Chronic Stress Induces Sex-Specific Renal Mitochondrial Dysfunction in Mice

Abstract

Chronic psychological stress has been linked to renal disease and is also associated with the development of hypertension. However, the mechanisms by which chronic stress alters renal function and promotes hypertension is unclear. This study tested the hypothesis that chronic stress causes impaired renal mitochondrial function that can lead to increased arterial pressure. Adult male and female C57BL/6 mice were exposed to a chronic unpredictable stress (CUS), or non-stress control, protocol for 28 consecutive days. The protocol models mild, persistent, and variable stress that is a common occurrence in daily life. The CUS protocol induced anxiety relevant behaviors in both male and female mice. CUS increased blood pressure in both sexes, but the increase was greater in female mice. Renal mitochondrial function was unchanged by CUS in male mice. In contrast, renal mitochondrial function was impaired in the proestrus phase of the estrous cycle in female mice. Female mice exposed to CUS had low renal progesterone. Impaired mitochondrial function correlated with low renal progesterone, which correlated with increased blood pressure. Renal sex steroids were unchanged by CUS in males. Urinary albumin excretion was significantly increased in female mice exposed to CUS. CUS did not affect urinary albumin excretion in male mice exposed to CUS. These data show a direct role for CUS in causing an increase in blood pressure. The mechanisms causing increased pressure in CUS-exposed mice are sex-dependent, with low renal progesterone leading to impaired renal mitochondrial function as a potential mechanism underlying the elevated pressure in female mice.

Graphical Abstract

Graphical Abstract.

Introduction

Prolonged psychological stress is associated with an increased risk for many diseases including kidney disease¹ and hypertension.² The prevalence of kidney disease (acute and chronic) and hypertension is growing with the increasingly aged population.³ The annual economic burden in the United States is estimated at >$120 billion and $219 billion for chronic and end stage kidney disease and hypertension, respectively. While there is a clear association between stress and renal disease⁴ or hypertension,⁵ the direct impact of chronic stress on the kidneys is understudied. Understanding how stress alters renal function has direct implications for developing better therapeutic strategies to manage renal and cardiovascular risk.

To date, studies examining the link between stress and renal function are commonly focused on early life stress,⁶ acute stress stimuli,⁷ or stressors that mimic trauma.^8-10 Whether chronic, mild, psychological stress directly impacts renal function to promote hypertension is unclear. In addition, the differential impact of chronic stress on the kidneys in males and females are unknown even as divergent sex-dependent responses to chronic psychological stress are clear.¹¹

Mitochondria are dynamic organelles that are essential for energy production in multicellular organisms. Kidneys are enriched with mitochondria to meet the large demand for ATP coupled transport of molecules across the nephron, including sodium, potassium, and glucose.¹² Mitochondria are fundamental for renal control of blood pressure and renal mitochondrial dysfunction contributes to the development of hypertension in part through the production of reactive oxygen species.¹³ Evidence shows that chronic stress impairs mitochondrial function in cardiovascular control regions of the brain.^{14, 15} However, whether chronic stress impairs renal mitochondrial function and leads to increased blood pressure has not been tested.

Because of the known association between chronic stress and hypertension, and the central role of the kidneys in blood pressure control, we hypothesized that exposing mice to chronic stress will lead to impaired renal mitochondrial function that is associated with an increase in blood pressure. Moreover, due to established sex-dependent responses to chronic psychological stress,¹¹ the hypothesis will be tested in both male and female mice to better understand potentially disparate mechanisms between the sexes.

Methods

Animals

Adult (6-8-wk-old) male (n = 32) and female (n = 32) C57BL/6 mice were purchased from Jackson Laboratories (Jackson Laboratories, Bar Harbor, ME, USA). Mice were housed under standard laboratory conditions and maintained on a 12-h light/dark cycle in temperature-controlled rooms with access to chow and water ad libitum. Mice were habituated to the animal vivarium for 1 wk and then gently handled for 3 consecutive days to habituate to experimenters prior to the start of the experimental protocol. Mice were randomly assigned to either a control (CON) group or a group subjected to a chronic unpredictable stress (CUS) protocol. CON animals were socially housed (4-5 animals per cage), while CUS animals were single housed beginning on the first day of the stress protocol through the end of the experiment. At the conclusion of the study, mice were rapidly euthanized by cervical dislocation and decapitation. All procedures conformed with the University of South Carolina Institutional Animal Care and Use Committee and the US National Institutes Health Guide for the Care and Use of Laboratory Animals.

CUS Protocol

Male and female mice were exposed to 28 consecutive days of random unpredictable stressors adapted from Monteiro et al.¹⁶ Once daily, under home cage isolation, the mice were exposed to either a physical or psychological stressor. The protocol is intended to model mild, persistent, and variable stress that is a common occurrence in daily life. The experimental timeline is shown in Figure 1A and the stressors are listed in Supplemental Table S1. To maximize unpredictability, time of day and stress duration varied with each daily stressor and there was no repetition of the same stressor within a given week. CON animals were gently handled by experimenters weekly. Body weight was recorded weekly for all animals. Three behavioral tests were conducted (elevated plus maze, open field, and forced swim). The elevated plus maze and open field tests were used to determine whether CUS induced anxiety- or avoidance-like behavior. The forced swim test was used to determine stress coping behavior. Results from these tests, coupled with changes in body weight, were used to generate a behavioral z-score where negative z-scores are indicative of stress-induced behavioral responses.

Figure 1.

CUS does not cause renal injury in male or female mice. (A) Experimental timeline. Created in BioRender. Hollis, F. (2025) https://BioRender.com/9rk21pl (B) Urinary albumin excretion was increased equally in control non-stressed males (in µg/24 h, control pre 23 ± 3 vs. post 35 ± 3, P < 0.01) and CUS males (stress pre 31 ± 3 vs. post 40 ± 4, P = 0.02). Male urinary albumin did not have a significant interaction between time and stress [F(1,25) = 0.49, P = 0.49], but there was an overall significant effect of time [F(1,25) = 18.79, P < 0.01] and CUS [F(1,25) = 3.24, P = 0.08]. (C) In females, there was an overall effect of stress [F(1,24) = 9.50, P = 0.01], and the interaction of time and stress was [F(1,24) = 4.00, P = 0.06]. The overall effect of time was not significant [F(1,24) = 3.63, P = 0.07]. There was a significant increase in urinary albumin excretion in CUS females (in µg/24 h, 19 ± 2 vs. 27 ± 3, P < 0.01 pre vs. post CUS). (D) No changes in glomerular injury score were detected in males or females exposed to stress. (E) Representative images of glomeruli from each group. Statistical significance was assessed by two-way ANOVA followed by uncorrected Fisher’s LSD. All data are represented as mean ± SEM. *P ≤ 0.05.

Blood Pressure

Systolic blood pressure was recorded in a subset of mice at the end of the study using tail plethysmography (Kent Scientific). Mice were lightly anesthetized with isoflurane during the measurement. Mice were placed on an infrared heating pad to raise their body temperature to 37.5°C and tail cuffs were positioned to read for 5 cycles. Average systolic, diastolic, and mean arterial pressure (MAP) was calculated from the cycles. Because the tail cuff method is a potential mild stressor,¹⁷ an attempt to measure pressure was made only once per mouse. No additional attempts were made, or cycles added, if a pressure value was not recorded to avoid undue stress for the animal. This limited the experimental number in Figure 3D.

Figure 3.

Renal mitochondrial respiration is decreased during the proestrus phase of CUS females. (A) Renal mitochondrial respiration was unchanged in female mice exposed to CUS. Statistical significance was assessed by unpaired t-test at each complex. All data are represented as mean ± SEM (B). Complex I showed a significant interaction of estrous and CUS, but [F(2,22) = 4.02, P = 0.03] there was no significant main effect of stress [F(2,22) = 0.01, P = 0.99] or estrous [F(1,22) = 0.21, P = 0.64]. CUS decreased mitochondrial respiration in CUS females in the proestrus phase (in pmol O2/mg protein, 118 ± 20 vs. 70 ± 11, P = 0.04 control vs. CUS), but not in the estrus (P = 0.99) or diestrus/metestrus (P = 0.45) phases. Statistical significance was assessed by two-way ANOVA followed by Šídák's multiple comparisons test. (C) Complex IV showed a significant interaction of estrous and CUS [F(2,22) = 3.28, P = 0.05], but no significant main effect of stress [F(2,22) = 0.23, P = 0.80] or estrous [F(1,22) = 1.54, P = 0.23]. CUS decreased mitochondrial respiration in CUS females in the proestrus phase (in pmol O2/mg protein, 329 ± 38 vs. 240 ± 9, P = 0.04 control vs. CUS), but not in the estrus (P = 0.88) or diestrus/metestrus phase (P = 0.68) phases. Statistical significance was assessed by two-way ANOVA followed by Šídák’s multiple comparisons test. All data are represented as mean ± SEM. *P ≤ 0.05. (D) Correlation between renal complex I respiration and systolic blood pressure in non-stressed control females was not significant, measured by simple linear regression and Pearson correlation [F(1,2) = 0.06, P = 0.84, r = −0.16]. (E) Correlation between renal complex I respiration and systolic blood pressure in stressed females was not significant, measured by simple linear regression and Pearson correlation [F(1,9) = 4.41, P = 0.07, r = −0.57].

Mitochondrial Respirometry

The left kidney was rapidly removed, cleaned of visceral fat, and transversely sectioned into thirds. The pole ends of the kidney were weighed and placed in a 24-well plate with 1 mL of relaxing solution (2.8 mm Ca2K2EGTA, 7.2 mm K2EGTA, 5.8 mm ATP, 6.6 mm MgCl2, 20 mm taurine, 15 mm sodium phosphocreatine, 20 mm imidazole, 0.5 mm dithiothreitol, and 50 mm MES, pH = 7.1) until further processing. Tissue samples were gently homogenized in ice-cold respirometry medium [MiR05: 0.5 mm EGTA, 3 mm MgCl2, 60 mm potassium lactobionate, 20 mm taurine, 10 mm KH2PO4, 20 mm HEPES, 110 mm sucrose and 0.1% (w/v) BSA, pH = 7.1] using a Dounce homogenizer. Samples were normalized to wet weight and 2 mg of homogenized tissue was loaded into a 2-mL chamber at 37°C to measure mitochondrial respiration rates via high-resolution respirometry (Oroboros Oxygraph 2 K, Oroboros Instruments, Innsbruck, Austria). To measure respiration due to oxidative phosphorylation and sequentially measure mitochondrial respiratory capacity at different steps of electron transport, a multi-substrate protocol was used as previously described.^{18, 19} Oxygen flux due to coupled complex I respiration was stimulated through the addition of ADP (5 mm) to a mixture of malate (2 mm), pyruvate (10 mm), and glutamate (20 mm; denoted as “complex I”). This was followed by the addition of succinate (10 mm) to stimulate coupled complex II respiration (“complex I + II”). Respiration was then uncoupled to examine the maximal capacity of the electron transport system (“ETS”) using the protonophore, carbonylcyanide m-chlorophenyl hydrazone (CCCP; successive titrations of 0.2 µm until maximal respiration rates were reached). The sole activity of complex II was then examined in the uncoupled state by inhibiting complex I by the addition of rotenone (0.1 µm; “ETS complex II”). Complex III electron transport was then inhibited by adding antimycin (2 µm) to obtain the level of residual oxygen consumption (ROX) due to oxidating side reactions outside of mitochondrial respiration. The O2 flux obtained in each step of the protocol was normalized by the wet weight of the tissue sample used for the analysis and corrected for ROX. Complex IV activity was measured by the addition of ascorbate (2 mm) to keep N, N, N′, N′-Tetramethyl-p-phenylenediamine dihydrochloride (TMPD; 0.5 mm) in a reduced state, as previously published.²⁰ Inhibition of complex IV was induced by the addition of sodium azide (100 mm) and chemical background oxygen consumption induced by auto-oxidation of TMPD and ascorbate reactions was assessed (“complex IV”). Oxygen-concentration in the chambers was sufficiently maintained to avoid limiting oxygen respiration throughout the respirometry protocol.

Estrous Cycle

Cells were collected via vaginal lavage with purified milliQ water at the time of euthanasia, placed on a microscope slide, allowed to dry for 24 h, and then stained with hematoxylin and eosin (H&E) stain for cell quantification.^21-23

Renal Injury

Overnight urine samples were collected in mice using metabolic cages prior to the start of the protocol and at the end of the protocol on day 27. Urinary albumin was measured using a commercial ELISA (Alpha Diagnostics International) according to manufacturer instructions. Data are presented as an excretion rate (urinary albumin/24 h) as previously described.²⁴ Glomerular injury was assessed by histology. The middle section of the left kidney was fixed in 10% neutral buffered formalin for 1 wk followed by transfer to and storage in 70% ethanol until further processing. Fixed tissues were embedded in paraffin wax, sectioned (5 µm sections), and stained with H&E. Two independent observers scored >25 glomeruli per slide blinded to the experimental group. Data are presented as a glomerular injury score as previously described.²⁵

Renal Sex Steroid Measurement

Half of the right kidney was flash frozen in chilled isopentane and stored at −80°C. Tissue weight was recorded, and the tissue was then homogenized in 1 mL of mass spectrometry grade acetonitrile (Fisher Scientific, Waltham, MA, USA) following the addition of stable isotope-labeled internal standards for each analyte, as previously described.²¹ Homogenization lasted for 3 to 5 min using a bead beater (BioSpec Products, Bartlesville, OK, USA) and 3.5-mm stainless steel UFO beads (Next Advance, Raymertown, NY, USA). Samples were centrifuged and the resulting supernatant was removed. The samples were resuspended in 1 mL of acetonitrile, centrifuged, and the supernatant collected for a second time. The supernatants were dried and resuspended in 200 mm sodium acetate. MTBE was added to the sample for liquid-liquid extraction of the steroids twice, then removed and dried as previously described.²⁶ After derivatization, the samples were placed in 0.2 PVDF microspin filters (Fisher Scientific, Waltham, MA, USA) and centrifuged to remove any insoluble material prior to mass spectrometry analysis. Calibration curves utilizing certified reference material (testosterone, Sigma Aldrich, Catalog# T037; E2, Sigma Aldrich, Catalog# E-060; progesterone, Sigma Aldrich, Catalog# P-069; and androstenedione, Sigma Aldrich, Catalog# A-075) were used to determine the quantity of each steroid. Chromatography and mass spectrometry analysis was carried out on a Q Exactive HF-X hybrid quadrupole-orbitrap mass spectrometer with a Vanquish HPLC on the front end (Thermo Electron, Waltham, MA, USA) as previously described.²¹

Data Analysis

This study is part of a multi-laboratory collaborative project focusing on the impact of chronic stress on the kidneys (current study), the brain and behavior (coauthor F. Hollis),²⁷ and cardiac function (coauthor F. Spinale). The behavioral outcomes and blood pressure measurements are central to all three projects. This is consistent with humane experimental principles to reduce the number of animals involved in experimental research. In this study, behavioral outcomes are utilized to validate the model of stress and correlate the behavior with blood pressure, renal mitochondrial function, and renal sex steroids. Because this was a multi-laboratory project, endpoints were measured in tissues collected from a randomized subset of animals. The data underlying this article will be shared at reasonable request to the corresponding author.

Sample sizes are indicated in the figure legends and experiments were run in 2 independent cohorts of n = 8-10/group. One female mouse was removed from sex steroid data as the sex steroid measurements were determined to be a statistical anomaly (using Grubbs Outlier Test). Data are presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism 10 (GraphPad software Inc., San Diego, CA, USA). Two-way ANOVA was used to analyze sex and stress as between subject factors, followed by Šídák’s or uncorrected Fisher’s LSD multiple comparison post hoc test where appropriate. An unpaired t-test was used to analyze the differences between stressed and control mice when appropriate. Correlation data were analyzed by simple linear regression and P-values are reported on the figures and in figure legends. A summary of statistical results is listed in Supplemental Table S2.

Results

Validation of Stress Protocol

Three behavioral tests were conducted in the last week of the CUS protocol to affirm the efficacy of the CUS protocol. Elevated plus maze was conducted on CUS day 24. There was a main effect of stress with animals spending less time in the open arms of the maze (n = 16/group, P = 0.01, two-way ANOVA) compared to non-stressed controls. However, there were no interaction (stress vs. sex, P = 0.65) and no individual group differences on post hoc analysis (Šídák’s). The open field test was conducted on CUS day 25, and neither male nor female mice displayed changes in time spent in open spaces. The forced swim test was conducted on CUS day 26. Female mice, but not males, exposed to CUS displayed a decreased latency time to immobility (n = 16-18/group, P = 0.04, two-way ANOVA) compared to non-stressed controls. These data suggest that the CUS protocol induces anxiety-relevant and passive stress coping behaviors in male and female mice, and that there are sex-dependent responses to different behavioral tests.

Chronic Stress Increases Blood Pressure in Male and Female Mice

The impact of CUS on systolic blood pressure was measured by tail cuff in males and females. The stress exposed mice showed an increase in systolic blood pressure in both males (in mmHg, n = 11-12/group, 101±3 vs. 122 ± 4, P < 0.01 control vs. stress) and females (in mmHg, n = 4-9/group, 109±11 vs. 146 ± 6, P < 0.01, main effect of stress) compared to non-stressed controls. The blood pressure response to CUS was different between males and females (main effect of sex, P < 0.01). While there was a main effect of stress on blood pressure, there was no statistical difference in the interaction of sex and stress (P = 0.11) given that pressure increased in both males and females. However, there was a main effect of sex (P < 0.01) with a greater increase in blood pressure caused by CUS in the female mice compared to males. The increased pressure in response to CUS is consistent with the effect of stress on blood pressure in other rodent models.^28-31

Chronic Stress Does Not Cause Renal Injury

Chronic stress is associated with worse outcomes for patients with renal disease; however, limited data directly assess the impact of chronic stress on markers of renal injury. Urinary albumin excretion was calculated by measuring overnight urine samples collected prior to the beginning of the CUS protocol (Pre) and on day 27 of the CUS protocol (Post) using a commercial ELISA and correcting for urine volume/24 h. Urinary albumin excretion was increased equally in male mice exposed to CUS and non-stressed controls (Figure 1B). Urinary albumin excretion was statistically increased in female mice subjected to CUS but was unchanged in the non-stressed control females (Figure 1C). Renal Kim-1 protein expression, a sensitive early marker of tubular injury,³² was not different between CUS and control groups in either sex and was not different across the phases of the estrous cycle in females (Supplemental Figure S1). The impact of CUS on glomerular histology was assessed in renal sections stained with H&E. An index of glomerular injury was calculated in sections from a subset of mice. Neither males nor females showed significant evidence of glomerular injury when compared to stressed animals (Figure 1D an E, P = 0.69). When the data was analyzed by the number of scored glomeruli in individual categories (ie, 0 = glomeruli with no injury, 1 = glomeruli with up to 25% of area injury, 2 = up to 50% injury, 3 = up to 75%, 4 = up to 100%), subtle evidence of injury emerged (Supplemental Figure S2). Female mice subjected to stress had more glomeruli in category 3, whereas male mice subjected to stress had more glomeruli in category 2.

Chronic Stress Impairs Renal Mitochondrial Respiration Only in Female Mice

Because of the central role for the kidneys in long-term blood pressure control, and the prominent role for mitochondria in maintaining renal function, we investigated whether chronic stress impaired mitochondrial function as a potential contributing mechanism to the elevated pressure. The effects of stress and sex on renal mitochondrial function were evaluated using high-resolution respirometry. In male mice subjected to CUS, there was no impact on renal mitochondrial respiration across all complexes (Figure 2A). However, there was a significant negative correlation between complex I coupled respiration and systolic blood pressure in male stressed animals, such that as complex I respiration decreased, systolic blood pressure increased (P = 0.01, r = −0.76) (Figure 2C). This correlation was not observed in control males (P = 0.94, r = 0.044) (Figure 2B).

Figure 2.

Renal mitochondrial respiration is unchanged by CUS in males. (A) No changes in male renal mitochondrial function were observed overall in control vs. CUS. Statistical significance was assessed by unpaired t-test at each complex. All data are represented as mean ± SEM. (B) No correlation was observed between control males and systolic blood pressure [Simple linear regression and Pearson correlation, F(1,8) < 0.01, P = 0.90, r = 0.04]. (C) A significant correlation was observed in male mice exposed to CUS between systolic blood pressure and mitochondrial function at complex I [simple linear regression and Pearson correlation, F(1,8) = 11.08, P = 0.01, r = −0.76].

Renal mitochondrial respiration did not appear to be changed in female mice subjected to CUS (Figure 3A). However, when the data was analyzed by estrous cycle phase, mitochondrial respiration was significantly impaired at complex I (Figure 3B) and complex IV (Figure 3C) only during the proestrus phase of the cycle. There was an overall significant effect of the estrous cycle phase on renal mitochondrial respiration in stressed animals at both complex I and complex IV (interaction, P < 0.05) (Figures 3B and C). Renal oxygen consumption at complex I negatively correlates with blood pressure in stressed females (Figure 3E), but not in control females (Figure 3D).

Renal Mitochondrial Oxidative Phosphorylation Complexes Are Not Altered By Chronic Stress

Protein analysis via western blot was used to assess relative renal mitochondrial content and mitochondrial complex number from the same left kidney homogenate used for mitochondrial respirometry. Translocase of the Outer Membrane 20 (TOM20) is an outer mitochondrial membrane protein used as an indicator of mitochondrial content. There was no change in mitochondrial content in either stressed males or females compared to non-stressed controls (Supplemental Figure S3A-D). When female TOM20 protein analysis was analyzed by estrous phase, there were no significant differences at any phase of the cycle (Supplemental Figure S3E). Renal mitochondrial oxidative phosphorylation proteins were assessed to determine if the CUS protocol affected the relative expression of specific complexes in the mitochondrial respiratory chain. There was a significant increase in complex I protein expression in the males exposed to CUS (Supplemental Figure S4A and C, P = 0.02), but no significant changes were observed at any other complexes (Supplemental Figure S4A and D-G). There were no significant changes at any complex, even when analyzed by estrous cycle phase, in females exposed to CUS compared to non-stressed controls (Supplemental Figure S4B and H-L).

Chronic Stress Alters Renal Sex Steroids Only in Female Mice

While, chronic stress can alter circulating sex steroid levels, the influence of chronic stress on renal sex steroids is unclear. Both progesterone and estrogen reportedly promote and preserve normal mitochondrial function.^33-35 In male mice, renal sex steroids were not altered by exposure to the CUS protocol (Figure 4A-B), and there was no relationship between renal progesterone and blood pressure, or renal respiration at complex I (Figure 4C-D). In female mice, renal testosterone and estrogen were unchanged in the CUS-exposed animals, while renal progesterone was decreased (Figure 5A-C). However, because hormone levels change across the estrous cycle,³⁶ we analyzed the data by estrous cycle phase. In kidneys from female mice exposed to CUS, testosterone was unchanged during proestrus and diestrus/metestrus phases compared to non-stressed controls. However, renal testosterone was lower during the estrus phase in female mice exposed to the CUS protocol (Figure 5D). Renal 17β-estradiol was not different between control and stressed females in any phase of the estrous cycle (Figure 5F). Renal progesterone was lower in kidneys from female mice subjected to CUS across the estrous cycle (Figure 5E). A significant negative correlation exists between blood pressure and renal progesterone in female mice, whereby lower renal progesterone correlates with higher blood pressure (P < 0.01, r = −0.78) (Figure 5G). A significant positive correlation exists between renal progesterone and complex I respiration, whereby impaired complex I function correlates with lower renal progesterone (P = 0.03, r = 0.40) (Figure 5H).

Figure 4.

Renal sex steroids are not altered by CUS in males. (A and B) Neither renal testosterone [F(7,7) = 30.25, P = 0.21] nor progesterone [F(7,7) = 1.79, P = 0.88] were altered by the CUS protocol in males. Statistical significance was assessed by unpaired t-test. All data are represented as mean ± SEM. (C) Renal progesterone does not correlate with systolic pressure in males [simple linear regression and Pearson correlation, F(1,7) = 0.95, P = 0.36, r = 0.35]. (D) Renal complex I respiration does not correlate with renal progesterone in males [simple linear regression and Pearson correlation, F(1,10) = 3.05, P = 0.11, r = −0.48].

Figure 5.

Renal sex steroids are altered by CUS in females. (A) No statistical difference was seen in renal testosterone in female mice [F(13,13) = 4.10, P = 0.88]. Statistical significance was assessed by unpaired t-test. (B) Renal progesterone was decreased in female mice exposed to stress [F(13,13) = 1249, P = 0.09]. (C) No statistical difference was seen in renal estrogen in female [F(13,13) = 1.32, P = 0.94]. Statistical significance was assessed by unpaired t-test. (D) Renal testosterone was decreased in the CUS females compared to controls in the estrus phase [F(4,3) = 72.38, P = 0.02 in pg/g, 10.4 ± 2.9 vs. 0.39 ± 0.38, control vs. CUS]. There was no significant effect of stress on testosterone levels in female mice in proestrus [F(5,4) = 6.71, P = 0.23], or diestrus/metestrus [F(3,3) = 3.27, P = 0.21]. Statistical significance was assessed by unpaired t-test. (E) Renal progesterone was lower in the diestrus/metestrus phase [F(3,3) = 23.49, P = 0.02 in pg/g, 1555 ± 328 vs. 482 ± 68, control vs. CUS], and in the proestrus [F(4,5) = 1971, P = 0.14], and estrus [F(4,3) = 133.8, P = 0.14] phases in CUS females. Statistical significance was assessed by unpaired t-test. (F) Renal estrogen levels were unchanged by CUS in proestrus [F(5,4) = 1.43, P = 0.75], estrus [F(4,3) = 1.27, P = 0.88], or diestrus/metestrus [F(3,3) = 2.91, P = 0.40]. Statistical significance was assessed by unpaired t-test. (G) There was a significant correlation between blood pressure and renal progesterone in female mice. Statistical significance was assessed by simple linear regression and Pearson correlation [F(1,26) = 7.05, P = 0.01, r = −0.78]. (H) There was a significant correlation between log transformed renal progesterone and renal mitochondrial function at complex I. [Simple linear regression and Pearson correlation, F(1,27) = 5.27, P = 0.03, r = 0.40]. All data are represented as mean ± SEM. *P ≤ 0.05.

Because renal progesterone was consistently low across the estrous cycle in females exposed to CUS, we assessed the relationship between renal progesterone and a composite behavioral z-score. There was no relationship between behavioral z-score and renal progesterone or mitochondrial respiration at complex I in male mice (Figure 6A-B). A positive correlation was found between renal progesterone and behavioral z-score in female mice, where an increased z-score indicates a less stressed animal (P < 0.01, r = 0.55) (Figure 6C). A positive correlation was also found between mitochondrial complex I respiration and behavioral z-score in female mice, where an increased z-score (lower stress) correlated with a higher complex I respiration (P = 0.05, r = 0.37) (Figure 6D).

Figure 6.

Behavioral phenotype correlates with renal progesterone and renal mitochondrial function in females but males. (A) Renal progesterone levels do not correlate with behavioral z-score in males [simple linear regression and Pearson correlation, F(1,11) = 0.14, P = 0.72, r = 0.01]. (B) Renal complex I respiration does not correlate with behavioral z-score in males [simple linear regression and Pearson correlation, F(1,25) < 0.01, P = 0.97, r = 0.01]. (C) Renal progesterone levels correlated with the behavioral z-score. A higher z-score is associated with lower stress [simple linear regression and Pearson correlation, F(1,28) = 9.89, P < 0.01, r = 0.55]. (D) Mitochondrial function at complex I in females correlates with the behavioral z-score [simple linear regression and Pearson correlation, F(1,27) = 4.33, P = 0.05, r = 0.37, n = 29]. All data are represented as mean ± SEM. *P ≤ 0.05.

Discussion

Psychological stress has been cited as a major risk factor for the development of renal disease and hypertension; however, the mechanisms linking stress with changes in renal function that can cause an increase in pressure remain poorly understood. The major new findings of this study are that (1) chronic stress impairs renal mitochondrial function in females in an estrous cycle dependent way, but stress does not impair renal mitochondrial function in males; (2) exposure to chronic stress increases blood pressure in both male and female mice, and there is a sex difference in the blood pressure response to stress; (3) chronic stress alters renal sex steroids in females, and these changes correlate with impaired renal mitochondrial function and increased blood pressure; (4) impaired renal mitochondrial function directly correlates with a stressed behavioral phenotype; and (5) chronic stress does not cause apparent renal injury in either male or female mice.

It has long been recognized that psychological stress can evoke acute changes in blood pressure and renal hemodynamic function. A study from 1950³⁷ directly showed that subjecting normotensive and hypertensive individuals to emotional conflict caused an increase in mean blood pressure and decrease in renal plasma flow with a greater effect in the hypertensive group. Another early study utilized an epidemiological approach to assess the impact of multiple environmental factors, including indices of anxiety, on blood pressure variance in men and found only a very small contribution of these factors.³⁸ Studies in rodent models like the spontaneously hypertensive rat showed that mental stress caused by a jet of air blown at the rats for 20 min, causes an acute increase in arterial pressure accompanied by reduced sodium excretion.³⁹ In mice, a study showed that males on a low salt diet exposed to chronic psychosocial stress for up to 4 mo caused an increase in systolic pressure, independent of renal injury.⁴⁰ More recent studies continue to support the link between chronic stress and hypertension in both humans and experimental models. For example, Inoue et al. showed that patients enrolled in the Multi-Ethnic Study of Atherosclerosis (MESA) who had higher circulating levels of stress hormones, such as cortisol, were more likely to have hypertension.⁴¹ Similarly, stress induced by restraint or social defeat in rats, consistently causes an increase in pressure.^8-10 The utilization of the CUS protocol in the present study is intended to model the exposure to random life stressors experienced by humans.⁴² Our data are consistent with blood pressure effects caused by other models of stress in rodents, and they expand the knowledge in two important ways. First, the data show that CUS directly increases blood pressure in both sexes, with a greater increase in the females. While this may not be surprising, previous studies largely focused on male responses to stress. Second, CUS causes sex-dependent changes in renal mitochondrial function thus providing new evidence that the mechanisms driving CUS induced blood pressure changes are different between males and females. Although not measured in this study, evidence shows that CUS activates the renin angiotensin aldosterone system (RAAS) in rodents and that behavioral changes induced by chronic stress can be mitigated by RAAS blockade.^43-47 A recent study characterized the dose dependent effects of angiotensin II (AngII) on renal mitochondrial function in mice.⁴⁸ A low pressor dose of 400 ng/kg/min stimulated renal mitochondrial respiration whereas a high pressor dose of 1000 ng/kg/min impaired mitochondrial respiration. These previous studies did not analyze data by sex, even in those that included females in the experimental design, and highlight the complex role of renal RAAS in the stress response. Future studies are needed to determine the potential role of RAAS in the model used here.

While significant data links behavioral responses to stress with mitochondrial dysfunction in the brain,^{15, 18, 19, 42, 49} the impact of chronic stress on mitochondrial function and its role in blood pressure control is unclear. In male C57BL6 mice subjected to chronic stress induced by daily intermittent foot shocks, MAP was increased in association with impaired mitochondrial function in the rostral ventral lateral medulla.⁵⁰ However, the effect of chronic stress on renal mitochondrial function as it relates to hypertension is unknown. The kidneys are enriched with mitochondria to meet the large metabolic demand required for ATP-coupled transport of electrolytes across the nephron. Impaired renal mitochondrial function is associated with both experimental and human hypertension and renal injury.^{51, 52} The present study addresses an important knowledge gap by showing that CUS causes impaired renal mitochondrial respiration in females, but not males. Our protein expression data suggest that CUS-induced changes in renal mitochondrial function are not due to a decrease in mitochondrial content, as TOMM20 expression levels were not different between groups. The differences in females cannot be explained by decreases in mitochondrial complex protein levels either. In contrast, CUS males exhibited significantly increased complex I protein expression compared to their non-stressed counterparts, suggesting a potential compensatory mechanism for protecting renal mitochondrial function from CUS-induced declines. Renal mitochondrial function is generally protected by progesterone and estrogens in female rodents, whereas male rodents are more susceptible to androgen mediated renal mitochondrial dysfunction and renal disease.⁵³ Our data suggest divergent mechanistic pathways for stress induced hypertension in males and females.

The impact of stress on renal sex steroids was considered in this study because of the known effects of stress on sex steroids, the role of mitochondria in their synthesis, and mitochondrial regulation by sex steroids. We previously showed that renal sex steroids track with circulating sex steroids across the estrous cycle in female C57BL6 mice.²¹ Because the kidneys play a central role in the long-term control of blood pressure, and sex steroids regulate mitochondrial function, we hypothesized that altered renal sex steroids in response to CUS might be a contributing factor to the impaired mitochondrial function and associated pressure increase. In the present study, renal progesterone was consistently lower across all phases of the estrous cycle in female mice exposed to CUS when compared to non-stressed female controls. Progesterone is important for preserving normal renal hemodynamic function, electrolyte and water transport, thus providing protection against the development of hypertension.⁵⁴ Progesterone also can preserve mitochondrial function in models of kidney disease,⁵⁵ traumatic brain injury,⁵⁶ and in cardiomyocytes.³⁴ While progesterone receptors are expressed in renal tubular epithelial and vascular smooth muscle cells,^{57, 58} and in mitochondria,³⁴ whether mitochondria isolated directly from the kidneys have progesterone receptors does not appear to have been tested. Whether stress-induced decreases in mitochondrial function are the cause or consequence of reduced progesterone will be an important point of future studies. It is also possible that impaired renal mitochondria could be responsible for lower tissue levels of progesterone. Our data showing that stressed females have lower renal progesterone is consistent with reports in both humans and rats where circulating progesterone is reduced in response to stress.^{59, 60} It also provides a potential mechanistic link between CUS and renal mitochondrial dysfunction that may explain why the blood pressure response to CUS is greater in female mice than the CUS-exposed male mice in which there were no changes in renal sex steroids.

To gain additional mechanistic insight, the relationship between renal progesterone and blood pressure or mitochondrial respiration was examined. Lower renal progesterone correlated with impaired mitochondrial respiration at complex I, and with increased blood pressure in female mice. These data suggest that stress-induced decreases in renal progesterone may contribute to the decreased renal mitochondrial function and increased blood pressure in females. Similarly, a stressed behavioral phenotype (low behavioral z-score) in female mice correlated with low renal progesterone and decreased mitochondrial respiration at complex I. This data further supports the link between CUS and low renal progesterone leading to impaired renal mitochondrial function, and the subsequent increase in pressure. The reported relationship between chronic stress and progesterone levels is variable in humans. In the Swiss Perimenopause Study, higher levels of progesterone in women are associated with fewer symptoms of depression such as lower perceived stress.⁶¹ In women experiencing symptoms of post-traumatic stress disorder, no correlation was observed between stress and progesterone.⁶² A meta-analysis assessing sex steroids in response to psychosocial stress suggests that progesterone is either unchanged or increased.⁶³ It is difficult to experimentally control for chronic stress in human studies, and information on tissue levels of sex steroids in response to stress are not available. Experimental animal models offer the controlled environment needed to more directly assess these relationships. The exposure of male rats to a CUS protocol for 4 or 5 wk is associated with a reduction in prefrontal cortex and hippocampal progesterone levels.^{64, 65} In a model of maternal separation with subsequent exposure to a CUS protocol in male mice, CUS was associated with reduced hippocampal progesterone.⁶⁶ The present study is the first to directly assess the relationship between CUS exposure in females with renal sex steroids levels.

There is a clear relationship between chronic kidney disease and social and psychological stressors.^{41, 67} The CUS protocol used in this study resulted in no significant renal injury (albumin excretion or glomerular injury) in either males or females. Although albumin excretion increased in males, it did so equally in the CUS-exposed and non-stressed mice. In females, there was a significant increase in urinary albumin excretion directly attributable to the CUS protocol; however, the levels of albumin are not consistent with microalbuminuria. The absence of renal injury in response to CUS may not be surprising given that the C57Bl/6 strain is notably resistant to renal injury.⁶⁸ However, it is possible that the 28-day CUS exposure leaves the mice susceptible to a “second hit.”

In conclusion, the present study advances the field toward a more complete understanding of how chronic stress can alter renal physiology and potentially promote hypertension. By itself, the collection of data from both male and female mice is an important advance for the field. The value of this data is even greater given that disparate sex-specific mechanisms appear to be underlying the changes in renal mitochondrial function and blood pressure increase. Nevertheless, there are limitations that should be considered. First, the correlations presented herein do not prove causation. Second, among inbred mouse strains, C57BL6 mice are notably resistant to both renal injury⁶⁹ and the effects of chronic stress.^{70, 71} Therefore, this study forms the foundation to address future questions surrounding the intersection of chronic stress with other hypertensive stimuli.

Author Contributions

Noelle I. Frambes (Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Writing – original draft, Writing – review & editing), Alexia M. Crockett (Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing), Amelia M. Churillo (Investigation, Methodology, Project administration, Writing – review & editing), Alaina Mullaly (Formal analysis, Investigation, Methodology, Project administration, Writing – review & editing), Molly Maranto (Data curation, Formal analysis, Investigation, Methodology, Project administration, Writing – review & editing), Cameron Folk (Formal analysis, Investigation, Methodology, Project administration, Writing – review & editing), Lisa A. Freeburg (Data curation, Formal analysis, Investigation, Methodology, Project administration, Writing – review & editing), Reilly T. Enos (Data curation, Formal analysis, Investigation, Methodology, Project administration, Writing – review & editing), Eliana Cavalli (Data curation, Investigation, Methodology, Project administration, Writing – review & editing), Susan K. Wood (Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Writing – review & editing), Francis G. Spinale (Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Writing – review & editing), Fiona Hollis (Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Writing – review & editing), and Michael J. Ryan (Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing).

Funding

National Institutes of Health (NIH) U54HL169191 and U.S. Department of Veterans Affairs (VA) BX002604 (to M.J.R.), University of South Carolina Office of the Vice President for Research (to M.J.R., F.H., F.G.S.), VA Merit Award 5I01-BX000168 (to F.G.S.), VA Merit Award 5I01-BX005320 (to F.G.S.), BX005661 (to S.K.W.), NIH R01HL13765 (to F.G.S.), R01HL67994 (to F.G.S.), P20GM155896 (to F.G.S.), VA BX006218-02 (to F.H.).

Conflict of Interest Statement

The contents do not represent the views of the US Department of Veterans Affairs or the United States Government. F.H. receives limited funding for research conducted in collaboration with MitoQ. The data presented here are not a part of that collaboration nor were they associated in any way by MitoQ.

Data Availability

The data underlying this article will be shared at reasonable request to the corresponding author