Male kidney-specific BMAL1 knockout mice are protected from K+-deficient, high-salt diet-induced blood pressure increases.

The circadian clock protein basic helix-loop-helix aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1) is a transcription factor that impacts kidney function, including blood pressure (BP) control. Previously, we have shown that male, but not female, kidney-specific cadherin Cre-positive BMAL1 knockout (KS-BMAL1 KO) mice exhibit lower BP compared with littermate controls. The goal of this study was to determine the BP phenotype and immune response in male KS-BMAL1 KO mice in response to a low-K+ high-salt (LKHS) diet. BP, renal inflammatory markers, and immune cells were measured in male mice following an LKHS diet. Male KS-BMAL1 KO mice had lower BP following the LKHS diet compared with control mice, yet their circadian rhythm in pressure remained unchanged. Additionally, KS-BMAL1 KO mice exhibited lower levels of renal proinflammatory cytokines and immune cells following the LKHS diet compared with control mice. KS-BMAL1 KO mice were protected from the salt-sensitive hypertension observed in control mice and displayed an attenuated immune response following the LKHS diet. These data suggest that BMAL1 plays a role in driving the BP increase and proinflammatory environment that occurs in response to an LKHS diet.NEW & NOTEWORTHY We show here, for the first time, that kidney-specific BMAL1 knockout mice are protected from blood pressure (BP) increases and immune responses to a salt-sensitive diet. Other kidney-specific BMAL1 knockout models exhibit lower BP phenotypes under basal conditions. A salt-sensitive diet exacerbates this genotype-specific BP response, leading to fewer proinflammatory cytokines and immune cells in knockout mice. These data demonstrate the importance of distal segment BMAL1 in BP and immune responses to a salt-sensitive environment.

Aldosterone: Renal Action and Physiological Effects.

Aldosterone exerts profound effects on renal and cardiovascular physiology. In the kidney, aldosterone acts to preserve electrolyte and acid-base balance in response to changes in dietary sodium (Na+ ) or potassium (K+ ) intake. These physiological actions, principally through activation of mineralocorticoid receptors (MRs), have important effects particularly in patients with renal and cardiovascular disease as demonstrated by multiple clinical trials. Multiple factors, be they genetic, humoral, dietary, or otherwise, can play a role in influencing the rate of aldosterone synthesis and secretion from the adrenal cortex. Normally, aldosterone secretion and action respond to dietary Na+ intake. In the kidney, the distal nephron and collecting duct are the main targets of aldosterone and MR action, which stimulates Na+ absorption in part via the epithelial Na+ channel (ENaC), the principal channel responsible for the fine-tuning of Na+ balance. Our understanding of the regulatory factors that allow aldosterone, via multiple signaling pathways, to function properly clearly implicates this hormone as central to many pathophysiological effects that become dysfunctional in disease states. Numerous pathologies that affect blood pressure (BP), electrolyte balance, and overall cardiovascular health are due to abnormal secretion of aldosterone, mutations in MR, ENaC, or effectors and modulators of their action. Study of the mechanisms of these pathologies has allowed researchers and clinicians to create novel dietary and pharmacological targets to improve human health. This article covers the regulation of aldosterone synthesis and secretion, receptors, effector molecules, and signaling pathways that modulate its action in the kidney. We also consider the role of aldosterone in disease and the benefit of mineralocorticoid antagonists. © 2023 American Physiological Society. Compr Physiol 13:4409-4491, 2023.

Circadian clocks of the kidney: function, mechanism, and regulation.

An intrinsic cellular circadian clock is located in nearly every cell of the body. The peripheral circadian clocks within the cells of the kidney contribute to the regulation of a variety of renal processes. In this review, we summarize what is currently known regarding the function, mechanism, and regulation of kidney clocks. Additionally, the effect of extrarenal physiological processes, such as endocrine and neuronal signals, on kidney function is also reviewed. Circadian rhythms in renal function are an integral part of kidney physiology, underscoring the importance of considering time of day as a key biological variable. The field of circadian renal physiology is of tremendous relevance, but with limited physiological and mechanistic information on the kidney clocks this is an area in need of extensive investigation.

Acclimation to a High-Salt Diet Is Sex Dependent.

Background Premenopausal women are less likely to develop hypertension and salt-related complications than are men, yet the impact of sex on mechanisms regulating Na+ homeostasis during dietary salt challenges is poorly defined. Here, we determined whether female rats have a more efficient capacity to acclimate to increased dietary salt intake challenge. Methods and Results Age-matched male and female Sprague Dawley rats maintained on a normal-salt (NS) diet (0.49% NaCl) were challenged with a 5-day high-salt diet (4.0% NaCl). We assessed serum, urinary, skin, and muscle electrolytes; total body water; and kidney Na+ transporters during the NS and high-salt diet phases. During the 5-day high-salt challenge, natriuresis increased more rapidly in females, whereas serum Na+ and body water concentration increased only in males. To determine if females are primed to handle changes in dietary salt, we asked the question whether the renal endothelin-1 natriuretic system is more active in female rats, compared with males. During the NS diet, female rats had a higher urinary endothelin-1 excretion rate than males. Moreover, Ingenuity Pathway Analysis of RNA sequencing data identified the enrichment of endothelin signaling pathway transcripts in the inner medulla of kidneys from NS-fed female rats compared with male counterparts. Notably, in human subjects who consumed an Na+-controlled diet (3314-3668 mg/day) for 3 days, women had a higher urinary endothelin-1 excretion rate than men, consistent with our findings in NS-fed rats. Conclusions These results suggest that female sex confers a greater ability to maintain Na+ homeostasis during acclimation to dietary Na+ challenges and indicate that the intrarenal endothelin-1 natriuretic pathway is enhanced in women.

Circadian Rhythm Effects on the Molecular Regulation of Physiological Systems.

Nearly every system within the body contains an intrinsic cellular circadian clock. The circadian clock contributes to the regulation of a variety of homeostatic processes in mammals through the regulation of gene expression. Circadian disruption of physiological systems is associated with pathophysiological disorders. Here, we review the current understanding of the molecular mechanisms contributing to the known circadian rhythms in physiological function. This article focuses on what is known in humans, along with discoveries made with cell and rodent models. In particular, the impact of circadian clock components in metabolic, cardiovascular, endocrine, musculoskeletal, immune, and central nervous systems are discussed. © 2021 American Physiological Society. Compr Physiol 11:1-30, 2021.

Endothelin B receptors impair baroreflex function and increase blood pressure variability during high salt diet.

Baroreflex function is an integral component maintaining consistent blood pressure. Hypertension is often associated with baroreflex dysfunction, and environmental risk factors such as high salt diet exacerbate hypertension in subjects with baroreflex dysfunction. However, the interactions between high salt diet, baroreflex dysfunction, and hypertension are incompletely understood. The endothelin system is another potent mediator of blood pressure control especially in response to a high salt diet. We hypothesized that the endothelin B (ETB) receptor activation on adrenergic nerves decreases baroreflex sensitivity. We utilized male ETB receptor deficient (ETB-def) rats that express functional ETB receptors only on adrenergic nerves and transgenic (TG) controls to evaluate baroreflex function during normal (0.49% NaCl) and high (4.0% NaCl) salt diets. In conscious rats equipped with telemetry, ETB-def rats had an increased lability of systolic blood pressure (SBP) compared to TG controls as indicated by higher standard deviation (SD) of SBP under both normal (10.2 ± 0.6 vs. 12.4 ± 0.9 mmHg, respectively, p = 0.0001) and high (11.7 ± 0.6 vs. 16.1 ± 1.0 mmHg, p = 0.0001) salt diets. In anesthetized preparations, ETB-def rats displayed reduced heart rate (p genotype = 0.0167) and renal sympathetic nerve (p genotype = 0.0022) baroreflex sensitivity. We then gave male Sprague-Dawley rats the selective ETB receptor antagonist, A-192621 (10 mg/kg/day), to block ETB receptors. Following ETB receptor antagonism, even though SBP increased (131 ± 7 before vs. 152 ± 8 mmHg after, p < 0.0001), the lability (standard deviation) of SBP decreased (9.3 ± 2.0 vs. 7.1 ± 1.1 mmHg, p = 0.0155). These data support our hypothesis that ETB receptors on adrenergic nerves contribute to baroreflex dysfunction.

Diurnal Control of Blood Pressure Is Uncoupled From Sodium Excretion.

The diurnal rhythms of sodium handling and blood pressure are thought to be regulated by clock genes, such as Bmal1. However, little is known about the regulation of these factors by Bmal1, especially in rats. Using a novel whole-body Bmal1 knockout rat model (Bmal1-/-), we hypothesized that time of day regulation of sodium excretion is dependent on Bmal1. Using telemetry to continuously record mean arterial pressure, we observed that male and female Bmal1-/- rats had significantly reduced mean arterial pressure over the course of 24 hours compared with littermate controls. The circadian mean arterial pressure pattern remained intact in both sexes of Bmal1-/- rats, which is in contrast to the Bmal1-/- mouse model. Male Bmal1-/- rats had no significant difference in baseline sodium excretion between 12-hour active and inactive periods, indicating a lack of diurnal control independent of maintained mean arterial pressure rhythms. Female Bmal1-/- rats, however, had significantly greater sodium excretion during the active versus inactive period similar to controls. Thus, we observed a clear dissociation between circadian blood pressure and control of sodium excretion that is sex dependent. These findings are consistent with a more robust ability of females to maintain control of sodium excretion, and furthermore, demonstrate a novel role for Bmal1 in control of diurnal blood pressure independent of sodium excretion.

Differences in renal BMAL1 contribution to Na+ homeostasis and blood pressure control in male and female mice.

The renal circadian clock has a major influence on the function of the kidney. Aryl hydrocarbon receptor nuclear translocator-like protein 1 [ARNTL; also known as brain and muscle ARNT-like 1 (BMAL1)] is a core clock protein and transcription factor that regulates the expression of nearly half of all genes. Using male and female kidney-specific cadherin BMAL1 knockout (KS-BMAL1 KO) mice, we examined the role of renal distal segment BMAL1 in blood pressure control and solute handling. We confirmed that this mouse model does not express BMAL1 in thick ascending limb, distal convoluted tubule, and collecting duct cells, which are the final locations for solute and fluid regulation. Male KS-BMAL1 KO mice displayed a substantially lower basal systolic blood pressure compared with littermate control mice, yet their circadian rhythm in pressure remained unchanged [male control mice: 127 ± 0.7 mmHg (n = 4) vs. male KS-BMAL KO mice: 119 ± 2.3 mmHg (n = 5), P < 0.05]. Female mice, however, did not display a genotype difference in basal systolic blood pressure [female control mice: 120 ± 1.6 mmHg (n = 5) vs. female KS-BMAL1 KO mice: 119 ± 1.5 mmHg (n = 7), P = 0.4]. In addition, male KS-BMAL1 KO mice had less Na+ retention compared with control mice in response to a K+-restricted diet (15% less following 5 days of treatment). However, there was no genotype difference in Na+ handling after a K+-restricted diet in female mice. Furthermore, there was evidence indicating a sex-specific response to K+ restriction where female mice reabsorbed less Na+ in response to this dietary challenge compared with male mice. We propose that BMAL1 in the distal nephron and collecting duct contributes to blood pressure regulation and Na+ handling in a sex-specific manner.

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.

Greater natriuretic response to ENaC inhibition in male versus female Sprague-Dawley rats.

Genes for the epithelial sodium channel (ENaC) subunits are expressed in a circadian manner, but whether this results in time-of-day differences in activity is not known. Recent data show that protein expression of ENaC subunits is higher in kidneys from female rats, yet females are more efficient in excreting an acute salt load. Thus, our in vivo study determined whether there is a time-of-day difference as well as a sex difference in the response to ENaC inhibition by benzamil. Our results showed that the natriuretic and diuretic responses to a single dose of benzamil were significantly greater in male compared with female rats whether given at the beginning of the inactive period [Zeitgeber time 0 (ZT0), 7 AM] or active period (ZT12, 7 PM). However, the response to benzamil was not significantly different between ZT0 and ZT12 dosing in either male or female rats. There was no difference in renal cortical α-ENaC protein abundance between ZT0 and ZT12 or males and females. Given previous reports of flow-induced stimulation of endothelin-1 (ET-1) production and sex differences in the renal endothelin system, we measured urinary ET-1 excretion to assess the effects of increased urine flow on intrarenal ET-1. ET-1 excretion was significantly increased following benzamil administration in both sexes, but this increase was significantly greater in females. These results support the hypothesis that ENaC activity is less prominent in maintaining Na+ balance in females independent of renal ET-1. Because ENaC subunit genes and protein expression vary by time of day and are greater in female rat kidneys, this suggests a clear disconnect between ENaC expression and channel activity.

Diurnal pattern in skin Na+ and water content is associated with salt-sensitive hypertension in ETB receptor-deficient rats.

Impairment in the ability of the skin to properly store Na+ nonosmotically (without water) has recently been hypothesized as contributing to salt-sensitive hypertension. Our laboratory has shown that endothelial production of endothelin-1 (ET-1) is crucial to skin Na+ handling. Furthermore, it is well established that loss of endothelin type B receptor (ETB) receptor function impairs Na+ excretion by the kidney. Thus we hypothesized that rats lacking functional ETB receptors (ETB-def) will have a reduced capacity of the skin to store Na+ during chronic high-salt (HS) intake. We observed that ETB-def rats exhibited salt-sensitive hypertension with an approximate doubling in the diurnal amplitude of mean arterial pressure compared with genetic control rats on a HS diet. Two weeks of HS diet significantly increased skin Na+ content relative to water; however, there was no significant difference between control and ETB-def rats. Interestingly, HS intake led to a 19% increase in skin Na+ and 16% increase in water content (relative to dry wt.) during the active phase (zeitgeber time 16) versus inactive phase (zeitgeber time 4, P < 0.05) in ETB-def rats. There was no significant circadian variation in total skin Na+ or water content of control rats fed normal or HS. These data indicate that ETB receptors have little influence on the ability to store Na+ nonosmotically in the skin during long-term HS intake but, rather, appear to regulate diurnal rhythms in skin Na+ content and circadian blood pressure rhythms associated with a HS diet.

Circadian regulation of renal function.

The kidneys regulate many vital functions that require precise control throughout the day. These functions, such as maintaining sodium balance or regulating arterial pressure, rely on an intrinsic clock mechanism that was commonly believed to be controlled by the central nervous system. Mounting evidence in recent years has unveiled previously underappreciated depth of influence by circadian rhythms and clock genes on renal function, at the molecular and physiological level, independent of other external factors. The impact of circadian rhythms in the kidney also affects individuals from a clinical standpoint, as the loss of rhythmic activity or clock gene expression have been documented in various cardiovascular diseases. Fortunately, the prognostic value of examining circadian rhythms may prove useful in determining the progression of a kidney-related disease, and chronotherapy is a clinical intervention that requires consideration of circadian and diurnal rhythms in the kidney. In this review, we discuss evidence of circadian regulation in the kidney from basic and clinical research in order to provide a foundation on which a great deal of future research is needed to expand our understanding of circadian relevant biology.

High dietary sodium causes dyssynchrony of the renal molecular clock in rats.

Speed JS, Hyndman KA, Roth K, Heimlich JB, Kasztan M, Fox BM, Johnston JG, Becker BK, Jin C, Gamble KL, Young ME, Pollock JS, Pollock DM. High dietary sodium causes dyssynchrony of the renal molecular clock in rats. Am J Physiol Renal Physiol 314: F89-F98, 2018. First published September 27, 2017; doi:10.1152/ajprenal.00028.2017.-Dyssynchrony of circadian rhythms is associated with various disorders, including cardiovascular and metabolic diseases. The cell autonomous molecular clock maintains circadian control; however, environmental factors that may cause circadian dyssynchrony either within or between organ systems are poorly understood. Our laboratory recently reported that the endothelin (ET-1) B (ETB) receptor functions to facilitate Na+ excretion in a time of day-dependent manner. Therefore, the present study was designed to determine whether high salt (HS) intake leads to circadian dyssynchrony within the kidney and whether the renal endothelin system contributes to control of the renal molecular clock. We observed that HS feeding led to region-specific alterations in circadian clock components within the kidney. For instance, HS caused a significant 5.5-h phase delay in the peak expression of Bmal1 and suppressed Cry1 and Per2 expression in the renal inner medulla, but not the renal cortex, of control rats. The phase delay in Bmal1 expression appears to be mediated by ET-1 because this phenomenon was not observed in the ETB-deficient rat. In cultured inner medullary collecting duct cells, ET-1 suppressed Bmal1 mRNA expression. Furthermore, Bmal1 knockdown in these cells reduced epithelial Na+ channel expression. These data reveal that HS feeding leads to intrarenal circadian dyssynchrony mediated, in part, through activation of ETB receptors within the renal inner medulla.

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.

Endothelin and renal ion and water transport.

The renal tubular epithelial cells produce more endothelin-1 (ET-1) than any other cell type in the body. Moving down the nephron, the amount of ET-1 produced appears fairly consistent until reaching the inner medullary collecting duct, which produces at least 10 times more ET-1 than any other segment. ET-1 inhibits Na(+) transport in all parts of the nephron through activation of the ETB receptor, and, to a minor extent, the ETA receptor. These effects are most prominent in the collecting duct where ETB-receptor activation inhibits activity of the epithelial Na(+) channel. Effects in other parts of the nephron include inhibition of Na(+)/H(+) exchange in the proximal tubule and the Na(+), K(+), 2Cl(-) co-transporter in the thick ascending limb. In general, the renal epithelial ET-1 system is an integral part of the body's response to a high salt intake to maintain homeostasis and normal blood pressure. Loss of ETB-receptor function results in salt-sensitive hypertension. The role of renal ET-1 and how it affects Na(+) and water transport throughout the nephron is reviewed.