Timing of food intake has become a critical factor in determining overall cardiometabolic health. We hypothesized that timing of food intake entrains circadian rhythms of blood pressure and renal excretion in mice. Male C57BL/6J mice were fed ad libitum or reverse feeding (RF) where food was available at all times of day or only available during the 12-hour lights-on period, respectively. Mice eating ad libitum had a significantly higher mean arterial pressure (MAP) during lights-off compared to lights-on (113 ± 2 vs 100 ± 2 mmHg, respectively; p < 0.0001); however, RF for 6 days inverted the diurnal rhythm of MAP (99 ± 3 vs 110 ± 3 mmHg, respectively; p < 0.0001). In contrast to MAP, diurnal rhythms of urine volume and sodium excretion remained intact after RF. Male Bmal1 knockout mice (Bmal1KO) underwent the same feeding protocol. As previously reported, Bmal1KO mice did not exhibit a diurnal MAP rhythm during ad libitum feeding (95 ± 1 vs 92 ± 3 mmHg, lights-off vs lights-on; p > 0.05); however, RF induced a diurnal rhythm of MAP (79 ± 3 vs 95 ± 2 mmHg, lights-off vs lights-on phase; p < 0.01). Transgenic PERIOD2::LUCIFERASE knock-in mice were used to assess the rhythm of the clock protein PERIOD2 in ex vivo tissue cultures. The timing of the PER2::LUC rhythm in the renal cortex and suprachiasmatic nucleus was not affected by RF; however, RF induced significant phase shifts in the liver, renal inner medulla and adrenal gland. In conclusion, the timing of food intake controls blood pressure rhythms in mice independent of Bmal1, urine volume or sodium excretion.
Histone deacetylase (HDAC) enzymes regulate transcription through epigenetic modification of chromatin structure, but their specific functions in the kidney remain elusive. We discovered that the human kidney expresses class I HDACs. Kidney medulla-specific inhibition of class I HDACs in the rat during high-salt feeding results in hypertension, polyuria, hypokalemia, and nitric oxide deficiency. Three new inducible murine models were used to determine that HDAC1 and HDAC2 in the kidney epithelium are necessary for maintaining epithelial integrity and maintaining fluid-electrolyte balance during increased dietary sodium intake. Moreover, single-nucleus RNA-sequencing determined that epithelial HDAC1 and HDAC2 are necessary for expression of many sodium or water transporters and channels. In performing a systematic review and meta-analysis of serious adverse events associated with clinical HDAC inhibitor use, we found that HDAC inhibitors increased the odds ratio of experiencing fluid-electrolyte disorders, such as hypokalemia. This study provides insight on the mechanisms of potential serious adverse events with HDAC inhibitors, which may be fatal to critically ill patients. In conclusion, kidney tubular HDACs provide a link between the environment, such as consumption of high-salt diets, and regulation of homeostatic mechanisms to remain in fluid-electrolyte balance.
Exposure to early life stress (ELS) is associated with a greater risk of chronic disease development including depression and cardiovascular disease. Altered gut microbiota has been linked to both depression and cardiovascular disease in mice and humans. Rodent models of early life neglect are used to characterize the mechanistic links between early life stress (ELS) and the risk of disease later in life. However, little is understood about ELS exposure and the gut microbiota in the young mice and the influence of the maternal inheritance of the gut microbiota. We used a mouse model of ELS, maternal separation with early weaning (MSEW), and normally-reared mice to determine whether the neonate microbiota is altered, and if so, are the differences attributable to changes in dam microbiota that are then transmitted to their offspring. Individual amplicon sequence variants (ASVs) displayed differential abundance in the microbiota of MSEW compared to normally-reared pups at post-natal day (PD)28. Additionally, ELS exposure reduced the alpha diversity and altered microbial community composition at PD28. The composition, levels of alpha diversity, and abundance of individual ASVs in the microbiota of dams were similar from MSEW or normally-reared cohorts. Thus, the observed shifts in the abundance of individual bacterial ASVs in the neonates and young pups are likely driven by endogenous effects of MSEW in the offspring host and are not due to inherited differences from the dam. This knowledge suggests that exposure to ELS has a direct effect on microbial factors on the risk of chronic disease development.
Exposure to early life stress (ELS) is associated with a greater risk of chronic disease development including depression, cardiovascular disease (CVD), and inflammatory bowel disease (IBD). Changes in the gut microbiota have been linked to IBD and CVD. Rodent models of early life neglect are used to characterize the mechanistic links between ELS exposure and the risk of disease later in life. However, little is understood about ELS exposure and the gut microbiota. We used a mouse model of ELS, maternal separation with early weaning (MSEW) and normally‐reared (NR) mice to determine whether the neonate microbiota is altered, and if so, whether the MSEW protocol induces changes in the dam microbiota that are then transmitted to their offspring. MSEW mice (n = 12) were subjected to maternal separation for 4 h per day on postnatal days (PDs) 2–5, and 8 h per day on PDs 6–16, and were subsequently weaned at PD17. NR mice (n = 13) remained undisturbed with dams until weaning on PD21. All mice were maintained on standard chow (NIH‐31; NSN 8710‐01‐005‐8438) following weaning. Paired‐end Illumina sequencing was conducted on colonic content from PD28 mice and DADA2 was utilized to infer and assign taxonomy to individual amplicon sequence variants (ASVs). The fecal microbiota of dams (n= 5) was also sequenced. Analysis of composition of microbiomes (ANCOM) detected 32 ASVs that had statistically different abundance in MSEW compared to NR mice at PD28. Five of the nine ASVs that increased in MSEW mice were classified as Clostridium senso stricto 1 or Lachnoclostridium, while none the of the 23 ASVs enriched in NR animals belonged to these taxa. Fifty‐seven percent (13 of 23) of the ASVs that had greater abundance in NR animals were classified as Lachnospiracea (other than Lachnoclostrium) or Ruminococcaceae. Compared to NR animals, MSEW PD28 mice had reduced phylogenetic diversity (18.7 ± 0.3 compared to 20.3 ± 0.3 in NR; P = 0.003) and Chao1ASV richness (175 ± 8 compared to 198 ± 8 in NR; P = 0.04). Microbial community composition was also altered at PD28 based permutational analysis of variance of Aitchison distance (F(1,24) = 2.29, P = 0.002). The composition, levels of diversity, and abundance of individual ASVs in the microbiota of dams were no different between MSEW and NR cohorts. Thus, the observed microbiota shifts in the young pups are likely driven by endogenous effects of MSEW in the offspring and independent of maternal inheritance. MSEW mice had lower microbial diversity and a reduction in Lachnospiraceae and Ruminococcaceae, which are important fermenters of short‐chain fatty acids (SCFAs). Nine different SCFAs were measured in plasma of PD28 mice and MSEW animals had reduced butyrate (P = 0.02) compared to NR controls. Butyrate promote colonic homeostasis as well as regulates metabolic health and CVD risk. Studies are on‐going to determine the impact of altered SCFA levels with ELS exposure. This study suggests a direct effect of ELS‐exposure on the gut microbiota, which may impact the risk of chronic disease...
Early life stress (ELS) is associated with cardiovascular disease (CVD) risk in adulthood, but the underlying vascular mechanisms are poorly understood. Increased hemoglobin and heme have recently been implicated to mediate endothelial dysfunction in several vascular diseases. Chronic physiological stress is associated with alterations in the heme pathway that have been well-described in the literature. However, very little is known about the heme pathway with exposure to ELS or chronic psychosocial stress. Utilizing a mouse model of ELS, maternal separation with early weaning (MSEW), we previously reported that MSEW induces endothelial dysfunction via increased superoxide production. We reasoned that heme dysregulation may be one of the culprits induced by MSEW and sustained throughout adulthood; thus, we hypothesized that MSEW induces heme dysfunction. We investigated whether circulating levels of heme, a circulating pro-oxidant mediator, are increased by MSEW and examined the role of the heme metabolic pathway and heme homeostasis in this process.We found that circulating levels of heme are increased in mice exposed to MSEW and that plasma from MSEW mice stimulated higher superoxide production in cultured mouse aortic endothelial cells (MAECs) compared to plasma from normally reared mice. The heme scavenger hemopexin blunted this enhanced superoxide production. Splenic haptoglobin abundance was significantly lower and hemoglobin levels per red blood cell were significantly higher in MSEW versus control mice. These findings lead us to propose that ELS induces increased circulating heme through dysregulation of the haptoglobin-hemoglobin system representing a mechanistic link between ELS
Circadian clock genes are important for vascular homeostasis. Loss of Bmal1 , a clock gene, impairs vascular function and blood pressure rhythm in mice. We previously reported that hepatocyte-specific Bmal1 deletion (HBK) in the liver alters perivascular adipose tissue-mediated vascular function in young adult mice, yet aortic collagen content and wall thickness in 4- to 6- month old HBK mice is similar to control genotype flox mice. To our knowledge, this is some of the first evidence that liver circadian clock disruption distally affects function in another tissue. We hypothesized that Bmal1 deletion in liver leads to vascular disease in older adult mice. Studies were performed in 8- to 11-month old male HBK and flox control mice. Aortic stiffness, measured by pulse wave velocity, was significantly higher in HBK mice compared to flox control mice (Flox: 1.93 ± 0.2 m/s; HBK: 3.3 ± 0.5 m/s; n = 7-8, p = 0.02). Light phase systolic blood pressure (tail-cuff) was similar in both flox control and HBK mice (Flox: 101 ± 1 mm Hg; HBK: 103 ± 2 mm Hg; n = 3-5, p = 0.35). Plasma and aortas were collected at ZT10 for metabolite measurements and histological analysis. Circulating plasminogen activator inhibitor-1 (PAI-1) was not different between genotypes. Picrosirius red (PSR)-stained aortic sections were examined under bright field or polarized light to assess collagen content with Metamorph software analysis. Aortic collagen content was not different between flox control and HBK mice under bright or polarized light (bright light, % area stained positive for PSR, Flox: 28.5 ± 2.4%; HBK: 23.6 ± 3.3%, p = 0.30; polarized, Flox: 16.7 ± 0.8%; HBK: 16.3 ± 1.3%; n = 4-5, p=0.82). TUNEL staining showed increased cellular apoptosis in aortas of HBK mice (Flox: 0.72 ± 0.3%; HBK: 2.73 ± 0.7%; n = 4, p = 0.04). Aortic wall thickness was measured as the difference between the external elastic lamina and the internal elastic lamina with CellSens software. Interestingly, aortic wall thickness was significantly lower in older HBK mice compared to flox control mice (Flox: 70.0 ± 2.3 μm; HBK: 58.8 ±2 .3 μm; n = 4-5, p =0. 01). Thus, liver circadian clock disruption in older adult mice increases aortic stiffness with aortic apoptosis and reduced wall thickness, which may result in cardiovascular disease.
Circadian rhythm disruption similar to shift work experience increases the risk of cardiometabolic disease. Numerous studies have demonstrated that shift workers have an increased risk of developing hypertension, show decreased endothelial function, and demonstrate an impaired pattern of sodium excretion. Shift work has also been associated with higher incidence of metabolic syndrome. Desynchrony between environmental conditions, such as the light‐dark (LD) cycle, and the endogenous circadian clock has detrimental effects in humans and animals. We hypothesized that chronic environmental circadian disruption leads to vascular disease and increased blood pressure. Male 8‐week old mice (C57BL/6J) were maintained under a standard light/dark cycle (12 hr light, 12 hr dark, controls) or subjected to a chronic circadian disruption protocol (10 hr light, 10 hr dark), a T20 cycle, with ad libitum access to food and water. Weekly food intake was similar between control mice and mice subjected to a shortened light cycle. Aortic pulse wave velocity (PWV), an index of vascular stiffness, was significantly higher in mice under a T20 cycle for 6 weeks compared to control mice (Control: 1.60±0.1 m/s; T20: 2.25±0.1 m/s; n=6, p=0.0037). Mice subjected to a T20 cycle also showed significantly increased body weight gain after 6 weeks (Control: 1.7±0.3 g; T20: 2.9±0.4 g; n=6, p=0.03). Radiotelemetry revealed blood pressure, heart rate, and locomotor activity diurnal rhythms were disrupted in mice under 10 weeks of a T20 cycle. Control mice showed a significant difference in mean arterial pressure between the inactive and active periods, while this variation was absent in T20 mice (Control 119±3 mm Hg vs. 101±2 mm Hg, active vs. inactive, respectively, p=0.01; T20 115±15 mm Hg vs. 114±11 mm Hg, active vs. inactive, respectively, p=0.92; n=3‐4 in all groups). Time of day and the interaction between time of day and light cycle were significant between control and T20 mice (n=3‐4, p=0.02 and p=0.04, respectively). In conclusion, these findings demonstrate that chronic circadian disruption leads to vascular stiffness, increased weight gain, and impaired blood pressure rhythm suggesting development of cardiovascular disease and alterations in metabolism.
Previous studies have identified Th17 to be highly pathogenic during immune dysregulation. We have previously shown that chronic high fat diet (HFD) led to significant kidney medullary interstitial fibrosis and aortic hypertrophy compared to normal diet intake. Numerous studies have identified a connection between high fat and high salt diets and an increase in pathogenic Th17 cells. Although little is understood about Th17 activation in the gut and kidney when high fat and high salt diets are combined (HF/HS). Furthermore, little is known about whether there are sex differences in dietary immune responses. We hypothesized that chronic HF/HS diet compared to normal diet (ND) intake can exaggerate Th17 activation and reduce regulatory T cells (Treg) in the gut and kidney compared to ND in a sex dependent manner. The gut comprises the largest site of immune cells in the body and are ideally located for mediating immune responses to dietary challenges, thus we focused on the gut as well as the kidney. We immunophenotyped T cells with flow cytometry through cytokine production and transcriptional markers from 10‐week‐old C57Bl6/J mice provided ND (10% fat, 0.4% salt) or HF/HS (45% fat, 4% salt) diet for 10 weeks. Small intestines, colons, and kidneys were collected and isolated to single cell suspensions and stimulated for two hours before fluorescent antibody staining for flow cytometric analysis. Results are reported as percentage of CD4+ TCRb+. We first examined baseline immune cell (CD45+) and T cell populations (CD4+) and found no statistical differences between ND and HF/HS. After gating on CD4+ TCRb+ T cells, we gated on IL‐17A+ IFNg‐ cells as our Th17 population. In the small intestine of male mice, we found a significant increase of Th17 cells in the HF/HS group compared to ND mice (ND: 7.05 1.12, HF/HS: 18.32 2.13, p=0.0009). However, in the colon and kidney, we found no significant difference in Th17 cells in the HF/HS and ND groups (Kidney: ND: 1.060.08, HF/HS: 1.140.13, p=0.5967; Colon: ND: 2.87 0.48, HF/HS: 4.83 0.83, p=0.0610). In the colon, we found significantly decreased frequencies of effector Tregs (FoxP3+ RORgt+) with HF/HS compared to ND (ND: 12.95 1.30, HF/HS: 6.400.72, p=0.0023). There were no significant differences in effector Tregs in the small intestine and kidney (Small Intestine: ND: 8.941.26, HF/HS: 7.080.78, p=0.2378; Kidney: ND:0.47 0.19, HF/HS: 0.43 0.20, p=0.9057). We found no significant differences between ND and HF/HS groups in all tissue sources and T cell phenotypes in female mice (Th17: Small Intestine: ND: 18.20 3.86, HF/HS: 16.361.90, p=0.6983; Colon: ND: 3.611.23, HF/HS: 3.020.71, p= 0.6886; Kidney: ND: 27.484.44, HF/HS: 18.963.11, p=0.1666; Treg: Small Intestine: ND: 9.581.20, HF/HS: 8.63 1.07, p=0.5762; Colon: ND: 5.701.14, HF/HS: 4.63 0.61, p=0.4237; Kidney: ND:0.84 0.31, HF/HS: 0.31 0.04, p=0.1505). In conclusion, HF/HS diet induced increased small intestinal Th17 cells and decreased colonic effector Tregs in male mice and not in female mice. These results indicate th...
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