Involvement of ENaC in the development of salt-sensitive hypertension

Salt-sensitive hypertension is associated with renal and vascular dysfunctions, which lead to impaired fluid excretion, increased cardiac output, and total peripheral resistance. It is commonly accepted that increased renal sodium handling and plasma volume expansion are necessary factors for the development of salt-induced hypertension. The epithelial sodium channel (ENaC) is a trimeric ion channel expressed in the distal nephron that plays a critical role in the regulation of sodium reabsorption in both normal and pathological conditions. In this mini-review, we summarize recent studies investigating the role of ENaC in the development of salt-sensitive hypertension. On the basis of experimental data obtained from the Dahl salt-sensitive rats, we and others have demonstrated that abnormal ENaC activation in response to a dietary NaCl load contributes to the development of high blood pressure in this model. The role of different humoral factors, such as the components of the renin-angiotensin-aldosterone system, members of the epidermal growth factors family, arginine vasopressin, and oxidative stress mediating the effects of dietary salt on ENaC are discussed in this review to highlight future research directions and to determine potential molecular targets for drug development.

Loss of endothelin B receptor function impairs sodium excretion in a time- and sex-dependent manner

Recent studies suggested a direct link between circadian rhythms and regulation of sodium excretion. Endothelin-1 (ET-1) regulates sodium balance by promoting natriuresis through the endothelin B receptor (ETB) in response to increased salt in the diet, but the effect that the time of day has on this natriuretic response is not known. Therefore, this study was designed to test the hypothesis that ETB receptor activation contributes to the diurnal control of sodium excretion and that sex differences contribute to this control as well. Twelve-hour urine collections were used to measure sodium excretion. On day 3 of the experiment, a NaCl load (900 μeq) was given by oral gavage either at Zeitgeber time [ZT] 0 (inactive period) or ZT12 (active period) to examine the natriuretic response to the acute salt load. Male and female ETB-deficient (ETB def) rats showed an impaired natriuretic response to a salt load at ZT0 compared with their respective transgenic controls (Tg cont). Male ETB def rats showed a delayed natriuretic response to a salt load given at ZT12 compared with male Tg cont, a contrast to the prompt response shown by female ETB def rats. Treatment with ABT-627, an ETA receptor antagonist, improved the natriuretic response seen within the first 12 h of a ZT0 salt load in both sexes. These findings demonstrate that diurnal excretion of an acute salt load 1) requires ET-1 and the ETB receptor, 2) is more evident in male vs. female rats, and 3) is opposed by the ETA receptor.

Abstract 095: Ovariectomy Restores Purinergic Receptor Activation of Endothelin-dependent Natriuresis

We have recently reported interplay between renal medullary endothelin-1 (ET-1) and purinergic (P2) systems, which play central roles in controlling Na + homeostasis in male rats. Evidence suggests that sex hormones regulate ET-1 and P2 systems. To test whether activation of the renal medullary P2 receptors promotes ET-dependent natriuresis in females and whether ovariectomy (OVX) modulates this potential interaction, we studied the effect of medullary NaCl loading on Na + excretion in adult intact female and OVX SD rats in the presence and absence of P2 or ET receptor antagonism. Isosmotic saline (284 mOsmol/kg H 2 O) was infused into the renal medullary interstitium during a baseline urine collection period, followed by isosmotic or hyperosmotic saline (1800 mOsmol/kg H 2 O) infusion. Blood pressure, renal blood flow, urine Na + , K + and osmolality were measured. Medullary NaCl loading significantly enhanced Na + excretion in intact females and OVX (from 0.8±0.2 to 6.2±1.6 and from 0.7±0.1 to 5.6±0.8 μmol/min, respectively, n=6-8, p<0.05). The natriuretic effect of NaCl loading in intact females was not attenuated by P2 or ET receptor blockade. Whereas, intramedullary infusion of the P2 receptor antagonist, suramin, inhibited the natriuresis induced by medullary NaCl loading in OVX (from 0.4±0.2 to 0.9±0.4 μmol/min, n=6). Additionally, combined ET A/B receptor blockade (ABT-627 + A-192621) abolished the natriuretic response to medullary NaCl load in OVX rats (from 0.2±0.1 to 1.4±0.7 μmol/min, n=4). Activation of medullary purinergic (P2Y 2/4 ) receptors by UTP infusion had no significant effect in intact females, but enhanced Na + excretion in OVX rats (from 0.5±0.1 to 2.3±0.8 μmol/min, n=5, p<0.05). Combined ET A/B receptor blockade significantly inhibited the natriuretic response to UTP observed in OVX rats. These data suggest that increased medullary NaCl loading induces ET-independent and P2-independent natriuresis in intact females. In OVX, activation of medullary P2 receptors promotes ET-dependent natriuresis, similar to our previous findings in male rats and suggests that OVX restores the interplay between the renal ET-1 and purinergic (P2) signaling systems to facilitate Na + excretion. Funded by AHA 15POST25090329 to EYG and P01 HL95499 to DMP

Angiotensin II-induced hypertension blunts thick ascending limb NO production by reducing NO synthase 3 expression and enhancing threonine 495 phosphorylation

Thick ascending limbs reabsorb 30% of the filtered NaCl load. Nitric oxide (NO) produced by NO synthase 3 (NOS3) inhibits NaCl transport by this segment. In contrast, chronic angiotensin II (ANG II) infusion increases net thick ascending limb transport. NOS3 activity is regulated by changes in expression and phosphorylation at threonine 495 (T495) and serine 1177 (S1177), inhibitory and stimulatory sites, respectively. We hypothesized that NO production by thick ascending limbs is impaired by chronic ANG II infusion, due to reduced NOS3 expression, increased phosphorylation of T495, and decreased phosphorylation of S1177. Rats were infused with 200 ng·kg−1·min−1ANG II or vehicle for 1 and 5 days. ANG II infusion for 5 days decreased NOS3 expression by 40 ± 12% ( P < 0.007; n = 6) and increased T495 phosphorylation by 147 ± 26% ( P < 0.008; n = 6). One-day ANG II infusion had no significant effect. NO production in response to endothelin-1 was blunted in thick ascending limbs from ANG II-infused animals [ANG II −0.01 ± 0.06 arbitrary fluorescence units (AFU)/min vs. 0.17 ± 0.02 AFU/min in controls; P < 0.01]. This was not due to reduced endothelin-1 receptor expression. Phosphatidylinositol 3,4,5-triphosphate (PIP3)-induced NO production was also reduced in ANG II-infused rats (ANG II −0.07 ± 0.06 vs. 0.13 ± 0.04 AFU/min in controls; P < 0.03), and this correlated with an impaired ability of PIP3 to increase S1177 phosphorylation. We conclude that in ANG II-induced hypertension NO production by thick ascending limbs is impaired due to decreased NOS3 expression and altered phosphorylation.

Physiology and pathophysiology of the renal Na-K-2Cl cotransporter (NKCC2)

The Na-K-2Cl cotransporter (NKCC2; BSC1) is located in the apical membrane of the epithelial cells of the thick ascending limb of the loop of Henle (TAL). NKCC2 facilitates ∼20–25% of the reuptake of the total filtered NaCl load. NKCC2 is therefore one of the transport proteins with the highest overall reabsorptive capacity in the kidney. Consequently, even subtle changes in NKCC2 transport activity considerably alter the renal reabsorptive capacity for NaCl and eventually lead to perturbations of the salt and water homoeostasis. In addition to facilitating the bulk reabsorption of NaCl in the TAL, NKCC2 transport activity in the macula densa cells of the TAL constitutes the initial step of the tubular-vascular communication within the juxtaglomerular apparatus (JGA); this communications allows the TAL to modulate the preglomerular resistance of the afferent arteriole and the renin secretion from the granular cells of the JGA. This review provides an overview of our current knowledge with respect to the general functions of NKCC2, the modulation of its transport activity by different regulatory mechanisms, and new developments in the pathophysiology of NKCC2-dependent renal NaCl transport.

Angiotensin II-induced hypertension increases plasma membrane Na pump activity by enhancing Na entry in rat thick ascending limbs

Thick ascending limbs (TAL) reabsorb 30% of the filtered NaCl load. Na enters the cells via apical Na-K-2Cl cotransporters and Na/H exchangers and exits via basolateral Na pumps. Chronic angiotensin II (ANG II) infusion increases net TAL Na transport and Na apical entry; however, little is known about its effects on the basolateral Na pump. We hypothesized that in rat TALs Na pump activity is enhanced by ANG II-infusion, a model of ANG II-induced hypertension. Rats were infused with 200 ng·kg−1·min−1 ANG II or vehicle for 7 days, and TAL suspensions were obtained. We studied plasma membrane Na pump activity by measuring changes in 1) intracellular Na (Nai) induced by ouabain; and 2) ouabain-sensitive oxygen consumption (QO2). We found that the ouabain-sensitive rise in Nai in TALs from ANG II-infused rats was 12.8 ± 0.4 arbitrary fluorescent units (AFU)·mg−1·min−1 compared with only 9.9 ± 1.1 AFU·mg−1·min−1 in controls ( P < 0.024). Ouabain-sensitive oxygen consumption was 17 ± 5% ( P < 0.043) greater in tubules from ANG II-treated than vehicle rats. ANG II infusion did not alter total Na pump expression, the number of Na pumps in the plasma membrane, or the affinity for Na. When furosemide (1.1 mg·kg−1·day−1) was coinfused with ANG II, no increase in plasma membrane Na pump activity was observed. We concluded that in ANG II-induced hypertension Na pump activity is increased in the plasma membrane of TALs and that this increase is caused by the chronically enhanced Na entry occurring in this model.

Regulation of the renal thiazide-sensitive Na-Cl cotransporter, blood pressure, and natriuresis in obese Zucker rats treated with rosiglitazone

Previously, we showed an increase in protein abundance of the renal thiazide-sensitive Na-Cl cotransporter (NCC) in young, prediabetic, obese Zucker rats relative to lean age mates (Bickel CA, Verbalis JF, Knepper MA, and Ecelbarger CA. Am J Physiol Renal Physiol 281: F639–F648, 2001). To test whether this increase correlated with increased thiazide sensitivity (NCC activity) and blood pressure, and could be modified by insulin-sensitizing agents, we treated lean and obese Zucker rats (9 wk old) with either a control diet or this diet supplemented with 3 mg/kg body wt rosiglitazone (RGZ), a peroxisomal proliferator-activated receptor subtype γ agonist and potent insulin-sensitizing agent, for 12 wk ( n = 9/group). The rise in blood pressure, measured continuously by radiotelemetry, was significantly blunted in the RGZ-treated obese rats. Similarly, blood glucose and urinary albumin were markedly decreased in these rats. RGZ-treated rats whether lean or obese excreted a NaCl load faster but excreted less sodium in response to hydrochlorothiazide, applied as a novel in vivo measure of NCC activity. Obese rats had increased renal protein abundance and urinary excretion of NCC; however, this was not significantly reduced by RGZ (densitometry in cortex homogenate − %lean control): 100 ± 9, 93 ± 4, 124 ± 9, and 141 ± 14 for lean control, lean RGZ, obese control, and obese RGZ, respectively. Subcellular localization, as evaluated by confocal microscopy and immunoblotting following differential centrifugation, of NCC was not different between rat groups. Overall, RGZ reduced blood pressure and thiazide sensitivity; however, the mechanism(s) did not seem to involve a decrease in NCC protein abundance or cellular location. Decreased NCC activity may have contributed to the maintenance of normotension in RGZ-treated obese rats.