Antinatriuretic Actions Research Articles

Proximal Tubule‐Specific Deletion of Angiotensin II Type 1a Receptors Augments Natriuretic Response to Atrial Natriuretic Peptide via NPRA/cGMP/NO Signaling Pathways in the Kidney

We and others have previously shown that the proximal tubules of the kidney expressed high levels of angiotensin II (Ang II) type 1 (AT1) receptors and atrial natriuretic peptide (ANP) type A receptors (NPRA). In the proximal tubules, Ang II binds and activate AT1 (AT1a) receptors to stimulate proximal tubule Na+reabsorption, whereas ANP binds and activates NPRA receptors to inhibit Ang II‐induced proximal tubule Na+reabsorption. Interactions between Ang II and ANP systems play important counteracting roles in the proximal tubules to control proximal tubule Na+ reabsorption in the kidney and maintain blood pressure homeostasis. It is not known, however, whether the hypotensive and natriuretic responses to ANP are altered by the absence of AT1 (AT1a) receptor‐mediated antinatriuretic action of Ang II in the proximal tubules. The present study used a novel mouse model with proximal tubule‐specific knockout of AT1a receptors in the kidney, PT‐Agtr1a−/−, to test the hypothesis that deletion of AT1a receptors selectively in the proximal tubules augments the hypotensive and natriuretic responses to ANP. Two groups (n=9‐12 per group) of adult male wild‐type and PT‐Agtr1a−/− mice were infused without (time controls) or with ANP via osmotic minipump (10 ng/min, i.p.) continuously for 2 weeks. Basal blood pressure was about 16 ± 3 mmHg lower (P<0.01), fractional proximal tubule Na+ reabsorption was lower (P<0.05), and 24 h urinary Na+ excretion was higher in PT‐Agtr1a−/− mice (P<0.01), compared with wild‐type mice. ANP infusion decreased blood pressure and increased the natriuretic response in PT‐Agtr1a−/− mice further by inhibiting proximal tubule Na+ reabsorption, compared with wild‐type controls (P<0.01). The augmented hypotensive and natriuretic responses to ANP infusion in PT‐Agtr1a−/− mice was associated with increased plasma and kidney cGMP levels (P<0.01), kidney NPRA and NPRC expression (P<0.05), total eNOS and phosphorylated eNOS proteins (P<0.01), and urinary NO excretion (P<0.01), respectively. Taken together, the results of the present study provide further new insights into the important physiological roles for intratubular Ang II/AT1a and ANP/NPRA in the proximal tubules to regulate proximal tubule Na+ reabsorption and help maintain blood pressure homeostasis.

The Role of the Mineralocorticoid Receptor and Mineralocorticoid Receptor-Directed Therapies in Heart Failure.

Mineralocorticoid receptor (MR) antagonists (MRA), also referred to as aldosterone blockers, are now well-recognized for their clinical benefit in patients who have heart failure (HF) with reduced ejection fraction (HFrEF). Recent studies have also shown MRA can improve outcomes in patients with HFpEF, where the ejection fraction is preserved but left ventricular filling is reduced. While the MR is a steroid hormone receptor best known for antinatriuretic actions on electrolyte homeostasis in the distal nephron, it is now established that the MR has many physiological and pathophysiological roles in the heart, vasculature, and other nonepithelial tissue types. It is the impact of MR activation on these tissues that underpins the use of MRA in cardiovascular disease, in particular HF. This mini-review will discuss the origins and the development of MRA and highlight how their use has evolved from the "potassium-sparing diuretics" spironolactone and canrenone over 60 years ago, to the more receptor-selective eplerenone and most recently the emergence of new nonsteroidal receptor antagonists esaxerenone and finerenone.

Drugs That Ameliorate Epicardial Adipose Tissue Inflammation May Have Discordant Effects in Heart Failure With a Preserved Ejection Fraction as Compared With a Reduced Ejection Fraction

Heart failure with a preserved ejection fraction (HFpEF) and heart failure with a reduced ejection fraction (HFrEF) have distinctive pathophysiologies, and thus, therapeutic approaches to the 2 disorders should differ. Neurohormonal activation drives the progression of HFrEF, and neurohormonal antagonists are highly effective in HFrEF, but not in HFpEF. Conversely, a broad range of chronic systemic inflammatory or metabolic disorders cause an expansion and inflammation of epicardial adipose tissue; the secretion of adipocytokines may lead to microvascular dysfunction and fibrosis of the underlying myocardium, which (if the left atrium is affected) may lead to atrial fibrillation (AF) and (if the left ventricle is affected) may lead to HFpEF. Anti-inflammatory drugs (such as statins and anticytokine agents) can ameliorate epicardial adipose tissue dysfunction. Statins appear to ameliorate the development of atrial myopathy (both experimentally and clinically), and in randomized controlled trials, they reduce the incidence of new-onset and recurrent AF and decrease the risk of heart failure with the features of HFpEF; yet, they have no benefits in HFrEF. Similarly, anticytokine agents appear to prevent heart failure in patients with or prone to HFpEF, but adversely affect HFrEF. Several antihyperglycemic agents also reduce epicardial fat mass and inflammation, but this benefit may be offset by additional actions to cause sodium retention and neurohormonal activation. Thiazolidinediones have favorable effects on experimental AF and HFpEF, but their antinatriuretic actions negate these benefits, and they worsen the clinical course of HFrEF. Glucagon-like peptide-1 receptor agonists also ameliorate AF and HFpEF in laboratory models, but their positive inotropic and chronotropic effects may be deleterious in HFrEF. By contrast, metformin and sodium-glucose cotransporter 2 inhibitors alleviate epicardial adipose tissue dysfunction and may reduce the risk of AF and HFpEF; yet, they may have additional actions to promote cardiomyocyte survival that are useful in HFrEF. The concordance of the benefits of anti-inflammatory and antihyperglycemic drugs on AF and HFpEF (but not on HFrEF) supports the paradigm that epicardial adipose tissue is a central pathogenetic mechanism and therapeutic target for both AF and HFpEF in patients with chronic systemic inflammatory or metabolic diseases.

Stress‐induced Salt Sensitivity is Modulated by Angiotensin II

IntroIncreased sensitivity of blood pressure (BP) to dietary sodium (“salt‐sensitivity”) has been described to partially explain the etiology of hypertension (HTN) in African‐Americans (AAs). Our group has demonstrated a form of salt sensitivity that is induced by mental stress rather than by dietary sodium in a significant percentage of AAs. The underlying mechanism for stress‐induced salt‐sensitivity in AAs has yet to be described. However, animal models provide evidence that it results from increased renal sympathetic nerve activity (SNA) that triggers renal sodium reabsorption through the action of angiotensin II (Ang II).AimOur study sought to test the hypothesis that mental stress‐induced impaired regulation of sodium and subsequent changes in BP are mediated by the action of Ang II in normotensive AAs.MethodsUtilizing a double‐blind, randomized and cross‐over study design, 132 normotensive AAs (25±7 yr) were placed on a controlled sodium diet for 7 days and were treated concomitantly with either a placebo or irbesartan (an Ang II receptor blocker; ARB) in random order. A mental stress by competitive video game challenges were used to provoke the stress response on the final day of each treatment. Urinary sodium excretion (UNaV), blood pressure (BP), total peripheral resistance (TPR) and heart rate (HR) were collected before, during, and 1‐hr after the stress.ResultsBP measured every 15 minutes stayed elevated throughout the stress period on placebo, whereas the stress‐induced increase in BP was delayed on ARB day (p=0.024 for Treatment × Time interaction). ARB treatment also resulted in lower BP and TPR at rest, stress and recovery (p>0.05 for all). Notably, systolic BP at the end of recovery on ARB was significantly lower compared to the placebo (109.8 ±13.3 vs 117.4 ± 12.3 mmHg respectively; p<0.001) and was also significantly decreased from the pre‐stress level (mean difference; −1.9 ±9.4 mmHg; p=0.02). In response to stress, UNaV, BP and HR were all increased in each of placebo and ARB day. However, 30.2% of subjects retained sodium during stress despite an increase in BP on placebo. ARB treatment increased ΔUNaVstress − rest among the sodium retainers (−1.14 ±5.95 mEq/min on placebo, 7.63±9.64 mEq/min on ARB, p=0.001) without changes in UNaV at rest and recovery.ConclusionThe use of ARBs in AAs is controversial despite an unsatisfactory efficacy of the current first line of HPT therapy in AAs. Our data demonstrates that suppression of Ang II, by using ARB, blunted the stress‐induced sodium retention, attenuated BP reactivity to stress and improved post‐stress BP recovery process in AAs. These confirm the Ang II‐dependent antinatriuretic actions in the process of stress responses on renal SNA. Thus, treatment approaches that antagonize Ang II's actions could have significant relevance to potentially lower susceptibility to stress‐induced dysregulation of sodium and BP and eventually the premature development of salt‐sensitive HTN in AAs.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Have we really demonstrated the cardiovascular safety of anti-hyperglycaemic drugs? Rethinking the concepts of macrovascular and microvascular disease in type 2 diabetes.

A primary goal of the treatment of type 2 mellitus is the prevention of morbidity and mortality associated with cardiovascular disease; however, anti-hyperglycaemic drugs have the capacity to cause deleterious effects on the circulation, a risk that is not adequately reflected by the endpoints selected for emphasis in large-scale clinical trials that are designed to evaluate cardiovascular safety. The primary endpoint of the large-scale studies mandated by regulatory authorities focuses only on 3 to 4 events that depict only a limited view of the circulatory system. One of the most serious adverse effects of many glucose-lowering drugs is new-onset or worsening heart failure. Most antidiabetic drugs can aggravate heart failure because they exert anti-natriuretic actions, and possibly, adverse effects on the myocardium. In addition, certain anti-hyperglycaemic agents may worsen peripheral vascular disease and trigger cardiac arrhythmias that may lead to sudden death. Initiation of treatment with antidiabetic medications may also cause deterioration of the function of the kidneys, retina and peripheral nerves, which are typically regarded as reflecting microvascular disease. The current confusion about the cardiovascular effects of glucose-lowering drugs may be exacerbated by conceptual uncertainties about the classification of large and small vessel disease in determining the clinical course of diabetes. Physicians should not be falsely reassured by claims that a new treatment appears to have passed a narrowly defined regulatory test. The management of people with diabetes often carries with it the risk of important cardiovascular consequences, even for drugs that do not overtly increase the risk of myocardial infarction or stroke.

Abstract 047: 20-SOLA, A Novel Water-soluble 20-HETE Antagonist, Elicits Natriuresis And Abrogates Hypertension In Cyp4a14 Knockout Mice.

20-HETE (20-hydroxyeicosatetraenoic acid) is a cytochrome P450 (CYP) 4A-derived arachidonate metabolite. In the vasculature, 20-HETE increases smooth muscle contraction, endothelial dysfunction/activation and sensitivity to constrictor stimuli; all of which contribute to hypertension. In renal tubules, 20-HETE inhibits ion transport and has pro-natriuretic functions in salt-sensitive models of hypertension. The pro-natriuretic effect of 20-HETE has not been reported in mouse models of 20-HETE-dependent hypertension. The CYP4a14 knockout (KO) male mice exhibit androgen-dependent upregulation of Cyp4a12, the sole 20-HETE synthase in mice, and display 20-HETE-dependent hypertension; female KO mice show no increase in Cyp412-20-HETE and are normotensive. We examined the effect of 20-SOLA (2,5,8,11,14,17-hexaoxanonadecan-19-yl-20-hydroxyeicosa-6(Z),15(Z)-dienoate), a water-soluble 20-HETE antagonist, and determined its effect on blood pressure (BP) and natriuresis in the KO mice. Administration of 20-SOLA (10mg/kg/day in drinking water) normalized BP in male KO mice; a significant reduction was observed on day 4 (146±1 vs153±2 mmHg in untreated littermates) reaching BP of the normotensive female KO mice (124±1 and 126±1 mmHg for treated and untreated, respectively) at day 10 of treatment (124±1 vs. 153±2 mmHg in untreated male KO; p<0.05). 20-SOLA decreased vascular reactivity to phenylephrine in male KO renal microvessels by 7-fold (EC50: 1.58±0.23 vs 0.22±0.03 μM). Moreover, treatment with 20-SOLA increased urine output in KO male mice as compared to untreated littermates (1.25±0.04 vs. 1.06±0.04 mL; p<0.05). The urine output to water intake ratio was significantly elevated in 20-SOLA-treated KO vs. untreated (0.25±0.01 vs. 0.19±0.01, p<0.05). Importantly, treatment with 20-SOLA increased urinary sodium excretion in the KO male mice (12.33±0.44 vs. 8.33±0.63 μmol/gr BW/24Hrs; p<0.05). These results suggest that 20-HETE has an anti-natriuretic effect in the kidneys of KO mice and that antagonism of 20-HETE’s action using 20-SOLA elicited natriuresis concurrent with reduction in blood pressure. The potential vascular and tubular mechanisms underlying 20-HETE anti-natriuretic action are being currently explored.

The renal functional response to Ang(1–7) infusion in DOCA‐salt hypertensive rats.

Angiotensin 1–7 (Ang1–7) has natriuretic properties at the kidney that counterbalance the anti‐natriuretic actions of angiotensin II. This study investigated whether the renal actions of Ang1–7 are deranged in Deoxycorticosterone (DOCA) salt hypertensive states where the renin‐angiotensin system (RAS) is suppressed. Groups of anaesthetised male Wistar rats (n=6) were prepared for renal interstitial infusion at 1ml/h of Ang 1–7 (3×10–9 M) into the left kidney and cannulation of its ureter. Data, means±SEM were subject to one‐way ANOVA. Ang 1–7 had no effect on blood pressure, at 118mmHg for control rats and 153mmHg for DOCA Hypertensive rats. In control rats, urine volune (UV), significantly increased from 100.9±6.9 to139.1±4.5μl/min, sodium excretion (UVNa), from 16.0±1.3 to 24.3±1.0μmol/min and fractional sodium excretion (FENa) by 2.3±0.5 to 2.9±0.5% (all P<0.05). Intra‐renal infusion of the same dose of Ang 1–7 in the DOCA model had no effect on the excretory variables. The reasons for the lack of response to the Ang (1–7) are uncertain but one possibility is that the action of Ang(1–7) may correlate with the level of RAS activation.

Abstract 344: Intrarenal Ghrelin Receptors Mediate Sodium Reabsorption via an ENaC-Dependent Pathway

Intrarenal ghrelin infusion stimulates distal nephron-dependent sodium (Na+) reabsorption in normal rats, but the mechanism is unknown. The main Na+ transporters of the distal nephron segment are the Na+-Cl- co-transporter (NCC) and the epithelial Na+ channel (ENaC). To determine which of these transporters is involved in the antinatriuretic actions of intrarenal ghrelin receptors, uninephrectomized Sprague-Dawley rats received 3 cumulative 1h renal interstitial (RI) infusions of ghrelin (0.3-3 μg/min, N=8) or vehicle (5% dextrose in water, D5W, N=8), prior to harvesting the infused kidney for determination of NCC and ENaC protein expression. Ghrelin-infused rats demonstrated significantly reduced Na+ excretion rates (UNaV) compared to D5W-infused rats (66.7±7.1% of baseline, P<0.01) and significantly increased cortical collecting duct ENaC expression (2.46±0.17 and 1.56±0.19 densitometric units respectively, P<0.01). Renal NCC expression did not change in response to either ghrelin or D5W infusion (4.21±0.58 and 4.13±0.44 densitometric units respectively, P=NS). To test whether the ghrelin-induced increase in collecting duct ENaC expression was responsible for the antinatriuretic actions of ghrelin, we infused ghrelin into the kidney in the presence of amiloride, a selective inhibitor of ENaC activity. Following uninephrectomy, rats were implanted with either a subcutaneous osmotic minipump which systemically delivered amiloride (1.4 ng/min) for 72h to block ENaC activity (N=6), or a minipump that was filled with D5W (N=6). On the day of the study, RI ghrelin (0.3-3 μg/min) or D5W infusion was initiated. RI ghrelin infusion (in the absence of amiloride) significantly reduced UNaV to 58.9±7.2% of baseline, P<0.01; however, in the presence of amiloride, RI ghrelin failed to reduce UNaV, demonstrating values identical to rats that did not receive RI ghrelin. In contrast, studies carried out in the presence of chlorothiazide pumps (to block NCC activity, 1.1 μg/min, N=6), continued to demonstrate ghrelin-induced antinatriuresis (UNaV decreased to 65.3±8.8% of baseline, P<0.01). Mean arterial pressures did not change during any of the acute RI infusions. Thus, intact ENaC function is necessary for ghrelin-induced Na+ reabsorption.

Chronic sodium-retaining action of insulin in diabetic dogs

Insulin-mediated sodium retention is implicated as a mechanism for hypertension in metabolic syndrome and type II diabetes. However, there is no direct experimental evidence for a sustained antinatriuretic effect of insulin outside of rodents, and all previous studies in dogs have been negative. This study used a novel approach to test for a chronic sodium-retaining action of insulin in dogs, by testing the hypothesis that natriuresis in type I diabetes is dependent on the decrease in insulin, rather than being due solely to osmotic actions of hyperglycemia. Dogs were chronically instrumented and housed in metabolic cages. Fasting blood glucose in alloxan-treated dogs was maintained at ~65 mg/dl by continuous intravenous insulin infusion. Then, a 6-day diabetic period was induced by either 1) decreasing the insulin infusion to induce type I diabetes (D; blood glucose = 449 ± 40 mg/dl) or 2) clamping the insulin infusion and infusing glucose continuously (DG; blood glucose = 470 ± 56 mg/dl). Control urinary sodium excretion (UnaV) averaged 70 ± 5 (D) and 69 ± 5 (DG) meq/day and increased on day 1 in both groups. UnaV remained elevated in the D group (115 ± 15 meq/day days 2-6), but it returned to control in the DG group (69 ± 11 meq/day days 2-6) and was accompanied by decreased lithium clearance. Thus, insulin had a sustained antinatriuretic action that was triggered by increased glucose, and it was powerful enough to completely block the natriuresis caused by hyperglycemia. These data may reveal an unrecognized physiologic function of insulin as a protector against hyperglycemia-induced salt wasting in diabetes.

Chronic Glucose Infusion Increases Tubular Sodium Reabsorption and Renal Blood Flow in Dogs

It is not known whether hyperinsulinemia and/or hyperglycemia in metabolic syndrome contribute to the associated hypertension through chronic antinatriuretic actions. We tested this using chronic iv. glucose infusion to increase plasma insulin and glucose concentrations comparable to those measured in metabolic syndrome, and used 24 hr/day methods for measuring mean arterial pressure (MAP; DSI) and renal blood flow (RBF; Transonic). Blood glucose increased ~ 10 mg/dl over the 6‐day glucose infusion period, and plasma insulin increased from 28±4 to 45±12 µU/ml. Urinary sodium excretion averaged 70±8 mEq/d during control, decreased to 49±5 mEq/d on day 1 of glucose, and averaged 51±6 mEq/d over the 6 days. RBF averaged 123±17 ml/min during control, increased to 137±19 on day 1, and averaged 153±17 by day 6 of glucose. Plasma renin activity increased 3‐fold, and all variables returned to control values during recovery. MAP did not increase significantly. However, there was no tendency for MAP to decrease, eliminating pressure natriuresis as a mechanism for the sodium retention. This confirms our earlier report that glucose infusion causes sustained antinatriuresis in dogs, and the renal vasodilation indicates that tubular reabsorption is the mechanism. The renin and RBF responses are consistent with a role for the macula densa, but that and the reason why MAP did not increase require further study. HL56259, HL75625

Epithelial Sodium Channel Is a Key Mediator of Growth Hormone-Induced Sodium Retention in Acromegaly

Acromegalic patients present with volume expansion and arterial hypertension, but the renal sites and molecular mechanisms of direct antinatriuretic action of GH remain unclear. Here, we show that acromegalic GC rats, which are chronically exposed to very high levels of GH, exhibited a decrease of furosemide-induced natriuresis and an increase of amiloride-stimulated natriuresis compared with controls. Enhanced Na(+),K(+)-ATPase activity and altered proteolytic maturation of epithelial sodium channel (ENaC) subunits in the cortical collecting ducts (CCDs) of GC rats provided additional evidence for an increased sodium reabsorption in the late distal nephron under chronic GH excess. In vitro experiments on KC3AC1 cells, a murine CCD cell model, revealed the expression of functional GH receptors and IGF-I receptors coupled to activation of Janus kinase 2/signal transducer and activator of transcription 5, ERK, and AKT signaling pathways. That GH directly controls sodium reabsorption in CCD cells is supported by: 1) stimulation of transepithelial sodium transport inhibited by GH receptor antagonist pegvisomant; 2) induction of alpha-ENaC mRNA expression; and 3) identification of signal transducer and activator of transcription 5 binding to a response element located in the alpha-ENaC promoter, indicative of the transcriptional regulation of alpha-ENaC by GH. Our findings provide the first evidence that GH, in concert with IGF-I, stimulates ENaC-mediated sodium transport in the late distal nephron, accounting for the pathogenesis of sodium retention in acromegaly.

Hemodynamic-independent anti-natriuretic effect of urotensin II in spontaneously hypertensive rats

The present study aims to test the hypothesis that U-II might have a direct anti-natriuretic action in spontaneously hypertensive rats (SHR). Bolus U-II injection (15 nmol kg(-1)) caused a transient decrease in glomerular filtration rate (GFR), urine flow rate (UV), urinary sodium (UNaV) and potassium excretion (U(K)V) that corresponded with a committed decrease in mean arterial pressure (MAP) and renal blood flow (RBF) during the first 30 min. Continuous U-II infusion (0.2 nmol kg(-1)h(-1)) following a bolus U-II injection (0.3 nmol kg(-1)) caused an anti-natriuretic effect without any significant change in MAP, RBF, GFR, UV and UKV during the entire 1.5-h perfusion period in SHR. The levels of aldosterone and angiotensin II were not altered in the plasma and kidney, while plasma antidiuretic hormone decreased in response to U-II injection (15 nmol kg(-1)). Protein levels of U-II receptors (UT) were significantly increased in the kidney of 17-week-old SHR when compared with the age-matched WKY rats, while mRNA transcripts of both U-II and UT were increased in the kidney, left ventricle and thoracic aorta. In conclusion, U-II exerts a hemodynamic-independent anti-natriuretic action in adult SHR. The anti-natriuretic action of U-II in SHR is probably associated with an increased expression of the U-II-UT system in the kidney, suggesting a potential renal role of U-II in the pathogenesis of hypertension.

Aldosterone-mediated apical targeting of ENaC subunits is blunted in rats with streptozotocin-induced diabetes mellitus

Diabetes mellitus (DM) is associated with a significant polyuria and natriuesis as well as increased plasma aldosterone and anti-diuretic hormone arginine vasopressin (AVP). This study aimed to determine whether diabetic kidneys compensate for the urinary sodium and water losses by increasing apical targeting of epithelial sodium channel (ENaC) subunits and aquaporin-2 (AQP2) in the collecting duct, in addition to the previously observed changes in ENaC subunit protein expression in different kidney zones. Female rats were investigated 2 weeks after induction of DM by streptozotocin administration. Kidneys were examined by immunohistochemisty and semiquantitative immunoblotting. We demonstrated that the protein expression of renal AQP2, Ser-256 phosphorylated AQP2, AQP3, beta- and gamma-ENaC (but not alpha-ENaC) increased consistently with an increased AVP response. In contrast, there were no significant changes in the relative apical targeting of beta-, gamma- and alpha-ENaC, and the shift in the molecular weight of gamma-ENaC from 85 kDa to 70 kDa was not observed despite increased plasma aldosterone levels. These results were supported by changes in the functional data showing increased solute-free water reabsorption, increased fractional excretion of sodium and an unchanged ratio of potassium to sodium in the urine. The data demonstrate that diabetic kidneys have a reduced sensitivity to the anti-natriuretic action of elevated plasma aldosterone levels with no relative increase in ENaC subunit apical targeting, whereas there is increased expression of beta- and gamma-ENaC, which alone may play a role in the increased sodium reabsorption in the kidney in DM.

Insulin's impact on renal sodium transport and blood pressure in health, obesity, and diabetes

Insulin has been shown to have antinatriuretic actions in humans and animal models. Moreover, endogenous hyperinsulinemia and insulin infusion have been correlated to increased blood pressure in some models. In this review, we present the current state of understanding with regard to the regulation of the major renal sodium transporters by insulin in the kidney. Several groups, using primarily cell culture, have demonstrated that insulin can directly increase activity of the epithelial sodium channel, the sodium-phosphate cotransporter, the sodium-hydrogen exchanger type III, and Na-K-ATPase. We and others have demonstrated alterations in the expression at the protein level of many of these same proteins with insulin infusion or in hyperinsulinemic models. We also discuss how this regulation is perturbed in type I and type II diabetes mellitus. Finally, we discuss a potential role for regulation of insulin receptor signaling in the kidney in contributing to sodium balance and blood pressure.

Acute insulin infusion effects on ENaC subunits in mice

Perfused tubule and cell culture studies have revealed that insulin positively regulates the activity of the epithelial sodium channel (ENaC). We investigated the effect of acute insulin injection to mice on the trafficking of ENaC subunits by immunobased approaches. Six-month-old male C57BL/CBA mice were randomly assigned one of three intraperitoneal treatments (n = 5/group): 1) control, 0.3 ml saline; 2) glucose (50% glucose in 0.3 ml saline); or 3) insulin (0.0078 units/kg·bw of insulin in 0.3 ml 50% glucose). Blood glucose levels were measured every 20 minutes by glucometer on tail blood. Blood glucose was significantly reduced by insulin (group 3) after 40 and 60 minutes, but mice were not markedly hypoglycemic (mg/dl)at 60 minutes: 126 ± 6, 256 ± 33, 87 ± 22, for groups 1–3, respectively. After 60 minutes, mice were anesthetized with pentobarbital, right kidneys were clamped and removed, and left kidneys were perfusion fixed. Immunoperoxidase labeling showed increased apical localization of γ-ENaC in the collecting duct principal cells of group 3, relative to groups 1 and 2. In agreement, western blotting of a plasma-membrane enriched fraction from whole kidney revealed an increase in the density of bands for γ-ENaC (both 85 and 70-kDa) in group 3, relative to 1 and 2, suggesting increased apical membrane residency and activation of this protein. No clear differences were found between groups for α- or β-ENaC, although α-ENaC appeared more tightly apical than did β-ENaC. These studies provide in vivo evidence of trafficking of γ-ENaC in response to acute insulin stimulation in mouse kidney, and may be part of the mechanism underlying the anti-natriuretic actions of this hormone. Funded by NIH.

Independent and Combined Effects of Testosterone and Growth Hormone on Extracellular Water in Hypopituitary Men

Symptoms of fluid retention in GH-deficient patients during GH replacement are greater in men than in women, suggesting that testosterone may augment or estradiol may attenuate the antinatriuretic actions of GH. The mechanisms underlying the sodium-retaining effects of GH are poorly understood. The aim of this study was to investigate the effects of GH and testosterone, alone and in combination, on extracellular water (ECW) and the hormonal mechanisms involved. Two separate, open-label, randomized, two-period, crossover studies were performed; the first compared the effects of GH alone with those of GH and testosterone, and the second compared the effects of testosterone alone with those of GH and testosterone. Twelve hypopituitary men with GH deficiency and hypogonadism were studied. During the weeks of intervention, GH (0.5 mg/d) and testosterone enanthate (250 mg) were administered by im injection. The outcome measures were ECW, IGF-I, plasma renin activity (PRA), aldosterone (Aldo), and atrial natriuretic peptide (ANP). GH treatment significantly increased (P < 0.05) both IGF-I and ECW, and these changes were enhanced by cotreatment with testosterone (P = 0.07 for both). PRA, Aldo, and ANP levels did not change. Testosterone treatment alone did not change the IGF-I concentration, whereas cotreatment with GH induced a marked increase. Testosterone alone increased (P < 0.05) ECW, and the effect was augmented (P < 0.01) by cotreatment with GH. Although PRA and ANP did not change, plasma Aldo decreased after single and combined treatments. GH and testosterone exerted independent and additive effects on ECW. The mechanisms of fluid retention for both hormones are likely to be exerted on the renal tubules. This is the first direct evidence that testosterone increases ECW.

Insulin receptors and renal sodium handling in hypertensive fructose-fed rats

Insulin resistance and hypertension are present in Sprague-Dawley rats fed a fructose-enriched diet. In these rats, insulin might elevate blood pressure via an antinatriuretic action. To investigate the sodium-insulin interaction in fructose-fed rats, we compared insulin sensitivity, insulin receptor binding, and insulin receptor mRNA levels in the kidney and skeletal muscle of rats that were fed standard rat chow or a fructose-enriched diet (66%) with either low (0.07%), normal (0.3%), or high (7.5%) NaCl concentrations for 3 weeks. Systolic blood pressure increased in the fructose-fed rats receiving the normal and high-salt diet, but not the low-salt diet. When the rats were fed the low-salt diet, the rate of glucose infusion required to maintain euglycemia during a hyperinsulinemic clamp and insulin receptor number and mRNA levels in skeletal muscle were lower in fructose-fed than control rats. High-salt diet decreased significantly the rate of glucose disposal during the clamp and muscular insulin receptor number and mRNA levels in control, but not fructose-fed rats. During the low-salt diet, renal insulin receptor number and mRNA levels were comparable in fructose-fed and control rats and hyperinsulinemia had comparable acute antinatriuretic effects in the two groups; when the rats were maintained on the high-salt diet, the expected decrease in renal insulin receptor number and mRNA levels occurred in control but not fructose-fed rats and, consistent with this finding, the antinatriuretic response to hyperinsulinemia was blunted only in controls. An inverse relationship between dietary NaCl content and renal insulin receptor mRNA levels was observed in control but not fructose-fed rats. Fructose-fed rats appear to have lost the feedback mechanism that limits insulin-induced sodium retention through a down-regulation of the renal insulin receptor when the dietary NaCl content is increased. This abnormality might possibly contribute to the elevation of blood pressure in these rats.

Role of superoxide in modulating the renal effects of angiotensin II.

Angiotensin II is known to stimulate NADPH oxidase-dependent superoxide (O2-) generation, which may contribute to the acute renal vasoconstrictor and antinatriuretic actions of this peptide. To evaluate this hypothesis, the effects of a superoxide dismutase mimetic (tempol) or a NADPH inhibitor (apocynin) on the angiotensin renal actions were studied. Renal cortical nitric oxide (NO) was measured electrochemically in vivo. Tempol increased sodium excretion and NO levels. Apocynin raised renal blood flow, glomerular filtration rate, sodium excretion, and NO levels. These results indicate the presence of an endogenous NADPH oxidase-dependent O2- generation that may modulate renal function by scavenging NO. Angiotensin II infusion reduced renal blood flow, glomerular filtration, sodium excretion, and NO levels in a dose-dependent manner. The angiotensin receptor antagonist valsartan, tempol, or apocynin blunted the angiotensin effects on renal excretion and NO, suggesting that angiotensin receptors stimulation induces the NADPH oxidase-dependent O2- generation that might reduce NO bioavailability. This idea is supported by the finding that angiotensin increased O2- generation in renal homogenates, and this effect was prevented by valsartan, apocynin, or tempol. These results indicate that some of the acute renal effects of angiotensin II may be enhanced by an increased NADPH oxidase-derived O2- production that reduces renal NO bioavailability.

The effects of the potassium channel opener minoxidil on renal electrolytes transport in the loop of henle.

ATP-sensitive potassium channels (K(ATP)) in the thick ascending limb of the loop of Henle play an important role in apical K(+) recycling, a mechanism essential for maintaining the activity of the Na/2Cl/K-cotransporter. We have previously demonstrated that inhibition of K(ATP) decreases Na(+) and K(+) absorption in the loop of Henle and induces diuretic and natriuretic effects. In the present study, we used renal clearance and in vivo microperfusion techniques to evaluate the effects of the K(ATP) opener minoxidil on the urinary excretion and absorption in the loop of Henle of Na(+), K(+), Ca(2+), and Mg(2+). Intravenous injection of minoxidil (1.5 mg/kg) significantly decreased fractional Na(+) (FENa) and Mg(2+) (FEMg) excretion and urine volume with a moderate decrease in blood pressure (12%) and glomerular filtration rate (15%). Urine volume decreased 63%, and FENa and FEMg decreased 58 and 37%, respectively. In contrast, K(+) and Ca(2+) excretion did not change significantly. In the microperfusion of the loop of Henle, addition of minoxidil to the perfusion fluid significantly increased fluid (J(v)), Na(+) (J(Na)), Cl(-) (J(Cl)), and K(+) (J(K)) absorption. J(v) increased 44% (from 8.32 to 11.95 nl/min), J(Na) increased 14% (from 1.96 to 2.34 nmol/min), J(Cl) increased 21% (from 1.72 to 2.08 nmol/min), and J(K) increased 57% (from 35.8 to 56.4 pmol/min). We conclude that the activation of K(ATP) leads to stimulation of Na/2Cl/K-cotransporter activity and increases the rates of Na(+), Cl(-), and K(+) absorption in the loop of Henle, an effect contributing to the antidiuretic and antinatriuretic action of this K channel opener.

The serine/threonine kinases SGK2 and SGK3 are potent stimulators of the epithelial Na+ channel alpha,beta,gamma-ENaC.

The serum- and glucocorticoid-inducible kinase 1 (SGK1) has been identified as a signalling molecule up-regulated by aldosterone, which stimulates the renal epithelial Na(+) channel ENaC. It is therefore thought to participate in the antinatriuretic action of this hormone. More recently, two isoforms, SGK2 and SGK3, have been cloned. The present study was performed to establish whether SGK2 and SGK3 influence ENaC activity similarly to SGK1. Dual-electrode voltage-clamp experiments in Xenopus laevis oocytes expressing alpha,ss,gamma-ENaC with or without SGK1, SGK2 or SGK3 revealed a stimulatory effect of all three kinases on the amiloride-sensitive current (I(Na)). To establish whether the SGK isoforms exert their effects through direct phosphorylation, we replaced the serine at the SGK consensus site of alphaENaC (alpha(S622A)ENaC) by site-directed mutagenesis. alpha(S622A),beta,gamma-ENaC was up-regulated similar to wild-type ENaC, suggesting that SGK isoforms do not act via direct phosphorylation of the transport proteins. In conclusion, SGK2 and SGK3 mimic the function of SGK1 and are likely to participate in the regulation of ENaC activity.