Renal Endothelin Research Articles

Abstract P146: Sex-Dependent Regulation of the Endothelin System in Multi-End Organ Inflammation in Lupus-Associated Cardiovascular Disease

Lupus is a chronic autoimmune disease characterized by IgG deposition, Toll-like receptor 7 (TLR7) overactivation, and autoantibody production resulting in end organ inflammation. SLE disproportionally affects women, SLE disproportionally affects women to men (9:1 ratio), with a high prevalence in African American women. Highly vascularized organs are severely impacted by lupus, specifically the kidneys and brain. Recent literature has shown that cardiovascular disease (CVD) has significant detrimental impacts on organ crosstalk. Endothelin-1 (ET-1), a potent vasoconstricting peptide, has been shown elevated in patients with CVD and in patients with lupus independent of disease severity. However, the extent to which ET-1 promotes lupus-associated CVD and end organ cross talk remains to be elucidated. Therefore, we hypothesized that TLR7 overactivation in a murine model of SLE-associated CVD would lead to a sex-dependent regulation of the renal and cerebral ET systems. Female and male lupus-prone B6.Nba2 mice were subjected to bi-weekly treatment with the Resiquimod (R848; TLR7/8 agonist; 100ug/30ul) or vehicle control (Acetone) for one month and subsequently studied for 12 weeks. At 16 weeks, the kidneys and brains were collected for histology and ET system (ET-1, ET A, and ET B ) analysis. Exploration of specific end organ damage demonstrates a profound increase in renal and cerebral IgG complex deposition as well as renal macrophages and T cells in both females and males receiving R848. Renal ET-1 levels displayed no time nor treatment-dependent effects in females, while males exhibited a significant time-dependent increase in expression at the 4 and 16-week timepoints. Interestingly, at the receptor level, neither sex demonstrated differences in ET A nor ET B levels in the kidney. While renal ET-1 level did not change, females demonstrated a significant increase in cerebral ET-1 at the 16-week timepoint, while males trended for increased cerebral ET-1. At the receptor level, females had a trend for increased cerebral ET A expression, while there was no change in males, and neither sex displayed a change in ET B receptor expression. These findings suggest a potential shift towards a pro-inflammatory state via the cerebral ET-1/ET A signaling axis in the R848-treated females. These findings warrant further exploration into the end-organ specific pathophysiological role ET-1/ET A signaling in multiorgan crosstalk in lupus-associated CVD.

Sex-specific role for the clock protein PER1 in renal ET-1 regulation in salt-sensitive hypertension

Global knockout (KO) of the clock protein PERIOD1 (PER1) in male, but not female, C57BL/6J mice displayed both blunted blood pressure and urinary endothelin-1 (ET-1) rhythms. More recently, we showed that Dahl salt-sensitive (SS) Per1 KO male rats had exacerbated SS hypertension, alongside increased renal injury, renal ET-1 gene ( Edn1) expression, and ET-1 peptide expression from kidneys collected during the rat’s inactive period (2 PM). Additionally, we see increased long non-coding RNA EDN1-AS, which has been suggested to modulate ET-1 production. However, it is unknown whether PER1 regulates rhythms of ET-1 in SS rats and whether PER1 regulates ET-1 in a sex-dependent way. The study objective was to test the hypothesis that PER1 negatively regulates renal EDN1-AS and Edn1 to modulate ET-1 in a sex- and time-dependent manner. To test this hypothesis, kidneys were collected during rat’s active period (2 AM) from male and female SS Per1 KO and SS control rats on a normal salt (0.4% NaCl) or 3 weeks high salt (4% NaCl) diet ( N=5-6). Renal ET-1 protein levels were measured by ELISA and semi- and quantitative PCR was performed to quantify the levels of EDN1-AS and Edn1. Renal ET-1 levels were significantly higher at 2 AM than our previously reported data at 2 PM in both SS Per1 KO and control male rats ( p<0.0001). Renal ET-1 levels were significantly increased in male SS Per1 KO rats following a high salt diet ( p=0.0362) but not in females ( p=0.9522). This was also reflected in the Edn1 mRNA levels (male, p=0.0005; female, p=0.0814). EDN1-AS levels were also increased in males ( p=0.0123) but not in females. Interestingly, significant sex differences were seen in EDN1-AS levels, where EDN1-AS levels were higher in males than females following both low and high salt diets, suggesting a potential novel sex-dependent mechanism for ET-1 regulation. Together, these data suggest sex-specific action of PER1 in the regulation of ET-1 in a model of salt-sensitive hypertension. Future work aims to investigate blood pressure and renal injury in female SS Per1 KO rats and whether this apparent negative regulation of ET-1 by PER1 contributes to the SS hypertensive and renal injury phenotype seen in male SS Per1 KO rats. This work was supported by the National Institutes of Health grants R35 HL135749 (to A.S.), R01 342 DK109570 (to M.G. and A.S.), and Department of Veteran Affairs grants I01 BX004024 (to A.S.). 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.

AT1-AA Induced Placental Ischemia Contributes to Hypertension in Female Offspring

Objective: Preeclampsia (PE), new-onset hypertension during the third trimester of pregnancy alongside other organ dysfunction, is associated with chronic inflammation and fetal growth restriction. Agonistic autoantibodies against the angiotensin II type 1 receptor (AT1-AA) are produced in PE women and induce a PE-like phenotype when infused into pregnant rats. AT1-AA is still present in maternal circulation up to eight years postpartum. We have shown that AT1-AA exposure during pregnancy contributes to impairment of cerebral blood flow in the postpartum period. Moreover, PE offspring are at higher risk for cardiovascular disease and hypertension but AT1-AA’s role in this elevated risk is unknown. The objective of this study was to determine if AT1-AA contributes to the risk for hypertension in PE offspring. Therefore, we hypothesized that AT1-AA infusion during pregnancy would cause offspring to have low birth weight and hypertension in adulthood. Methods: We infused AT1-AA (1:40) starting on gestational day (GD) 14 and allowed the dams to give birth on GD21. Birth weight was measured within 12 hours. Offspring were aged to 4 months. At that time, one male and female per litter were randomly selected to undergo carotid catheterization. The following day mean arterial pressure (MAP) was measured and blood and tissues were collected. Immune cells were measured by flow cytometry. Sex hormones were measured by mass spectroscopy. AT1-AA was measured by cardiomyocyte bioassay. Renal endothelin was measured by ELISA and RT-PCR. A two-way ANOVA was used for statistical analysis. Results: AT1-AA male and female offspring (6.1±0.1g, n=19, p<0.05; 5.8±0.1g, n=19, p<0.05) were larger at birth than NP male and female offspring (5.8±0.1g, n=11; 5.5±0.1g, n=11). Female AT1-AA offspring had elevated MAP (124±2 mmHg, n=14, p<0.05) compared to NP female offspring (110±5 mmHg, n=8) while male AT1-AA offspring had similar MAP to male NP offspring. Circulating cytolytic NK cells trended higher in female AT1-AA offspring (2.73±0.37 % gated, n=9, p=0.0949) compared to female NP offspring (1.66±0.31 % gated, n=6). Female AT1-AA offspring also had elevated progesterone (41.3±6.0 pg/μL, n=8, p=0.0556) compared to female NP offspring (26.9±4.9 pg/μL, n=7). Renal PPET expression was increased in female AT1-AA offspring (2.6±0.69 fold increase, n=4, p<0.05) compared to female NP offspring. Renal endothelin-1 was increased in female AT1-AA offspring (1.02±0.23 pg/mg, n=7, p=0.062) compared to female NP offspring (0.59±0.06 pg/mg, n=6). Male and female AT1-AA offspring had increased circulating AT1-AA (9.5±2.2 ΔBPM, n=6, p<0.0001; 7.9±0.9 ΔBPM, n=8, p<0.001) compared to male and female NP offspring (-0.4±.4 ΔBPM, n=7; -0.8±0.4 ΔBPM, n=4). Conclusion: These data demonstrate that AT1-AA infusion during pregnancy can predispose female offspring to have elevated blood pressure in adulthood. This study was supported by NIH grants RO1HD067541 (BL), F31HD110230 (NC), P20GM121334 (BL, LMA), HL151407 (DCC) and American heart association (AHA) early career award 19CDA34670055 (LMA). 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 mediates sex-differences in acclimation to high salt diet in rats

IntroductionCurrent understanding of sodium (Na+) handling is based on studies done primarily in males. Contrary to the gradual increase in high salt (HS) induced natriuresis over 3–5 days in males, female Sprague Dawley (SD) rats have a robust natriuresis after 1 day of HS. Renal endothelin-1 (ET-1) signaling, through ET receptor A and B, is an important natriuretic pathway and was implicated in our previous dietary salt acclimation studies, however, the contribution of ET receptors to sex-differences in acclimation to dietary Na+ challenges has yet to be clarified. We hypothesized that ET receptors mediate the augmented natriuretic capacity of female rats in response to a HS diet.MethodsTo test our hypothesis, male and female SD rats were implanted with telemeters and randomly assigned to treatment with A-182086, a dual ETA and ETB receptor antagonist, or control. 24-h urine samples were collected and assessed for electrolytes and ET-1. Studies were performed on a normal salt (NS, 0.3% NaCl) diet and after challenging rats with HS (4% NaCl) diet for 1 day.ResultsWe found that A-182086 increased blood pressure in male and female SD rats fed either diet. Importantly, A-182086 eliminated sex-differences in natriuresis on NS and HS. In particular, A-182086 promotes HS-induced natriuresis in male rats rather than attenuating the natriuretic capacity of females. Further, the sex-difference in urinary ET-1 excretion in NS-fed rats was eliminated by A-182086.ConclusionIn conclusion, ET receptors are crucial for mediating sex-difference in the natriuretic capacity primarily through their actions in male rats.

Myoglobin-driven ET-1 production and downstream activation of TRPC3- but not TRPC6-dependent vasoconstriction contributes to rhabdomyolysis-induced AKI

Acute kidney injury (AKI) is the most severe and life-threatening complication of rhabdomyolysis, with 10-40% of patients developing some form of kidney insufficiency within days. Experimental evidence indicates that renal vasoconstriction plays a significant role in the pathophysiology of rhabdomyolysis-induced AKI. Rodents subjected to glycerol-induced rhabdomyolysis exhibited increased plasma concentrations of endothelin-1 (ET-1). Treating glomerular mesangial cells with myoglobin has also been shown to increase the cellular production of ET-1. The downstream effectors of ET-1-induced vasoconstriction are smooth muscle Ca2+ permeable transient receptor potential canonical (TRPC) channels. There are seven members of the TRPC gene family (TRPC1-7). Whereas TRPC2 and 7 are absent, TRPC1, 3, 4, 5, and 6 are expressed in renal vessels, with TRPC3 predominant. TRPC3, TRPC6, and TRPC7 are highly homologous. Although the ET system is known to be associated with rhabdomyolysis-induced AKI, the function of renal vascular ion channels in the disease is unclear. Here, we investigated the involvement of TRPC3 and TRPC6 in glycerol-induced rhabdomyolysis in rats. ET-1 production is increased in myoglobin-treated epithelial cells, the primary source of renal ET. Rhabdomyolysis also increased renal ET-1 production, decreased renal blood flow (RBF), and increased renal vascular resistance, effects attenuated by ET receptor and TRPC channel blocker Pyr3. Furthermore, we used TRPC3 and TRPC6 knockout (KO) rats to support the pharmacological approaches. Basal day and night arterial pressure, heart rate, GFR, plasma creatinine, and BUN were unchanged in WT vs. KO rats. Twenty-four h rhabdomyolysis led to comparable increases in urinary ET-1 production in WT and KO rats. However, rhabdomyolysis-induced decreases in GFR, elevations in BUN, and plasma creatinine were all attenuated in the TRPC3 but not TRPC6 KO rats. Similarly, morphological kidney damage (tubular necrosis, casts, and dilatation) was mitigated in the TRPC3 KO with no protection offered by the KO of TRPC6 channels. Together, our data suggest that myoglobin-driven ET-1 production and downstream activation of TRPC3- but not TRPC6-dependent vasoconstriction contributes to rhabdomyolysis-induced AKI. American Heart Association Predoctoral Fellowship This is the full abstract presented at the American Physiology Summit 2023 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.

G protein-coupled estrogen receptor 1 regulates renal endothelin-1 signaling system in a sex-specific manner.

Demographic studies reveal lower prevalence of hypertension among premenopausal females compared to age-matched males. The kidney plays a central role in the maintenance of sodium (Na+) homeostasis and consequently blood pressure. Renal endothelin-1 (ET-1) is a pro-natriuretic peptide that contributes to sex differences in blood pressure regulation and Na+ homeostasis. We recently showed that activation of renal medullary G protein-coupled estrogen receptor 1 (GPER1) promotes ET-1-dependent natriuresis in female, but not male, rats. We hypothesized that GPER1 upregulates the renal ET-1 signaling system in females, but not males. To test our hypothesis, we determined the effect of GPER1 deletion on ET-1 and its downstream effectors in the renal cortex, outer and inner medulla obtained from 12-16-week-old female and male mice. GPER1 knockout (KO) mice and wildtype (WT) littermates were implanted with telemetry transmitters for blood pressure assessment, and we used metabolic cages to determine urinary Na+ excretion. GPER1 deletion did not significantly affect 24-h mean arterial pressure (MAP) nor urinary Na+ excretion. However, GPER1 deletion decreased urinary ET-1 excretion in females but not males. Of note, female WT mice had greater urinary ET-1 excretion than male WT littermates, whereas no sex differences were observed in GPER1 KO mice. GPER1 deletion increased inner medullary ET-1 peptide content in both sexes but increased outer medullary ET-1 content in females only. Cortical ET-1 content increased in response to GPER1 deletion in both sexes. Furthermore, GPER1 deletion notably increased inner medullary ET receptor A (ETA) and decreased outer medullary ET receptor B (ETB) mRNA expression in male, but not female, mice. We conclude that GPER1 is required for greater ET-1 excretion in females. Our data suggest that GPER1 is an upstream regulator of renal medullary ET-1 production and ET receptor expression in a sex-specific manner. Overall, our study identifies the role of GPER1 as a sex-specific upstream regulator of the renal ET-1 system.

The circadian clock protein PER1 is important in maintaining endothelin axis regulation in Dahl salt-sensitive rats.

Endothelin-1 (ET-1) is a peptide hormone that acts on its receptors to regulate sodium handling in the kidney's collecting duct. Dysregulation of the endothelin axis is associated with various diseases, including salt-sensitive hypertension and chronic kidney disease. Previously, our lab has shown that the circadian clock gene PER1 regulates ET-1 levels in mice. However, the regulation of ET-1 by PER1 has never been investigated in rats. Therefore, we used a novel model where knockout of Per1 was performed in Dahl salt-sensitive rat background (SS Per1 -/-) to test a hypothesis that PER1 regulates the ET-1 axis in this model. Here, we show increased renal ET-1 peptide levels and altered endothelin axis gene expression in several tissues, including the kidney, adrenal glands, and liver in SS Per1 -/- compared with control SS rats. Edn1 antisense lncRNA Edn1-AS, which has previously been suggested to be regulated by PER1, was also altered in SS Per1 -/- rats compared with control SS rats. These data further support the hypothesis that PER1 is a negative regulator of Edn1 and is important in the regulation of the endothelin axis in a tissue-specific manner.

Abstract P045: Endothelin Receptor Blockade Eliminates Sex-differences In Natriuresis In Rats Acclimating To A High Salt Diet

The current understanding of sodium (Na + ) handling, which is critical for blood pressure regulation, is based on studies done primarily in male (M) subjects. Our recent studies showed that female (F) Sprague Dawley (SD) rats reach a steady state in urinary Na + excretion after 1 day of increasing dietary Na + intake while M rats take 3-5 days. RNA sequencing data suggests that renal endothelin-1 (ET-1) signaling plays an important role in this difference, however, the role of ET receptors in sex-differences in acclimation to a high salt (HS) diet is not clear. We hypothesized that ET receptors mediate the advanced natriuretic capacity of F rats, compared to M rats, during acclimation to a HS diet. To test our hypothesis, M and F SD rats were implanted with telemeters and randomly assigned to treatment with A-182086, a dual antagonist of ET receptor A and B, or vehicle. After 2-3 days of treatment, rats were placed into metabolic cages and 24-hour urine samples were collected on a normal salt (NS, 0.3% NaCl) diet and after challenging rats with a HS (4% NaCl) diet for 1 day. Treatment with the ET receptor antagonist increased serum ET-1 and mean arterial pressure (MAP) to a comparable extent in both sexes (ET-1: M; 4.17 ± 0.65 vs 0.32 ± 0.02 pg/ml P=0.0004, F; 4.94 ± 1.01 vs 0.30 ± 0.01 pg/ml P<0.0001; MAP: M; 126 ± 2 vs 116 ± 3 mmHg P<0.0001, F; 114 ± 3 vs 107 ± 3 mmHg P<0.0043). On a NS and HS diet, F rats treated with A-182086 consumed more food than corresponding M rats (NS; P=0.0377, HS; P<0.0001), whereas vehicle-treated rats have no sex-difference in food intake. In vehicle-treated rats, urinary Na + excretion was higher in F compared to M rats on either NS or HS diet. Interestingly, ET receptor antagonism eliminates these sex differences in natriuresis during both dietary phases. In particular, A-182086 promotes urinary Na + excretion in M rats fed a HS diet (65.0 ± 2.3 vs 28.2 ± 4.6 % of Na + intake, P=0.0004). In conclusion, ET receptor antagonism eliminates sex-differences in natriuresis on NS and day 1 of HS. In opposition to our hypothesis, ET receptor antagonism promotes HS-induced natriuresis in M rats rather than attenuating the natriuretic capacity of F rats. A better mechanistic understanding of M-F differences in natriuresis could reveal sex-specific targets for hypertension treatment.

Sex Differences in Diurnal Sodium Handling During Diet-Induced Obesity in Rats.

Emerging evidence over the past several years suggests that diurnal control of sodium excretion is sex dependent and involves the renal endothelin system. Given recent awareness of disruptions of circadian function in obesity, we determined whether diet-induced obesity impairs renal handling of an acute salt load at different times of day and whether this varies by sex and is associated with renal endothelin dysfunction. Male and female Sprague-Dawley rats were placed on a high-fat diet for 8 weeks before assessing renal sodium handling and blood pressure. Male, but not female, rats on high fat had a significantly reduced natriuretic response to acute NaCl injection at the beginning of their active period that was associated with lower endothelin 1 (ET-1) excretion, lower ET-1 mRNA expression in the cortex and outer medulla as well as lower ETB receptor expression in the outer medulla of the high-fat rats. Obese males also had significantly higher blood pressure (telemetry) that was exacerbated by adding high salt to the diet during the last 2 weeks. While female rats developed hypertension with a high-fat diet, they were not salt sensitive and ET-1 excretion was unchanged. These data identify diet-induced obesity as a sex-specific disruptive factor for maintaining proper sodium handling. Although high-fat diets induce hypertension in both sexes, these data reveal that males are at greater risk of salt-dependent hypertension and further suggest that females have more redundant systems that can be productive against salt-sensitive hypertension in at least some circumstances.

Endothelin system expression in the kidney following cisplatin-induced acute kidney injury in male and female mice.

The chemotherapeutic agent cisplatin accumulates in the kidney and induces acute kidney injury (AKI). Preclinical and clinical studies suggest that young female mice and women show greater recovery from cisplatin-AKI compared to young male mice and men. The endothelin (ET) and ET receptors are enriched in the kidney and may be dysfunctional in cisplatin-AKI; however, there is a gap in our knowledge about the putative effects of sex and cisplatin on the renal ET system. We hypothesized that cisplatin-AKI male and female mice will have increased expression of the renal ET system. As expected, all cisplatin-AKI mice had kidney damage and body weight loss greater than control mice. Cisplatin-AKI mice had greater cortical Edn1, Edn3, Ednra, and Ednrb, while outer medullary Ednra was significantly suppressed in both sexes. Of the ∼25 000 genes sequenced from the inner medulla, only 91 genes (comparing saline mice) and 134 genes (comparing cisplatin-AKI mice) were differentially expressed and they were unrelated to the ET system. However, Edn1 was significantly greater in the inner medulla of male and female cisplatin-AKI mice. Thus, RNA profiles of the ET system were significantly affected by cisplatin-AKI throughout the kidney regardless of sex and this may help determine the therapeutic potential of targeting the ET receptors in cisplatin-AKI.

Global PER1 knockout Dahl Salt Sensitive rats show increased expression of renal Edn1 mRNA and Endothelin‐1 peptide

BackgroundPreviously, we showed that global knockout of the circadian clock protein PERIOD1 (PER1) in Dahl Salt Sensitive (SS) rats resulted in worsening of the SS hypertensive phenotype (1). PER1 KO rats also exhibited evidence of renal injury and increased plasma aldosterone levels after three weeks on a high salt diet (4% NaCl). Because we previously showed that PER1 negatively regulates the peptide hormone Endothelin‐1 (ET‐1) via transcriptional control of the ET‐1‐encoding gene Edn1, we wanted to investigate ET‐1 regulation in this novel rat model. Interestingly, Edn1 is also positively regulated by aldosterone through the mineralocorticoid receptor (MR).HypothesisGiven that ET‐1 signaling can be pro‐hypertensive and profibrotic in the kidney, we hypothesized that ET‐1 peptide levels in the kidney may be upregulated as part of the phenotype of the PER1 KO SS rats.MethodsProtein and total RNA were isolated from the kidney samples of control and PER1 KO SS rats on normal diet (0.4% NaCl) or after three weeks on a high salt (4% NaCl) diet. Renal ET‐1 peptide and plasma aldosterone levels were measured by ELISA and quantitative PCR was performed to quantify the renal mRNA levels of ET‐1 and its receptors ETA and ETB, MR, and GR. Renal MR protein levels were measured by Western Blot and quantified by relative densitometry. Data were evaluated for genotype or diet differences using ANOVA.ResultsRenal ET‐1 peptide levels were significantly higher in PER1 KO rats on high salt diet when compared to control SS rats (p=0.0003), as were plasma aldosterone levels (p=0.0156). There was also a significant genotype difference in Edn1 mRNA levels, with increased levels in PER1 KO rats on a high salt diet (p=0.0389). In contrast, significant differences were not observed in the mRNA levels of ET‐1 receptors Ednra and Ednrb between genotypes or diets. Importantly, quantification of renal MR protein levels showed marked increase with genotype and treatment effects (genotype: p=0.0002, treatment: p=0.0133).ConclusionsThese data suggest that PER1 KO results in increased ET‐1 in the kidney, due to increased transcription of the Edn1 gene, with increased plasma aldosterone and renal MR protein levels. This apparent negative regulation of ET‐1 by PER1, coupled with increased MR and aldosterone levels, is likely to contribute to the hypertensive and renal injury phenotype of PER1 KO SS rats on a high salt diet. Together these data show an important role for PER1 and its target ET‐1 in salt‐sensitive hypertension and the accompanying renal injury.Reference1 Spires DR, Zietara A, Levchenko V, Gumz ML, Staruschenko A. Knockout of Per1 Exacerbates the Hypertensive Phenotype of the Dahl Salt Sensitive Rat. FASEB J. 2020;34(S1):1.

Bevacizumab Increases Endothelin-1 Production via Forkhead Box Protein O1 in Human Glomerular Microvascular Endothelial Cells In Vitro.

Molecular mechanisms underlying the nephrotoxicity associated with bevacizumab are unclear. Endothelin-1 (ET-1) is involved in podocyte injury and proteinuria, and its level increases in most cases of kidney disorders. Forkhead box protein O1 (FoxO1), a transcription factor, is a major determinant of ET-1 promoter activation and is regulated by protein kinase B (Akt) phosphorylation-dependent nuclear exclusion. We evaluated the effect of bevacizumab on ET-1 production in human glomerular microvascular endothelial cells (hGECs). We analyzed the changes in the mRNA and protein levels of ET-1 in hGECs treated with bevacizumab using real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Changes in the protein levels and phosphorylation status of Akt and FoxO1 in hGECs treated with bevacizumab were analyzed by western blotting. After cell lysis, FoxO1 protein was isolated from the cytoplasmic and nuclear fractions. We also investigated the effects of AS1842856 (a FoxO1 inhibitor) on bevacizumab-induced ET-1 production. Bevacizumab significantly and dose-dependently increased the mRNA and protein levels of ET-1 in hGECs (p < 0.05). Bevacizumab treatment also led to a decrease in phosphorylated Akt protein levels. Inhibition of Akt activity by LY294002 promoted ET-1 production. Bevacizumab also induced an increase in FoxO1 protein levels in the nucleus. Inhibition of FoxO1 activity by AS1842856 resulted in decreased ET-1 levels in bevacizumab-treated hGECs. ET-1 axis activation, Akt inactivation, and FoxO1 nuclear localization are the molecular mechanisms underlying bevacizumab-induced nephrotoxicity. Therefore, inhibition of renal ET-1 production could be a promising approach to protect against or treat bevacizumab-induced nephrotoxicity.

Study of transcranial Doppler ultrasound and endothelin-1 in children with sickle cell anemia: a single-center study

To assess the impact of our transcranial Doppler (TCD) screening program on the incidence of a first stroke in children with sickle cell anemia and to study the role of elevated serum endothelin-1 (an inflammatory mediator) in these children. Background: stroke is a major complication of sickle cell disease (SCD), even in very young children. About 11% of children with homozygous sickle cell anemia (SS) develop stroke by the end of the second decade of life. The underlying etiology in most cases is an ischemic stroke caused by large-vessel stenosis or occlusion. Transcranial Doppler (TCD) recommended as a routine screening test to identify children at high risk of developing a stroke, measures flow velocities within large intracranial arteries. TCD should be routinely performed in children between 2 and 16 years as this age group is at the highest risk of sickle cerebral vasculopathy. We carried out a prospective case-control study which included 2 groups: a patient group consisted of 30 children with sickle cell anemia and sickle thalassemia and a group of 30 healthy children of matched age and sex. Each group included 11 males (36.5%) and 19 females (63.5%); the age range was 2 to 17 years. Both groups underwent a thorough clinical examination and laboratory tests (CBC, liver and renal function, serum ferritin and endothelin-1). Additionally, TCD was performed in all children included in the patient group. According to the results of TCD, time-averaged mean of the maximum velocity (TAMMX) was &lt; 170 cm/s (normal), 170–200 cm/s (conditional), ≥ 200 cm/s (high risk) in 20 (66.7%), 4 (13.3%) 6 (20%) patients, respectively. The level of endothelin-1 was significantly higher in the patients (57.1 ± 91.3) than in the controls (21.9 ± 14.8). Hemoglobin concentration was significantly lower in the patient group than in the control group, but the levels of reticulocytes, WBCs and serum ferritin were significantly higher in the patients than in the healthy controls. Serum Endotheline-1 level was higher in patients with sickle cell anemia than control group.

Effects of systemic and renal intramedullary endothelin-1 receptor blockade on tissue NO and intrarenal hemodynamics in normotensive and hypertensive rats

Endothelin 1 (ET-1) seems essential in salt-dependent hypertension, and activation of ETA receptors causes renal vasoconstriction. However, the response in the renal medulla and the role of tissue NO availability has never been adequately explored in vivo. We examined effects of ETA and ETB receptor blockade (atrasentan and BQ788) on blood pressure (MAP), medullary blood flow (MBF) and medullary tissue NO. Effects of systemic and intramedullary blocker application were compared in anesthetized normotensive ET-1-pretreated Sprague-Dawley rats (S-D), in salt-dependent hypertension (HS/UNX) and in spontaneously hypertensive rats (SHR). Total renal blood flow (RBF) was measured using a Transonic renal artery probe, MBF as laser-Doppler flux, and tissue NO signal using selective electrodes.In normotensive rats ET-1 significantly increased MAP, decreased RBF (−20%) and renal medullary NO. In HS/UNX rats atrasentan decreased MAP and increased medullary NO, earlier and more profoundly with intravenous infusion. In SHR atrasentan decreased MAP, more effectively with intravenous infusion; the increase in tissue NO (∼10%) was similar with both routes; however, only intramedullary atrasentan increased MBF. No consistent responses to BQ788 were seen.We confirmed dominant role of ETA receptors in regulation of blood pressure and renal hemodynamics in normotensive and hypertensive rats and provided novel evidence for the role of ETA in control of intrarenal NO bioavailability in salt-dependent and spontaneous hypertension. Under conditions of activation of the endothelin system ETB stimulation preserved medullary perfusion.

Endothelin receptors in renal interstitial cells do not contribute to the development of fibrosis during experimental kidney disease

Renal interstitial fibrosis is characterized by the development of myofibroblasts, originating from resident renal and immigrating cells. Myofibroblast formation and extracellular matrix production during kidney damage are triggered by various factors. Among these, endothelins have been discussed as potential modulators of renal fibrosis. Utilizing mouse models of adenine nephropathy (AN) and unilateral ureter occlusion (UUO), this study aimed to investigate the contribution of endothelin signaling in stromal mesenchymal resident renal interstitial cells. We found in controls that adenine feeding and UUO caused marked upregulations of endothelin-1 (ET-1) gene expression in endothelial and in tubular cells and a strong upregulation of ETA-receptor (ETA-R) gene expression in interstitial and mesangial cells, while the gene expression of ETB-receptor (ETB-R) did not change. Conditional deletion of ETA-R and ETB-R gene expression in the FoxD1 stromal cell compartment which includes interstitial cells significantly reduced renal ETA-R gene expression and moderately lowered renal ETB-R gene expression. ET receptor (ET-R) deletion exerted no apparent effects on kidney development nor on kidney function. Adenine feeding and UUO led to similar increases in profibrotic and proinflammatory gene expression in control as well as in ETAflflETBflfl FoxD1Cre+ mice (ET-Ko). In summary, our findings suggest that adenine feeding and UUO activate endothelin signaling in interstitial cells which is due to upregulated ETA-R expression and enhanced renal ET-1 production Our data also suggest that the activation of endothelin signaling in interstitial cells has less impact for the development of experimentally induced fibrosis.

Evaluation of left ventricle dysfunction and endothelin-1 level in arterial hypertension of renal genesis under influence of therapy

Background and Aims: Purpose: to study the aspects of left ventricle dysfunction (LVD) in patients with renal arterial hypertension (AH) and endothelin-1 level, to evaluate the effectiveness of ramipril.

G Protein‐Coupled Estrogen Receptor 1 is Required for Greater Endothelin‐1 Excretion in Female Mice

Estrogen signaling via G protein-coupled estrogen receptor 1 (GPER1) can elicit cardiovascular and renal protective actions. Endothelin-1 (ET-1) is a pro-natriuretic peptide that contributes to sex differences in blood pressure regulation and Na+ homeostasis. We recently showed that activation of GPER1 in the renal medulla promotes ET-1-dependent natriuresis in female but not male rats. Also, renal GPER1 expression is significantly higher in female rats than in male rats. In the current study, we hypothesized that genetic deletion of GPER1 would elevate blood pressure and impair renal ET-1 production/release in a sex-specific manner. Twelve-week-old male and female GPER1-knockout (KO) and wild-type (WT) littermate mice were implanted with telemeters to monitor blood pressure. Parallel groups of mice were placed into metabolic cages, and 24-h urine samples were collected while food and water intake were monitored. Then, animals were euthanized to obtain plasma. Urinary levels of electrolytes and ET-1 were measured. GPER1 deletion did not significantly affect mean arterial blood pressure in female (KO: 102±1 mmHg; WT: 102±2 mmHg; n=7-8/group, p=0.9) or male mice (KO: 106±3 mmHg; WT: 106±2 mmHg; n=6-10/group, p=1). No genotypic differences were observed in food intake, water intake, or urine flow rate. GPER1 deletion did not significantly affect urinary Na+excretion in female (KO: 0.25±0.05 μg/24 h; WT: 0.17±0.05 μg/24 h; n=9-12/group, p=0.52) or male mice (KO: 0.34±0.06 μg/24 h; WT: 0.35±0.04 μg/24 h; n=10-12/group, p=1.00). Similarly, GPER1 deletion did not affect urinary K+ excretion in either sex. However, GPER1 deletion decreased urinary ET-1 excretion in females (KO: 75.16±16.35 ng/24 h; WT: 172.83±24.67 ng/24 h; n=6-10/group, p<0.005) but not males (KO: 95.18±17.52 ng/24 h; WT: 76.03± 9.90 ng/24 h; n=10-12/group, p=0.69). Of note, WT females had significantly greater urinary ET-1 excretion than WT males (p=0.001), whereas KO mice had no sex differences in ET-1 excretion. Importantly, urinary excretion of ET-1 reflects intrarenal ET-1 production/release. GPER1 deletion did not significantly change plasma ET-1 concentration in either sex. Finally, GPER1 deletion did not affect urinary aldosterone excretion in females (KO: 315.98±73.13 pg/24 h; WT: 306.79±81.45 pg/24 h; n=7-8, p=1.00) or males (KO: 544.09±130.20 pg/24 h; WT: 481.84±68.30 pg/24 h; n=8/group, p=0.87). However, male mice had significantly greater urinary aldosterone excretion than female mice (p=0.04), regardless of genotype. Overall, we demonstrate that WT female mice have higher urinary ET-1 excretion than WT males, and that GPER1 deletion eliminates this sex-related difference in intrarenal ET-1 production/release. Our data suggest that GPER1 may contribute to the enhanced capacity of the female kidney to maintain Na+ homeostasis and have led us to hypothesize this as a mechanism for protecting females against salt-sensitive hypertension.

Therapeutic Effect of Endothelin-Converting Enzyme Inhibitor on Chronic Kidney Disease through the Inhibition of Endoplasmic Reticulum Stress and the NLRP3 Inflammasome.

Chronic inflammation and oxidative stress significantly contribute to the development and progression of chronic kidney disease (CKD). The NOD-like receptor family pyrin containing domain-3 (NLRP3) inflammasome plays a key role in the inflammatory response. The renal endothelin (ET) system is activated in all cases of CKD. Furthermore, ET-1 promotes renal cellular injury, inflammation, fibrosis and proteinuria. Endothelin-converting enzymes (ECEs) facilitate the final processing step of ET synthesis. However, the roles of ECEs in CKD are not clear. In this study, we investigated the effects of ETs and ECEs on kidney cells. We found that ET-1 and ET-2 expression was significantly upregulated in the renal tissues of CKD patients. ET-1 and ET-2 showed no cytotoxicity on human kidney tubular epithelial cells. However, ET-1 and ET-2 caused endoplasmic reticulum (ER) stress and NLRP3 inflammasome activation in tubular epithelial cells. The ECE inhibitor phosphoramidon induced autophagy. Furthermore, phosphoramidon inhibited ER stress and the NLRP3 inflammasome in tubular epithelial cells. In an adenine diet-induced CKD mouse model, phosphoramidon attenuated the progression of CKD by regulating autophagy, the NLRP3 inflammasome and ER stress. In summary, these findings showed a new strategy to delay CKD progression by inhibiting ECEs through autophagy activation and restraining ER stress and the NLRP3 inflammasome.

Circulating and Renal Endothelin-1 Expression Lead to Glomerular Impairment in High Fat-Induced Diet Wistar Rats

Circulating and Renal Endothelin-1 Expression Lead to Glomerular Impairment in High Fat-Induced Diet Wistar Rats

17-Hydroxyprogesterone caproate improves hypertension and renal endothelin-1 in response to sFlt-1 induced hypertension in pregnant rats.

Preeclampsia (PE) is characterized by new onset hypertension in association with elevated soluble fms-like tyrosine kinase-1 (sFlt-1) and preproendothelin-1 (PPET-1) levels. Currently there is no effective treatment for PE except for early delivery of the fetal placental unit, making PE a leading cause for premature births worldwide. Administration of 17-hydroxyprogesterone caproate (17-OHPC) is used for prevention of recurrent preterm birth. This study was designed to test the hypothesis that 17-OHPC improves hypertension and ET-1 in response to elevated sFlt-1 in pregnant rats. sFlt-1 was infused into normal pregnant (NP) Sprague-Dawley rats (3.7μg·kg-1·day-1 for 6days, gestation days 13-19) in the presence or absence of 17-OHPC (3.32mg/kg) administered via intraperitoneal injection on gestational days 15 and 18. Mean arterial blood pressure (MAP), pup and placenta weights, renal cortex PPET-1 mRNA levels and nitrate-nitrite levels were measured on GD 19. Infusion of sFlt-1 into NP rats elevated mean arterial pressure (MAP) compared with control NP rats: 115±1 (n=13) vs. 99±2mmHg (n=12, p<0.05). 17-OHPC attenuated this hypertension reducing MAP to 102±3mmHg in sFlt-1 treated pregnant rats (n=8). Neither pup nor placental weight was affected by sFlt-1 or 17-OHPC. Importantly, renal cortex PPET-1 mRNA levels were elevated 3 fold in NP+sFlt-1 rats compare to NP rats, which decreased with 17-OHPC administration. Plasma nitrate-nitrite levels were 44±9µM in NP rats (n=9), 20±3µM in NP+sFlt-1 (n=7), which increased to 42±11µM NP+sFlt-1+17OHPC (n=6). Administration of 17-OHPC improves clinical characteristics of preeclampsia in response to elevated sFlt-1 during pregnancy.