days, 90 days post oVx. The long period between oVx and experimental studies seems to be relevant to this particular model.

Ovariectomized rats, when consuming a normal sodium diet (NS, 0.24% NaCl), have a mean blood pressure (MBP) lower than that present in IF rats. The latter finding is probably due to a higher activity of renal kallikrein in oVx rats together with an increase in renal plasma flow. As a consequence, oVx rats have a reduced renal vascular resistance. When exploring the behavior of sodium transporters in the renal epithelium, we found that oVx rats have a higher expression of total and dephosphorylated state of Na+,K+-ATPase (NKA). Current knowledge indicates that NKA is more active when it is dephosphorylated. The NKA overexpression was corroborated by immunohistochemistry, which localized the increment of NKA at the thick ascending limb of Henle´s loop (renal medulla). Despite the increment in total NKA expression and dephosphorylation state in oVx rats, under a normal sodium intake, sodium balance did not show variations between IF and oVx rats. So, oVx rats under NS intake are able to achieve a normal sodium balance.

 

Figure 1 WBF

Figure 1: Relationship sodium intake/sodium excretion in IF and oVx rats. oVx excrete less sodium than IF rats.

 

However, when an extra sodium load is administered (1% NaCl in drinking water), oVx rats develop salt sensitive hypertension. After five days on high salt intake (HS), and in opposition to IF rats under HS, oVx rats show a positive sodium balance (Figure 1) and their MBP significantly increases. IF rats, instead, excrete all the sodium they ingest, and so they keep a normal mean blood pressure (Figure 2).

We then explored probable variables that may explain the altered response to a HS load observed in oVx rats, which means an altered regulation of the extracellular fluid volume. Some findings taken from our initial results prompted us to investigate the renal dopaminergic system, dopamine receptors and dopamine signaling. A defect in the phosphorylation state of NKA was found at the Ser 23 residue. Dopamine inhibits NKA activity by phosphorylation of the Ser 23 residue, which is located in the catalytic alpha 1 subunit of the pump. The Ser 23 residue is specifically phosphorylated by PKC, a very well-known second messenger for dopamine effects. So, the synthesis and effects of endogenous renal dopamine and the effects of infused dopamine were explored.

 

 

Figure 2 WBF

Figure 2: Pressure-Natriuresis curve in IF and oVx rats. Upon HS intake MBP increases and Pressure-Natriuresis curve shifts rightward in oVx rats.

 

Main findings when rats receive a normal sodium intake:

Ovariectomized rats had a decreased expression of dopamine 1 receptor (D1R) and an increment of D3R compared to IF rats while no changes were observed in D2 receptors. Remarkably, the difference in the expression of dopamine receptors between IF and oVx rats was already present under NS intake.

Another important aspect is the fact that oVx rats, contrary to IF rats, did not increase the excretion of water or sodium upon treatment with exogenously administered dopamine. The classical natriuretic response to dopamine infusion was lost in oVx rats. When further studying dopamine´s pathway under NS intake, we did not find other differences between IF and oVx rats neither in mediators such as Cytochrome P450 pathway or PKC alpha expression nor in urinary dopamine excretion. In short, when compared to IF rats, oVx rats under NS intake show a lower MBP, a decrease in D1R expression, an increase in D3R expression, and an overexpression of total and dephosphorylated NKA together with a higher urinary kallikrein activity and a higher RPF.

Which is the response to an HS intake in oVx rats compared to IF rats?

Upon a high salt intake (NaCl 1% in drinking water), oVx rats increase synthesis and excretion of renal dopamine as IF do. D1receptors remain low and the expression of D3R decreases.

When exploring other messengers of the dopamine pathway in IF rats, we found that HS intake caused an increase in the expression and activity of Cytochrome P450 pathway. as well as in the Cyp450 4A expression and in the 20-HETE synthesis. This is not observed in oVx rats. One of the most remarkable differences between IF and oVx rats upon HS intake is that NKA becomes robustly phosphorylated in IF (so, inhibited) but not in oVx

in which NKA remains dephosphorylated and more active. These studies were performed in vivo, and therefore changes reflect local and systemic variations when the sodium load is increased. As stated before, the response of MBP to a HS intake is quite different in IF and in oVx rats. oVx rats become hypertensive upon HS, they are unable to handle a sodium load and develop salt sensitive hypertension. IF rats keep MBP at normal levels. Thus, it seems that the whole picture of defective D1R expression, lack of response of the Cytochrome P450 pathway and the concurrent less phosphorylated NKA leads oVx rats to a condition in which they need more blood pressure to excrete sodium and, consequently, the pressure-natriuresis curve shifts rightward (Figure 2).

 

Figure 3 WBF

Figure 3: Schematic picture of differences between IF (panel A) and oVx (panel B) rats in the renal dopamine system and in the Na+, K+-ATPase dephosphorylation state (d-NKA) during HS intake. In oVx rats the expression of D1R is decreased and d-NKA remains dephosphorylated with or without treatment with D1-like receptors blockers (panel B). The opposite is observed in IF rats (panel A).

 

Conclusion:

It is worth noting that oVx rats, even on NS intake, already have a reduced abundance of D1R together with an increase in D3R expression, and a lack of response to infused dopamine. These findings may indicate the presence of a prehypertensive condition uncovered by sodium load. The picture shown in Figure 3 illustrates the similarities and differences in the response of the dopamine system and NKA alpha 1 phosphorylation state to a high sodium intake in IF and oVx rats.

The renal dopamine system is greatly distorted in ovary hormone deprived rats and, consequently, they are unable to handle a high sodium intake. Then, a normotensive rat strain develops salt sensitive hypertension. Data support the crucial role of gonadal steroids in regulating renal function and systemic hemodynamics as well as renal sodium handling, particularly under a high salt intake. A better understanding of the renal dopamine system in postmenopausal women as related to renal disease and blood pressure control could provide insights into new preventive and therapeutic strategies.

 

References:

1) Azurmendi PJ, Oddo EM, Obika LF, Corbera NL, Martín RS, Ibarra FR, Arrizurieta EE. Gonadectomy influences blood pressure through the kallikrein-kinin system. Kidney Blood Press Res. 2009;32(5):342-8.

2) Di Ciano LA, Azurmendi PJ, Toledo JE, Oddo EM, Zotta E, Ochoa F, Arrizurieta EE, Ibarra FR. Ovariectomy causes overexpression of renal Na(+),K(+)-ATPase and sodium-sensitive hypertension in adult Wistar rats. Clin Exp Hypertens. 2013;35(7):475-83.

3) Di Ciano LA, Azurmendi PJ, Colombero C, Levin G, Oddo EM, Arrizurieta EE, Nowicki S, Ibarra FR. Defective renal dopamine function and sodium-sensitive hypertension in adult ovariectomized Wistar rats: role of the cytochrome P-450 pathway. Am J Physiol Renal Physiol. 2015 Jun 15;308(12):F1358-68.

 

Contact:

Fernando R Ibarra, MD, PhD

Kidney Laboratory, Instituto A Lanari and

Departamento de Fisiología, Facultad de Medicina

Universidad de Buenos Aires

e-mail: ibarraf@hotmail.com

 

 

 

Multiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier SchönmannMultiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier Schönmann