Virginia A Aparicio1*, Daniel Camiletti-Moirón1, Mohammed Tassi2, Elena Nebot1, Carlos de-Teresa1,3 and Pilar Aranda1
1Department of Physiology, Faculty of Pharmacy and Institute of Nutrition and Food Technology, University of Granada, Spain
2Department of Pathologic Anatomy and Institute of Regenerative Biomedicine, School of Medicine, University of Granada, Spain
3Andalusian Centre of Sport Medicine, Granada, Spain
Received: 08 June, 2017; Accepted: 24 June, 2017; Published: 26 June, 2017
Virginia A Aparicio, Department of Physiology, Faculty of Pharmacy, Campus de la Cartuja s/n, 18011, Granada, Spain, Tel: 34-958-243882; Fax: 34-958-248959; E-mail:
Aparicio VA, Camiletti-Moirón D, Tassi M, Nebot E, de-Teresa C, et al. (2017) Effects of Anabolic Androgenic Steroids on Renal Morphology in Rats. Arch Renal Dis Manag 3(2): 034-037. DOI: 10.17352/2455-5495.000024
© 2017 Aparicio VA, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Corrosive injury; Lye; Acid; Management
Aim: To investigate the long-term effects of anabolic androgenic steroids (AAS) on renal status in rats.
Methods: Twenty Wistar rats were distributed into 2 groups: AAS or placebo, for 3 months. The animal received 10mg/kg body weight of Stanozonol once a week by intramuscular injection in the gluteus, or saline solution as placebo.
Results:Urinary pH was more acidic in the AAS compared to the placebo group (p<0.05). Kidney weight was 15% higher in the AAS compared to the placebo group (p<0.001). Renal glomerular area was 12% higher in the AAS compared to the placebo group (p=0.001). Animals injected with AAS also displayed a no significant but clinically relevant ~20% higher kidney interstitial connective tissue, glomerular tufts and mesangiums.
Conclusions: Overall, AAS negatively affected urinary pH and kidney morphology leading to a worse renal status. More attention should be taken to the use of AAS among populations at high risk for kidney disease.
Anabolic androgenic steroids (AAS) are one of the most used performance-enhancing substances among professional athletes as well as recreational body builders . Chronic use of AAS has been known to cause several alterations. Among these disorders, renal diseases have received less attention probably because they are less frequent while heart disease, altered lipid profile and hepatotoxicity have been extensively explored [2,3]. Nephrotoxic side effects have been documented, such as renal failure or Wilms’ tumours [3-7].
As use of AAS is illicit, much of the knowledge of their effects is derived from case reports or retrospective studies. Another limitation of human studies is represented by the fact that information about AAS is generally self-reported, and it is difficult to know the exact dosage. Furthermore, AAS are often used in combination with other substances (e.g. growth hormone) and it is complicated to separate its effects. Consequently, experimental studies conducted in animal models are mandatory given the complexity of carrying out long-term and well-controlled interventional studies in humans. The present study aimed to examine the renal effects of AAS.
A total of 20 male Wistar rats were allocated into two groups: AAS vs. placebo. The animals, with an initial body weight of 152±8g were housed in group cages. The cages were located in a well-ventilated thermostatically controlled room (21±2°C) with a relative humidity ranging 40-60% and a reverse 12h light-12h dark cycle (08:00-20:00h). Throughout the experimental period (12 weeks) all rats had free access to type 2 water and consumed the diet ad-libitum. Experimental diets were formulated to meet the nutrient requirements of rats based on the AIN-93M formulation.
The rats’ body weights were measured weekly at the same time, and the amount of food consumed by each rat was registered daily.
On week 11, a 12-hour urine sample from each animal was collected for biochemical analysis. At the end of the experimental period, animals were anaesthetized with ketamine-xylazine and sacrificed by cannulation of the abdominal aorta. Blood was collected and centrifuged at 4500rpm to separate plasma that was frozen in liquid N and stored at -80ºC. Carcass weight was recorded. Kidneys were extracted, weighed, and the left one was introduced in formalin for the posterior histological analysis.
All experiments were performed according to the Directional Guides Related to Animal Housing and Care, and all procedures were approved by the Animal Experimentation Ethics Committee of the University of Granada (ref:2011-343).
The animals from the experimental group received 10mg/kg body weight of Stanozonol once a week by intramuscular injection in the gluteus (alternating the lateral side). This dosage is comparable to which has been reported as being frequently used by athletes . We used a commercially available Stanozolol solution of 50mg/mL (Winstrol Depot, Zambon) that was diluted to appropriate concentrations and to keep the volume of injection constant. Control groups were injected with saline solution as placebo.
Urinary pH was analysed with a bench pH-meter (Crison, Barcelona). Plasma urea, albumin and creatinine concentrations were measured with an autoanalyzer (Hitachi-Roche p800, Hoffmann-La Roche Ltd).
The left-kidney samples were fixed in 4% buffered formalin and embedded in paraffin. Subsequently, three micrometer sections were cut for a hematoxylin eosin stain and four-micrometer-thick sections were obtained and stained with 1% Picrosirius red F3BA (Gurr, BDH Chemicales Ltd, Poole, United Kingdom) . This technique facilitates the visualization of connective fibers as deep red stains on a pale yellow background . The sections were assessed by optical microscopy. Forty images per sample were captured: 20 of the glomerulus to determine the morphometry and the intraglomerular connective tissue and 20 of the tubulointerstitial area to measure the interstitial connective tissue. All images were acquired using the 20× objective and analyzed with the Fibrosis HR® software . This image analysis application allowed us to automatically quantify morphometric parameters by using various image-processing algorithms.
The following eight morphological variables were stimated: a) Percentage of interstitial connective tissue in reference to the image area, excluding the glomerular area (the connective tissue that is in the gap over the Bowman’s capsule). b) The area of interstitial connective tissue (including Bowman’s capsule). The Fibrosis HR® software divides glomerular tufts into two categories: “glomerular tuft I” and “glomerular tuft II”. The variable “glomerular tuft I” correspond to the renal corpuscle excluding the Bowman’s capsule. The variable “glomerular tuft II” corresponds to the renal corpuscle excluding the Bowman’s capsule and considering the area of the capillary lumens and urinary spaces in the glomerulus. c) Glomerular tuft I area. d) Glomerular tuft II area. e) Glomerular tuft I percentage (percentage of glomerular tuft I related to the glomerular area). f) Glomerular tuft II percentage (percentage of glomerular tuft II related to the glomerular area). g) Mesangial area. h) Glomerular area.
Analysis of variance (ANOVA) test was used to compare AAS and placebo groups. Additionally, effect size between groups was calculated by using the Cohen’s d (standardised mean differences) statistic.
All analyses were performed using the Statistical Package for Social Sciences (IBM-SPSS, version 20.0 for Windows), and the level of significance was set at p<0.05.
The effects of AAS on final body weight, carcass weight, food intake, plasma, urinary and renal parameters are shown in table 1. Urinary pH was lower and volume higher in the AAS compared to the placebo group (p<0.05). No differences between groups were observed on body weight, carcass weight, food intake and plasma urea, creatinine and albumin (all, p>0.05).
Kidney weight was ~15% higher in the AAS compared to the placebo group (p=0.009). Renal glomerular area was ~12% higher in the AAS compared to the placebo group (p=0.001). Despite no statistically significant, but with large effect size, AAS group showed ~20% enlarged interstitial connective tissue, glomerular tufts I and II, and mesangiums (Figure 1).
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