ETB-deficient Rats Research Articles

Profound Sex Differences in the Kidney Expression of Mitochondria-Related Genes in Endothelin-1 Receptor B (ETB) Deficient Rats Fed a High Salt Diet

Elevated levels of endothelin-1 (ET-1), overactivation of the ETA receptor and oxidative stress are critical in the development of hypertensive kidney disease. Females are more resistant than males to kidney damage; however, it is unclear if overactivation of the ETA receptor disrupts mitochondrial homeostasis similarly in both sexes. With these studies we aimed to determine if overactivation of the ET-1/ETA axis has a differential effect on mitochondrial homeostasis in the male and female kidney during high salt consumption. Male and female ETB deficient (ETB def) and transgenic (TG) control rats were placed on a high salt diet (4.0% NaCl) for 3 weeks. Kidneys were collected and the expression of 84 mitochondria-related genes in renal cortex and outer medulla was assessed with qRT-PCR arrays. In response to high salt, male ETB def rats displayed cortical overexpression of 13 genes when compared to male TG controls, while only 7 genes were upregulated in cortex of female ETB def rats compared to female TG controls. Genes upregulated in male ETB def rats are involved in apoptosis, mitochondrial biogenesis and transport, energy metabolism and ROS handling (selected genes, male ETB def vs. TG controls, n=3/group: Ucp1 (19-fold increase, p=0.03), Bnip3 and Scl25a21 (6-fold, p=0.003 and p=0.004, respectively), and Mfn1 (5-fold, p=0.03). Genes upregulated in females are involved in apoptosis, ROS handling and mitochondrial transport (selected examples, female ETB def vs. TG control, n=3/group: 2-fold increase in Sod1 (p=0.03), Akt1 (p=0.003) and Grpel1 (p=0.019). No common genes were changed between the sexes. When comparisons were made in the outer medulla, we found that deficiency of the ETB receptor in males resulted in a ≥30% reduction in the expression of 29 genes involved in apoptosis, mitochondrial biogenesis and transport, energy metabolism, ROS handling and other mitochondrial functions (p<0.05). On the other hand, the expression of 8 genes involved in mitochondrial transport and apoptosis was reduced by ≥80% in the female ETB def rat outer medulla vs. TG controls, while 3 genes involved in mitochondrial transport and energy metabolism had a 2-fold overexpression (p<0.05). Altogether, our results demonstrate that salt-sensitive hypertension affects mitochondrial homeostasis differentially in distinct regions of the kidney, and that the male and female kidney respond to salt in a disparate manner: with overactivation of the ET-1/ETA axis resulting in greater dysregulation of mitochondria-related genes in the male versus the female kidney during a high salt diet. Funded by UAB-UCSD O’Brien Center Summer Research Pathway for Students P30 DK079337 and KURE R25 DK115353 to BSM, NIGMS UAB CORD BBRT to GJC, and NIH K01HL145324 and Sapphire North America Return to Science Research grant to CDM. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Endothelin Modulates Rhythm Disturbances and Autonomic Responses to Acute Emotional Stress in Rats.

The ubiquitous peptide endothelin is currently under investigation as a modulatory factor of autonomic responses to acute emotional stress. Baseline plasma levels of endothelin alter blood pressure responses, but it remains unclear whether autonomic activity and arrhythmogenesis (i.e., brady- or tachyarrhythmias) are affected. We recorded sympathetic and vagal indices (derived from heart rate variability analysis), rhythm disturbances, voluntary motion, and systolic blood pressure after acute emotional stress in conscious rats with implanted telemetry devices. Two strains were compared, namely wild-type and ETB-deficient rats, the latter displaying elevated plasma endothelin. No differences in heart rate or blood pressure were evident, but sympathetic responses were blunted in ETB-deficient rats, contrasting prompt activation in wild-type rats. Vagal withdrawal was observed in both strains at the onset of stress, but vagal activity was subsequently restored in ETB-deficient rats, accompanied by low voluntary motion during recovery. Reflecting such distinct autonomic patterns, frequent premature ventricular contractions were recorded in wild-type rats, as opposed to sinus pauses in ETB-deficient rats. Thus, chronically elevated plasma endothelin levels blunt autonomic responses to acute emotional stress, resulting in vagal dominance and bradyarrhythmias. Our study provides further insights into the pathophysiology of stress-induced tachyarrhythmias and syncope.

Elevated renal afferent nerve activity in a rat model of endothelin B receptor deficiency.

Renal nerves have been an attractive target for interventions aimed at lowering blood pressure; however, the specific roles of renal afferent (sensory) versus efferent sympathetic nerves in mediating hypertension are poorly characterized. A number of studies have suggested that a sympathoexcitatory signal conveyed by renal afferents elicits increases in blood pressure, whereas other studies identified sympathoinhibitory afferent pathways. These sympathoinhibitory pathways have been identified as protective against salt-sensitive increases in blood pressure through endothelin B (ETB) receptor activation. We hypothesized that ETB-deficient (ETB-def) rats, which are devoid of functional ETB receptors except in adrenergic tissues, lack appropriate sympathoinhibition and have lower renal afferent nerve activity following a high-salt diet compared with transgenic controls. We found that isolated renal pelvises from high salt-fed ETB-def animals lack a response to a physiological stimulus, prostaglandin E2, compared with transgenic controls but respond equally to a noxious stimulus, capsaicin. Surprisingly, we observed elevated renal afferent nerve activity in intact ETB-def rats compared with transgenic controls under both normal- and high-salt diets. ETB-def rats have been previously shown to have heightened global sympathetic tone, and we also observed higher total renal sympathetic nerve activity in ETB-def rats compared with transgenic controls under both normal- and high-salt diets. These data indicate that ETB receptors are integral mediators of the sympathoinhibitory renal afferent reflex (renorenal reflex), and, in a genetic rat model of ETB deficiency, the preponderance of sympathoexcitatory renal afferent nerve activity prevails and may contribute to hypertension.NEW & NOTEWORTHY Here, we found that endothelin B receptors are an important contributor to renal afferent nerve responsiveness to a high-salt diet. Rats lacking endothelin B receptors have increased afferent nerve activity that is not responsive to a high-salt diet.

Endothelin‐1 Receptor B (ETB) Deficient Rats Fed High Salt Diet Display Differential Mitochondria‐Related Gene Expression in the Kidney Cortex and Outer Medulla

Levels of the vasoactive peptide endothelin‐1 (ET‐1) are persistently elevated in patients and animal models of salt‐sensitive hypertension. Our group recently reported that loss of ETB receptor function and the consequent overactivation of the ET‐1/ETA receptor axis are associated with exaggerated kidney damage, T cell inflammation and apoptosis under high salt conditions. Importantly, the molecular mechanisms by which ET‐1 leads to high salt‐induced renal damage remain unclear. These studies aimed to determine if the overactivation of the ET‐1/ETA axis alters the kidney expression of genes involved in mitochondrial biogenesis and function during high salt consumption. Male ETB deficient (ETB def) and transgenic (TG) control rats were placed on a high salt diet (HSD, 4.0% NaCl) for 3 weeks. At the end of this HSD period, 24h urine was collected and kidneys were separated in cortex and outer medulla. Urinary protein excretion was measured as a marker of kidney damage and the kidney expression of 84 mitochondria‐related genes was assessed by qRT‐PCR arrays. Compared to TG controls, high salt‐fed ETB def rats presented with greater kidney damage as indicated by elevated protein excretion (TG control vs. ETB def, 73.9±8.0 vs. 121.4±10.0 mg/day; n=5/group; p=0.01). In addition, ETB def rats fed HSD displayed cortical overexpression of 13 genes involved in energy metabolism, apoptosis, mitochondrial biogenesis, and regulation of the production of reactive oxygen species (selected examples of ETB def vs. TG control genetic expression (n=3/group): Ucp1, 19‐fold increase (p=0.03); Bnip3 (p=0.003) and Scl25a21 (p=0.004), 6‐fold; Mfn1,5‐fold (p=0.03); and Dnm1l, 4‐fold (p=0.004)). On the other hand, when the same comparisons were made in the kidney outer medullary region, we found that deficiency of the ETB receptor resulted in a 50% reduction in the expression of 29 genes (among them, genes involved in mitochondrial membrane polarization, transport and dynamics; p<0.05). These results demonstrate a differential effect of ETAoveractivation on mitochondrial homeostasis in the kidney cortical and outer medullary regions during high salt consumption, with overactivation of the ET‐1/ETA axis leading to exaggerated mitochondrial function in the cortex and downregulation of this function in the outer medulla.

Functional Interaction of Endothelin Receptors in Mediating Natriuresis Evoked by G Protein-Coupled Estrogen Receptor 1.

The G protein–coupled estrogen receptor 1 (GPER1) mediates rapid estrogenic signaling. We recently reported that activation of GPER1 in the renal medulla evokes endothelin-1–dependent natriuresis in female, but not male, rats. However, the involvement of the ET receptors, ETA and ETB, underlying GPER1 natriuretic action remain unclear. In this study, we used genetic and pharmacologic methods to identify the contributions of ETA and ETB in mediating this female-specific natriuretic effect of renal medullary GPER1. Infusion of the GPER1-selective agonist G1 (5 pmol/kg per minute) into the renal medulla for 40 minutes increased Na+ excretion and urine flow in anesthetized female ETB-deficient (ETB def) rats and littermate controls but did not affect blood pressure or urinary K+ excretion in either group. Pretreatment with the selective ETA inhibitor ABT-627 (5 mg/kg, intravenous) abolished G1-induced natriuresis in ETB def rats. To further isolate the effects of inhibiting either receptor alone, we conducted the same experiments in anesthetized female Sprague-Dawley (SD) rats pretreated or not with ABT-627 and/or the selective ETB inhibitor A-192621 (10 mg/kg, intravenous). Neither antagonism of ETA nor antagonism of ETB receptor alone affected the G1-induced increase in Na+ excretion and urine flow in SD rats. However, simultaneous antagonism of both receptors completely abolished these effects. These data suggest that ETA and ETB receptors can mediate the natriuretic and diuretic response to renal medullary GPER1 activation in female rats.SIGNIFICANCE STATEMENTActivation of G protein–coupled estrogen receptor 1 (GPER1) in the renal medulla of female rats evokes natriuresis via endothelin receptors A and/or B, suggesting that GPER1 and endothelin signaling pathways help efficient sodium excretion in females. Thus, GPER1 activation could be potentially useful to mitigate salt sensitivity in females.

Loss of endothelin type B receptor function improves insulin sensitivity in rats.

High salt intake (HS) is associated with obesity and insulin resistance. ET-1, a peptide released in response to HS, inhibits the actions of insulin on cultured adipocytes through ET-1 type B (ETB) receptors; however, the in vivo implications of ETB receptor activation on lipid metabolism and insulin resistance is unknown. We hypothesized that activation of ETB receptors in response to HS intake promotes dyslipidemia and insulin resistance. In normal salt (NS) fed rats, no significant difference in body mass or epididymal fat mass was observed between control and ETB deficient rats. After 2 weeks of HS, ETB-deficient rats had significantly lower body mass and epididymal fat mass compared to controls. Nonfasting plasma glucose was not different between genotypes; however, plasma insulin concentration was significantly lower in ETB-deficient rats compared to controls, suggesting improved insulin sensitivity. In addition, ETB-deficient rats had higher circulating free fatty acids in both NS and HS groups, with no difference in plasma triglycerides between genotypes. In a separate experiment, ETB-deficient rats had significantly lower fasting blood glucose and improved glucose and insulin tolerance compared to controls. These data suggest that ET-1 promotes adipose deposition and insulin resistance via the ETB receptor.

Tauroursodeoxycholic acid (TUDCA) abolishes chronic high salt-induced renal injury and inflammation.

Chronic high salt intake exaggerates renal injury and inflammation, especially with the loss of functional ETB receptors. Tauroursodeoxycholic acid (TUDCA) is a chemical chaperone and bile salt that is approved for the treatment of hepatic diseases. Our aim was to determine whether TUDCA is reno-protective in a model of ETB receptor deficiency with chronic high salt-induced renal injury and inflammation. ETB -deficient and transgenic control rats were placed on normal (0.8% NaCl) or high salt (8% NaCl) diet for 3weeks, receiving TUDCA (400mg/kg/d; ip) or vehicle. Histological and biochemical markers of kidney injury, renal cell death and renal inflammation were assessed. In ETB -deficient rats, high salt diet significantly increased glomerular and proximal tubular histological injury, proteinuria, albuminuria, excretion of tubular injury markers KIM-1 and NGAL, renal cortical cell death and renal CD4+ T cell numbers. TUDCA treatment increased proximal tubule megalin expression as well as prevented high salt diet-induced glomerular and tubular damage in ETB -deficient rats, as indicated by reduced kidney injury markers, decreased glomerular permeability and proximal tubule brush border restoration, as well as reduced renal inflammation. However, TUDCA had no significant effect on blood pressure. TUDCA protects against the development of glomerular and proximal tubular damage, decreases renal cell death and inflammation in the renal cortex in rats with ETB receptor dysfunction on a chronic high salt diet. These results highlight the potential use of TUDCA as a preventive tool against chronic high salt induced renal damage.

Abstract 001: Loss of ET B Receptor Function Activates Inflammasome Signaling Pathways and Dendritic Cells in the Renal Outer Medulla During Type 1 Diabetes

Renal inflammation is a hallmark of diabetic kidney disease. Endothelin-1 (ET-1), a potent vasoactive peptide that acts through two receptors (ET A and ET B ), has been implicated in diabetes and is upregulated in patients with diabetic nephropathy (DN) and in animal models of diabetic kidney injury. ET B receptors are highly expressed in renal outer medulla (OM). ET-1 exerts pro-inflammatory actions in the kidney; however, the mechanisms by which ET-1 mediate these effects are unclear. The present studies were designed to determine the role of the ET B receptor in priming inflammasome signaling pathways, as well as its role in the activation of dendritic cells (DCs), in the renal OM during type 1 diabetes. Diabetes was induced in ET B deficient (ET B def) and transgenic (TG) control rats by i.v. injection of streptozotocin (STZ). 10 wk later, 24-h urine and kidneys were collected for analysis of kidney damage. Diabetic ET B def rats presented exaggerated kidney damage compared to diabetic TG controls, with greater excretion of protein, albumin and ET-1, cortical expression of KIM-1 (respectively: 90±12 vs. 45±6 mg/day, 41±8 vs. 12±4 mg/day, 18±1 vs. 15±1 pg/day, 12±1 vs. 7±1 pg/mg prot; n=4- 6/group, p<0.05), and excessive kidney fibrosis, tubular dilation and cell death. Immunohistochemistry for the DC activation marker CD83 also demonstrated exaggerated activation of DCs in renal OM of diabetic ET B def rats. Expression of inflammasome genes in renal OM was assessed by RT-PCR array. Diabetes led to upregulation of NLRP5 (4-fold increase vs. TG controls; n=3/group; p<0.05) and IL-1β (~3-fold increase vs. TG controls; n=3/group; p<0.05) in OM of ET B def rats. In addition, PSTPIP1, a negative regulator of the inflammasome, was decreased in OM of diabetic ET B def rats vs. TG controls. Together, these results demonstrate that dysfunction of the ET B receptor leads to worsening of diabetic kidney injury and overactivation of the inflammasome and DCs specifically in the renal OM. These results highlight the protective role of ET B receptors against the development of diabetes- induced kidney inflammation and damage. Funded by NIH T32 DK007545 to CDM and P01 HL69999, P01HL136267, AHA SFRN 24450002 and U01HL117684 to DMP and JSP.

Endothelin receptor-specific control of endoplasmic reticulum stress and apoptosis in the kidney

Endothelin-1 (ET-1) promotes renal damage during cardiovascular disease; yet, the molecular mechanisms involved remain unknown. Endoplasmic reticulum (ER) stress, triggered by unfolded protein accumulation in the ER, contributes to apoptosis and organ injury. These studies aimed to determine whether the ET-1 system promotes renal ER stress development in response to tunicamycin. ETB deficient (ETB def) or transgenic control (TG-con) rats were used in the presence or absence of ETA receptor antagonism. Tunicamycin treatment similarly increased cortical ER stress markers in both rat genotypes; however, only ETB def rats showed a 14–24 fold increase from baseline for medullary GRP78, sXBP-1, and CHOP. Pre-treatment of TG-con rats with the ETA blocker ABT-627 for 1 week prior to tunicamycin injection significantly reduced the ER stress response in cortex and medulla, and also inhibited renal apoptosis. Pre-treatment with ABT-627 failed to decrease renal ER stress and apoptosis in ETB def rats. In conclusion, the ET-1 system is important for the development of tunicamycin-induced renal ER stress and apoptosis. ETA receptor activation induces renal ER stress genes and apoptosis, while functional activation of the ETB receptor has protective effects. These results highlight targeting the ETA receptor as a therapeutic approach against ER stress-induced kidney injury.

Activation of neuronal endothelin B receptors mediates pressor response through alpha-1 adrenergic receptors.

Abnormalities in activity of the endothelin (ET) system have been widely reported in a number of cardiovascular disease states such as hypertension and heart failure. Although the vascular responses to ET are well established, the interaction between ET and other important modulators of blood pressure, such as the sympathetic nervous system, are less understood. Previous reports implicate ET signaling through ET type B (ETB) receptors in increasing neuronal activity. Therefore, we hypothesized that activation of ETB receptors on sympathetic nerves would increase blood pressure through an adrenergic‐mediated mechanism. Thus, we used anesthetized ETB‐deficient rats, which only express functional ETB receptors on adrenergic neurons, and genetic controls, which express functional ETB receptors in vascular tissue and kidney epithelium. We determined the pressor response to the selective ETB receptor agonist sarafotoxin c (S6c). Separate groups of rats were treated with the α 1‐adrenergic receptor antagonist prazosin or the β‐adrenergic receptor antagonist propranolol to elucidate the role of adrenergic signaling in mediating the blood pressure response. We observed a dose‐dependent pressor response to S6c in ETB‐deficient rats that was reversed by prazosin treatment and augmented by propranolol. In genetic control rats, the effects of S6c on sympathetic neurons were mostly masked by the direct activity of ETB receptor activation on the vasculature. Heart rate was mostly unaffected by S6c across all groups and treatments. These results suggest that ETB activation on sympathetic neurons causes an increase in blood pressure mediated through α 1‐adrenergic receptor signaling.

Abstract 060: Tauroursodeoxycholic Acid (TUDCA) Prevents High Salt-Induced Renal Damage in ET B Deficient Rats in a Blood Pressure-Independent Manner

The molecular mechanisms by which the vasoactive peptide endothelin-1 (ET-1) leads to high salt-induced renal damage remain uncertain. These studies were designed to determine if the ET system induces renal damage by increasing renal cellular stress. ET B deficient (ET B def) and transgenic (TG) control rats were placed on normal (NSD, 1% NaCl) or high salt (HSD, 8% NaCl) diet for 3 weeks, and received daily i.p. injections of the cellular stress reliever tauroursodeoxycholic acid (TUDCA, 400 mg/kg/day) or vehicle. Blood pressure was monitored by telemetry. At the end of the study, renal inflammation, apoptosis and markers of kidney damage were assessed. In ET B def rats, HSD significantly increased blood pressure (NSD vs. HSD: 131.2±1.1 vs. 153.9±5.9 mmHg, p<0.05), albuminuria, excretion of cortical tubular injury markers (NSD vs. HSD: KIM-1: 14.2±3.4 vs. 104.1±20.6 pg/day; NGAL: 43.6±17.6 vs. 215.6±27.7 pg/day; n=4-7/group; p<0.05), cortical T cell infiltration (5.7±0.3 vs. 17.4±4.5 CD3 + cells/field; p<0.05) and cortical apoptosis (4.0±1.0 vs. 18.0±4.2 TUNEL+ cells/field; n=3/group; p<0.05). No changes were seen in TG controls on HSD. TUDCA treatment prevented HSD-induced cortical damage in ET B def rats, as indicated by reduced excretion of kidney injury markers (albumin: 2.3±1.5 ng/day; KIM-1: 55.7±13.8 pg/day; NGAL: 114.2±18 pg/day; p<0.05), cortical T cell infiltration (10.8±0.5 CD3 + cells/field; p<0.05) and cortical tubular apoptosis (1.9±0.3 TUNEL+ cells/field; p<0.05). TUDCA had no effect on renal medullary apoptosis or blood pressure. Nephrin excretion remained elevated in both genotypes despite TUDCA treatment. In conclusion, loss of ET B receptor leads to exaggerated salt-mediated renal cortical damage that is attenuated by treatment with TUDCA. These results highlight the protective role of ET B receptors against the development of renal damage, especially in renal cortex. Funded by NIH T32 DK007545 to CDM and P01 HL95499 and P01 HL69999 to DMP and JSP.

Local conduction during acute myocardial infarction in rats: Interplay between central sympathetic activation and endothelin

We investigated the effects of autonomic dysfunction and endothelin on local conduction and arrhythmogenesis during myocardial infarction. We recorded ventricular tachyarrhythmias, monophasic action potentials, and activation sequences in wild-type and ETB-deficient rats displaying high endothelin levels. Central sympathetic inputs were examined after clonidine administration. Clonidine mitigated early and delayed arrhythmogenesis in ETB-deficient and wild-type rats, respectively. The right ventricular activation delay increased in clonidine-treated ETB-deficient rats and slightly decreased in wild-type rats. The left ventricular voltage rise decreased in all groups, whereas the activation delay increased mainly in clonidine-treated ETB-deficient rats. Central sympathetic activation and endothelin modulate ischemia-induced arrhythmogenesis. Ischemia alters excitability, whereas endothelin impairs local conduction, an action partly counterbalanced by central sympathetic activity.

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 P639: Attenuation of Renal Inner Medullary Circadian Clock Gene Expression in Response to High Salt Intake is Dependent on the Endothelin B Receptor

Our lab has recently shown that ETB deficient (ETB def) rats have a time of day dependent impairment in their ability to excrete a Na+ load. These observations suggest an interaction between renal ETB receptors and circadian mechanisms that regulate renal tubular Na+ transport and excretion. Given that knockout of the circadian clock gene Bmal1 reduces blood pressure in mice, we hypothesized that a high salt intake impairs the clock mechanism in the renal inner medulla in an ETB dependent manner. Transgenic control (Tg con) or ETB def rats were fed normal (NS, 0.8% NaCl) or high (HS, 4% NaCl) salt for two weeks. In one group, rats were euthanized every 4 hours beginning at zeitgeber time 0 (lights on) for tissue collection (and subsequent assessment of circadian clock genes), while in a second group of rats urine was collected in 12-hour intervals (active vs. inactive). Consistent with our hypothesis, we observed that HS abolished the normal oscillation in Bmal1 expression in the renal inner medulla of Tg con rats, and effect not observed in ETB def rats. Interestingly, renal production of ET-1, was significantly higher during the active period vs. inactive period in both NS (3.6±1.1 vs. 0.8±0.2 pg/12hr respectively) and HS (9.2±4.1 vs. 1.6±0.3 pg/12hr respectively) fed Tg con rats. There was no time-of-day-dependent difference in ET-1 excretion in ETB def rats on NS (6.6±2.2 vs. 4.6±1.7 pg/12hr respectively), although this pattern was restored in ETB def rats fed HS (2.2±1.0 vs. 9.2±2.5 pg/12hr inactive vs. active). Taken together, these data indicate that an increase in renal ET-1/ETB activation in response to HS modulates inner medullary clock gene expression to promote renal Na+ excretion.

Endothelin contributes to blunted renal autoregulation observed with a high-salt diet.

Autoregulation of renal blood flow (RBF) is an essential function of the renal microcirculation that has been previously shown to be blunted by excessive dietary salt. Endogenous endothelin 1 (ET-1) is increased following a high-salt (HS) diet and contributes to the control of RBF but the differential effects of ET-1 on renal microvessel autoregulation in response to HS remain to be established. We hypothesized that a HS diet increases endothelin receptor activation in normal Sprague-Dawley rats and blunts autoregulation of RBF. The role of ET-1 in the blunted autoregulation produced by a HS diet was assessed in vitro and in vivo using the blood-perfused juxtamedullary nephron preparation and anesthetized rats, respectively. Using highly selective antagonists, we observed that blockade of either ETA or ETB receptors was sufficient to restore normal autoregulatory behavior in afferent arterioles from HS-fed rats. Additionally, normal autoregulatory behavior was restored in vivo in HS-fed rats by simultaneous ETA and ETB receptor blockade, whereas blockade of ETB receptors alone showed significant improvement of normal autoregulation of RBF. Consistent with this observation, autoregulation of RBF in ETB receptor-deficient rats fed HS was similar to both ETB-deficient rats and transgenic control rats on normal-salt diets. These data support the hypothesis that endogenous ET-1, working through ETB and possibly ETA receptors, contributes to the blunted renal autoregulatory behavior in rats fed a HS diet.

ET B Receptors in High Salt Diet‐Induced Decline of Renal Autoregulation in Rats

High salt (HS) diet is linked to increased endothelin-1 (ET-1) and salt-dependent hypertension. Previous studies showed that HS treatment increased renal microvascular ETB receptor expression and impaired renal blood flow (RBF) autoregulation. We hypothesized that HS leads to a decline of renal autoregulation via activation of ETB receptors. RBF autoregulation was assessed in anesthetized ETB deficient (ETB) and intact control littermates (Con) rats fed normal salt (NS) or HS (8% NaCl) for 2 weeks. RBF was monitored using an ultrasonic flow probe and autoregulation was assessed during step-decrements of arterial pressure imposed using an aortic occluder placed between the two renal arteries. ETB deficient rats (n=6) exhibited significantly elevated baseline mean arterial pressure (MAP, 132±5 vs. 116±1 mmHg in Con+NS, n=8, P<0.05). HS did not change MAP in either group but significantly increased RBF in Con rats (n=8, 12.5±0.7 vs. 9.2±0.5 ml/min.gkw in Con+NS, P<0.05) while decreasing RBF in ETB rats (6.8±0.6 ml/min.gkw vs. Con+NS, P<0.05). HS led to attenuated autoregulation of RBF in Con rats. Decreasing renal perfusion pressure from ambient to 95 mmHg resulted in an 11±2% decrease in RBF in Con+HS versus a 3±1% in Con+NS (P<0.05). Accordingly, autoregulatory index (AI) was 0.63±0.08 in Con+HS versus 0.18±0.09 in Con+NS (P<0.05). In contrast, RBF remained fairly stable in ETB+NS and ETB+HS rats during a stepwise reduction of renal perfusion pressure from ambient to 95 mmHg. AI averaged 0.26±0.14 and 0.12±0.24, respectively, suggesting intact autoregulation in ETB deficient rats. These data suggest that HS leads to attenuated RBF autoregulation and increased RBF via ETB receptoractivation.

TUDCA Attenuates High Salt‐Induced Renal Injury in ET B Deficient sl/sl Rats

Endothelin (ET-1) mediates sodium excretion via the ETB receptor pathway, however the ET-1/ETA receptor pathway plays a role in salt-dependent hypertension. The mechanisms by which ET-1 leads to high salt-induced renal injury are unclear. Reports have associated endoplasmic reticulum (ER) stress with renal injury, thus we hypothesized that the loss of functional ETB receptors increases high salt-induced renal injury via activation of ER stress. ETB deficient (sl/sl) rats and transgenic controls were placed on normal (1% NaCl,NSD) or high salt (8%, HSD) diet for three weeks (n=5-7/group) with daily i.p. injections of saline or the ER stress inhibitor tauroursodeoxycholic acid (TUDCA, 400 mg/kg/day). Histological injury assessments, urinary injury biomarkers, renal immune cell infiltration, renal apoptosis by TUNEL staining, and renal ER stress gene expression by qRT-PCR were studied. In sl/sl rats, HSD slightly increased glomerulosclerosis and significantly increased urinary markers of renal injury (KIM-1: NSD 14.2±3.4 to HSD 104.1±20.6 pg/day; NGAL: NSD 43.6±17.6 to HSD 215.6±27.7 pg/day, p<0.001) with no difference in control rats. TUDCA significantly decreased renal tubular damage in sl/sl rats (KIM-1: 55.7±13.8 pg/day; NGAL: 114.2±18.0 pg/day, p<0.05 vs. HSD alone). Renal ER stress markers, immune cell infiltration, and apoptosis were not different amongst groups. Thus, our data suggest that HSD contributes to renal tubular injury via cellular stress pathways and ETB receptor activation is reno-protective. Further investigation in the HSD-induced renal tubular stress pathways is warranted. Supported by NIH T32 DK007545 to CDM and P01 HL95499 to DMP and JSP.

Central Sympathetic Activation and Arrhythmogenesis during Acute Myocardial Infarction: Modulating Effects of Endothelin-B Receptors.

Sympathetic activation during acute myocardial infarction (MI) is an important arrhythmogenic mechanism, but the role of central autonomic inputs and their modulating factors remain unclear. Using the in vivo rat-model, we examined the effects of clonidine, a centrally acting sympatholytic agent, in the presence or absence of myocardial endothelin-B (ETB) receptors. We studied wild-type (n = 20) and ETB-deficient rats (n = 20) after permanent coronary ligation, with or without pretreatment with clonidine. Cardiac rhythm was continuously recorded for 24 h by implantable telemetry devices, coupled by the assessment of autonomic and heart failure indices. Sympathetic activation and arrhythmogenesis were more prominent in ETB-deficient rats during the early phase post-ligation. Clonidine improved these outcomes throughout the observation period in ETB-deficient rats, but only during the delayed phase in wild-type rats. However, this benefit was counterbalanced by atrioventricular conduction abnormalities and by higher incidence of heart failure, the latter particularly evident in ETB-deficient rats. Myocardial ETB-receptors attenuate the arrhythmogenic effects of central sympathetic activation during acute MI. ETB-receptor deficiency potentiates the sympatholytic effects of clonidine and aggravates heart failure. The interaction between endothelin and sympathetic responses during myocardial ischemia/infarction and its impact on arrhythmogenesis and left ventricular dysfunction merits further investigation.

Renal endoplasmic reticulum stress is induced via endothelin A receptor activation (857.7)

The endothelin (ET‐1) system has been recently implicated in renal endoplasmic reticulum (ER) stress development, a type of cellular stress caused by misfolded protein accumulation within this organelle. To clarify the role of the ETA receptor in renal ER stress, we hypothesized that pharmacological blockade of this receptor via ABT‐627 leads to reduced renal response to the ER stress inducer tunicamycin (TM). WT and ETB deficient (sl/sl) rats (n=3‐4/group) were pre‐treated with ABT‐627 (5mg/kg/day; drinking water) for 1 week and given a single injection of TM (2ug/g bwt; i.p.). Kidneys, plasma and urine were collected 24h later. ER stress markers in renal cortical and medullary regions were determined by qRT‐PCR. Treatment of WT animals with ABT‐627 significantly reduced the ER stress response to TM in cortex and outer medulla (fold change/WT+H2O; GRP78 (0.12±0.02 and 0.04± 0.01, respectively), ATF‐4 (0.20± 0.02 and 0.15± 0.02), ATF‐6 (0.28± 0.08 and 0.18± 0.04), spliced XBP‐1 (0.03+0.01 and 0.05± 0.01) and CHOP (0.02± 0.01 and 0.04± 0.01). On the contrary, ABT‐627 pre‐treatment failed to decrease cortical and medullary ER stress development in sl/sl rats. These results indicate that ETA receptor activation leads to induction of renal ER stress genes, while ETB receptor activation has protective effects. Modulation of the ET system may have therapeutic value in protecting against ER stress‐induced renal injury.Grant Funding Source: Supported by NIH and AHA

Endothelin B-receptors and sympathetic activation: Impact on ventricular arrhythmogenesis during acute myocardial infarction

AimsWe investigated the role of endothelin-B receptors on sympathetic activation originating from the adrenal gland or from the myocardium and its impact on arrhythmogenesis during acute myocardial infarction. Main methodsWe studied two groups of rats (n=120, 284±2g), namely wild-type and ETB-deficient. Myocardial infarction was induced by permanent ligation of the left coronary artery and ventricular tachyarrhythmias were evaluated from continuous electrocardiographic recordings. Sympathetic activation, measured by indices of heart rate variability, was evaluated after adrenalectomy or catecholamine depletion induced by reserpine. Acute left ventricular failure was assessed by total animal activity. Key findingsAdrenalectomy decreased the total duration of tachyarrhythmias in ETB-deficient rats, but their incidence remained higher, compared to wild-type rats. After reserpine, heart rate variability indices and tachyarrhythmias were similar in the two groups during the initial, ischaemic phase. During evolving infarction, tachyarrhythmia duration was longer in ETB-deficient rats, despite lower sympathetic activation. Heart rate was lower in ETB-deficient rats throughout the 24-hour observation period, whereas activity was comparable in the two groups. SignificanceEndothelin-B receptors modulate sympathetic activation during acute myocardial infarction not only in the ventricular myocardium, but also in the adrenal gland. Sympathetic activation markedly increases early-phase ventricular tachyarrhythmias, but other mechanisms involving the endothelin system underlie delayed arrhythmogenesis.