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Morphometric changes and AQP2 expression in kidneys of young male rats exposed to chronic stress and a high-sucrose diet
Biomedicine & Pharmacotherapy 105 (2018) 1098–1105
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Morphometric changes and AQP2 expression in kidneys of young male rats exposed to chronic stress and a high-sucrose diet
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Cristhian Neftaly Sánchez-Solísa, Estela Cuevas-Romerob, Alvaro Munozc, ⁎ Margarita Cervantes-Rodríguezd, Jorge Rodríguez-Antolínb, Leticia Nicolás-Toledob, a
Doctorado en Ciencias Biológicas, Universidad Autónoma de Tlaxcala, Tlaxcala, Mexico Centro Tlaxcala de Biología de la Conducta, Universidad Autónoma de Tlaxcala, Tlaxcala, Mexico c Centro Universitario del Norte, Universidad de Guadalajara, Jalisco, Mexico d Facultad de Nutrición, Universidad Autónoma de Tlaxcala, Tlaxcala, Mexico b
A R T I C LE I N FO
A B S T R A C T
Keywords: Creatinine Mesangial cell Mesangial matrix Puberty rats Restriction stress High sucrose diet
Objective: Consumption of a cafeteria-like diet and chronic stress have a negative impact on kidney function and morphology in adult rats. However, the interaction between chronic restraint stress and high-sucrose diet on renal morphology in young rats is unknown. A high-sucrose diet does not modify serum glucose levels but reduces serum corticosterone levels in stressed young rats, in this way it is confusing a possible potentiate or protector effect of this diet on kidney damage induced by stress. Methods: Wistar male rats at 4 weeks of age were randomly assigned into 4 groups: control (C), stressed (St), high-sucrose diet (S30), and chronic restraint stress plus a 30% sucrose diet (St + S30). Rats were fed with a standard chow and tap water (C group) or 30% sucrose diluted in water (S30 group). Chronic restraint stress consisted of 1-h daily placement into a plastic cylinder, 5 days per week, and for 4 weeks. Results: Stressed rats exhibited a low number of corpuscles, glomeruli, high number of mesangial cells, major deposition of mesangial matrix and aquaporin-2 protein (AQP-2) expression, and low creatinine levels. Meanwhile, high-sucrose diet ameliorated AQP-2 expression and avoided the reduction of creatinine levels induced by chronic stress. The combination of stress and high-sucrose diet maintained similar effects on the kidney as stress alone, although it induced a greater reduction in the area of proximal tubules. Conclusions: Our results show that both chronic stress and a high-sucrose diet induce histological changes, but chronic stress may generate an accelerated glomerular hypertrophy associated with functional changes before puberty.
1. Introduction Consumption of soft drinks contribute to obesity-related chronic kidney disease (CKD) in humans [1,2] and rats [3,4]. Particularly, the consumption of a cafeteria diet induces inflammation and advanced glycation end products in the kidneys of adult rats [5] which induce glomerulosclerosis in adult mice [6], as well as glomerular hypertrophy associated with an excessive mesangial matrix [7,8], proximal tubular hypertrophy [9,10], and glomerular hyperfiltration [11]. Additionally, hyperglycemia induced by streptozotocin affects the aquaporin-2 protein (AQP-2) expression in the collecting duct principal cells of rats [12]. However, it is unknown whether a high-sucrose diet in young rats could increase enough to affect renal function, considering that it does not modify serum glucose levels [13].
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Stress is considered an important cause for the development of metabolic syndrome [14], which also induces morphometric [15], and functional renal alterations [16]. In this regard, glucocorticoids increase AQP-2 protein expression and glomerular filtration rate in adult [17] and infant [18] rats, decrease proteoglycans synthesis in mesangial cells [19], and regulate renal transport [20,21], which have been associated with hypertension [22]. The physiological renal effects of combining a rich-sucrose diet and stress on the kidneys is unknown, and may be confuse because a highsucrose diet reduces corticosterone levels in serum and the activity of the hepatic 11-beta hydroxysteroid dehydrogenase, which coincides with a reduced response to chronic restraint stress [13]. For these reasons, we investigated the histological changes at glomeruli level, expression of AQP-2, and serum creatinine levels in young
Corresponding author at: Leticia Nicolás Toledo, Ph. D., Centro Tlaxcala de Biología de la Conducta, Universidad Autónoma de Tlaxcala, 90000-Tlaxcala, México. fax: (52) 246 46 215
57. E-mail address: [email protected] (L. Nicolás-Toledo). https://doi.org/10.1016/j.biopha.2018.06.086 Received 10 April 2018; Received in revised form 13 June 2018; Accepted 14 June 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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Imager A1, Thornwood, NY) equipped with a digital camera (Tuczon Olympus, Tucson, AZ). Reconstructions of the images from each cortical and medulla zones kidney were performed. The number of glomeruli was determined in the reconstruction of the right kidney. Density of glomeruli was obtained with a virtual square of 250,000 μm2 drawn into the kidney reconstructions [23]. Using a grid of 10 × 12 cm on the computer screen, some glomerulus were randomly chosen and photographed at 40x to measure the area renal corpuscle (Vtuft), the glomerular area (Vglom) and the proximal tubules area using Axiovision 4.8 (Carl Zeiss MicroImaging, Inc.). Bowman’s space (VBS) was evaluated using a semi-quantitative score method calculated as VBowman’s space = Vglom-Vtuft [11]. The glomerular area was categorized in two groups, small size (< 4000 μm2) and large size (> 4001 μm2). The renal corpuscle area was classified in two groups, small size (< 5000 μm2) and large size (> 5001 μm2). Finally, the Bowmanʼs space area was categorized in two groups, small size (< 900 μm2) and large size (> 901 μm2). Mesangial cells and the mesangial matrix inside the glomeruli was scored with a scale ranging from 0 to 4 (0 for no change; 1 for changes affecting < 25%; 2 for changes affecting 25–50%; 3 for changes affecting 50–75%; and 4 for changes affecting > 75% of the glomeruli) using the Axiovision 4.3 program [24].
rats exposed to chronic stress and a high-sucrose diet. Our hypothesis is that either chronic stress or a high-sucrose diet induce histological changes in the kidney of young rats, whereas both factors together may potentiate kidney injury independently of glucocorticoids levels. 2. Materials and methods 2.1. Animals Thirty-two 21-day-old and 60–80 g body weight post-weaned Wistar male rats (Rattus norvegicus) were housed in individual 37 cm × 27 cm × 16 cm polypropylene cages and maintained at 20 ± 2 °C with a 12 h light/12 h dark cycle (light on at 12:00 h) at the Centro Tlaxcala de Biología de la Conducta from the Universidad Autónoma de Tlaxcala. Rats were randomly assigned into four experimental groups: Control-standard chow (C, n = 6–8), chronic restraint stress (St, n = 6–8), 30%-sucrose diet (S30, n = 6–8), and chronic restraint stress plus a 30% sucrose diet (St + S30, n = 6–8). The Research Ethics Committee of the Universidad Autónoma de Tlaxcala approved all experimental procedures according to the Mexican Guidelines for Animal Care, which are based on recommendations by The Association for Assessment and Accreditation of Laboratory Animal Care International.
2.6. AQP-2 immunohistochemistry
2.2. Dietary protocol
Additional kidney sections were deparaffinized and incubated in a microwave-heated 10 mM sodium citrate pH 6 solution to retrieve the antigens. Endogenous peroxidases were quenched with 0.3% hydrogen peroxide diluted in phosphate-buffered saline (PBS) during 30 min. Afterwards, tissue sections were blocked with 5% donkey serum diluted in PBS containing 0.3% Triton X100 (PBST) during 1 h. Thereafter, sections were first incubated with a primary antibody anti-AQP-2 (1:100; Santa Cruz Biotechnology, Inc.) during 72 h at 4 °C, rinsed and then with a secondary antibody (mouse anti-goat IgG-HRP) for 2 h at 37 °C. Sections were rinsed with PBS, and the immunostaining was developed according to the directions of a Vectastain ABC kit (Vector Labs, USA), using 0.05% 1.3′-diaminobenzidine (Sigma-Aldrich, St. Louis, MO) and 0.01% H2O2 as enzymatic substrate. Slides were washed and counterstained with Mayer’s hematoxylin, dehydrated and mounted with histological resin. Photomicrographs were obtained with a 10× magnification objective using an optical microscope (Zeiss Axio Imager A1, USA) equipped with a digital camera (Tuczon Olympus, USA). To comparative ends, two photomicrographs from the upper and lower portion of the renal cortex and medulla were taken. The percentage of the positive area for AQP2 cells stained in the collecting ducts was semi-quantitatively calculated using a true color image analysis system with Axio Vision Rel 4.6 (Zeiss Software Inc.), and applying a segmentation method with fixed thresholds. An overall mean value of the estimations was determined and used for comparisons among groups [25].
All rats were fed with a standard diet (Purina Laboratory chow 5001). Rats in groups C and St had access to tap water ad libitum, whereas those in groups S30 and St + S30 groups had access to 30% sucrose-sweetened tap water ad libitum for 4 weeks [13]. 2.3. Restraint stress procedure The protocol used to induce stress by motility restraint was previously reported [13]. Briefly, rats were placed in a 16 cm long by 6 cm in diameter plastic tube that limited their movement; one end of the tube was kept open to allow the animal to breathe. Rats were exposed daily for 1 h to this procedure at 11:00 h for five consecutive days/ week, for 4 weeks; rats were not subjected to this stressful stimulus during the weekends. Rats were returned to their home cages immediately after stress sessions. Rats in the C and S30 groups were not exposed to restraint stress and were maintained in their home cages throughout the experimental period. 2.4. Kidney weight and serum creatinine levels At the end of the experimental period, rats were decapitated using a rodent guillotine (Harvard-Apparatus, Holliston, MA) between 08:00 and 09:00. Trunk blood was collected and serum was obtained by centrifugation [13]. Kidneys were immediately removed and their weight means are reported in milligrams kidney tissue/100 g body weight [13]. Serum creatinine levels were measure using a commercially available method using enzymatic-colorimetric kits, and in accordance with the manufacturer directions (Elitech Clinical Systems, México).
2.7. Statistical analysis Data are presented as mean ± SEM unless otherwise is stated. Oneway ANOVA followed by a Bonferroni’s test was carried out to determine significant differences between groups. Factors considered for the former analysis were the stress, high-sucrose diet, and interactions between them. In all cases, p ≤ 0.05 was considered statistically significant. All statistical analyses were carried out using GraphPad Prism Software (Version 5.01) for Windows.
2.5. Histology of kidney Right kidneys were immersed in Bouin-Duboscq fixative for 24 h at room temperature. The tissue then was dehydrated through 60, 70, 80, 96 and 100% ethyl alcohol, cleared in xylene, embedded in paraplast Plus (Sigma-Aldrich, St. Louis, MO) and sectioned longitudinally with a microtome (Leica RM2135, Bensheim, Germany) at 5 μm, and three kidney sections mounted per slide. Series of slides were stained with Periodic Acid-Schiff (PAS) to evaluate the renal morphology. Stained sections were photographed with a light microscopy at 40× (Zeiss Axio
3. Results 3.1. Characteristics of corpuscles Compared to the C group, the kidney weight was significantly lower in the rats that drank the 30% sucrose solution (p < 0.0001; Fig. 1a). 1099
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Fig. 1. Effect of chronic stress and sucrose consumption on characteristics of kidney and renal corpuscles evaluated in longitudinal PAS stained kidney sections. C, control; St, chronic stress; S30, 30% sucrose diet; St + S30, chronic stress + 30% sucrose diet. n = 6–8. Values are expressed as mean ± SEM. *p < 0.05 for C vs. St; &&&p < 0.0001 for C vs. S30 and #p < 0.05 St vs. St + S30; groups.
the high-sucrose diet groups (Fig. 1f).
The treatment combination (St + S30 group) also decreased the kidney weight compared to the St group (p < 0.05; Fig. 1a), but was similar to the S30 group. No differences were found by the interaction vs. the St and S30 groups (Fig. 1a). The number of corpuscles was decreased by the chronic stress (St group) when compared to the C group (p < 0.05; Fig. 1b). No effects were found in the number of corpuscles by highsucrose diet or its interaction with chronic stress (Fig. 1b). The cross-sectional area of the renal corpuscle was unaffected by either chronic stress or a high-sucrose diet when compared to the C group, but it was decreased by the chronic stress in the St + S30 group in comparison with the C group (p < 0.05; Fig. 1c). No differences were found by the interaction vs. the chronic stress or high-sucrose diet (Fig. 1c). The classification of corpuscles in small and large show that the percentage of large renal corpuscles (> 5001 μm2) was decreased in the stressed groups when compared with the C group (p < 0.05; Fig. 1d). No effects were found by high-sucrose diet in comparison with the C group or its interaction with chronic stress (Fig. 1d). The mean area of the Bowmanʼs space remained unaffected on any experimental condition (Fig. 1e). The classification by size of renal corpuscles with a large Bowmanʼs space (> 901 μm2), was significantly reduced in the stressed groups when compared with the C group (p < 0.05; Fig. 1f). No effects on large Bowmanʼs space were found by high-sucrose diet in comparison with the C group, or after comparing the chronic stress vs.
3.2. Changes in glomerular size and proximal tubes The kidney sections from the C group showed a detailed cortical parenchyma and the renal corpuscles appeared as dense rounded structures with the glomerulus surrounded by a narrow Bowmanʼs space (Fig. 2a,b). In the St group, we observed a decrease in the area for the Bowmanʼs space with adhesions of the Bowman capsule, the presence of hypercellularity at the glomerular level, and mesangial matrix expansion (Fig. 2c,d). The kidney sections from the S30 group only showed a reduction of the Bowmanʼs space (Fig. 2e,f). Finally, the observation in the St + S30 group indicated a reduction in the Bowmanʼs space, as well as glomerular hyperplasia, mesangial matrix expansion and the presence of adherences (Fig. 2g,h). The mean value for the glomeruli area was unaffected by chronic stress or the high-sucrose diet compared to the C group, but it was significantly decreased by the interaction vs. the chronic stress or highsucrose diet when compared to the C group (p < 0.001; Fig. 2i). The analysis of classification by size showed that the percentage of glomeruli with a large area (> 4001μm2) in the stressed groups was lower than that in the C group (p < 0.05; p < 0.001, respectively; Fig. 2j). No differences were found by the high-sucrose diet or when comparing 1100
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Fig. 2. Effect of chronic stress and sucrose consumption on the glomerular area, mesangial cells number, and mesangial matrix expansion visualized in longitudinal PAS stained kidney sections. C, control; St, chronic stress; S30, 30% sucrose diet; St + S30, chronic stress + 30% sucrose diet. n = 6–8. Values are expressed as mean ± SEM. *p < 0.05 for C vs. St and **p < 0.001 for C vs. St + S30; ***p < 0.0001 C vs. St; @@p < 0.001 for S30 vs. St + S30 groups. Yellow line defines the glomerular area, white arrows define the mesangial matrix expansion and white circle define the mesangial cells. Scale bars 50 μm (a, c, e, g) and 20 μm (b, d, f, h).
3.3. AQP-2 expression
chronic stress vs. high-sucrose diet groups (Fig. 2j). The mean number of mesangial cells in small glomeruli was only increased by chronic stress (p < 0.0001; Fig. 2k), and no effects were observed by highsucrose diet or the chronic stress and sucrose diet combination (Fig. 2k). Mesangial matrix expansion was higher in the glomeruli of rats belonging to the stressed groups in comparison to the C group (p < 0.05; Fig. 2l). No differences were found in the mesangial matrix by high-sucrose diet, but a higher significant value was observed for the chronic stress and high-sucrose diet (St + S30 group) when compared to the S30 group (p < 0.001; Fig. 2l). Compared to the C group, rats belonging to the stressed groups presented dilated tubules and reduced sinusoidal spaces (Fig. 3a–h). The S30 group only showed an increased value for the sinusoidal spaces (Fig. 3a–h). The mean value of the crosssectional area for proximal tubules was unaffected by chronic stress and high-sucrose diet when compared to the C group (Fig. 3i). The area of proximal tubules in the St + S30 group was significantly lower than in the C group (p < 0.05; Fig. 3i). No effects were found by the interaction between stress chronic and high-sucrose diet (Fig. 3i).
The immunohistochemical evaluation for AQP-2 uncovered the protein presence in principal cells of collecting ducts in both the cortex and the medulla in the kidneys from rats in the C group (Fig. 4a–d). In the St group, we observed an intense immunoreactivity for AQP-2 in both cortex and medulla when compared to the C group (Fig. 4e–h). In either S30 or St + S30 groups, we detected a low immunoreactivity of AQP-2 in the kidney in comparison with the St group (Fig. 4i–p). In the kidney cortex, when compared to the control group, the immunoreactivity for AQP-2 was significantly increased by chronic stress (p < 0.0001; Fig. 4q). No differences were uncovered for AQP-2 immunoreactivity by high-sucrose diet (Fig. 4q). Surprisingly, the highsucrose diet was able to reduce the effects caused by chronic stress on the immunoreactivity for AQP-2 in the St + S30 group (p < 0.0001; Fig. 4q). In the kidney medulla, the immunoreactivity for AQP-2 was notoriously increased by chronic stress (p < 0.0001; Fig. 4r). No differences were found in the immunoreactivity in the AQP-2 by highsucrose diet (Fig. 4r). Remarkably, the high-sucrose diet reduced the effect generated by chronic stress on the medulla immunoreactivity for 1101
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Fig. 3. Effect of chronic stress and sucrose consumption on proximal tubules area evaluated in longitudinal PAS stained kidney sections. C, control; St, chronic stress; S30, 30% sucrose diet; St + S30, chronic stress + 30% sucrose diet. n = 6–8. Values are expressed as mean ± SEM. &p < 0.05 for C vs. St + S30. Black arrows show the loss of sinusoidal spaces between tubule. Asterisks show increased sinusoidal spaces between tubules. Scale bars 50 μm (a, c, e, g), and 20 μm (b, d, f, h).
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Fig. 4. Effect of chronic stress and sucrose consumption on expression of Aquoporin-2 in collecting duct principal cells of the kidney. C, control; St, chronic stress; S30, 30% sucrose diet; St + S30, chronic stress + 30% sucrose diet. (4q) AQP-2 expression in collecting duct of renal cortex and (4 r) AQP-2 expression in medullar collecting duct. Values are expressed as a percentage (%) of positive stained area ± SEM; n = 6; (Scale bars 200 μm (a, c, e, g, i, k, m, o) and 50 μm (b, d, f, h, j, l, n, p). ***p < 0.0001 C vs. St; ###p < 0.0001 St vs. St + S30; &p < 0.05 C vs. S30 groups.
AQP-2 in the St + S30 group (p < 0.0001), and furthermore, the area was substantially lower in comparison with the C group (p < 0.05; Fig. 4r). 3.4. Serum creatinine levels When compared with the C group, chronic stress significantly reduced serum creatinine levels (p < 0.001; Fig. 5). This value was unaffected by high-sucrose diet (Fig. 5). The serum creatinine levels in the St + S30 group were significantly higher in comparison with the St group (p < 0.001), but not vs. the S30 group. No differences were found for combination of chronic stress and high-sucrose diet (Fig. 5). 4. Discussion Fig. 5. Effect of chronic stress and sucrose consumption on serum creatinine levels. C, control; St, chronic stress; S30, 30% sucrose diet; St + S30, chronic stress + 30% sucrose diet. n = 6–8. Values are expressed as mean ± SEM. **p < 0.001 for C vs. St and @@p < 0.001 for St vs. St + S30.
We found that chronic stress decreased the number of corpuscles and the percentage of large renal corpuscles in young rats, as well as reducing the Bowman’s space area in the glomerulus. Additionally, small glomerulus had an increase in the number of mesangial cells and 1103
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mesangial matrix, while collecting duct cells had a major expression of AQP-2. All these changes were accompanied by a dysfunction on renal function evaluated through the serum concentrations of creatinine. Previously, we reported that the stress protocol used in the present study is able to increase serum concentration of corticosterone [13]. Considering that the kidney in rats matures from birth to a postnatal age of approximately 40 days [26,27], our results may involve a harmful effect of glucocorticoid levels during the development of the kidney [28]. The reduction in renal mass has been associated with glomerular hyperfiltration [29], and tubules atrophy [30] which is a key factor for future renal damage. In agreement, an exposure to chronic stress before puberty reduce the number of nephrons and the glomerular volume [15]. Furthermore, the administration of dexamethasone promotes an increase in the mRNA expression for Connective Tissue Growth Factor that has been associated with promoting renal fibrogenesis [31], and the expression of renal proteoglycans in the mesangial matrix [32]. In this regard, glucocorticoids receptors are present in renal proximal tubules, and both 11beta-hydroxysteroid dehydrogenase (11β-HSD) type 1 and 2 are also expressed in proximal and distal tubules, respectively [33]. Furthermore, glucocorticoids influence renal sodium reabsorption and water retention [34], regulating both mineralocorticoid and glucocorticoid receptors [35]. With respect to the major expression of AQP-2 observed in the kidney and promoted by stress, it has been described that corticotropinreleasing hormone (CRH) increases vasopressin levels [36], which favor the expression AQP-2 and associated with water retention [17,37]. It is possible to suggest that the histologic changes of the kidney promoted by stress were associated with glomerular hyperfiltration, thus decreasing creatinine levels in serum. In support of this idea, it has been shown that the administration of dexamethasone also promotes proteinuria [38]. Thus, glucocorticoids may have a dual effect on kidney physiology, being beneficial for a short time depending on the dosage, but can be toxic when used for a long period of time [39]. Results show that one month of a high-sucrose diet intake was not enough to affect renal structure and function. This discrepancy may be related to the lack of effects on serum corticosterone concentrations that these rats show during this protocol for sucrose ingestion [13]. Perhaps a longer treatment with a high-sucrose diet could induce a major damage on the kidney, as has been reported in previous reports [40]. In agreement, other studies have showed a tubular hyperplasia after six weeks of fructose but not of glucose diet, without affecting the glomerulus [41]. Our present findings show that a high-sucrose diet decrease the expression of the AQP-2 protein in the kidney, probably due to the reduction in the levels of serum corticosterone previously observed in these animals [13]. The combination of both factors (restrain stress and high-sucrose diet) does not potentiate the damage on the renal corpuscles, although it maintains the damage provoked by the stress on almost all evaluated parameters. However, the area of proximal tubules was significantly reduced in the St + S30 group, possibly as a summatory effect of sucrose intake [41]. In conclusion, chronic stress before puberty may represent a factor for an accelerated glomerular hypertrophy associated with functional changes; while the high-sucrose diet was not enough to affect the renal structure, the combination of stress and high-sucrose diet induced similar effects on the kidney as those generated by stress alone. Possibly a longer period of sucrose intake combined with restrain stress could markedly enhance the dysfunctional characteristics of the stressed kidney. Our results may be important for better understanding the complex development of kidney illnesses in-juvenile individuals, while considering psychological stress as a possible associative cause.
Acknowledgments A pre-doctoral fellowship (Reg. 487305) from the Consejo Nacional de Ciencia y Tecnología of México was received by CN. Authors thank the expert technical assistance of Laura García. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References [1] V.P. Karalius, D.A. Shoham, Dietary sugar and artificial sweetener intake and chronic kidney disease: a review, Adv. Chronic Kidney Dis. 20 (2013) 157–164. [2] E. Yuzbashian, G. Asghari, P. Mirmiran, A. Zadeh-Vakili, F. Azizi, Sugar-sweetened beverage consumption and risk of incident chronic kidney disease: Tehran lipid and glucose study, Nephrology 21 (2016) 608–616. [3] A. Alkhedaide, M.M. Soliman, A.E. Salah-Eldin, T.A. Ismail, Z.S. Alshehiri, H.F. Attia, Chronic effects of soft drink consumption on the health state of Wistar rats: a biochemical, genetic and histopathological study, Mol. Med. Rep. 13 (2016) 5109–5117. [4] G. Cao, J. González, A. 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Conflicts of interest None of the authors have any potential conflicts of interest with this 1104
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mesangial cells, Kidney Int. 62 (2002) 780–789. [33] B.L. Roland, Z.S. Krozowski, J.W. Funder, Glucocorticoid receptor, mineralocorticoid receptors, 11 beta-hydroxysteroid dehydrogenase-1 and -2 expression in rat brain and kidney: in situ studies, Mol. Cell. Endocrinol. 111 (1995) R1–R7. [34] R.W. Hunter, J.R. Ivy, M.A. Bailey, Glucocorticoids and renal Na+ transport: implications for hypertension and salt sensitivity, J. Physiol. 592 (2014) 1731–1744. [35] K. Rafiq, D. Nakano, G. Ihara, H. Hitomi, Y. Fujisawa, N. Ohashi, H. Kobori, Y. Nagai, H. Kiyomoto, M. Kohno, A. Nishiyama, Effects of mineralocorticoid receptor blockade on glucocorticoid-induced renal injury in adrenalectomized rats, J. Hypertens. 29 (2011) 290–298. [36] H.K. Caldwell, E.A. Aulino, K.M. Rodriguez, S.K. Witchey, A.M. Yaw, Social context, stress, neuropsychiatric disorders, and the vasopressin 1b receptor, Front. Neurosci. 11 (2017) 567. [37] G. Aguilera, C. Rabadan-Diehl, Vasopressinergic regulation of the hypothalamicpituitary-adrenal axis: implications for stress adaptation, Regul. Pept. 96 (2000) 23–29. [38] A. Chen, L.F. Sheu, Y.S. Ho, Y.F. Lin, W.Y. Chou, J.Y. Wang, W.H. Lee, Administration of dexamethasone induces proteinuria of glomerular origin in mice, Am. J. Kidney Dis. 31 (1998) 443–452. [39] C. Ponticelli, F. Locatelli, Glucocorticoids in the treatment of glomerular diseases: pitfalls and pearls, Clin. J. Am. Soc. Nephrol. 13 (2018) 815–822. [40] A.P. Arikawe, I.C. Udenze, M.F. Akinwolere, A.O. Ogunsola, R.T. Oghogholosu, Effects of streptozotocin, fructose and sucrose-induced insulin resistance on plasma and urinary electrolytes in male Sprague-Dawley rats, Nig. Q. J. Hosp. Med. 22 (2012) 224–230. [41] T. Nakayama, T. Kosugi, M. Gersch, T. Connor, L.G. Sanchez-Lozada, M.A. Lanaspa, C. Roncal, S.E. Perez-Pozo, R.J. Johnson, T. Nakagawa, Dietary fructose causes tubulointerstitial injury in the normal rat kidney, Am. J. Physiol. Renal Physiol. 298 (2010) F712–F720.
islet size, Endocrine 48 (2015) 811–817. [24] S. Bernardi, B. Toffoli, C. Zennaro, F. Bossi, P. Losurdo, A. Michelli, R. Carretta, P. Mulatero, F. Fallo, F. Veglio, B. Fabris, Aldosterone effects on glomerular structure and function, J. Renin Angiotensin Aldosterone Syst. 16 (2015) 730–738. [25] L. Nicolás-Toledo, M. Cervantes-Rodríguez, E. Cuevas-Romero, D.L. CoronaQuintanilla, E. Pérez-Sánchez, E. Zambrano, F. Castelán, J. Rodríguez-Antolín, Hitting a triple in the non-alcoholic fatty liver disease field: sucrose intake in adulthood increases fat content in the female but not in the male rat offspring of dams fed a gestational low-protein diet, J. Dev. Orig. Health Dis. 9 (2018) 151–159. [26] L. Larsson, M. Horster, Ultrastructure and net fluid transport in isolated perfused developing proximal tubules, J. Ultrastruct. Res. 54 (1976) 276–285. [27] H.Y. Chang, Y.L. Tain, Postnatal dexamethasone-induced programmed hypertension is related to the regulation of melatonin and its receptors, Steroids 108 (2016) 1–6. [28] R.R. Singh, K.M. Moritz, J.F. Bertram, L.A. Cullen-McEwen, Effects of dexamethasone exposure on rat metanephric development: in vitro and in vivo studies, Am. J. Physiol. Renal Physiol. 293 (2007) F548–F554. [29] D. Fong, K.M. Denton, K.M. Moritz, R. Evans, R.R. Singh, Compensatory responses to nephron deficiency: adaptive or maladaptive? Nephrology (Carlton) 19 (2014) 119–128. [30] R. Lan, H. Geng, P.K. Singha, P. Saikumar, E.P. Bottinger, J.M. Weinberg, M.A. Venkatachalam, Mitochondrial pathology and glycolytic shift during proximal tubule atrophy after ischemic AKI, J. Am. Soc. Nephrol. 27 (2016) 3356–3367. [31] H. Okada, T. Kikuta, T. Inoue, Y. Kanno, S. Ban, T. Sugaya, M. Takigawa, H. Suzuki, Dexamethasone induces connective tissue growth factor expression in renal tubular epithelial cells in a mouse strain-specific manner, Am. J. Pathol. 168 (2006) 737–747. [32] M. Kuroda, H. Sasamura, R. Shimizu-Hirota, M. Mifune, H. Nakaya, E. Kobayashi, M. Hayashi, T. Saruta, Glucocorticoid regulation of proteoglycan synthesis in
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Morphometric changes and AQP2 expression in kidneys of young male rats exposed to chronic stress and a high-sucrose diet
T
Cristhian Neftaly Sánchez-Solísa, Estela Cuevas-Romerob, Alvaro Munozc, ⁎ Margarita Cervantes-Rodríguezd, Jorge Rodríguez-Antolínb, Leticia Nicolás-Toledob, a
Doctorado en Ciencias Biológicas, Universidad Autónoma de Tlaxcala, Tlaxcala, Mexico Centro Tlaxcala de Biología de la Conducta, Universidad Autónoma de Tlaxcala, Tlaxcala, Mexico c Centro Universitario del Norte, Universidad de Guadalajara, Jalisco, Mexico d Facultad de Nutrición, Universidad Autónoma de Tlaxcala, Tlaxcala, Mexico b
A R T I C LE I N FO
A B S T R A C T
Keywords: Creatinine Mesangial cell Mesangial matrix Puberty rats Restriction stress High sucrose diet
Objective: Consumption of a cafeteria-like diet and chronic stress have a negative impact on kidney function and morphology in adult rats. However, the interaction between chronic restraint stress and high-sucrose diet on renal morphology in young rats is unknown. A high-sucrose diet does not modify serum glucose levels but reduces serum corticosterone levels in stressed young rats, in this way it is confusing a possible potentiate or protector effect of this diet on kidney damage induced by stress. Methods: Wistar male rats at 4 weeks of age were randomly assigned into 4 groups: control (C), stressed (St), high-sucrose diet (S30), and chronic restraint stress plus a 30% sucrose diet (St + S30). Rats were fed with a standard chow and tap water (C group) or 30% sucrose diluted in water (S30 group). Chronic restraint stress consisted of 1-h daily placement into a plastic cylinder, 5 days per week, and for 4 weeks. Results: Stressed rats exhibited a low number of corpuscles, glomeruli, high number of mesangial cells, major deposition of mesangial matrix and aquaporin-2 protein (AQP-2) expression, and low creatinine levels. Meanwhile, high-sucrose diet ameliorated AQP-2 expression and avoided the reduction of creatinine levels induced by chronic stress. The combination of stress and high-sucrose diet maintained similar effects on the kidney as stress alone, although it induced a greater reduction in the area of proximal tubules. Conclusions: Our results show that both chronic stress and a high-sucrose diet induce histological changes, but chronic stress may generate an accelerated glomerular hypertrophy associated with functional changes before puberty.
1. Introduction Consumption of soft drinks contribute to obesity-related chronic kidney disease (CKD) in humans [1,2] and rats [3,4]. Particularly, the consumption of a cafeteria diet induces inflammation and advanced glycation end products in the kidneys of adult rats [5] which induce glomerulosclerosis in adult mice [6], as well as glomerular hypertrophy associated with an excessive mesangial matrix [7,8], proximal tubular hypertrophy [9,10], and glomerular hyperfiltration [11]. Additionally, hyperglycemia induced by streptozotocin affects the aquaporin-2 protein (AQP-2) expression in the collecting duct principal cells of rats [12]. However, it is unknown whether a high-sucrose diet in young rats could increase enough to affect renal function, considering that it does not modify serum glucose levels [13].
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Stress is considered an important cause for the development of metabolic syndrome [14], which also induces morphometric [15], and functional renal alterations [16]. In this regard, glucocorticoids increase AQP-2 protein expression and glomerular filtration rate in adult [17] and infant [18] rats, decrease proteoglycans synthesis in mesangial cells [19], and regulate renal transport [20,21], which have been associated with hypertension [22]. The physiological renal effects of combining a rich-sucrose diet and stress on the kidneys is unknown, and may be confuse because a highsucrose diet reduces corticosterone levels in serum and the activity of the hepatic 11-beta hydroxysteroid dehydrogenase, which coincides with a reduced response to chronic restraint stress [13]. For these reasons, we investigated the histological changes at glomeruli level, expression of AQP-2, and serum creatinine levels in young
Corresponding author at: Leticia Nicolás Toledo, Ph. D., Centro Tlaxcala de Biología de la Conducta, Universidad Autónoma de Tlaxcala, 90000-Tlaxcala, México. fax: (52) 246 46 215
57. E-mail address: [email protected] (L. Nicolás-Toledo). https://doi.org/10.1016/j.biopha.2018.06.086 Received 10 April 2018; Received in revised form 13 June 2018; Accepted 14 June 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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Imager A1, Thornwood, NY) equipped with a digital camera (Tuczon Olympus, Tucson, AZ). Reconstructions of the images from each cortical and medulla zones kidney were performed. The number of glomeruli was determined in the reconstruction of the right kidney. Density of glomeruli was obtained with a virtual square of 250,000 μm2 drawn into the kidney reconstructions [23]. Using a grid of 10 × 12 cm on the computer screen, some glomerulus were randomly chosen and photographed at 40x to measure the area renal corpuscle (Vtuft), the glomerular area (Vglom) and the proximal tubules area using Axiovision 4.8 (Carl Zeiss MicroImaging, Inc.). Bowman’s space (VBS) was evaluated using a semi-quantitative score method calculated as VBowman’s space = Vglom-Vtuft [11]. The glomerular area was categorized in two groups, small size (< 4000 μm2) and large size (> 4001 μm2). The renal corpuscle area was classified in two groups, small size (< 5000 μm2) and large size (> 5001 μm2). Finally, the Bowmanʼs space area was categorized in two groups, small size (< 900 μm2) and large size (> 901 μm2). Mesangial cells and the mesangial matrix inside the glomeruli was scored with a scale ranging from 0 to 4 (0 for no change; 1 for changes affecting < 25%; 2 for changes affecting 25–50%; 3 for changes affecting 50–75%; and 4 for changes affecting > 75% of the glomeruli) using the Axiovision 4.3 program [24].
rats exposed to chronic stress and a high-sucrose diet. Our hypothesis is that either chronic stress or a high-sucrose diet induce histological changes in the kidney of young rats, whereas both factors together may potentiate kidney injury independently of glucocorticoids levels. 2. Materials and methods 2.1. Animals Thirty-two 21-day-old and 60–80 g body weight post-weaned Wistar male rats (Rattus norvegicus) were housed in individual 37 cm × 27 cm × 16 cm polypropylene cages and maintained at 20 ± 2 °C with a 12 h light/12 h dark cycle (light on at 12:00 h) at the Centro Tlaxcala de Biología de la Conducta from the Universidad Autónoma de Tlaxcala. Rats were randomly assigned into four experimental groups: Control-standard chow (C, n = 6–8), chronic restraint stress (St, n = 6–8), 30%-sucrose diet (S30, n = 6–8), and chronic restraint stress plus a 30% sucrose diet (St + S30, n = 6–8). The Research Ethics Committee of the Universidad Autónoma de Tlaxcala approved all experimental procedures according to the Mexican Guidelines for Animal Care, which are based on recommendations by The Association for Assessment and Accreditation of Laboratory Animal Care International.
2.6. AQP-2 immunohistochemistry
2.2. Dietary protocol
Additional kidney sections were deparaffinized and incubated in a microwave-heated 10 mM sodium citrate pH 6 solution to retrieve the antigens. Endogenous peroxidases were quenched with 0.3% hydrogen peroxide diluted in phosphate-buffered saline (PBS) during 30 min. Afterwards, tissue sections were blocked with 5% donkey serum diluted in PBS containing 0.3% Triton X100 (PBST) during 1 h. Thereafter, sections were first incubated with a primary antibody anti-AQP-2 (1:100; Santa Cruz Biotechnology, Inc.) during 72 h at 4 °C, rinsed and then with a secondary antibody (mouse anti-goat IgG-HRP) for 2 h at 37 °C. Sections were rinsed with PBS, and the immunostaining was developed according to the directions of a Vectastain ABC kit (Vector Labs, USA), using 0.05% 1.3′-diaminobenzidine (Sigma-Aldrich, St. Louis, MO) and 0.01% H2O2 as enzymatic substrate. Slides were washed and counterstained with Mayer’s hematoxylin, dehydrated and mounted with histological resin. Photomicrographs were obtained with a 10× magnification objective using an optical microscope (Zeiss Axio Imager A1, USA) equipped with a digital camera (Tuczon Olympus, USA). To comparative ends, two photomicrographs from the upper and lower portion of the renal cortex and medulla were taken. The percentage of the positive area for AQP2 cells stained in the collecting ducts was semi-quantitatively calculated using a true color image analysis system with Axio Vision Rel 4.6 (Zeiss Software Inc.), and applying a segmentation method with fixed thresholds. An overall mean value of the estimations was determined and used for comparisons among groups [25].
All rats were fed with a standard diet (Purina Laboratory chow 5001). Rats in groups C and St had access to tap water ad libitum, whereas those in groups S30 and St + S30 groups had access to 30% sucrose-sweetened tap water ad libitum for 4 weeks [13]. 2.3. Restraint stress procedure The protocol used to induce stress by motility restraint was previously reported [13]. Briefly, rats were placed in a 16 cm long by 6 cm in diameter plastic tube that limited their movement; one end of the tube was kept open to allow the animal to breathe. Rats were exposed daily for 1 h to this procedure at 11:00 h for five consecutive days/ week, for 4 weeks; rats were not subjected to this stressful stimulus during the weekends. Rats were returned to their home cages immediately after stress sessions. Rats in the C and S30 groups were not exposed to restraint stress and were maintained in their home cages throughout the experimental period. 2.4. Kidney weight and serum creatinine levels At the end of the experimental period, rats were decapitated using a rodent guillotine (Harvard-Apparatus, Holliston, MA) between 08:00 and 09:00. Trunk blood was collected and serum was obtained by centrifugation [13]. Kidneys were immediately removed and their weight means are reported in milligrams kidney tissue/100 g body weight [13]. Serum creatinine levels were measure using a commercially available method using enzymatic-colorimetric kits, and in accordance with the manufacturer directions (Elitech Clinical Systems, México).
2.7. Statistical analysis Data are presented as mean ± SEM unless otherwise is stated. Oneway ANOVA followed by a Bonferroni’s test was carried out to determine significant differences between groups. Factors considered for the former analysis were the stress, high-sucrose diet, and interactions between them. In all cases, p ≤ 0.05 was considered statistically significant. All statistical analyses were carried out using GraphPad Prism Software (Version 5.01) for Windows.
2.5. Histology of kidney Right kidneys were immersed in Bouin-Duboscq fixative for 24 h at room temperature. The tissue then was dehydrated through 60, 70, 80, 96 and 100% ethyl alcohol, cleared in xylene, embedded in paraplast Plus (Sigma-Aldrich, St. Louis, MO) and sectioned longitudinally with a microtome (Leica RM2135, Bensheim, Germany) at 5 μm, and three kidney sections mounted per slide. Series of slides were stained with Periodic Acid-Schiff (PAS) to evaluate the renal morphology. Stained sections were photographed with a light microscopy at 40× (Zeiss Axio
3. Results 3.1. Characteristics of corpuscles Compared to the C group, the kidney weight was significantly lower in the rats that drank the 30% sucrose solution (p < 0.0001; Fig. 1a). 1099
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Fig. 1. Effect of chronic stress and sucrose consumption on characteristics of kidney and renal corpuscles evaluated in longitudinal PAS stained kidney sections. C, control; St, chronic stress; S30, 30% sucrose diet; St + S30, chronic stress + 30% sucrose diet. n = 6–8. Values are expressed as mean ± SEM. *p < 0.05 for C vs. St; &&&p < 0.0001 for C vs. S30 and #p < 0.05 St vs. St + S30; groups.
the high-sucrose diet groups (Fig. 1f).
The treatment combination (St + S30 group) also decreased the kidney weight compared to the St group (p < 0.05; Fig. 1a), but was similar to the S30 group. No differences were found by the interaction vs. the St and S30 groups (Fig. 1a). The number of corpuscles was decreased by the chronic stress (St group) when compared to the C group (p < 0.05; Fig. 1b). No effects were found in the number of corpuscles by highsucrose diet or its interaction with chronic stress (Fig. 1b). The cross-sectional area of the renal corpuscle was unaffected by either chronic stress or a high-sucrose diet when compared to the C group, but it was decreased by the chronic stress in the St + S30 group in comparison with the C group (p < 0.05; Fig. 1c). No differences were found by the interaction vs. the chronic stress or high-sucrose diet (Fig. 1c). The classification of corpuscles in small and large show that the percentage of large renal corpuscles (> 5001 μm2) was decreased in the stressed groups when compared with the C group (p < 0.05; Fig. 1d). No effects were found by high-sucrose diet in comparison with the C group or its interaction with chronic stress (Fig. 1d). The mean area of the Bowmanʼs space remained unaffected on any experimental condition (Fig. 1e). The classification by size of renal corpuscles with a large Bowmanʼs space (> 901 μm2), was significantly reduced in the stressed groups when compared with the C group (p < 0.05; Fig. 1f). No effects on large Bowmanʼs space were found by high-sucrose diet in comparison with the C group, or after comparing the chronic stress vs.
3.2. Changes in glomerular size and proximal tubes The kidney sections from the C group showed a detailed cortical parenchyma and the renal corpuscles appeared as dense rounded structures with the glomerulus surrounded by a narrow Bowmanʼs space (Fig. 2a,b). In the St group, we observed a decrease in the area for the Bowmanʼs space with adhesions of the Bowman capsule, the presence of hypercellularity at the glomerular level, and mesangial matrix expansion (Fig. 2c,d). The kidney sections from the S30 group only showed a reduction of the Bowmanʼs space (Fig. 2e,f). Finally, the observation in the St + S30 group indicated a reduction in the Bowmanʼs space, as well as glomerular hyperplasia, mesangial matrix expansion and the presence of adherences (Fig. 2g,h). The mean value for the glomeruli area was unaffected by chronic stress or the high-sucrose diet compared to the C group, but it was significantly decreased by the interaction vs. the chronic stress or highsucrose diet when compared to the C group (p < 0.001; Fig. 2i). The analysis of classification by size showed that the percentage of glomeruli with a large area (> 4001μm2) in the stressed groups was lower than that in the C group (p < 0.05; p < 0.001, respectively; Fig. 2j). No differences were found by the high-sucrose diet or when comparing 1100
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Fig. 2. Effect of chronic stress and sucrose consumption on the glomerular area, mesangial cells number, and mesangial matrix expansion visualized in longitudinal PAS stained kidney sections. C, control; St, chronic stress; S30, 30% sucrose diet; St + S30, chronic stress + 30% sucrose diet. n = 6–8. Values are expressed as mean ± SEM. *p < 0.05 for C vs. St and **p < 0.001 for C vs. St + S30; ***p < 0.0001 C vs. St; @@p < 0.001 for S30 vs. St + S30 groups. Yellow line defines the glomerular area, white arrows define the mesangial matrix expansion and white circle define the mesangial cells. Scale bars 50 μm (a, c, e, g) and 20 μm (b, d, f, h).
3.3. AQP-2 expression
chronic stress vs. high-sucrose diet groups (Fig. 2j). The mean number of mesangial cells in small glomeruli was only increased by chronic stress (p < 0.0001; Fig. 2k), and no effects were observed by highsucrose diet or the chronic stress and sucrose diet combination (Fig. 2k). Mesangial matrix expansion was higher in the glomeruli of rats belonging to the stressed groups in comparison to the C group (p < 0.05; Fig. 2l). No differences were found in the mesangial matrix by high-sucrose diet, but a higher significant value was observed for the chronic stress and high-sucrose diet (St + S30 group) when compared to the S30 group (p < 0.001; Fig. 2l). Compared to the C group, rats belonging to the stressed groups presented dilated tubules and reduced sinusoidal spaces (Fig. 3a–h). The S30 group only showed an increased value for the sinusoidal spaces (Fig. 3a–h). The mean value of the crosssectional area for proximal tubules was unaffected by chronic stress and high-sucrose diet when compared to the C group (Fig. 3i). The area of proximal tubules in the St + S30 group was significantly lower than in the C group (p < 0.05; Fig. 3i). No effects were found by the interaction between stress chronic and high-sucrose diet (Fig. 3i).
The immunohistochemical evaluation for AQP-2 uncovered the protein presence in principal cells of collecting ducts in both the cortex and the medulla in the kidneys from rats in the C group (Fig. 4a–d). In the St group, we observed an intense immunoreactivity for AQP-2 in both cortex and medulla when compared to the C group (Fig. 4e–h). In either S30 or St + S30 groups, we detected a low immunoreactivity of AQP-2 in the kidney in comparison with the St group (Fig. 4i–p). In the kidney cortex, when compared to the control group, the immunoreactivity for AQP-2 was significantly increased by chronic stress (p < 0.0001; Fig. 4q). No differences were uncovered for AQP-2 immunoreactivity by high-sucrose diet (Fig. 4q). Surprisingly, the highsucrose diet was able to reduce the effects caused by chronic stress on the immunoreactivity for AQP-2 in the St + S30 group (p < 0.0001; Fig. 4q). In the kidney medulla, the immunoreactivity for AQP-2 was notoriously increased by chronic stress (p < 0.0001; Fig. 4r). No differences were found in the immunoreactivity in the AQP-2 by highsucrose diet (Fig. 4r). Remarkably, the high-sucrose diet reduced the effect generated by chronic stress on the medulla immunoreactivity for 1101
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Fig. 3. Effect of chronic stress and sucrose consumption on proximal tubules area evaluated in longitudinal PAS stained kidney sections. C, control; St, chronic stress; S30, 30% sucrose diet; St + S30, chronic stress + 30% sucrose diet. n = 6–8. Values are expressed as mean ± SEM. &p < 0.05 for C vs. St + S30. Black arrows show the loss of sinusoidal spaces between tubule. Asterisks show increased sinusoidal spaces between tubules. Scale bars 50 μm (a, c, e, g), and 20 μm (b, d, f, h).
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Fig. 4. Effect of chronic stress and sucrose consumption on expression of Aquoporin-2 in collecting duct principal cells of the kidney. C, control; St, chronic stress; S30, 30% sucrose diet; St + S30, chronic stress + 30% sucrose diet. (4q) AQP-2 expression in collecting duct of renal cortex and (4 r) AQP-2 expression in medullar collecting duct. Values are expressed as a percentage (%) of positive stained area ± SEM; n = 6; (Scale bars 200 μm (a, c, e, g, i, k, m, o) and 50 μm (b, d, f, h, j, l, n, p). ***p < 0.0001 C vs. St; ###p < 0.0001 St vs. St + S30; &p < 0.05 C vs. S30 groups.
AQP-2 in the St + S30 group (p < 0.0001), and furthermore, the area was substantially lower in comparison with the C group (p < 0.05; Fig. 4r). 3.4. Serum creatinine levels When compared with the C group, chronic stress significantly reduced serum creatinine levels (p < 0.001; Fig. 5). This value was unaffected by high-sucrose diet (Fig. 5). The serum creatinine levels in the St + S30 group were significantly higher in comparison with the St group (p < 0.001), but not vs. the S30 group. No differences were found for combination of chronic stress and high-sucrose diet (Fig. 5). 4. Discussion Fig. 5. Effect of chronic stress and sucrose consumption on serum creatinine levels. C, control; St, chronic stress; S30, 30% sucrose diet; St + S30, chronic stress + 30% sucrose diet. n = 6–8. Values are expressed as mean ± SEM. **p < 0.001 for C vs. St and @@p < 0.001 for St vs. St + S30.
We found that chronic stress decreased the number of corpuscles and the percentage of large renal corpuscles in young rats, as well as reducing the Bowman’s space area in the glomerulus. Additionally, small glomerulus had an increase in the number of mesangial cells and 1103
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mesangial matrix, while collecting duct cells had a major expression of AQP-2. All these changes were accompanied by a dysfunction on renal function evaluated through the serum concentrations of creatinine. Previously, we reported that the stress protocol used in the present study is able to increase serum concentration of corticosterone [13]. Considering that the kidney in rats matures from birth to a postnatal age of approximately 40 days [26,27], our results may involve a harmful effect of glucocorticoid levels during the development of the kidney [28]. The reduction in renal mass has been associated with glomerular hyperfiltration [29], and tubules atrophy [30] which is a key factor for future renal damage. In agreement, an exposure to chronic stress before puberty reduce the number of nephrons and the glomerular volume [15]. Furthermore, the administration of dexamethasone promotes an increase in the mRNA expression for Connective Tissue Growth Factor that has been associated with promoting renal fibrogenesis [31], and the expression of renal proteoglycans in the mesangial matrix [32]. In this regard, glucocorticoids receptors are present in renal proximal tubules, and both 11beta-hydroxysteroid dehydrogenase (11β-HSD) type 1 and 2 are also expressed in proximal and distal tubules, respectively [33]. Furthermore, glucocorticoids influence renal sodium reabsorption and water retention [34], regulating both mineralocorticoid and glucocorticoid receptors [35]. With respect to the major expression of AQP-2 observed in the kidney and promoted by stress, it has been described that corticotropinreleasing hormone (CRH) increases vasopressin levels [36], which favor the expression AQP-2 and associated with water retention [17,37]. It is possible to suggest that the histologic changes of the kidney promoted by stress were associated with glomerular hyperfiltration, thus decreasing creatinine levels in serum. In support of this idea, it has been shown that the administration of dexamethasone also promotes proteinuria [38]. Thus, glucocorticoids may have a dual effect on kidney physiology, being beneficial for a short time depending on the dosage, but can be toxic when used for a long period of time [39]. Results show that one month of a high-sucrose diet intake was not enough to affect renal structure and function. This discrepancy may be related to the lack of effects on serum corticosterone concentrations that these rats show during this protocol for sucrose ingestion [13]. Perhaps a longer treatment with a high-sucrose diet could induce a major damage on the kidney, as has been reported in previous reports [40]. In agreement, other studies have showed a tubular hyperplasia after six weeks of fructose but not of glucose diet, without affecting the glomerulus [41]. Our present findings show that a high-sucrose diet decrease the expression of the AQP-2 protein in the kidney, probably due to the reduction in the levels of serum corticosterone previously observed in these animals [13]. The combination of both factors (restrain stress and high-sucrose diet) does not potentiate the damage on the renal corpuscles, although it maintains the damage provoked by the stress on almost all evaluated parameters. However, the area of proximal tubules was significantly reduced in the St + S30 group, possibly as a summatory effect of sucrose intake [41]. In conclusion, chronic stress before puberty may represent a factor for an accelerated glomerular hypertrophy associated with functional changes; while the high-sucrose diet was not enough to affect the renal structure, the combination of stress and high-sucrose diet induced similar effects on the kidney as those generated by stress alone. Possibly a longer period of sucrose intake combined with restrain stress could markedly enhance the dysfunctional characteristics of the stressed kidney. Our results may be important for better understanding the complex development of kidney illnesses in-juvenile individuals, while considering psychological stress as a possible associative cause.
Acknowledgments A pre-doctoral fellowship (Reg. 487305) from the Consejo Nacional de Ciencia y Tecnología of México was received by CN. Authors thank the expert technical assistance of Laura García. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References [1] V.P. Karalius, D.A. Shoham, Dietary sugar and artificial sweetener intake and chronic kidney disease: a review, Adv. Chronic Kidney Dis. 20 (2013) 157–164. [2] E. Yuzbashian, G. Asghari, P. Mirmiran, A. Zadeh-Vakili, F. Azizi, Sugar-sweetened beverage consumption and risk of incident chronic kidney disease: Tehran lipid and glucose study, Nephrology 21 (2016) 608–616. [3] A. Alkhedaide, M.M. Soliman, A.E. Salah-Eldin, T.A. Ismail, Z.S. Alshehiri, H.F. Attia, Chronic effects of soft drink consumption on the health state of Wistar rats: a biochemical, genetic and histopathological study, Mol. Med. Rep. 13 (2016) 5109–5117. [4] G. Cao, J. González, A. 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