Behavioral, metabolic, and renal outcomes of 1-month isolation in adolescent male Dahl salt-sensitive rats

Abstract

Social contact deficit is considered a stressful circumstance associated with various neural, hormonal, genetic, immune, and behavioral effects. A growing body of clinical and basic science evidence suggests that social isolation is linked to a higher risk of various neurological, cardiovascular, and metabolic diseases, including hypertension, diabetes mellitus, and obesity. However, the impact of the deficit of social interaction on kidney function is not well established. The Dahl salt-sensitive (SS) rat is a classical model of salt-induced hypertension and associated kidney injury. In this study, we investigated the effect of 30 days of social isolation (SI) on blood and urine electrolytes and metabolic, physiological, and behavioral parameters in adolescent male Dahl SS rats fed a normal 0.4% NaCl diet. SI rats demonstrated increased behavioral excitability compared with rats kept in groups. We also observed increased food consumption and a decrease in plasma leptin levels in the SI group without differences in water intake and weight gain compared with grouped animals. No changes in the level of blood and urine electrolytes, 24-h urine output, creatinine clearance, and albumin/creatinine ratio were identified between the SI and grouped rats. These findings indicate that 30 days of social isolation of adolescent Dahl SS rats affects metabolic parameters but has no apparent influence on kidney function.

INTRODUCTION

A large body of evidence suggests that social support is critical for health and psychological well-being. In humans, loneliness is associated with a higher incidence of cardiovascular disease, type 2 diabetes and metabolic dysregulation, immune dysfunctions, cognitive and affective disorders, and all-cause mortality (2). Animal studies have shown consistency with human findings regarding the adverse effects of social isolation (SI) on cardiovascular health (5, 29), type 2 diabetes, and obesity progression (20, 24). Considering that these conditions increase the incidence of the development of chronic kidney disease (CKD) (9), it can be expected that SI might mediate the adverse effects on the kidney function of vulnerable individuals. It has been identified that SI is a critical risk factor for diabetic kidney disease (DKD) (11), while a high social network score is associated with reduced incidence and progression of DKD in patients with type 2 diabetes (8). Social deprivation is also one of the strong independent predictors of mortality in patients with CKD (25). In contrast, social support has been associated with improved outcomes and increased survival rates in patients with end-stage renal disease (4). However, the causal link in these observational studies remains unclear. Thus, experimental animal studies are needed to better understand this relationship.

To date, most studies have focused on the effects of SI in adults animals (33). However, there is a lack of animal studies focused on the impact of SI on the progression of cardiovascular and renal disorders in the adolescent age (16). Such studies are critical, considering current trends with adolescent and young adults, when a new generation has less human-to-human interactions. Another important factor playing a crucial role is the COVID-19 pandemic, which is creating massive isolation of billions of people for several weeks to months. As reported, the kidney is one of the critical targets of SARS-CoV-2 (1, 18). Adolescents are especially vulnerable during the pandemic since they lose the ability to socialize, which is critical for the development of the healthy neuroendocrine system.

The present study aimed to evaluate the effect of 1-mo SI on renal function, metabolic parameters, blood, and urine electrolyte balance in adolescent Dahl salt-sensitive (SS) rats housed in home cages. The SS rat is a widely used model to study salt-induced hypertension. In addition to elevated blood pressure, this strain exhibits an elevated level of urinary albumin at a young age due to the intrinsic renal abnormalities and slowly progressed albuminuria with age, regardless of diet (26). As SI stress was shown to induce various hormonal, immune, and neural alterations that could potentially affect renal function, we hypothesized that SI in the adolescent age might exacerbate the progression of renal damage in SS rats.

MATERIALS AND METHODS

Experimental protocol and animals.

Male Dahl SS rats were used throughout the study and treated in compliance with the animal use and welfare guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and following protocol review and approval by the Medical College of Wisconsin Institutional Animal Care and Use Committee. Animals were fed a normal salt diet (0.4% NaCl; 0.36% KCl; AIN-76 purified rodent chow; Dyets No. 113755, Bethlehem, PA) with water and food provided ad libitum. At 5 wk of age, animals were separated randomly into two groups: isolated (SI; 1 rat/cage) or group-housed (GH; 3 rats/cage) for 30 days. SI and GH animals were kept in the same room to sustain a uniform visual, auditory, and olfactory social contact. Twenty-four-hour food and water intake and body weight measures were done 24 h after separation and every 10 days.

Electrolyte measurements and albumin assay.

Before the start of the isolation procedure and at the end of the isolation protocol, rats were placed in metabolic cages (40615; Laboratory Products) for 24-h acclimation and then for 24-h urine collection. Blood was collected from the tail vein on the 32nd day of the experimental period after urine collection. Blood and urine electrolytes were analyzed using the ABL800 FLEX analyzer. The albumin blue fluorescent assay kit was acquired from Active Motif (Carlsbad, CA) and the creatinine assay kit was acquired from Cayman Chemical (Ann Arbor, MI).

Creatinine clearance was estimated as described previously (21).

Leptin ELISA.

Leptin study was performed on blood plasma collected from SI or GH animals at the end of the isolation protocol and stored in −80°C (for current experiments, plasma samples were stored for ∼9 mo). Plasma leptin levels were determined using a Rat Leptin ELISA Kit (ab100773, Abcam, Cambridge, MA) at a 3× dilution, according to manufacturer’s instructions.

Behavioral tests.

A pick-up test was performed on the 30th day of the experimental period to assess behavioral excitability. Animal behavior in response to grasping around the body was scored as it was reported previously (13).

For the sociability test, animals were kept in groups or isolated in cages for one more week after final blood and urine collection. Social behavior was assessed using a three-chamber apparatus (122 cm × 61 cm × 30 cm), divided by walls with central openings, into three areas (12). Before the experiment, each animal was placed into the empty apparatus for a 5-min acclimation time and then returned to the home cage. Thereafter, an age-matched male SS rat unfamiliar to the test rat was placed in a wire cage in one of the peripheral chambers of the apparatus. An empty cage in the opposite chamber was considered as an object. The test animal was then placed to the apparatus again for 10 min to assess the preference between an unfamiliar rat or an object. Each exploration session was video-recorded, and the time spent sniffing the rat and the object cages was analyzed.

Statistical analysis.

All data were presented as means ± SE. Analysis of all data was performed by an experimenter blinded to a treatment group. Statistical significance was verified with a Student’s t test or a nonparametric Mann–Whitney test where appropriate. To evaluate changes in metabolic and urine parameters, we used a repeated-measures ANOVA with post hoc Šidák correction for multiple comparisons.

RESULTS AND DISCUSSION

To assess the effect of SI on behavioral excitability, a pick-up test was performed in GH (grouped) and SI (isolated) rats at the age of 9 wk. During the pick-up test, all GH animals were easily grasped and elevated without vocalization or aggressive behavior. At the same time, most SI group rats demonstrated audible vocalizations and/or struggling when being elevated. Figure 1A shows that SI rats exhibited significantly more excitable behavior than GH controls (GH: 1.0 ± 0.0 vs. SI: 2.2 ± 0.3; P < 0.02, Mann–Whitney test). In a three-chamber sociability test, both groups of rats spent significantly more time interacting with an unfamiliar rat compared with an object (Fig. 1B). There were no differences in relative time spent with a rat or with an object between experimental groups.

Fig. 1.

Effect of 30-day social isolation on the behavior of adolescent salt-sensitive (SS) rats. A: behavioral excitability of the rats was evaluated using pick-up test. B: time spent interacting with a rat or an object in a three-chamber sociability test. n = 5–6 animals/group. *P < 0.05.

Over the 30 days of the experimental period, GH and SI rats were gradually gaining body weight (BW) and increasing food intake. We did not observe the difference in the BW change between experimental groups (Fig. 2A). Both groups increased their water intake toward the end of the experiment, but no significant differences were found between the two housing environments (Fig. 2B). Individually housed SS rats consumed significantly more food than GH ones, as evidenced as early as 10 days of isolation [P = 0.01, repeated-measures (RM) ANOVA test, Fig. 2C]. Figure 2D shows that the plasma leptin level in rats reared 30 days in isolation was significantly lower than that in GH rats (P < 0.03, Mann–Whitney test).

Fig. 2.

Effect of social isolation on metabolic measures. Summary graphs of the effect of 30-days social isolation on body weight gain (A), water intake (B), food consumption (C), and leptin plasma level (D) in adolescent salt-sensitive (SS) rats on a low salt diet. n = 4–9 animals/group. *P < 0.05, **P < 0.01.

Blood sodium, potassium, and chloride levels were not significantly different between SI and GH rats at the end of the experiment (Table 1). Glucose levels were also similar between experimental groups (Table 1). After 30 days of the experimental period, both groups demonstrated a significant increase in 24-h urine volume (P < 0.05, RM ANOVA, Fig. 3A). However, there were no significant effects of SI on this parameter in SS rats. Urine creatinine levels were similar between SI and GH rats, and there were no significant changes in values before and after the experimental period in both groups (Fig. 3B). We also did not find any differences in the albumin/creatinine ratio and creatinine clearance between SI and GH animals (Fig. 3, C and D). Furthermore, there were no significant differences in urine glucose levels between groups (GH: 2.8 ± 0.7 mmol/L; SI: 3.9 ± 1.3 mmol/L) as well. Finally, Fig. 4 shows that SI had no significant effect on 24-h sodium, potassium, and chloride excretion levels and electrolyte to creatinine ratios (Fig. 4).

Table 1.

Blood electrolytes and glucose values in adolescent isolated and group-housed salt-sensitive rats over a 30-day experimental period

Parameter, mmol/L	Group
	Grouped (n = 9)	Isolated (n = 8)
Na+	141.0 ± 0.5	140.6 ± 0.9
K+	4.0 ± 0.1	3.9 ± 0.1
Cl−	107.9 ± 1.7	109.4 ± 1.1
Glucose	9.7 ± 0.8	9.5 ± 0.3

Data are means ± SE; n, number of rats.

Fig. 3.

Urinary output, urine creatinine level, albumin-to-creatinine ratio, and creatinine clearance in adolescent-grouped and socially isolated salt-sensitive (SS) rats. A: twenty-four-hour urine volume in isolated and group-housed rats. B: urine creatinine level. C and D: summary graphs of albumin-to-creatinine ratio and creatinine clearance in isolated and grouped rats at the end of the experimental period. n = 6–9 animals/group. *P < 0.05.

Fig. 4.

Effect of 30-day social isolation urine electrolytes level in adolescent salt-sensitive (SS) rats. A–C: daily electrolyte excretion normalized to 100 g body weight. D–F: urine electrolyte/creatinine ratios. n = 9 animals/group.

Briefly summarizing, our data revealed that 1-mo SI induced behavioral and metabolic alterations in adolescent male Dahl SS rats but did not affect the blood and urine electrolyte balance and renal function. One of the potential limitations of the present study is that collecting some data such as performing pick-up tests, measuring water/food intake, and body weight changes was not performed blindly. To limit observer-related bias, the data analyses were performed at the end of the experiment by other investigators completely blinded to the treatment protocols used.

Since the early 1960s, long-term SI has been generally considered as a chronic stress-related condition due to its physiological effects, such as increase in adrenal hypertrophy and reduction of thymus weight, as well as behavioral abnormalities, including increased reactivity to handling, aggression, and depressive and anxiety-like behavior (3, 11a, 32). SI-induced changes in young individuals are more pronounced and could be irreversible due to the ongoing maturation of the neuroendocrine system (3). In our study, SI in the adolescent period increased behavioral excitability in SS rats, which is in general agreement with increased aggressiveness and anxiety-like behavior of isolated animals reported previously. It was also shown that long-term isolation, especially at a young age, may affect social-related behavior resulting either in decrease or increase in sociability depending on the strain, the age of the animals when experiments started, and the duration of the isolation procedure (19, 28, 34). In the present study, we did not find any difference in SI and GH Dahl SS rats’ behavior during the three-chamber sociability test.

SI rats consumed significantly more food, while their water consumption and body weight were not different from those of GH rats. These data agree with the systematic review and meta-analysis study of the effect of rodent SI on energy balance regulation and metabolic health (24). The study revealed that despite the contradictory data on the effects of individual housing on the body weight, increased food consumption in isolated animals is more consistent. Interestingly, the plasma level of leptin, a hormone that plays a critical role in energy homeostasis and hunger control, was significantly downregulated in SI rats, which may explain the increase in the overall food intake in the SI group. One of the potential explanations is that SI rats experience a lack of group thermoregulation. Downregulation of leptin production was demonstrated in response to cold exposure in human and animal studies (7). Therefore, alteration of leptin level and subsequent stimulation of food intake may play a compensatory role in a cold-induced increase of energy expenditure. Stress-related conditions by themself were also shown to affect the expression of genes involved in energy metabolism, including leptin, due to hypothalamic-pituitary-adrenal (HPA) axis hyperactivation (14, 17). Further studies need to determine the specific mechanisms of SI’s effects on the metabolic parameters of adolescent SS rats observed in our study.

It was shown previously that individual housing exacerbates the onset of age-related proteinuria and increases the severity of glomerular pathology in adult Wistar rats (33). It is well documented that young SS rats show increased urinary albumin excretion compared with other strains, which worsens during aging and does not depend on salt loading (10, 22, 26). It is also established that, in young SS rats, hypertension is maintained due to sympathetic nervous system hyperactivation (35); thus, SI stress might aggravate the existing kidney pathology in this strain. Our results show that neither blood/urine electrolyte balance, albumin excretion, or creatinine clearance were affected in SS rats by SI, suggesting that 30 days of individual housing has no apparent effect on water electrolyte balance and kidney function in this strain.

Perspectives and Significance

Human studies indicate the association between social deprivation and kidney health. However, there is a lack of animal studies exploring this relationship. Our data indicate that, although 1-mo isolation of adolescent male Dahl SS rats in home cages results in behavioral and metabolic alterations, it has no apparent effect on kidney function. It has been shown previously that the gender difference in the Dahl salt-sensitive rat does not impact the variability in over 100 phenotypes, including metabolic and renal parameters (6). On the other hand, human and animal studies consistently show that isolation experienced in adolescence results in a striking difference on endocrine and behavioral end points in males and females (15, 23, 27, 30). So, further studies are needed to better understand the impact of gender in experimental settings described in the present study. Finally, as Dahl SS rats represent a classical model for study salt-induced hypertension and SI is linked to a higher risk of developing hypertension, additional studies are required to test whether a high-salt diet will contribute to kidney injury and blood pressure in the setting of isolation.

GRANTS

This research was supported by the National Institutes of Health Grant R35 HL135749 and by the Department of Veteran Affairs Grant I01 BX004024 (to A. Staruschenko).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

E.I., O.P., and A.S. conceived and designed research; E.I. and V.L. performed experiments; O.N. and E.I. analyzed data; E.I. interpreted results of experiments; O.N. and E.I. prepared figures; O.N. and E.I. drafted manuscript; O.N., E.I., O.P., and A.S. edited and revised manuscript; O.N., E.I., V.L., O.P., and A.S. approved final version of manuscript.