INTRODUCTION

Hypertension is the most prevalent chronic disease with worldwide incidence and one of the cardiovascular risk factors that takes part in the definition of metabolic syndrome. (^{1, 2}) It has been shown that altered renal excretion of sodium has vital importance in the pathogenesis of hypertension. (³) Renal dopamine and the atrial natriuretic peptide (ANP) are two natriuretic systems capable of modifying renal sodium management through the regulation of various sodium ion transporters in tubular cells. (^{3, 4}) Among these cells, proximal tubular cells are able to synthesize dopamine from the uptake and subsequent decarboxylation of the L-dopa precursor by dopa decarboxylase. (⁵) Under basal conditions, more than 50% of the renal excretion of sodium is regulated by the activation of dopamine- mediated D1 receptors. (⁶) On the other hand, ANP exerts its natriuretic action by activating the A-type natriu-retic peptide receptor (NPRA) in the kidneys. (⁷) The atrial natriuretic peptide can also bind to the C-type natriuretic peptide receptor (NPRC), which is consid-ered a clearing receptor that mediates the degradation of this peptide. (8) During the last decade, numerous studies have suggested the possible existence of an in-teraction between natriuretic peptides and the renal dopaminergic system. (9, 10). In this sense, it has been reported that renal dopamine and ANP can achieve their natriuretic effects through a common pathway that involves the reversible deactivation of renal Na+/ K+-ATPase, an enzyme whose alteration is closely re-lated to saline retention. (11, 12)

On the other hand, it is estimated that about 30-50% of hypertensive patients have insulin resistance. (1, 13) Several studies have established an association between insulin resistance and alterations in the renal dopaminergic system in the genesis of hypertension. (14) It has been demonstrated that rats with insulin-resistance due to fructose overload (FO) have lower urinary excretion of dopamine, which inversely cor-relates with blood pressure levels. (15) Likewise, sev-eral studies have confirmed a correlation between low levels of ANP and high levels of glucose and insulin, as well as of cholesterol and plasma triglycerides. (16, 17) However, there is little evidence about the interac-tion between renal dopamine and ANP in the physi-opathology of hypertension induced by a high fructose diet. Moreover, the kidney is one of the target organs of hypertension, and microalbuminuria is currently an early indicator of nephropathy and a predictor of ischemic heart disease in essential hypertension. (18) In this context, the association between metabolic syndrome, insulin resistance and renal damage has been demonstrated. (19, 20)

The aim of this work was to determine whether alterations in the natriuretic systems of ANP and renal dopamine in insulin-resistant rats due to high fructose diet are associated with changes in systolic blood pressure and renal tubular function, increased activity or expression of renal Na+/K+-ATPase and the presence of microalbuminuria as a marker of renal structural damage.

METHODS experimental animals

Male Sprague-Dawley rats weighing 150 to 180 g were used, with 12:12 h light/dark cycles, at 22ºC, and fed with a bal-anced diet for rodents.

Systolic blood pressure assessment

The animals were trained three times a week in the proce-dure of blood pressure assessment. Systolic blood pressure (SBP) was determined before beginning the study and at the end of each experimental period through indirect ple-thysmographic method by means of a sphygmomanometer in the rat tail, using an inflatable cuff and a microphone connected to a Grass DC amplifier (model 7DAC, Grass In-struments Co.) coupled to a polygraph (model 79D, Grass Instruments Co.).

Collection and processing of urine and blood samples

At the end of each experimental period, 24-hour urine sam-ples were collected from the animals housed in metabolic cages, to determine 24-hour urine output and the urinary excretion of sodium, creatinine, albumin, L-dopa and dopa-mine. Before sacrifice and under intraperitoneal anesthesia with ketamine (80 mg/kg) and xylazine (12 mg/kg), blood samples were collected from the retroocular plexus and centrifuged at 2700 rpm for 20 minutes at 4°C. Plasma and urinary concentrations of sodium, creatinine and albumin were determined in an automated autoanalyzer (Spectrum CCX automated analyzer, diagnostic Abbott) by spectropho-tometric method. Plasma insulin levels were determined by ELISA (Millipore Corporation) and plasma levels of ANP by ELISA (R & D Systems), after extraction by reverse phase chromatography with Sep-Pak C18 cartridges, according to the purification methodology of Sarda et al. (21)

l-dopa and dopamine concentration assessment

Assessment of the urinary concentration of L-dopa and do-pamine was performed by stabilization with 1 M acetic acid (pH 6.10), recovery in alumina and separation by reversed-phase high-performance liquid chromatography (RP-HPLC) using 4.6 mm × 25 cm columns (Beckman Instruments). Catecholamines were quantified by amperometric detection with 0.65 V Bioanalytical Systems electrode versus Ag/Ag reference electrodes.

renal function parameter assessment

Plasma and urinary sodium and creatinine values were used to assess renal function parameters. Glomerular filtration rate was estimated by creatinine clearance and tubular function by urinary sodium excretion.

renal tissue collection and processing

Immediately after sacrifice, both kidneys were removed from each animal and decapsulated. Sections of both kidneys were obtained and stored at -80ºC for western blot analyses and specific activity of Na+/K+-ATPase.

Protein expression analysis by western blot technique

Protein expression was semiquantitatively analyzed using western blot technique, as previously described. (22) Renal expression of Na+/K+-ATPase was determined using anti-Na+/K+-ATPase antibody (Amersham, dilution 1: 8000), renal expression of NPRA, using anti-NPRA antibody (Santa Cruz Biotechnology, dilution 1: 7500), and expression of NPRC, using anti-NPRC antibody (Santa Cruz Biotechnol-ogy, dilution 1: 400). Biotinylated IgG anti-rabbit antibody (GE Healthcare Life Sciences, dilution 1: 2000) and strepta-vidin conjugated with radish peroxidase (GE Healthcare Life Sciences, dilution of 1: 2000) were used for secondary and tertiary reactions, respectively. The samples were revealed by chemiluminescence using ECL reagent (Amersham Phar-macia Biotech). The density of the bands was quantified with Image J software (RSB). Anti-GAPDH antibody was used as internal standard loading control (Santa Cruz Bio-technology, dilution 1: 1500).

na+/K+-aTPase specifc activity analysis

Fifty mg samples of renal cortex were homogenized (1:10 weight/volume) in a solution containing 25 mM imidazole/1 mM EDTA/0.25 M sucrose and centrifuged at 4700 g at 4°C for 15 minutes. ATPase activity was measured by colorimet-ric determination of the released orthophosphate and oua-bain was used to specifically inhibit the Na+/K+-ATPase ac-tivity according to Fiske-Subarrow’s method, as previously described. (22)

Statistical analysis

The data were processed with the GraphPad Prism pro-gram, version 2.0. Gaussian distribution was evaluated by the Kolmogorov and Smirnov method and ANOVA followed by Newman-Keuls or Tukey tests was used to compare be-tween the groups, as appropriate. Results were expressed as

mean±SEM. Results with p<0.05 were considered statisti-cally significant.

ethical considerations

All the experiments were carried out in accordance with the ethical norms of international regulations and the principles of care and use of experimental animals (protocol approved by CICUAL with Res. CD 2100-15, EXP-UBA: 0035638/15). At the beginning of the study rats were randomly assigned to 2 groups: a control group (C) with ad libitum tap water and a fructose group (F) with fructose (at 10% W/V) in the ad libitum drinking water for 4, 8 and 12 weeks (n=8 rats per group and period).

RESULTS

Metabolic, hemodynamic and renal function parameters

Plasma insulin values were significantly increased by the high fructose diet compared with control rats since week 4 of treatment, indicating presence of hy-perinsulinemia . Similarly, SBP values were significantly increased in the rats with high fructose diet compared with control rats since week 4 of treatment (). In the kidneys, fructose overload in the diet was associated with a significant increase in diuresis, as well as a reduction in sodium urinary excretion compared with control rats since week 4 of treatment ). Creatinine clearance was not sig-nificantly modified by fructose treatment in any experimental period. The presence of microalbuminuria as a marker of renal damage was observed in week 12 of fructose treatment .

renal dopaminergic system

A significant reduction in urinary dopamine excretion together with an increase of L-dopa excretion was ob-served in rats with high fructose diet compared with the control groups since week 4 of treatment. These changes caused a significant increase in the L-dopa/ urinary dopamine ratio since the same week . A positive correlation was found between SBP and L-dopa/urinary dopamine ratio during the 12-week study period (R2=0.7816, p=0.002).

Specifc activity and expression of renal na+/K+-aTPase

The specific activity of renal Na+/K+-ATPase was significantly increased since week 4 of fructose treat-ment with respect to control rats (Table 1). The in-crease in activity was accompanied by an increase in protein expression by western blot (Figure 1).

anP system

A significant decrease in plasma ANP concentration was observed in rats with high fructose diet at weeks 8 and 12 of treatment (Figure 2). In the kidneys, a decrease in NPRA expression was found since week 8 (Figure 3) together with an increase in NPRC expres-sion since week 4 of treatment (Figure 4).

DISCUSSION

Metabolic syndrome is an entity that comprises a set

Fructose (10% w/v) was administered Fructose; SBP: Systolic blood pressure; in the ad libitum drinking water. Values are indicated as mean ± SEM CrCl: Creatinine clearance, UNaV: Urinary sodium excretion. * p <0.05

Fructose (10% w/v) was administered in the ad libitum drinking water. Values are indicated as mean ± SEM (n=8 animals per group). C: Control; F: Fructose. * p <0.05 vs. C group.

Protein expression of renal Na+/K+-ATPase in control and high fructose diet rats at 4, 8 and 12 weeks of treatment.

of disorders or metabolic abnormalities considered risk factors for the development of diabetes and cardiovascular disease. (1) It has been shown that rats fed a high fructose diet exhibit a large number of metabolic syndrome characteristics, such as insulin resistance, hyperinsulinemia and high blood pressure. (23) In our experiments, rats with a high fructose diet pre-sented hyperinsulinemia, indicating the existence of insulin-resistance. The increase in SBP was detected after week 4 of fructose treatment and reached the highest blood pressure values at 8 and 12 weeks, with

levels close to 160 mmHg. Systolic blood pressure lev-els were associated with a decrease in urinary sodium excretion since the same week, reflecting the state of saline retention in this experimental model. Although the exact mechanism by which hypertension develops in insulin-resistance conditions is still unknown, some studies suggest that hyperinsulinemia could cause so-dium retention and an increase in sympathetic activ-ity, key factors in the development of this disease. (24) Other authors suggest that the increase in renal re-absorption of sodium induced by insulin is enhanced

Fructose as mean peptide.

(10% w/v) was administered in the ad libitum drinking water. Valúes are indicated ± SEM (n=8 animáis per group). C: Control; F: Fructose; ANP: Atrial natriuretic * p <0.05 vs. C group.

. A-type natriuretic peptide receptor protein expression in the kidneys of control and high fructose diet rats at 4, 8 and 12 weeks of treatment.

Fructose (10% w/v) was administered in the ad libitum drinking water. Valúes are indicated as mean ± SEM (n=8 animáis per group). C: Control; F: Fructose; NPRA: A-type natriuretic peptide receptor. * p <0.05 vs. C group.

in insulin resistance. Insulin stimulates all sodium transporters located in the proximal tubules, among them the Na+/K+-ATPase pump, the type 3 Na+/H+ exchanger (NHE3), and the electrogenic basolateral cotransporter Na+/HCO3-(NBCe1). (24) In our mod-el, an increase in the specific activity of Na+/K+-AT-Pase enzyme was observed since week 4 of treatment,

accompanied by an increase in its protein expression by western blot. These data are supported by other studies showing that Na+/K+-ATPase is stimulated in insulin-resistance conditions and chronic exposure of renal cells to increased plasma insulin, which leads to greater sodium and water reabsorption, and, there-fore, greater saline retention. (25)

Fig. 4. C-type natriuretic peptide receptor protein expression in the kidneys of control and high fructose diet rats at 4, 8 and 12 weeks of treatment. A high fructose diet was associated with altera-tions in the natriuretic systems of renal dopamine and ANP. Regarding the renal dopaminergic system, a progressive increase in urinary L-dopa/ dopamine ratio was observed after week 4 of treatment, at the expense of an increase in the urinary excretion of L-dopa and a reduction of dopamine excretion. These changes would indicate the presence of reduced tubular uptake of L-dopa, a deficit of L-dopa conversion into dopamine in the tubular cells or a modification in the tubular release of the amine. In this sense, it has been shown that insulin stimulates the process of tubular L-dopa uptake and that this effect is abolished in rat models with insulin resistance due to fructose overload. (26) In another model of obesity-induced hyperinsulinemia, the presence of insulin resistance together with an increase in sympathetic tone was associated with lower renal dopaminergic activity. (²⁷) The increase in urinary L-dopa/ dopamine ratio showed a positive correlation with the increase in SBP, thus being an adequate parameter that accompanies the increase in blood pressure levels in this model. Conversely, the ANP plasma concentration was sig-nificantly reduced since week 8 in rats with fructose overload, accompanied by a decrease in NPRA receptor expression. These results are consistent with the literature, where an association between low ANP levels, reduction in NPRA expression and states of in-sulin-resistance has been demonstrated. (16, 17, 28) A possible explanation for the reduction in plasma ANP

concentration would be an increase in its degradation by NPRC receptors. In our study, NPRC expression was increased in the kidneys. In models of obesity and insulin resistance, the increased levels of NPRC described in adipose tissue would be responsible for the reduction of natriuretic peptide plasma levels in these conditions. (28) Likewise, the alteration of both natriuretic systems preceded in time the presence of microalbuminuria as a marker of renal structural damage in week 12; therefore, it can be postulated that the alteration of these systems would be associ-ated with damage in this organ. Regarding this point, it has been described that renal dopamine regulates the inflammatory and oxidative condition through its D2 receptors, with an increase in proinflammatory and oxidative stress markers in models with receptor deficiency. (29) Additionally, it has been shown that ANP has antioxidant effects in rats by attenuating the levels of reactive oxygen species in a model of renal damage by ischemia-reperfusion. (30)

Our laboratory has shown that ANP stimulates dopamine uptake by tubular kidney cells, through the stimulation of NPRA receptors and protein kinase G (PKG) activation. (11, 12, 22) Moreover, it has been shown that ANP increases the synthesis of renal dopamine by stimulating the activity of dopa decar-boxylase. (31) In this insulin-resistance model, and as a result of low levels of plasma ANP, a reduction in both synthesis and uptake of dopamine by tubular cells would be expected as a consequence of reduced NPRA receptor expression in the kidneys. These ef-fects would explain the decrease in the urinary excre-tion of dopamine and, therefore, the increase in the L-dopa/dopamine ratio observed in our experiments.

The results of the present work provide new evi-dence of the interaction between ANP and renal do-pamine systems, so an association can be established between the reduction in urinary excretion of dopamine observed in this model of fructose overload in the diet and a depression in the ANP system. Similarly, it can be postulated that the alteration of both systems would be associated with the development of renal damage, taking into account that this preceded the appearance of renal structural injury evidenced by microalbuminuria at week 12 of fructose treatment.

Conficts of interest

None declared. (See authors’ conflicts of interest forms on the website/Supplementary material).