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.

Keywords: mitochondria; pressure; renal; sex; steroid; stress.

Graphical Abstract

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.

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].

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].

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.

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.