Time of day drives angiotensin II-dependent hypertension

T cells are important contributors to the pathogenesis of hypertension.¹ Many investigations have demonstrated that T cell dependent responses are modulated by circadian rhythms as well as impacted by the initial timing of challenges.² The chronic angiotensin II (AngII) infusion rodent model used to study mechanisms in hypertension is initiated in the light phase or inactive period. A recent report in healthy humans determined that the blood pressure responses to a one-time 7-hour infusion of AngII varies according to the timing of the initial AngII infusion.^3,4 We hypothesized that pro-hypertensive responses and T cell activation to AngII is dependent on the initial timing of the AngII continuous infusion in the preclinical mouse model (C57Bl6/J, Jackson Laboratories).

As expected, initiating AngII-infusion at ZT0 (490 ng/kg*min; beginning of the light period) significantly increased systolic blood pressure (SBP) and diastolic blood pressure (DBP) within 7 days as measured by telemetry in male mice. In contrast, initiating AngII-infusion at ZT12 (490 ng/kg*min; beginning of the dark period) did not significantly change SBP and DBP, demonstrating that the time of day when AngII infusion was initiated drives the hypertensive response (Panel A). Heart rate was not significantly different between the two groups, suggesting differences in peripheral resistance may be driving the blood pressure differential (Panel B). A potential confounder to this result may be the time of implantation of minipumps as opposed to when AngII infusion was initiated. To control for this possible effect, we implanted programmable iPrecio peristaltic minipumps in mice at ZT5, but initiated AngII infusion at either ZT2 or ZT14. The hypertensive response to AngII was dependent on the time of initiating AngII infusion independent of the time of minipump implantation (Panel C).

We identified significantly elevated numbers of renal cortical interstitial CD3⁺ T cells by immunohistochemical analysis after 7 days of AngII infusion initiated at ZT0 compared to when AngII infusion was initiated at ZT12 (Panel D). Flow cytometry also showed elevated TCRβ⁺, CD8⁺, and CD4⁺ T cell numbers in kidneys with AngII infusion at ZT0 compared to ZT12 (Panel E). In addition, we found that the initiation of AngII infusion at ZT0 significantly increased plasma IL-2, IL-17a, and MCP-1 but not IFNγ in samples obtained during the light endpoint but not dark endpoint (Panel F). While initiating AngII infusion at ZT12 did not change cytokine levels at either light or dark endpoints. Kidney fibrosis was not elevated in either Ang II infusion group at day 7 (p>0.05, Masson’s Trichrome or Picrosirius Red, n=6–10 kidneys); however, in-depth end-organ damage analysis is needed at later timepoints. These findings demonstrate that AngII-mediated hypertension, renal T cell infiltration, and cytokine production are dependent on the time of day that AngII infusion is initiated.

These studies focused on male mice, thus further experiments are needed to determine whether females respond similarly as males. Future experiments will also include utilizing distinct pro-hypertensive stimuli, such as aldosterone or high salt, to understand the mechanisms regulating these time of day outcomes. Interestingly, shift workers show elevated renin-angiotensin-aldosterone signaling which may contribute to the elevated blood pressure and pro-inflammatory immune responses observed in this population.² In conclusion, the time of day represents a critical variable to determine the contribution of mechanistic pathways in the pathogenesis of hypertension risk in preclinical models and in translation to human hypertension risk.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

A) The Institutional Animal Care and Use Committee at the University of Alabama at Birmingham approved our protocol. At 8 weeks of age, male C57Bl/6J mice were acclimated and housed in 12-hour light cycles (standard: 7 AM lights on, 7 PM lights off or reverse: 7 PM lights on, 7 AM lights off), fed ad libitum standard chow (Research Diets D12451) in our facility prior to experimentation at 12 weeks of age. Telemetry devices were implanted at 10 weeks of age to monitor 24-hour blood pressure (DSI). AngII (490 ng/kg*min, Sigma-Aldrich #A9525) infusions were conducted with osmotic minipump (Alzet) implantations initiated at ZT0–2 (blue circles) or ZT12–14 (red squares) at 12 weeks of age. Minipump implantations conducted at ZT12 or later were performed under red light with mouse eyes shielded from any light. SBP and DBP presented as 1-hour and 24-hour averages were compared within or between each group (n=8–9/group). These data indicate significantly lower SBP and DBP when AngII infusion initiated at ZT12 compared to initiation at ZT0. Analyzed via mixed-effects analysis (24-hour averages) and 2-way ANOVA (baseline vs. day 7 summary dot-plots).

B) Heart rates monitored from the same telemetry devices at 1 hour and 24-hour demonstrate no significant differences in response to AngII infusion initiated at ZT0 (blue circles) or ZT12 (red squares). Analyzed via mixed-effects analysis (24-hour averages) and 2-way ANOVA (baseline vs. day 7 summary dot-plots).

C) Experimental design schematic to determine whether the time of surgery was a confounding factor in the blood pressure outcomes. Mice were implanted with telemetry devices at 10 weeks of age at ZT1–7. Ten days later a programmable minipump (iPrecio SMP-310R) was implanted at ZT5–6. Continuous infusion of AngII (490 ng/kg*min, Sigma-Aldrich #A9525) was initiated 32 hours later at ZT14–16 (red squares). In a separate group of telemetry and programmable minipump implanted mice, AngII infusion was initiated after 44 hours later at ZT2–4 (blue circles). Presented as mean arterial pressure (MAP) in 1-hour and 24-hour averages show a significantly higher response in mice that received AngII infusion initiated at ZT2–4 compared to initiation at ZT14–16. Analyzed via mixed effects analysis (24-hour averages) and unpaired two-tailed t test (change from baseline).

D) CD3⁺ or pan-T cell immunostaining (DAB) of kidneys obtained from saline-infused (PBS vehicle initiated at either ZT0 or ZT12) or AngII-infused mice after 7 days as outlined above in panel A. Counts of positive stained cells (representative positive cells highlighted by arrows) in the renal cortical interstitium show significantly higher numbers of T cells/20X image when AngII infusion was initiated at ZT0–2 (blue symbols) compared to initiation at ZT12–14 (red symbols) or compared to sham (green symbols), specifically when the kidneys were harvested at ZT0 (white background) but not when harvested at ZT12 (gray background). Representative images are provided for kidneys harvested at ZT0. Analyzed via two-way ANOVA followed by post-hoc Tukey’s.

E) Flow cytometry analysis T cell populations from kidneys after 7 days of saline-infused or AngII-infused mice. Kidneys were digested in GentleMACs C-Tubes using 0.5mg/mL Collagenase IV, 200ug/mL DNAse1, 0,1 U/mL Dispase II, 0,125 mg/mL Collagenase Xi, 60 U/mL Hyaluronidase type I-s in 1640 RPMI media (Gibco 11875093) supplemented with 10% FBS, sodium pyruvate (Gibco 11360070), NEAA (Gibco 11140076), L-glutamine (Gibco 25030081), and HEPES (Gibco 15630130). Leukocytes were enriched via filtration followed by gradient centrifugation in Histopaque 1119 and stained with 1:200 dilution of CD4 [Biolegend 100406], CD8α [Thermofisher 47-0081-82], CD45 [Biolegend 103140], CD11B [Biolegend 101228], CD11C [ThemroFisher 45-0114-82], TCRβ [BD 563135], Live/Dead [Zombie NIR]. Initiation of AngII infusion at ZT0–2 (blue symbols) resulted in significantly elevated TCRβ+, CD8⁺, and CD4⁺ T cell numbers in kidneys obtained at ZT0 endpoint time (white background) compared to initiation of Ang II infusion at ZT12–14 (red symbols). There were no significant differences in renal TCRβ+, CD8⁺, and CD4⁺ T cell numbers at the ZT12 endpoint time (gray background). Analyzed via two-way ANOVA followed by Fisher’s LSD.

F) Plasma was obtained in mice at ZT0 or ZT12 endpoint times as outlined above in panel A harvested on day 7 after AngII initiation or saline and cytokines quantified (Luminex 200 system, #MCYTMAG-70K-PX32 kit) according to manufacturer protocol by the UAB Immunophenotyping core. Significantly higher IL-2 and MCP-1 levels were observed at ZT0 endpoint (white background), but not ZT12 endpoint (gray background), when AngII infusion was initiated at ZT0–2 (blue symbols) compared to AngII infusion initiated at ZT12–14 (red symbols). IL-17a was significantly elevated above baseline (green symbols) when AngII infusion began at ZT0–2 but not when infusion was initiated at ZT12–14. No significant changes were observed for IFNγ levels at any time points. Analyzed via two-way ANOVA followed by post-hoc Tukey’s.