Excess dietary salt and salt-sensitivity contribute to cardiovascular disease. Distinct T cell phenotypic responses to high salt and hypertension, as well as influences from environmental cues, are not well understood. The aryl hydrocarbon receptor (AhR) is activated by dietary ligands, promoting T cell and systemic homeostasis. We hypothesized that activating AhR supports CD4+ homeostatic functions, such as cytokine production and mobilization, in response to high salt intake while mitigating salt-sensitive hypertension. In the intestinal mucosa, we demonstrate that a high-salt diet (HSD) is a key driving factor, independent of hypertension, in diminishing interleukin 17A (IL-17A) production by CD4+ T (Th17) cells without disrupting circulating cytokines associated with Th17 function. Previous studies suggest that hypertensive patients and individuals on a HSD are deficient in AhR ligands or agonistic metabolites. We found that activating AhR augments Th17 cells during experimental salt-sensitive hypertension. Further, we demonstrate that activating AhR in vitro contributes to sustaining Th17 cells in the setting of excess salt. Using photoconvertible Kikume Green-Red mice, we also revealed that HSD drives CD4+ T cell mobilization. Next, we found that excess salt augments T cell mobilization markers, validating HSD-driven T cell migration. Also, we found that activating AhR mitigates HSD-induced T cell migration markers. Using telemetry in a model of experimental salt-sensitivity, we found that activating AhR prevents the development of salt-sensitive hypertension. Collectively, stimulating AhR through dietary ligands facilitates immunologic and systemic functions amid excess salt intake and restrains the development of salt-sensitive hypertension.

Disrupted feeding and fasting cycles as well as chronic high-fat diet-induced (HFD-induced) obesity are associated with cardiovascular disease risk factors. We designed studies that determined whether 2 weeks of time-restricted feeding (TRF) intervention in mice fed a chronic HFD would reduce cardiovascular disease risk factors. Mice were fed a normal diet (ND;10%fat) ad libitum or HFD (45% fat) for 18 weeks ad libitum to establish diet-induced obesity. ND or HFD mice were continued on ad libitum diet or subjected to TRF (limiting food availability to 12 hours only during the dark phase) during the final 2 weeks of the feeding protocol. TRF improved whole-body metabolic diurnal rhythms without a change in body weight. HFD mice showed reduced blood pressure dipping compared with ND, which was restored by TRF. Further, TRF reduced aortic wall thickness, decreased aortic stiffness, as well as increased kidney tubular brush border integrity, decreased renal medullaryfibrosis, and reduced renal medullaryT cell inflammation in HFD mice. These findings indicate that TRF may be an effective intervention for improving vascular and kidney health in a model of established diet-induced obesity.

Circadian rhythms have an important role in cardiovascular physiology. Circadian clock genes are critical for vascular homeostasis. Global loss of the circadian clock gene Bmal1 increases aortic stiffness with impaired vascular function and blood pressure rhythm in mice. We previously found that hepatocyte-specific Bmal1 deletion (HBK) in the liver changes perivascular adipose tissue-mediated vascular function in young adult mice, indicating that liver circadian clock disruption distally affects function in another tissue and potential cross-talk between the liver and vasculature. We hypothesized that liver-specific Bmal1 deletion leads to aortic stiffness and pathological vascular remodeling. Studies were performed in 4- to 6- month old male HBK and flox control mice. Aortic stiffness, measured by pulse wave velocity, was significantly elevated in HBK mice compared to flox control mice (Flox: 1.84 ± 0.1 m/s; HBK: 2.9 ± 0.2 m/s; n = 5, p < 0.01). Systolic blood pressure (tail-cuff) in the light phase 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). Aortas and plasma were collected at ZT2 for histological analysis and metabolite measurements. MetaMorph software analysis of Masson’s Trichrome-stained aortic sections revealed significantly increased aortic wall fibrosis in HBK mice (% fibrosis per area, Flox 17.0% ± 3.3%, HBK 27.4% ± 2.1%, p=0.03). The medial cell number in H&E stained-aortic sections was increased in HBK mice compared to flox control mice (Flox: 534.6 ± 31.9; HBK: 1069 ± 90.1; p < 0.01). TUNEL stained apoptotic cells were also counted and more apoptotic cells were observed in young HBK mice aortas compared to flox controls (Flox: 224.4 ± 56.1; HBK: 408.4 ± 20.8; p = 0.02). Aortic wall thickness was not different in young adult HBK mice compared to flox control mice. Aortic levels of the lipid peroxidation marker vascular 4-hydroxynonenal (4-HNE), a measure of oxidative stress, were similar between flox control and HBK mice. In plasma, the oxidative stress marker, 8-hydroxy-2’-deoxyguanosine (8-OHdG), a DNA damage product, and plasminogen activator inhibitor-1 (PAI-1) were not different between genotypes. In conclusion, our findings indicate that liver circadian clock disruption increases aortic stiffness and fibrosis with higher cell numbers as well as aortic apoptosis. These data suggest that altered vascular remodeling in HBK mice may contribute to aortic stiffness resulting in cardiovascular disease. Funded by P01HL136267 to JSP, R21AA026906 to SMB, and AHA 856877 to PP. 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.

Diet-induced obesity (DIO) is an epidemic in the United States and globally. Obesity is well known to exacerbate and/or promote kidney disease. Inflammation is a major factor in DIO and kidney disease. Utilizing a 20-week DIO mouse model (45% fat) and a normal diet (ND; 10% fat), our lab previously showed greater kidney medullary interstitial fibrosis as well as increased kidney medullary CD3+ T cells in DIO mice when compared to ND mice. Specifically, we found increased kidney CD8+ T cells, but similar CD4+ T cells, in DIO mice compared to ND mice. We hypothesized that CD8+ T cells in DIO mice are a primary factor in promoting kidney interstitial fibrosis. DIO mice were treated with anti-CD8 antibody or control anti-IgG antibody and ND mice were treated with control anti-IgG antibody for the final two weeks of the DIO protocol (n=7-9). DIO/anti-IgG and DIO/anti-CD8 mice had significantly higher body weight than ND/anti-IgG mice as expected (one-way ANOVA; p<0.0001). Administration of anti-CD8 treatment in DIO mice had null effects on body weight (one-way ANOVA, p=0.509) and kidney weight (one-way ANOVA; p=0.067). Utilizing flow cytometry, we verified that anti-CD8 treatment significantly reduced CD8+ T cells compared to anti-IgG in the kidney (one-way ANOVA; p=0.0077; DIO/anti-IgG vs DIO/anti-CD8, p=0.0066) and in the blood (one-way ANOVA; p=0.0034; DIO/anti-IgG vs DIO/anti-CD8, p=0.0048). Using Picrosirius Red histological staining to assess kidney fibrosis, we found that anti-CD8 treatment significantly blunted kidney medullary fibrosis (one-way ANOVA, p=0.0177, ND/anti-IgG vs DIO/anti-IgG, p=0.0329; DIO/anti-IgG vs DIO/anti-CD8, p=0.0318), whereas glomerular fibrosis was not affected by anti-CD8 treatment in DIO mice (one-way ANOVA, p=0.31). Urine was collected in 12-hour increments and analyzed for markers of kidney damage. All groups of mice showed similar urinary KIM-1 excretion (one-way ANOVA; p=0.62) and protein excretion (one-way ANOVA; p=0.69). DIO mice with anti-CD8 or anti-IgG treatment had significantly increased urinary NGAL excretion compared to ND mice (one-way ANOVA; p=0.0203). In summary, anti-CD8 treatment reduced circulating and kidney CD8+ T cells, as well as kidney medullary interstitial fibrosis but not urinary NGAL excretion. These results indicate that CD8+ T cells mediate kidney medullary interstitial fibrosis in a 20 week DIO mouse model. Funding: R01 DK134562, R25 DK115353, F31HL167626. 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.

Experiencing Early Life Stress (ELS), such as physical/verbal abuse before the age of 18, has become an increasingly apparent concern in the context of lifelong health. Systemic Lupus Erythematosus (SLE) is one autoimmune condition linked with ELS, predominantly affects women, and has high CVD burden. Exposure to ELS independently increases the risk of CVD and SLE, so we have developed a model to study this gap. The mouse model of ELS, maternal separation with early weaning (MSEW), combined with the pristane model of SLE mimics the accelerated disease (shown by earlier production of autoantibodies, increased vascular stiffness, and vascular dysfunction at 4 months post injection (p.i.) opposed to 7 months p.i.) in ELS. We hypothesized that the accelerated disease and CVD burden in MSEW+SLE mice, as opposed to normally reared (NR)+SLE mice, arose from severe kidney damage and increased blood pressure. MSEW mice were separated from dams for 4 hours/day (postnatal day 2 to 5) and 8 hours/day before weaning at day 17. NR litters were weaned at postnatal day 21. MSEW or NR female mice were randomly administered PBS or pristane through i.p. injection. Urine was collected at 4 months and 7 months p.i. to test for kidney damage markers and telemetry was utilized. MSEW animals develop increased Anti-Smith autoantibodies at 4 months p.i while SLE NR animals had no difference in titer levels compared to NR PBS mice. However, we found no difference in urine NGAL (Neutrophil Gelatinase-Associated Lipocalin) at 4 (p=0.32) or 7 months p.i. (p=0.6) or urine proteinuria 7 months p.i. (p=0.9) between groups. Blood pressure between MSEW+SLE and NR+SLE mice at 4, 5, and 6 months p.i. compared to control mice was not different. At 7 months p.i., SBP in NR+SLE (140.3 ± 10.22, n=4), MSEW+SLE (140.28 ±9.32, n=4) was not statistically greater than the NR/MSEW controls (128.1 ± 3.16, n=5 NR, n=7 MSEW). Ejection fraction in NR+SLE (66.04 ± 3.24, n=3), MSEW+SLE (75.17 ±3.19, n=2) compared to NR/MSEW controls (64.36 ± 2.49, n=5 NR, n=4 MSEW) was slightly changed over time. We see that at 6 months p.i. PWV and adventitial wall thickness is greater in MSEW+SLE (2.89 mm/sec ± 0.11) mice compared to both NR+SLE mice (2.047 mm/sec ± 0.16) and NR control mice (1.49 mm/sec ± 0.12, p=<0.001, F= 26.02), but at seven months PWV is not different between MSEW+SLE (2.94 mm/sec ± 0.13) mice compared to both NR+SLE mice (2.63 mm/sec ± 0.32, p=0.30). The acceleration of autoantibodies and PWV in MSEW+SLE mice, but not kidney or heart function indicates a specific impact on larger vessels and autoimmune cells in SLE. This work furthers the understanding of how ELS accelerates cardiovascular burden in autoimmune diseases and early identifying targets to prevent worsened outcomes in patients exposed to childhood trauma. Crohn’s and Colitis Foundation NIH R25 PROmoTE NIH T32 PRIME F31HL165863-0 NIH P01 HL158500. 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.

Obesity is a leading cause of chronic kidney disease (CKD). Our lab previously showed that a 20 week diet-induced obesity (DIO) protocol (45% high fat) significantly increased kidney medullary fibrosis in male mice. Further, we found that time restricted feeding (TRF, feeding during 12-hr dark phase only) during the final 2 weeks of the 20-week DIO protocol significantly reduced kidney fibrosis. DIO mice showed significantly greater CD8+ T cells, compared to mice on a normal diet (ND) only during the dark phase. TRF in the DIO mice normalized the CD8+ T cell to that similar to ND mice. We hypothesized that pro-fibrotic CD8+ T cells infiltrate the kidney and mediate damage in the DIO mice. We used anti (a)-CD8 treatment during the final 2 weeks of DIO to deplete all CD8+ T cells. Separate cohorts of ND and DIO mice received control a-IgG treatment during the final 2 weeks of the protocol. DIO mice treated with a-CD8 showed significantly reduced kidney fibrosis compared to DIO mice treated with a-IgG and similar to ND mice treated with a-IgG (assessed by blue trichrome and picrosirius red staining; one-way ANOVA, p=0.018; NDa-IgG vs DIOa-IgG: p=0.033; DIOa-IgG vs DIOa-CD8: p=0.032). We next investigated the intestinal mucosa (IM) as a source of the kidney-infiltrating CD8+ T cells in DIO mice since the IM is one of the primary non-lymphoid sites of T cells in the body. Previous studies have shown that migration of T cells from the IM to the kidney can drive acute kidney injury, but it is unclear if this occurs with chronic models such as DIO. We used the KikumeGreen (KikGr) mouse model to identify migrating cells into the kidney. When KikGR protein is exposed to 408 nm blue light it is photoconverted to a KikRed. We photoconverted the IM at the end of the ND, DIO, and DIO+TRF protocols. Kidneys were analyzed by flow cytometry at 2.5 days post-photoconversion. We detected significantly higher KikRed+ CD8+ T cells in the kidneys of DIO mice compared to ND mice indicating that CD8+ T cells migrate to the kidney from the IM. DIO+TRF mice showed reduced KikRed+ CD8+ T cells in the kidney compared to DIO mice (one-way ANOVA: p=0.006, ND vs DIO: p=0.008; DIO vs TRF: p=0.047). DIO did not have any significant effect on CD4+ T cell migration (one-way ANOVA: p=0.997). This study indicates that DIO induces migration and infiltration of pro-fibrotic CD8+ T cells from the IM to the kidney, while TRF specifically limits migration and infiltration of CD8+ T cells.

Histone deacetylase 1 (HDAC1) is an important regulator of cell differentiation and is associated with progression of cardiovascular disease (CVD). Our lab previously showed that over-expression of HDAC1 in cultured endothelial cells reduces NO production. Other investigators have shown increased HDAC1 protein in models of CVD. However, there is a gap in knowledge of whether endothelial-specific HDAC1 mediates vascular dysfunction in models of CVD. Diet induced obesity (DIO) is a leading risk factor for CVD, thus our studies are focused on a chronic high fat feeding mouse model. Our studies utilize a mouse model of DIO where mice are fed a normal diet (ND, 10% fat) or high fat diet (DIO, 45% fat) beginning at 8 weeks of age for 20 weeks. Previously, we showed that this model of DIO in male mice significantly increases aortic stiffness (measured by pulse wave velocity, PWV) and increases aortic thickness compared to ND (PWV: unpaired T test, n=9-12, normal diet (ND) vs DIO, p=<0.0001; Aortic Thickness: unpaired T test, n=6, ND vs DIO, p=0.005). We hypothesized that endothelial-specific HDAC1 mediates aortic dysfunction in a mouse model of DIO. We generated inducible endothelial-Scl-Cre-ER-HDAC1 knockout (KO) mice (iEC-HDAC1KO), which specifically deletes HDAC1 in endothelial cells after tamoxifen injection, and the flox control mice (iEC-HDAC1flox), which does not delete HDAC1 after tamoxifen injection. We assessed aortic stiffness of iEC-HDAC1 KO and iEC-HDAC1flox in ND and DIO mice. We treated with tamoxifen for 5 days when ND mice were 20 weeks old and measured PWV 2 weeks later. Interestingly, iEC-HDAC1KO mice had significantly reduced PWV compared to iEC-HDAC1flox mice on ND (unpaired T test; n=4-5, p=0.009). At week 18 of the DIO protocol, mice were given daily tamoxifen injections for 5 days. After week 20 of the DIO protocol, iEC-HDAC1KO DIO mice had significantly reduced PWV compared to iEC-HDAC1flox DIO mice (unpaired T test; n=4-5; p=0.008). These data indicate that endothelial-specific HDAC1 mediates vascular stiffness and remodeling in high fat fed mice. Further investigation is needed to understand the relationship of endothelial HDAC1 and NO in the context of DIO and aortic health. Funding: R01 DK134562, F31HL167626. 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.

Early life stress (ELS) is highly prevalent worldwide and has been associated with an elevated risk of developing cardiovascular disease (CVD) in adulthood. We previously showed that maternal separation with early weaning (MSEW), an established ELS model of neglect in mice, leads to blunted endothelium-dependent relaxation as well as increased histone deacetylase (HDAC) activity. We hypothesized that specific HDAC isoform(s) mediate the ELS-induced endothelial dysfunction. In a screen of all HDAC isoforms, HDAC1, 6, and 9 gene expression were significantly elevated in aortas from male adult MSEW and normally reared (NR) C57BL6/J mice (N=3/group, p<0.05). Western blot analysis found that only HDAC9 protein abundance was significantly elevated in aortas from male MSEW as compared to NR mice (N=6/group, p=0.01). Detection of HDAC9 via immunohistochemistry was greater in the aortic endothelium of MSEW mice compared to NR (N=5/group). Subsequently, vascular reactivity was measured with wire myography on tamoxifen inducible endothelium-specific HDAC9KO (Cdh5CreERT2-HDAC9) MSEW and NR adult mice to determine the role of HDAC9 in MSEW-induced endothelial dysfunction. Male mice exposed to MSEW displayed blunted aortic endothelial-dependent relaxation to acetylcholine compared to NR, whereas endothelial-specific knockout of HDAC9 (HDAC9KO) restored endothelial-dependent relaxation (NR: Emax=89.27%±4.15, N=5; MSEW: Emax=48.12%±22.12, N=2; NR HDAC9 KO: Emax=68.98%±4.73, N=5, MSEW HDAC9KO: Emax=70.50%±1.85, N=3). Females demonstrated no difference between MSEW and NR endothelial dependent relaxation, however, HDAC9KO MSEW displayed blunted relaxation compared to all other groups (NR: Emax= 77.14%±4.86, N=5; MSEW: Emax=84.30%±8.71, N=2; NR HDAC9 KO: Emax=70.80%±5.87, N=5, MSEW HDAC9KO: Emax=69.76%±9.10, N=4). Endothelial-independent relaxation to sodium nitroprusside (SNP) was similar in all groups in both sexes (Male: NR: Emax=96.92%±1.26, N=5; MSEW: Emax=100%±0, N=2; NR HDAC9 KO: Emax=96.52%±3.484, N=5; MSEW HDAC9KO: Emax=100%±0, N=3; Female: NR: Emax=98.58%±0.42, N=5; MSEW: Emax=100%±0, N=2; NR HDAC9 KO: Emax=98.48%±1.08, N=5; MSEW HDAC9KO: Emax=99.15%±0.85, N=4). These findings suggest that HDAC9 mediates ELS-induced endothelial dysfunction in males, however, HDAC9 may play a protective role in females. Future studies aim to further investigate sex-dependent differences in the specific role of HDAC9 in ELS related CVD. NIH P01 HL158500, F31HL165863-01, T32GM135028. 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.

Th17 cells are a major contributor to many pro-inflammatory disease conditions. Endothelin 1 (ET-1) through the ETA receptor promotes inflammation, however its role in T cell differentiation, especially T helper 17 (Th17) cells, is not fully understood. Importantly, ET-1 has been shown to be elevated in people with autoimmunity, diabetes, and salt-sensitive hypertension in which augmented Th17 cells are known to contribute to the pathogenesis. Therefore, we hypothesized that ETA receptor activation mediates Th17 cell differentiation. To test our hypothesis, we first treated C57BL/6J male mice (8–12-week-old, normal chow) with the ETA receptor antagonist (ABT-627, 10mg/kg/day) in the drinking water for five weeks. We measured Th17 cells in the colon and kidneys by flow cytometry. We found that ETA receptor blockade reduced the number of Th17 cells compared to that in vehicle control mice. Indeed, the absolute number of Th17 cells decreased from 461±61 Th17 cells/per two kidneys in vehicle control to 146±29 Th17 cells per two kidneys in the ABT-627 treated group (p=0.004), and from 8372±1454 Th17 cells to 3378±936 Th17 cells (p=0.028) in the whole colon. To determine whether this observation is a direct effect on IL17A induction, we evaluated ETA receptor antagonism under Th17 polarizing conditions in vitro. Naïve CD4+ T cells were isolated from the spleen and cultured under Th17 polarizing conditions (TGFβ and IL-6) in the presence or absence of ETA receptor antagonist (BQ-123, 1μM). We found that ETA receptor blockade directly attenuated Th17 differentiation in vitro. The frequency of IL17A-expressing cells within CD4+ T cells reduced from 11.37±0.68% in media control to 9.87±0.60% in BQ-123 treated cells (p=0.0006). Similarly, the relative mean fluorescence intensity (MFI) analysis revealed a reduction in IL17A production following ETA receptor blockade from 8937±871 relative units in media control to 7132±725 relative units in BQ-123 treated cells (p=0.0136). These data taken together support our hypothesis that the ETA receptor plays a key role in Th17 differentiation. Because of the critical role of Th17 cells in pathophysiology, our findings warrant further investigations for the use of ETA receptor antagonist in pro-inflammatory conditions. Tha Luong - U2C/TL1 Deep South KUH PRIME U2C DK133422 & TL1 DK139566 from the NIH/NIDDK Patrick Molina - F31HL151264 David & Jennifer Pollock - R01 DK134562. 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.

Sickle cell disease (SCD) is an autosomal recessive genetic disease caused by a point mutation in the hemoglobin gene. This substitution results in a “sickling” of red blood cells and subsequent vascular and organ damage. Current treatments, while increasing the lifespan of sickle cell patients, has resulted in a higher prevalence of chronic complications like SCD nephropathy. Our group has previously shown that time-restricted feeding (TRF) in chronic high fat fed mice reduces kidney damage. Given the increased kidney damage in SCD, we hypothesize that TRF in humanized SCD mice will attenuate sickle cell nephropathy. To test this hypothesis, we studied 18-22-week-old male SCD mice. Mice underwent 3 weeks of TRF, only having access to food during the active, dark period (Zeitgeber Time (ZT) 12 to 24). The control group had access to food ad libitum (ad-lib). At the end of the 3 weeks, at 21-25 week old ages, 12 hour urine was collected in metabolic cages as well as kidneys and plasma collection at ZT3 (inactive period). Excretion of urinary kidney damage markers (albumin and NGAL-1) showed no difference between TRF and ad-lib in both the active and inactive collection periods. Urinary excretion of endothelin-1 is known to be upregulated in SCD patients; however, TRF did not change this marker in SCD mice. TRF did not change plasma levels of cytokines or soluble adhesion molecules. Histological analysis of kidneys was conducted as an additional marker of kidney damage. We analyzed iron levels in the kidney with Prussian blue staining as well as fibrosis with Masson’s trichrome. TRF did not change kidney iron accumulation or kidney fibrosis. Immunohistochemical staining for CD3+ T cells did not show a difference in glomerular T cell infiltration in the TRF group compared to the ad-lib group. In conclusion, TRF does not appear to have an effect on renal inflammation or kidney function in SCD mice, but future studies need to be completed. R01 DK134562. 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.

Sickle cell disease (SCD) carries a significant risk for poor vascular health and vascular dysfunction. High levels of vascular reactive oxygen species (ROS) as well as elevated plasma endothelin-1 (ET-1), a potent vasoconstrictor with actions via the ETA receptor, are both common phenotypes in SCD. Alpha-1 adrenergic receptor activation is a major mediator of stress-induced vasoconstriction. However, the mechanism of the SCD enhanced vasoconstrictive response is unknown. We hypothesized that SCD induces enhanced alpha-1 adrenergic mediated vasoconstriction through the ET-1/ETA receptor pathway in arterial tissues. Utilizing humanized SCD (HbSS) and genetic control (HbAA) mice, alpha-1a, but not alpha-1b or alpha-1d, receptor expression was significantly greater in aortic tissue from HbSS mice compared to HbAA mice. Significantly enhanced vasoconstriction in aortic and carotid arterial segments were observed from HbSS mice compared with HbAA mice. Treatment with ambrisentan, a selective ETA receptor antagonist, and a ROS scavenger normalized the aortic vasoconstrictive response in HbSS mice. In a randomized translational study, patients with SCD were treated with placebo or ambrisentan for 3 months, with the treatment group showing an increase in the percent brachial arterial diameter. Taken together, these data suggest that the ETA receptor pathway interaction with the adrenergic receptor pathway contributes to enhanced aortic vasoconstriction in SCD. Findings indicate the potential of ETA antagonism as a therapeutic avenue for improving vascular health in SCD.

Recent studies suggest that patients with hypertension and individuals that consume a HSD are depleted of numerous tryptophan-derived indoles and other AHR-binding metabolites that are key in intestinal CD4+ T cell homeostasis. Thus, the inappropriate immunologic response by CD4+ T cells, and their role in hypertension, may be due in part to loss of AHR activation. Therefore, we hypothesized that AHR activation contributes to maintaining effector T cell status in salt-sensitive mice. To examine the influence of a representative homeostatic or proinflammatory environment and the subsequent response to salt, we cultured naïve CD4+ T cells under homeostatic cytokine or pathogenic cytokine conditions and exposed the cultures to increased NaCl conditions with or without the AHR-activating metabolite, FICZ (6-Formylindolo(3,2-b)-carbazole). TH17 cells cultured under homeostatic high salt conditions were depressed with a concomitant increase in response to FICZ. In contrast, TH17 cells cultured under pathogenic high salt conditions had augmented IL-17A-expressing cells without a response to AHR activation. These data suggest that excess salt is disrupting the ability to induce IL-17A and increasing AHR activity overcomes this disruption to promote IL-17A, without expansion of the pathogenic TH17 pool, and this observation was specific to non-pathogenic microenvironments (homeostatic TH17: media: 11±0.6%, +NaCl: 4.9±0.3%*, +NaCl/FICZ: 9.9±0.8%, of CD4; pathogenic TH17: media: 1.0±0.2%, +NaCl: 2.2±0.2%*, +NaCl/FICZ: 2.2±0.2%*, *vs respective media, n=8/group). Given these data, we then fed salt-sensitive mice (nitric oxide synthase inhibition) a HSD or a HSD supplemented with the AHR agonist indole-3-carbinol (I3C) and examined Peyer’s patches, a lymphoid tissue in close proximity to intestinal lumen contents enriched with salt and the I3C-metabolites and noted expansion of non-pathogenic TH17 cells. In conclusion, increasing AHR ligand availability through supplementing I3C may provide a gut-centric experimental approach in targeting experimental salt-sensitivity as it relates to immunologic disturbances. F31HL151264. 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.

Diet induced obesity (DIO) is one of the leading risk factors for chronic kidney disease (CKD). Many studies have shown a role for pro-inflammatory T cells in mediating kidney damage in DIO. We previously found a 20-week model of DIO with time restricted feeding (TRF) to the 12-hour active phase during weeks 18-20 of diet significantly reduces kidney fibrosis and urinary NGAL excretion. We also found that TRF decreases total T cells (CD3+) in the vasa recta of the kidney medulla in DIO compared to ad libitum DIO. Immune cells, including T cells, have rhythms of migration through tissues. We hypothesized that DIO drives increased T cells in the kidney and that TRF in DIO mice reduces T cell infiltration and kidney damage. Utilizing our 20-week DIO protocol (45% high fat diet) and DIO with TRF intervention (DIO+TRF), we collected kidneys at 6 timepoints and assessed changes in Cd3, a T cell marker, in the outer medulla over a 24-hour period via qPCR. DIO increased Cd3 expression at Zeitgeber time (ZT) 9 (inactive period) and ZT13 (active period) compared to DIO+TRF, with an overall effect of diet and time of day (2-way ANOVA, time of day: p=0.051, diet: p=0.050). To assess diurnal changes in T cells by flow cytometry, we collected kidneys from the normal diet (ND), DIO, and DIO+TRF groups at ZT3 (inactive period) and ZT13 (active period). We found a significant diurnal rhythm for total immune cells (CD45+) in the DIO group with increased immune cells at ZT13 that is not seen in the ND or DIO+TRF groups (2-way ANOVA, interaction: p=0.027, time of day: p=0.0003, Tukey’s post hoc: DIO ZT3 vs DIO ZT13: p=0.005). Similarly, we found increased T cells at ZT13 compared to ZT3 in DIO mice. Interestingly, at ZT13 but not ZT3, DIO has significantly more T cells than both the ND and DIO+TRF group (2-way ANOVA, Interaction: p=0.01, time of day: p=<0.0001, diet: p=0.009; Tukey’s post hoc: DIO ZT3 vs DIO ZT13: p=<0.0001, ND ZT13 vs DIO ZT13: p=0.049, DIO ZT3 vs DIO+TRF ZT13: p=0.005). We investigated the T cell subtypes and found that DIO led to a diurnal rhythm of CD4+ T cells, with an increase at ZT13 compared to ZT3 (2-way ANOVA, Interaction: p=0.02, time of day: p=0.01, Tukey’s Post Hoc: DIO ZT3 vs DIO ZT13: p=0.008). CCR6 and CXCR3 are trafficking markers of T cells. We found a significant diurnal effect in kidneys of DIO mice with greater frequency of CCR6+ and CXCR3+ CD4+ T cells at ZT13 compared to ZT3 (2-way ANOVA, time of day: CCR6+: p=0.004; CXCR3+: p=0.03). Interestingly, we found no diurnal changes in the proliferation marker Ki-67 of CD4+ T cells. Taken together, these data suggest that the diurnal variation of kidney CD4+T cells during DIO is driven through T cell trafficking. Also, TRF intervention in DIO mice reverses the diurnal effect in CD4+ T cell trafficking to the kidney and may drive the reduced kidney damage. In conclusion, these findings suggest that DIO driven kidney damage is mediated through infiltrating pro-inflammatory T cells. 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.

Diet induced obesity (DIO) is one of the leading risk factors for chronic kidney disease. Previous studies identified a role for pro-inflammatory T cells in mediating DIO-driven renal damage. We found that time restricted feeding (TRF; food available only in 12-hr dark phase) during the final 2 weeks of a 20-week DIO model (45% fat) significantly improves renal damage compared to DIO ad libitum fed male mice. DIO+TRF also significantly reduced total T cells in the kidney adjacent to vasa recta in the outer medulla compared to DIO ad libitum mice assessed at the start of the dark phase or Zeitgeber Time (ZT) 12. We hypothesized that DIO+TRF decreases renal and circulating pro-inflammatory CD4 + and CD8 + T cells compared to DIO ad libitum in a time-of-day dependent manner . Using flow cytometry, we found that DIO significantly increases total renal T cells compared to normal diet (ND; 10% fat) at ZT13-15 (dark phase) only. DIO+TRF significantly decreases total renal T cells compared to DIO in the dark phase (n=7-14; 2-way ANOVA, Interaction: p=0.016, Effect of Time of Day: p=0.010, Effect of Diet: p=0.001). Our data showed no significant changes in renal T cell numbers in the light phase. Compared to ND, DIO increased renal CD8 + T cells at ZT13-15 and DIO+TRF normalized renal CD8 + T cells (n=7-14; 2-way ANOVA, Interaction: p=0.007, Effect of Diet: p=0.004). DIO and DIO+TRF did not alter renal CD4 + T cells at either ZT1-3 or ZT13-15 compared to ND. We further examined the kidney CD8 + T cell phenotype with DIO and TRF. We also found no significant changes in CD8 + T cell activation markers, perforin and granzyme B, CD8 + T cell development marker EOMES, or CD8 + T cell pro-inflammatory cytokines, IFNγ and TNFα. We assessed circulating CD8 + and CD4 + T cells via flow cytometry to analyze systemic effects of TRF. DIO+TRF led to a diurnal rhythm of CD8 + T cells with increased circulating CD8 + T cells in the dark phase compared to the light phase that was not observed in the ND or DIO groups (n=7-17; 2-way ANOVA, Interaction: p=0.012). No significant changes were observed in circulating CD8 + T cell activation markers or cytokines. DIO and TRF had no significant effect on numbers of circulating CD4 + T cells. We conclude that DIO+TRF influences rhythms of circulating and renal CD8 + T cells, but not CD4 + T cells.

Shift workers have an elevated risk of developing hypertension and higher incidence of cardiovascular disease. Circadian rhythm disruption increases cardiometabolic disease risk in both humans and animals. We hypothesized that chronic circadian disruption (CCD) impairs cardiovascular rhythms and leads to vascular disease. Male 8-week old C57BL/6J mice were subjected to a standard light/dark cycle (12-h light, 12-h dark, control) or a CCD protocol (10-h light, 10-h dark, T20) with ad libitum food and water for 10 wks. T20 mice had an increased rate of weight gain (n=6, p=0.04; time: p<0.001, light cycle: p<0.001). Mean arterial pressure (MAP), heart rate (HR), and activity were measured by telemetry after 10 weeks of CCD. Control mice (12:12 LD) had significant diurnal variation in MAP, while T20 mice (10:10 LD) lacked diurnal variation (control 119±3 vs.101±2 mm Hg, dark vs. light, p<0.001; T20 114±13 vs.113±12 mm Hg, dark vs. light, p=0.86; n=3-4). Control mice had a diurnal variation in HR which was absent in T20 mice (Control 584±11 vs. 511±4 bpm, dark vs. light, p=0.001; T20- 537±14 vs. 526±5 bpm, dark vs. light, p=0.48; n=3-4). Dark phase HR was significantly lower in T20 mice (p=0.01, control vs. T20). Control mice had a light-dark difference in activity, while T20 mice lacked diurnal variation in activity with significantly higher light phase activity (p=0.04, control vs. T20). Aortic pulse wave velocity (PWV), a measure of vascular stiffness, was significantly higher after 6 weeks in T20 mice compared to control mice (Control: 1.60±0.1 m/s; T20: 2.25±0.1 m/s; n=6, p=0.0037), although aortic wall thickness was similar between groups. Wire myography in isolated aortic rings was used to assess vascular reactivity with cumulative concentration responses to phenylephrine (PE) and acetylcholine (ACh). CCD reduced sensitivity (EC50) to PE-induced vasoconstriction (Control: -6.92±0.06 M; T20: -6.74±0.04 M, n=6, p=0.03), while there were no significant changes seen in the ACh sensitivity or maximal response. These data show that CCD impairs cardiovascular and behavioral rhythms linked to the development of aortic stiffness.

Circadian rhythm disruption increases cardiometabolic disease risk. For example, night shift workers have an increased risk of developing metabolic syndrome, hypertension, and endothelial dysfunction. Loss of Bmal1, an essential circadian clock gene, impairs cardiometabolic rhythms and promotes endothelial dysfunction. We hypothesized that chronic light cycle disruption increases fat mass, blunts metabolic rhythms, and leads to cardiovascular disease in adult mice dependent on the molecular circadian clock. Littermate wild type (WT) and global Bmal1-KO mice (5-7 month old males) were maintained on a standard light/dark cycle (control, 12-h light, 12-h dark) or a chronic circadian disruption protocol (CCD,10-h light, 10-h dark for 14-18 weeks) with food and water available ad libitum. Food intake over 24-h was similar between all groups. Body weight was similar between WT control and WT CCD mice whereas weight was lower in Bmal1-KO mice with control and CCD conditions (p<0.01 WT vs Bmal1-KO, n=3-4). Body composition measured by quantitative magnetic resonance revealed lower fat mass in Bmal1-KO control as well as both WT CCD and Bmal1-KO CCD mice compared to WT on the control schedule (p<0.01 WT control vs WT CCD; p=0.02 WT vs Bmal1-KO; n=3-4). Lean mass was not different between control and CCD WT mice but was lower in Bmal1-KO mice regardless of light cycle (p<0.01 WT vs Bmal1-KO; n=3-4). Total body water was similar in control and CCD WT mice but significantly lower in both control and CCD Bmal1-KO mice (p<0.01 WT vs Bmal1-KO; n=3-4). Respiratory exchange ratio measured by indirect calorimetry during light and dark phases was not significantly different between groups although both groups of KO mice were significantly higher than controls (p=0.04; n=3-4). As expected, there was a light-dark phase difference in energy expenditure (EE) in WT control mice, whereas the light-dark phase difference in EE was absent in WT CCD mice (p=0.05 WT control dark vs light; p>0.05 WT CCD dark vs. light; n=3-4). Bmal1-KO control mice lacked a light-dark phase difference in EE. CCD did not affect the EE light-dark phase difference in Bmal1-KO mice. Aortic stiffness, measured by pulse wave velocity, was similar in WT under control and CCD and in Bmal1-KO mice in both conditions (p>0.05). Systolic blood pressure (tail-cuff) was similar in WT control and CCD mice, yet lower in both control and CCD Bmal1-KO mice (p=0.02 WT control vs Bmal1-KO; p=0.04 WT CCD vs Bmal1-KO CCD; n=4-5). In isolated vessels, aortic endothelial-dependent relaxation was not impaired by CCD in WT mice but attenuated in both control and CCD Bmal1-KO mice (p<0.01; n=4-5). Endothelial-independent relaxation was similar between all groups. In conclusion, we found that CCD decreases fat mass in older adult WT mice and dampens EE rhythm, while Bmal1-KO mice have reduced fat and lean mass, total water, and blunted EE rhythm regardless of light cycle. These data suggest that metabolic changes due to light cycle disruption may be dependent on the molecular clock.

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.

Exposure to early life stress (ELS) is associated with a greater risk of developing cardiovascular disease (CVD) later in life. Using a mouse model of ELS, we have recently showed that 4-week-old pre-pubertal mice exposed to ELS have lower microbial diversity and reduced abundances of Lachnospiraceae and Ruminococcaceae taxa, which are important producers of short-chain fatty acids (SCFAs). An essential mechanism by which gut microbes influence host physiology is through the production of SCFAs that act as vasoactive mediators, histone deacetylase inhibitors, and immunomodulators. Therefore, we hypothesized that ELS-induced changes in the gut microbiota would result in reduced circulating SCFAs. To test this hypothesis, we subjected mice to maternal separation (MaSep) in which pups underwent daily separation for 4 h on postnatal days (PDs) 2-5 and 8 h on PDs 6-16 until weaning on PD17. Normally reared (NR) mice remained undisturbed with dams until weaning on PD21. All mice were maintained on standard chow (NIH-31) following weaning. Plasma was collected at PD28 (n=17 MaSep mice from 7 litters and 13 NR mice from 7 litters) and at PD84 (n=8 MaSep from 3 litters and 11 NR mice from 3 litters) for analysis of lactate and the SCFAs acetate, butyrate, isovalerate, propionate, and succinate by gas chromatography/mass spectrometry. Acetate, succinate, and lactate were not different between MaSep and NR mice at either timepoint. However, ELS exposure resulted in significantly reduced butyrate plasma concentrations at PD28 (14.38 ± 0.50 µM in MaSep mice compared to 18.58 ± 1.87 µM in NR mice; P = 0.02) and PD84 (11.94 ± 0.18 µM in MaSep mice compared to 15.03 ± 0.21 µM in NR mice; P < 0.001). ELS also reduced propionate plasma concentrations at PD28 (35.96 ± 1.47 µM in MaSep mice compared to 43.70 ± 1.44 µM in NR mice; P = 0.001) and PD84 (32.16 ± 0.92 µM in MaSep mice compared to 42.53 ± 1.05 µM in NR mice; P < 0.001). Isovalerate plasma concentration was greater in MaSep mice at PD84 (1.11 ± 0.13 µM) compared to NR mice (0.45 ± 0.11 µM; P = 0.002), while there was no difference in isovalerate between MaSep and NR mice at PD28. These data indicate that exposure to stress in early life can result in sustained reductions in butyrate (~22%) and propionate (~21%) concentrations in circulation. Recent advances have determined that butyrate and propionate are involved in blood pressure regulation and vascular physiology. Much less is known about the branched SCFA isovalerate. Future studies will determine if decreases in circulating butyrate and propionate, or increases in circulating isovalerate, play a role in ELS-induced increased risk of CVD.