Role of Renin-Angiotensin System, Renal Nerve System, and Oxidative Stress in Chronic Stress-Induced Renal Expression of Aquaporin-1 in Rats

Research Article

Austin J Nephrol Hypertens. 2021; 8(1): 1093.

Role of Renin-Angiotensin System, Renal Nerve System, and Oxidative Stress in Chronic Stress-Induced Renal Expression of Aquaporin-1 in Rats

Hu L-X1#, Jiang W-Y2#, Wang Y-Y2#, Chen J-W3* and Zhang G-X2*

1Department of Pathophysiology, Jining Medical University, 133 He-Hua Road, PR China

2Department of Physiology, Medical College of Soochow University, PR China

3Department of Internal Medicine, Nanjing University of Chinese Medicine, PR China

#These authors are contributed equally to this work

*Corresponding author: Guo-Xing Zhang, Department of Physiology, Medical College of Soochow University, 199 Ren-Ai Road, Dushu Lake Campus, Suzhou Industrial Park, Suzhou 215123, PR China

Jing-Wei Chen, Department of Internal Medicine, the Suzhou TCM Hospital Affiliated to Nanjing University of Chinese Medicine, 18 Yang-Su Road, Suzhou 215003, P.R. China

Received: June 01, 2021; Accepted: July 02, 2021; Published: July 09, 2021


Aims: To investigate the renal aquaporin-1 (AQP1) expression under chronic stress (induced by foot shock) condition and possible mechanisms involved in rats.

Methods: The chronic stress model was established in male Sprague Dawley (SD) rats by foot shock for two weeks. Rats were randomly divided into control group, chronic stress group, renal denervation group, renal denervation plus chronic stress group, captopril (an angiotensin I converting enzyme inhibitor, ACEI) plus chronic stress group and tempol (a superoxide dismutase mimetic) plus chronic stress group. Body weight, food intake, water intake, blood pressure and heart rate were monitored. Real-time PCR was used to detect the mRNA level of AQP1 in the renal tissue. Immunohistochemistry stain was used to observe the expression and location of AQP1 in rat kidneys.

Results: Chronic stress reduced body weight gain and food intake, while it significantly increased systolic blood pressure and renal expressions of mRNA and protein of AQP1 (P<0.05) as compared with control group. Renal denervation and tempol treatments did not affect stress-induced decreases of body weight gain and food intake. Renal denervation, captopril and tempol treatments decreased systolic blood pressure. Compared with the chronic stress group, mRNA and protein expression of AQP1 was decreased (P<0.05) in renal denervation plus chronic stress group, captopril plus chronic stress group and tempol plus chronic stress group.

Conclusion: Chronic stress induces increase of the AQP1 expression in kidney, which is regulated by renal nerve system, renin-angiotensin system and oxidative stress.

Keywords: Aquaporin-1; Stress-induced hypertension; Oxidative stress; Renal nerve system; Renin-angiotensin system (RAS); Vasopressin


An individual’s response to stress should promote the ability of the body to maintain homeostasis. Physiological stress response occurs through several pathways involving the sympathetic nervous system, Hypothalamic-Pituitary-Adrenal (HPA) axis, and behavioral fight-or-flight response. However, chronic stress condition eventually disrupts the homeostasis, resulting in pathological and psychological sequela. The continuous exposure to chronic stress insults can induce maladaptive reactions, including depression, anxiety, cognitive impairment, and cardiovascular diseases [1-3]. Long-term stress affects the functioning of the body in several ways. For instance, overactivation of the autonomic nervous system and abnormal changes in the humoral factors have been reported to be responsible for stress-induced imbalance of the body [4,5], the excessive release of neuronal transmitters and changes in the humoral factors could disturb the homeostasis. Our previous observations have demonstrated that chronic stress (foot shock) activates the neuronal factors through the Renal Sympathetic Nerve System (RSNS), the neuronal and humoral factors through the Renin-Angiotensin System (RAS), and the humoral factors through oxidative stress. These factors eventually contribute to the development of hypertension [6,7]. Therefore, it is imperative to understand how chronic stress disrupts the homeostasis.

Water homeostasis is one of the fundamental regulatory systems in the body. The kidneys are vital for maintaining the water volume of the body. Several Aquaporin (AQP)-type water channels are expressed in this organ to regulate the water volume. AQP1, which is essential for water re-absorption and urinary concentration, has been shown to be abundantly expressed in the proximal tubule, descending thin limb Henle, and vasa recta [8,9]. Polyuria has been observed in mice with defective AQP1, indicating that AQP1 plays an essential role in urine hypertonicity [10,11]. AQP1 has also been demonstrated to protect the renal system from endotoxemia-induced acute kidney injury [12], besides retarding the renal cyst development in polycystic kidney disease [13]. Such findings reveal that AQP1 is important in maintaining the renal function and water homeostasis.

Hypertension is a disease involving multiple factors such as genetic, environmental, and lifestyle issues [14-16]. This condition increases the cardiovascular and cerebral events [17,18]. The expression of AQP1 in the kidneys varies from one hypertension model to another. For example, high expression of AQP1 has been noted in spontaneous [19] and angiotensin II-induced hypertension models [19], while low expression has been observed in Deoxycorticosterone Acetate (DOCA)-salt [20] and sodium-sensitive hypertension models [21]. The role of AQP1 and its contribution to the development of hypertension in the models remains largely unknown. Our previous observation revealed that RAS, RSNS and oxidative stress reciprocally potentiate to play important roles in the development of foot shockinduced hypertension [6,7]. Until date, there are no data available on the expression of renal AQP1 in chronic stress-induced hypertension model, and the mechanism that regulates its expression is also unclear.

In the present study, we explored the expression of AQP1 in the foot shock-induced hypertension model and investigated the possible mechanisms behind it.


Chronic stress animal model

For this study, 10-week-old male Sprague–Dawley rats were purchased from the Shanghai Laboratory Animal Center (Shanghai, China). The rats were housed under optimal conditions with standard hygiene at the animal center of the Soochow University. The temperature was maintained at 25°C with a 12/12 light-dark cycle. Stress was applied by placing the rats in a foot shock box with a metal grid floor for 4 h. The electric current was controlled using an electronic device (0.15 mA, 5–30 s with random current). The rats’ renal nerve was surgically denervated under anesthesia with 10% chloral hydrate according to previous reports [22]. Foot shock stress was commenced 1 week after the surgery. Captopril (an Angiotensin I-Converting Enzyme Inhibitor [ACEI], 100 mg/kg/ day) or Tempol (a superoxide dismutase mimetic, 10 mg/kg/day) was intraperitoneally injected daily after the stress exposure. Each group consisted of 15 rats. The parameters of body weight, water and food intake, and Systolic Blood Pressure (SBP) of the rats were monitored. SBP was monitored for 30 minutes every day 2 hours after foot shock during the test period for two-week by tail-cuff method as our previous report [7]. The animals were anesthetized and sacrificed on day 14. Their kidneys were harvested for further analysis. The study was reviewed and approved by the Animal Ethics Committee of Soochow University.

Enzyme-Linked Immunosorbent Assay (ELISA) for vasopressin in the serum

Serum level of vasopressin was measured using commercially available Arg8-vasopressin ELISA kits (Abcam Inc., Shanghai, China, Catalog No: ab205928). All steps were performed according to the manufacturer’s instructions.

Real-time PCR for AQP1 mRNA expression in the renal tissues

Total RNA was isolated from the renal tissues by guanidinium isothiocyanate-acid phenol extraction and quantified by measuring the absorbance at 260 nm. One microgram of the sample was used for reverse transcription, and the rat AQP1 was determined by realtime PCR (Prism 7000; Applied Biosystems, Foster, California). The primer pairs for rat AQP1 cDNA were forward 5'-GAC CTG ATG CTG TGG CTT CT-3' and reverse 5'-GAA TGT GGC TCT CGG TTC AC-3'. The primer pairs for GAPDH were forward 5'-GGA GAT TAC TGC CCT GGC TCC TA-3' and reverse 5'-GAC TCA TCG TAC TCC TGC TTG CTG-3'. The expression of AQP1 mRNAs was normalized using GAPDH mRNA.

Western blot for AQP1 protein expression in the renal tissue

Renal tissues were homogenized with RIPA buffer (50 mm Tris, pH 7.0, 150 mM NaCl, 1% Triton-X-100) containing phenylmenthanesulfonyl fluoride (R&D Systems Inc., Minneapolis, US). Homogenates were centrifuged at 12,000×g for 10 minutes at 4°C. Cell protein were separated by SDS-PAGE and transferred to PVDF membranes (Hybond TM-ECL; Amersham Pharmacia Biotech, Inc.). The membranes were blocked in 5% nonfat milk in PBS and 0.1% Tween-20 at room temperature. The blots were then incubated with primary antibody: Anti-AQP1 (1:1000, abcam, Inc., Catalog No: ab15080) or anti-GAPDH (Santa Cruz Biotech, Inc., Catalog No: sc-47724). Then the membranes were incubated for 1 hour with a secondary antibody (HRP-conjugated anti-rabbit Ig- G, 1:2000, abcam, Inc., Catalog No: ab205718). Excess antibody was washed off with TBS-T three times (15 minutes each) before incubation enhanced chemiluminescent reagent (ECL, R&D Systems Inc, Minneapolis, USA) for 1 min. Subsequently, the membrane was exposed to X-ray film. Immunoreactive bands were detected by the analysis of X-ray films using the software of Image J. The quantity of target proteins is normalized by GAPDH expression.

Immunohistochemical staining for AQP1 protein expression in the renal tissues

The renal tissues were fixed with 4% formaldehyde for 48 h, dehydrated, and then embedded in paraffin, and cut into 10-μm slices. Anti-AQP1 first antibody (1: 200 dilution, Abcam Inc., Shanghai, China, Catalog No: ab15080) was applied, following which the secondary antibody anti-rabbit IgG (1:2000, Abcam Inc., Shanghai, China, Catalog No: ab205718) was added. The protein expression was observed microscopically (Olympus, Japan). Ten areas close to the renal tubules were randomly selected from each section. The proportion of AQP1 positive cells in the renal tubular epithelium were calculated by the following equation:

Positive rate (%)=number of positive cells/(positive cells number + negative number)X 100%.

The immunohistochemical score of renal AQP1 expression was evaluated as per the criteria listed in Table 1, and the total score was obtained by adding the score of the positive ratio and that of the tinting intensity.