Loss of circadian gene Bmal1 in the collecting duct lowers blood pressure in male, but not female, mice

Kidney function follows a 24-h rhythm subject to regulation by circadian genes including the transcription factor Bmal1. A high-salt diet induces a phase shift in Bmal1 expression in the renal inner medulla that is dependent on endothelin type B (ETB) receptors. Furthermore, ETB receptor-mediated natriuresis is sex dependent. Therefore, experiments tested the hypothesis that collecting duct Bmal1 regulates blood pressure in a sex-dependent manner. We generated a mouse model that lacks Bmal1 expression in the collecting duct, where ETB receptor abundance is highest. Male, but not female, collecting duct Bmal1 knockout (CDBmal1KO) mice had significantly lower 24-h mean arterial pressure (MAP) than flox controls (105 ± 2 vs. 112 ± 3 mmHg for male mice and 106 ± 1 vs. 108 ± 1 mmHg for female mice, by telemetry). After 6 days on a high-salt (4% NaCl) diet, MAP remained significantly lower in male CDBmal1KO mice than in male flox control mice (107 ± 2 vs. 113 ± 1 mmHg), with no significant differences between genotypes in female mice (108 ± 2 vs. 109 ± 1 mmHg). ETB receptor blockade for another 6 days increased MAP similarly in both male and female CDBmal1KO and flox control mice. However, MAP remained lower in male CDBmal1KO mice than in male flox control mice (124 ± 2 vs. 130 ± 2 mmHg). No significant differences were observed between female CDBmal1KO and flox mice during ETB blockade (130 ± 2 vs. 127 ± 2 mmHg). There were no significant genotype differences in amplitude or phase of MAP in either sex. These data suggest that collecting duct Bmal1 has no role in circadian MAP but plays an important role in overall blood pressure in male, but not female, mice.

Oxidative stress induces BH4 deficiency in male, but not female, SHR

We previously published that female spontaneously hypertensive rats (SHR) have significantly greater nitric oxide (NO) bioavailability and NO synthase (NOS) enzymatic activity in the renal inner medulla (IM) compared with age-matched males, although the mechanism responsible remains unknown. Tetrahydrobiopterin (BH4) is a critical cofactor required for NO generation, and decreases in BH4 as a result of increases in oxidative stress have been implicated in the pathogenesis of hypertension. As male SHR are known to have higher levels of oxidative stress compared with female SHR, we hypothesized that relative BH4 deficiency induced by oxidative stress in male SHR results in lower levels of NOS activity in renal IM compared with females. Twelve-week-old male and female SHR were randomized to receive tempol (30 mg/kg/day via drinking water) or vehicle for 2 weeks. Tempol treatment did not affect blood pressure (BP) in either sex, but reduced peroxynitrite levels only in males. Females had more total biopterin, dihydrobiopterin (BH2), and BH4 levels in renal IMs than males, and tempol treatment eliminated these sex differences. Females had greater total NOS activity in the renal IM than males, and adding exogenous BH4 to the assay increased NOS activity in both sexes. This sex difference in total NOS and the effect of exogenous BH4 were abolished with tempol treatment. We conclude that higher oxidative stress in male SHR results in a relative deficiency of BH4 compared with females, resulting in diminished renal NOS activity in the male.

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

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.

Architecture of the human renal inner medulla and functional implications

The architecture of the inner stripe of the outer medulla of the human kidney has long been known to exhibit distinctive configurations; however, inner medullary architecture remains poorly defined. Using immunohistochemistry with segment-specific antibodies for membrane fluid and solute transporters and other proteins, we identified a number of distinctive functional features of human inner medulla. In the outer inner medulla, aquaporin-1 (AQP1)-positive long-loop descending thin limbs (DTLs) lie alongside descending and ascending vasa recta (DVR, AVR) within vascular bundles. These vascular bundles are continuations of outer medullary vascular bundles. Bundles containing DTLs and vasa recta lie at the margins of coalescing collecting duct (CD) clusters, thereby forming two regions, the vascular bundle region and the CD cluster region. Although AQP1 and urea transporter UT-B are abundantly expressed in long-loop DTLs and DVR, respectively, their expression declines with depth below the outer medulla. Transcellular water and urea fluxes likely decline in these segments at progressively deeper levels. Smooth muscle myosin heavy chain protein is also expressed in DVR of the inner stripe and the upper inner medulla, but is sparsely expressed at deeper inner medullary levels. In rodent inner medulla, fenestrated capillaries abut CDs along their entire length, paralleling ascending thin limbs (ATLs), forming distinct compartments (interstitial nodal spaces; INSs); however, in humans this architecture rarely occurs. Thus INSs are relatively infrequent in the human inner medulla, unlike in the rodent where they are abundant. UT-B is expressed within the papillary epithelium of the lower inner medulla, indicating a transcellular pathway for urea across this epithelium.

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.

Abstract P082: Mitochondrial Gene Expression is Decreased in Renal Inner Medulla of Non-human Primates with Spontaneous Hypertension

Mitochondrial gene expression may influence renal function and consequently, long-term blood pressure control. The African Green Monkey (Chlorocebus aethiops sabaeus; AGM) exhibits heritable, spontaneous hypertension and thus is a translational model for the study of human essential hypertension. We hypothesized that renal mitochondrial gene expression in hypertensive AGMs is decreased and may contribute to renal mitochondrial dysfunction in specific kidney regions. AGMs were phenotyped as normotensive (NT, systolic blood pressure; SBP <120 mmHg) or hypertensive (HT, SBP > 140 mmHg) by forearm plethysmography. Gene expression was determined using qRT-PCR with RNA extracted from renal cortex, outer medulla (OM), inner medulla (IM), and liver of 18 HT (mean SBP 166±7 mmHg) and 18 NT (mean SBP 98±3 mmHg) animals. In renal cortical and OM tissue of HT vervets, COX 3 (Complex IV), Cyt B (Complex III), NADH4 (Complex I) and ATP8 (Complex V) expression were similar in NT and HT AGMs. In IM of HT AGMs, COX3, NADH4, ATP8, and CYTB were downregulated by 4.7-fold (p=0.007), 4-fold (p=0.002), 4.1-fold (p=0.006), and 3-fold (p=0.018), respectively. In the liver, COX 3 expression was decreased 1.9-fold (p=0.04), 1.6-fold for ATP8 (p=0.03), and 2.0-fold for CYTB (p=0.01). Expression of SDH (Complex II) and COX4 (Complex IV), nuclear encoded subunits of the OXPHOS chain, was also assessed. SDH expression was up-regulated 8-fold in renal OM (p=0.005) but unchanged in liver, IM, and cortex. COX4 expression however, was down-regulated in OM by 8-fold (p=0.05), in IM by 5-fold (p=0.011) but unchanged in cortex and liver. Gene expression of the mitochondrial transcription factor TFAM was downregulated by 4-fold in renal OM, and unchanged in renal cortex and liver. Citrate synthase activity showed no difference in mitochondrial number between NT and HT AGMs (p=0.73). We suggest that reduced expression of mitochondrially encoded OXPHOS subunits in the renal IM may contribute to the development of hypertension in the AGM. Mitochondrial gene down-regulation in the liver may be a consequence of the systemic hypertension. We conclude that altered mitochondrial gene expression may be a cellular response to increased oxidative stress in the renal inner medulla of HT AGM.

Quercetin attenuates cyclooxygenase-2 expression in response to acute ureteral obstruction

Unilateral ureteral obstruction (UUO) is associated with increased hydrostatic pressure, inflammation, and oxidative stress in the renal parenchyma. Previous studies have demonstrated marked cyclooxygenase (COX)-2 induction in renal medullary interstitial cells (RMICs) in response to UUO. The aim of the present study was to evaluate the effect of quercetin, a naturally occurring antioxidant, on COX-2 induction in vivo and in vitro. Rats subjected to 24 h of UUO were treated intraperitoneally with quercetin (50 mg·kg−1·day−1). Quercetin partly prevented COX-2 induction in the renal inner medulla in response to UUO. Moreover, RMICs exposed to conditions associated with obstruction, inflammation (produced by IL-1β), oxidative stress (produced by H2O2), and mechanical stress (produced by stretch) showed increased COX-2 expression. Interestingly, quercetin reduced COX-2 induction in RMICs subjected to stretched. Similarly, PGE2 production was markedly increased in RMICs exposed to stretch and was reversed to control levels by quercetin treatment. Furthermore, stretch-induced phosphorylation of ERK1/2 was blocked by quercetin, and inhibition of ERK1/2 attenuated stretch-induced COX-2 induction in RMICs. These results indicate that quercetin attenuated the induction of COX-2 expression and activity in RMICs exposed to mechanical stress as a consequence of acute UUO and that the MAPK ERK1/2 pathway might be involved in this quercetin-mediated reduction in COX-2.