Published On: Mon, Apr 1st, 2019

Effect of intracerebroventricular epinephrine microinjection on blood pressure and urinary sodium handling in gestational protein-restricted male adult rat offspring [RESEARCH ARTICLE]


The interaction between environmental and genetic factors interfere in ontogenic development, leading to morphofunctional disorders in tissues and organs in adulthood. Gestational protein restriction is followed by low birthweight in rats which in turn, leads to gender-related changes in blood pressure, kidney function, glucose metabolism and anxiety-like behaviors in male compared to female offspring. Sex hormones contribute to a sexual phenotype dimorphism in the fetal programming model of adult disease by modulating regulatory pathways critical in the long-term control of neural, cardiovascular and metabolic functions (Ashton, 2000; Barker, 1998; Mesquita et al., 2010a,b; Dasinger et al., 2016; Vaccari et al., 2015; Sene et al., 2013; Custódio et al., 2017; Menegon et al., 2008; Kwong et al., 2000; Ozaki et al., 2001; Gillette et al., 2017; Torres et al., 2018). Thus, this study was conducted only in male rats to ward off interference from gender differences. The current study confirms a reduced birth weight of rats whose mothers were fed a gestational restricted-protein diet compared to an NP intake (Mesquita et al., 2010a,b; Lopes et al., 2013). However, beyond the fourth week of age, body mass in both groups was the same, a phenomenon known as catch-up growth. This effect was associated with a significant enhancement in arterial blood pressure in the LP group. The present investigation also confirmed a pronounced decrease in fractional urinary sodium excretion in maternal protein-restricted offspring (Fig. 3). The decreased FENa observed in LP offspring compared with the age-matched NP group was accompanied by reduced post-proximal tubule sodium rejection, although the creatinine clearance was unchanged and sodium was usually filtered (3,4,41). The decreased renal potassium excretion verified in LP offspring suggests that tubular sodium reabsorption in LP offspring occurs before the distal nephron segment.

The precise mechanism underlying the chronic arterial hypertension in offspring induced by maternal LP has not been identified. Arterial pressure is thought to be controlled by the renal-mediated regulation of fluid and electrolytes. A prior report from our lab has shown higher renal and plasma catecholamine levels in LP offspring when compared to age-matched NP rats (Custódio et al., 2017). Also, as demonstrated in the previous study (Custódio et al., 2017), the bilateral renal denervation reduced kidney catecholamine concentrations in both NP and LP groups, though the decreased arterial blood pressure was observed only in growth-restricted offspring relative to renal denervated control rats. The enhanced urinary sodium excretion in kidney-denervated LP offspring suggests an indirect but close relationship between enhanced renal nerve activity and attenuated sodium excretion in the development of hypertension in LP offspring. The decreased FENa+ in LP offspring may result from the interactions of a variety of mechanisms, such as renal arteriolar postglomerular vasoconstriction, renal sympathetic nervous system overexcitability and, by direct tubule transport effects; our previous study has demonstrated increased activity of the Na+/K+-ATPase pump in the basolateral membrane in LP rats (Mesquita et al., 2010a,b).

On the other hand, the adult kidney comprises several filtering units; in some species, total numbers of nephrons are determined before birth (Mesquita et al., 2010a; Hall et al., 2012). In rodents, the permanent metanephric kidney is very immature at birth and in rats about 20% of the total nephron number is present at birth. There is evidence that any insult, including maternal undernutrition offspring, alters the total number of nephrons and also causes late-onset hypertension (Mesquita et al., 2010a,b; Pinhal et al., 2013). However, in the present study, it does not seem that merely a reduced nephron number is responsible for the increased blood pressure, since we did not observe any significant difference between LP and NP glomerular filtration rate. Additionally, these findings are reiterated by data showing that impaired pelvic neurokinin expression associated with responsiveness of renal sensory receptors in 16-week-old LP offspring are conducive to excess renal reabsorption of sodium and development of hypertension in this programmed model (Custódio et al., 2017). Otherwise, investigators have demonstrated that administration of adrenergic agonists into different cerebral sites elicits a substantial increase in renal sodium excretion accompanied by decreasing arterial pressure (Gontijo et al., 1992; Pillar et al., 1977; Camargo et al., 1976; Saad et al., 1976; Lutaif et al., 2015). Thus, we hypothesized that enhanced blood pressure in maternal LP offspring could be associated, at least in part, with changes in renal neural control and reduced urinary sodium excretion that which may relate to imbalanced central adrenergic receptor modulation. Here we evaluated the effect of cerebro-LV administration of adrenergic agonists and/or antagonists on blood pressure and urinary sodium handling in 16-week-old LP offspring compared with appropriate age-matched NP controls in a concentration-dependent fashion. Of particular interest, we have confirmed results from different stimulation techniques (Gontijo et al., 1992; Pillar et al., 1977; Camargo et al., 1976; Saad et al., 1976; Lutaif et al., 2015; Kapusta et al., 1989; Koepke et al., 1988, 1987). This study reveals a rapid, transient but significant, blood pressure decrease after LV microinjection of Epi; this effect was, in turn, attenuated by α1-adrenoceptor antagonist and unchanged by α2-receptor antagonist ICV microinjections in NP rats. Surprisingly, only in LP offspring, the yohimbine LV microinjections, followed by Epi administration, cause a significant reduction in basal blood pressure, suggesting the centrally α1-adrenergic receptor participation in that pressure response.

Additionally, LV Epi microinjections, in a dose-dependent fashion, promoted an increase in urinary sodium and potassium excretion over 120 min in NP rats. Conversely, the natriuretic but not the pressure response to Epi microinjections into LV were significantly blunted in age-matched LP offspring. These findings confirm the participation of the CNS α1 and α2-adrenergic receptors in the regulation of renal sodium and potassium excretion. The increased natriuresis and kaliuresis response to LV Epi microinjections in NP rats were significantly attenuated by previous local injection of prazosin, an α1-adrenergic antagonist. Note that LV microinjections of prazosin inhibited fractional sodium excretion induced by Epi in NP and, to a lesser extent, in LP rats, whereas the current findings demonstrate that LV pre-injection of yohimbine, an α2-adrenergic antagonist, synergically potentiates and normalizes the action of ICV Epi administration on renal sodium excretion in age-matched LP offspring. Thus, this study confirms that Epi, when centrally microinjected in conscious rats, leads to a very predictable and reproducible natriuretic response accompanied by unchanged glomerular filtration rate and can be associated with an increased ion delivery from the proximal tubule, incompletely compensated by more distal nephron segments. This effect demonstrates diminished Epi graded-fashion responses with a rightward shift of the dose-response curve, providing evidence of downregulation of target organ responsiveness to LP cerebroventricular stimuli. Despite repeated demonstration of the natriuretic effect of central Epi administration to a variety of species, to the best of our knowledge, there has been no previous description of these effects among LP offspring.

However, the precise mechanism of this phenomenon remains unclear. Several possibilities could be considered to explain the natriuretic response in this study. First, the CNS directly affects renal sodium excretion via neural routes. Second, nephron hemodynamic changes are responsible for alteration of tubule electrolyte handling. Third, the natriuresis results from fluctuations in the level of presumable neural-borne factors which disrupt sodium and water transporters function in renal tubules. Fourth, the attenuated central response in LP relative age-matched NP offspring can supposedly be explained by a definite lack of control between centrally adrenergic and/or receptors activity that may blunt the peripheral kidney ion and salt excretion responses.

There is evidence of the importance of renal sympathetic nerve activity in the pathogenesis of experimental models of hypertension (DiBona, 2000; Johns et al., 2011; Boer et al., 2005). We previously demonstrated that the urinary sodium excretion response to central administration of insulin, angiotensin II, hypertonic saline and cholinergic and noradrenergic agonists were strikingly and similarly attenuated in different models of hypertensive rats when compared with age-matched normotensive controls (Custódio et al., 2017; Lutaif et al., 2015; Menegon et al., 2008; Guadagnini and Gontijo, 2006; Andersson et al., 1969). Thus, the significant reduced natriuretic response in LP compared to NP rats may reflect a hyperactive state in the peripheral sympathetic nervous system, including in the kidneys, at least in part caused by reduced sensory (afferent renal nerve activity) renal activity in LP offspring in adult life (Custódio et al., 2017; Boer et al., 2005; Oparil et al., 1987; Beierwaltes et al., 1982). This dysfunctional response in LP offspring could be essential to the development and maintenance of hypertension in LP offspring. In this way, it is well known that α2-adrenoceptors brainstem stimulation in the conscious rats causes a decrease in blood pressure and enhanced urinary sodium excretion. These effects are selectively mediated by downstream Gαi2, but not Gαi1, Gαi3, Gαo, or Gαs subunit GTP-binding regulatory protein signal transduction pathways (Kapusta et al., 1989, 2012; Koepke et al., 1988, 1987; Wainford and Kapusta, 2012). Studies revealed that the brain Gαi2 protein-mediated sympathetic inhibitory renal nerve-dependent path is of critical importance in the central neural mechanisms activated to maintain fluid and electrolyte homeostasis. The underlying mechanisms by which brain Gαi2-subunit protein-gated pathways induce α2-adrenoreceptor-evoked sodium and blood pressure control in vivo are unclear. Here, given the intimate association between fluid and electrolyte homeostasis and the long-term control of arterial pressure, we may speculate that downregulation of brain Gαi2 protein expression in LP offspring may lead to high kidney sympathetic drive, renal sodium retention and the development of renal nerve-dependent hypertension, effects partially disrupted by yohimbine LV microinjection. Our experiments furnished good evidence of the existence of a central adrenergic control mechanism consisting of α1 and α2 receptor signals, which work reciprocally on the regulation of blood pressure and renal sodium excretion. Speculatively, we may suppose that stimulation of CNS α2-adrenergic receptors by Gαi2 subunit GTP-binding regulatory protein, may prevent basal increased renal sympathetic overexcitability in conscious LP rats based on two main findings. First, the effect of LV administration of Epi on natriuresis is significantly attenuated in gestational protein-restricted offspring. Second, pretreatment with α2-adrenergic receptor antagonists reversed the impact of the LV Epi injection, which demonstrates that central α2-adrenergic receptors are involved in the diminished natriuresis observed for the LP lineage (Kapusta et al., 1989; Koepke et al., 1988, 1987). However, we cannot discount the possibility that LP neural synapses have more α2-adrenergic receptors than those of NP offspring. It is more likely that natriuresis is a result of reduced renal sympathetic nerve activity and a consequent decrease in renal tubular reabsorption of sodium, and a simultaneous consequent reduction in the blood pressure in LP offspring. Because adrenergic agonist or antagonists did not alter the glomerular filtration rate, changes in glomerular dynamics do not explain the natriuresis. Catecholamines administration into several CNS places in conscious rats increases urinary sodium excretion; the natriuresis is prevented by central α1-adrenergic receptor blockade and potentiated by α2-adrenergic receptor blockade (Gontijo et al., 1991, 1990; Kapusta et al., 1989; Koepke et al., 1988, 1987). Taking into account the current and previous studies, we may suggest an inhibitory effect of central α2-adrenergic receptors mediated, at least in part, by unbalanced downstream subunit GTP-binding regulatory protein on urinary sodium excretion and an excitatory effect of central α1-adrenoceptors.

In conclusion, our results suggest the striking participation of central adrenergic receptors in the renal pathogenesis of elevated blood pressure in LP offspring. Although the precise mechanism of the different natriuretic response of NP and LP rats is still uncertain, these results lead us to speculate that inappropriate neural adrenergic pathways may have significant effects on tubule sodium transport, resulting in the inability of the kidneys to control hydrosaline balance and, consequently, an increase in blood pressure.

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