Effects of Immobilization Stress on Kidneys of Wistar Male Rats: A Morphometrical and Stereological Analysis

This paper verifies the morphological changes induced by immobilization stress on the kidney of rats by using stereological methods. Fifteen 4-week-old Wistar male rats were randomly assigned to control (n = 7) and stressed (n = 8) groups. Stress stimuli were performed over 5 weeks by immobilization of the rats for 2 h daily in a rigid opaque plastic cylinder that restrained their movements. Increases in the adrenal mass index (p ! 0.05) and decreases in serum testosterone levels (p ! 0.05) demonstrated the efficacy of the stressor stimuli. Stressed rats presented diminished body weight gain when compared to controls (p ! 0.05). The mean values of kidney weight, kidney volume, kidney volume index and glomerular volume density were significantly lower in the stressed group (p ! 0.05); nevertheless, no significant difference was found in the cortical/medullar ratio or in the volume-weighted mean glomerular volume. The number of glomeruli per kidney was 45% lower in the stressed group (p ! 0.0001), but no change in serum creatinine levels was found. However, the morphological alterations may have serious implications predisposing individuals to renal disease and hypertension in adult life. Copyright © 2011 S. Karger AG, Basel Received: July 13, 2010 Accepted: April 1, 2011 Published online: June 28, 2011 Célia Martins Cortez Department of Applied Mathematics, IME, Rio de Janeiro State University Rua São Francisco Xavier, 524 (sexto andar) Maracanã, Rio de Janeiro, RJ 20559-900 (Brazil) Tel. +55 212 334 0144, E-Mail ccortez @ uerj.br © 2011 S. Karger AG, Basel 1420–4096/11/0346–0424$38.00/0 Accessible online at: www.karger.com/kbr D ow nl oa de d by : 54 .7 0. 40 .1 1 10 /6 /2 01 7 6: 47 :3 2 A M Immobilization Stress on Kidneys of Wistar Male Rats Kidney Blood Press Res 2011;34:424–429 425 al., 1975; Krishtal and Gozhenko, 1989; Chen et al., 2004]. Singh et al. [2007] observed that maternal GC treatment results in a nephron deficit and development of hypertension in the offspring of rats, suggesting increased risk of hypertension and other diseases in adult life [Seckl, 2004]. They suggested it may have important implications for women experiencing significant stress during pregnancy. On the other hand, some studies have demonstrated that postnatal exposure to prolonged stress and excess GC is also able to affect the anatomy and physiology of some organs [Krishtal and Gozhenko, 1989; Cortez et al., 2003]. Krishtal and Gozhenko [1989] showed that both stress and GC elevate the urinary excretion of hydrogen ions, and Hu et al. [2000] observed a significant increase in adrenal and kidney weight in repeated swimmingstressed animals when compared to controls. There is not much literature about the effects of the different kinds of chronic stress on kidney morphology, and further studies are required to clarify the role of renal structural changes induced by stress in the pathogenesis of renal disease. It is possible that prepubertal chronic immobilization stress leads to increased GC release and that this hormone causes a negative effect on renal tissue morphology. Thus, the aim of the present study was to verify the morphological changes induced by prepubertal immobilization stress in the kidney of rats by using stereological methods. Material and Methods Fifteen 4-week-old Wistar male rats were included in this study. The animals were kept with their mothers until the third week of life. Only male pups were used in the study. The rats were kept in a room with controlled temperature (25 8 1 ° C) with an artificial dark-light cycle (lights on from 7: 00 a.m. to 7: 00 p.m.) and were fed standard rat food and water ad libitum. The rats were weighed weekly until the day of death. All experiments were done according to Brazilian law for the scientific use of animals, and this project was formally approved by the local ethics committee. The rats were randomly assigned to two groups: control (n = 7) and stressed (n = 8). The control group was maintained under standard conditions until 9 weeks of age. The stressed group was submitted to stress stimuli daily until 9 weeks of age. Stress stimuli were performed by immobilization in a rigid opaque plastic cylinder that restrained the movements of the rats [Retana-Marquez et al., 2003]. The cylinders, with different diameters and length, were adjusted weekly depending on the animal’s weight. The animals did not experience any pain and were contained for 2 h daily in the morning from the 28th to the 63rd day of life. Adrenal mass index (weight of adrenal/body weight) and testosterone serum concentration (measured by radioimmunoassay) were used to assess the physiologic efficacy of the stress stimulus [Bauer et al., 2001; Hardy et al., 2005]. On the 64th day of life, under deep anesthesia, the blood was collected by heart puncture and the serum was separated by centrifugation and used for creatinine and testosterone determination. The rats were killed by an anesthetic overdose and the kidneys and adrenal glands were dissected, cleared of adipose tissue and weighed. The volume of the right kidneys was estimated by the water displacement method, [Scherle, 1970]. The kidneys were then sectioned frontally, fixed in 10% formaldehyde and routinely processed for paraffin embedding. Serial 5 m sections of the entire kidney were obtained and stained with hematoxylin and eosin. The cortical/medullar ratio was estimated by using the Cavalieri principle [Cruz-Orive and Weibel, 1990] and the absolute cortical volume was calculated by multiplying the cortical/medullary ratio by the renal volume. The left kidneys were sectioned sagittally into small fragments, fixed in 10% formaldehyde, routinely processed for paraffin embedding, sectioned in thicknesses of 5 m and stained with hematoxylin and eosin. From each animal, 26 histological fields obtained from different sections of the kidney cortex were acquired with a digital camera coupled to a microscope. A transparent M42 test-system was adjusted on the monitor and the glomerular volume density was estimated by the point-counting technique [Aguila et al., 2005]. The volume-weighted mean glomerular volume was estimated with the point-sampled intercepts method [Gundersen and Jensen, 1985], analyzing 50 glomeruli per animal. The estimation of the total number of glomeruli per kidney was calculated by multiplying the cortical volume by the glomerular volume density, divided by the volume-weighted mean glomerular volume [Aguila et al., 2005]. Additionally, the number of glomeruli corrected for kidney weight was calculated by dividing the total number of glomeruli of one kidney by the mean kidney weight of each animal [Aguila et al., 2005]. Student’s t test was used for mean comparisons. In all cases, significance was set at a probability value of 0.05. All analyses were performed using GraphPad Prism software.