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Changes in gene expressions of hypothalamic neuropeptides controlling feeding behaviors in bilateral nephrectomized rats
Neuroscience Letters 711 (2019) 134426
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Research article
Changes in gene expressions of hypothalamic neuropeptides controlling feeding behaviors in bilateral nephrectomized rats
T
Hiromichi Uenoa,b, Tetsu Miyamotob, Kenya Sanadab, Kazutoshi Nakazonob, Kentaro Tanakaa, Haruki Nishimuraa, Kazuaki Nishimuraa, Satomi Sonodaa, Mitsuhiro Yoshimuraa, ⁎ Takashi Maruyamaa, Ryota Serinoc, Yutaka Otsujib, Yoichi Uetaa, a
Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan The Second Department of Internal Medicine, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan c Department of Nephrology, Yoshino Hospital, Kitakyushu 808-0034, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Neuropeptides controlling feeding behaviors Anorexia Kidney dysfunction In situ hybridization Hypothalamus
Anorexia is one of the most widespread eating disorders that appears to contribute to malnutrition in patients with advanced kidney dysfunction. The changes of neuropeptides controlling feeding behaviors synthesized in the hypothalamus under several physiological condition could induce anorexia. While several mechanisms underlying uremic anorexia have been proposed, the changes of hypothalamic neuropeptides controlling feeding behaviors of uremic patients are poorly understood. The gene expressions of hypothalamic neuropeptides controlling feeding behaviors were evaluated after bilateral nephrectomy, which is a model of acute kidney dysfunction, by in situ hybridization histochemistry. Food consumption decreased markedly in bilateral nephrectomized rats. The mRNA levels of corticotrophin-releasing hormone, proopiomelanocortin, cocaine- and amphetamine-regulated transcript, which suppress feeding behavior, were significantly higher in bilateral nephrectomized rats than in sham-operated rats. On the other hand, the mRNA levels of Agouti-related peptide, neuropeptide Y, melanin-concentrating hormone, and orexin, which promote feeding behavior, were significantly lower in bilateral nephrectomized rats than in sham-operated rats. In addition, the plasma level of leptin, which has an anorexic effect, increased after bilateral nephrectomy. The results suggest that hypothalamic neuropeptides controlling feeding behaviors may be involved in the development of anorexia in bilateral nephrectomized rats. This report is the first step to elucidating the physiological mechanisms of anorexia in patients with kidney dysfunction.
1. Introduction It is well known that anorexia is a typical clinical manifestation of both acute and chronic kidney dysfunction. Anorexia is one of the most common disorders that contribute to malnutrition in patients with advanced kidney dysfunction. Accumulated evidence suggests that malnutrition is common and strongly associated with substantially increased morbidity and mortality in patients with kidney dysfunction, especially dialysis patients [1–3]. Although several strategies, including appetite stimulants, for prevention and treatment have been reported to improve nutritional status in patients with kidney dysfunction, there
are no nutritional interventional strategies that have shown effectiveness in large, randomized, clinical trials [4]. Feeding behaviors are controlled by complicated interactions of circulatory signals to maintain energy homeostasis or to satisfy the emotional desire for eating pleasure and are needed to acquire and consume food [5–7]. It is known that these complicated signals in feeding behaviors are integrated by the hypothalamus, which is an essential center for the endocrine system of the brain. The hypothalamus is an important area in which various neuropeptides controlling feeding behaviors are synthesized, and food intake is greatly affected by the expression levels of these neuropeptides. Representative hypothalamic neuropeptides that attenuate
Abbreviations: CRH, corticotrophin-releasing hormone; POMC, proopiomelanocortin; CART, cocaine- and amphetamine-regulated transcript; AgRP, agouti-related peptide (AgRP); NPY, neuropeptide Y; MCH, melanin-concentrating hormone; PVN, paraventricular nucleus (PVN); Arc, arcuate nucleus; LHA, lateral hypothalamic area ⁎ Corresponding author at: Department of Physiology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. E-mail address: [email protected] (Y. Ueta). https://doi.org/10.1016/j.neulet.2019.134426 Received 21 May 2019; Received in revised form 31 July 2019; Accepted 8 August 2019 Available online 08 August 2019 0304-3940/ © 2019 Elsevier B.V. All rights reserved.
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Table 1 The sequences of the probes used for in situ hybridization. Probe
sequence
CRH POMC CART AgRP NPY MCH Orexin
5’-CAG TTT CCT GTT GCT GTGAGC TTG CTG AGC TAA CTG CTC TGC CCT GGC-3’ 5’-TGG CTG CTC TCC AGG CAC CAG CTC CAC ACA TCT ATGGAG G-3’ 5’-TCC TTC TCG TGG GAC GCA TCA TCC ACG GCA GAG TAG ATG TCC AGG-3’ 5’-CGA CGC GGA GAA CGA GAC TCGCGG TTC TGT GGA TCT AGC ACC TCT GCC-3’ 5’-CAA ATG GAT GAT TGG TCA TTT CAA CAT AGA GTT GGG GGC TTGCT-3’ 5’-CCA ACA GGG TCG GTA GAC TCG TCC CAG CAT-3’ 5’-TCC TCA TAG TCT GGA GGC AGG TGG AAG GGTTCC CCA CTG CTA GTG-3’
2.4. In situ hybridization histochemistry for hypothalamic neuropeptides controlling feeding behaviors
feeding behaviors are corticotrophin-releasing hormone (CRH) in the paraventricular nucleus (PVN), proopiomelanocortin (POMC), and cocaine- and amphetamine-regulated transcript (CART) in the arcuate nucleus (Arc). On the other hand, representative hypothalamic neuropeptides that promote feeding behaviors are Agouti-related peptide (AgRP) and neuropeptide Y (NPY) in the Arc, and melanin-concentrating hormone (MCH) and orexin in the lateral hypothalamic area (LHA). We hypothesized that kidney dysfunction affected the synthesis of the hypothalamic neuropeptides controlling feeding behaviors, which could induce anorexia. To the best of our knowledge, these changes in hypothalamic neuropeptides in kidney dysfunction are poorly understood. In this study, the effects of bilateral nephrectomy, which is one of the classical animal models of acute kidney dysfunction [8–14], on food consumption and gene expressions of the hypothalamic neuropeptides were investigated. Furthermore, the change of plasma leptin levels after bilateral nephrectomy was examined.
To analyze the changes of hypothalamic neuropeptides controlling feeding behaviors by in situ hybridization histochemistry, rats were decapitated at 6, 12, and 24 h after treatment at 11:00-13:00 (n = 6 in each group at each time point). The procedure for in situ hybridization is described briefly, since the details of the procedure were provided in our previous reports [16,17].The brains were carefully removed, frozen on crushed dry, ice and stored at −80 °C. Blood was collected for measurement of plasma concentrations of blood urea nitrogen (BUN), creatinine (Cre), and leptin at decapitation. After centrifugation of the collected blood, plasma was extracted, and it was sent to a company (SRL, Tokyo, Japan) for analysis of plasma levels of BUN and Cre. Plasma leptin levels after treatment were quantified using a sandwich enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Morinaga Rat Leptin ELISA Kit; Morinaga, Yokohama, Japan). The frozen brains were cut to a thickness of 12 μm using a cryostat at −20 °C. The observation area was determined with reference to an atlas [18]. The probe sequences used for in situhybridization are shown in Table 1. The probe used in this experiment has been used before, and we believe that its reliability is guaranteed [16,19]. The hybridized sliced sections were exposed to autoradiography films (Amersham Hyperfilm, Buckinghamshire, UK) for 5 days for CRH, and 1 week for POMC, CART, AgRP, NPY, MCH, and orexin. Semi-quantitative analyses of gene expression were performed on the autoradiographic images using the Image J system (National Institutes of Health, Baltimore, MD, USA).
2. Materials and methods 2.1. Animals Adult male Wistar rats weighing 200–300 g at 7 weeks of age were used for this experiment. They were bred three per cage and were free to eat meals and drink tap water. The light and dark periods were alternated every 12 h (light on at 7:15 a.m.), and the room was kept at 23–25 °C. All experiments were carried out in accordance with the guidelines on the use and management of experimental animals of the Japan Physiological Society and were approved by the Animal Ethics Committee of the University of Occupational and Environmental Health (Kitakyushu, Japan).
2.5. Statistical analysis 2.2. Bilateral nephrectomy
Statistical analysis was performed using one-way analysis with the Bonferroni post hoc test for multiple comparisons. P < 0.05 was considered significant. The data are expressed as means ± standard error of the mean (SEM).
Wistar rats that underwent bilateral nephrectomy or sham surgery were sedated by a mixture of three anesthetic agents, medetomidine (0.3 mg/kg), midazolam (4.0 mg/kg), and butorphanol (5.0 mg/kg), during the operation [15]. Bilateral kidneys were removed after dorsal incision. The surgery was performed carefully so as not to disturb the adrenal glands and other intra-abdominal cavity organs.
3. Results 3.1. Changes in body weight
2.3. Measurements of body weight, water intake, and food intake
There were no changes in body weight before surgery in all groups (Fig. 1A). The untreated group then tended to have increased body weight, and the sham-operated and bilateral nephrectomized groups tended to have decreased body weight after surgery (Fig. 1A). However, there were no significant differences in weight change before and after surgery in all groups (Fig. 1A).
All rats were randomly divided into three groups, the bilateral nephrectomized group (n = 6), the sham-operated group (n = 6), and the untreated group (n = 6). All rats were put into a metabolic cage for measurements and adapted to the environment for 3 days before the experiment. All rats were able to consume food and tap water at any time during the experiment. Body weight was measured immediately before surgery and 24 h after surgery in the bilateral nephrectomized and sham-operated rats. In the untreated group, body weight was measured immediately before and 24 h after the start of measurement. Surgeries were performed from 11:00 am to 01:00 pm, and food and water intake were measured 0, 6, 12, and 24 h after bilateral nephrectomy or sham surgery.
3.2. Plasma levels of BUN and Cre After bilateral nephrectomy, the plasma levels of BUN and Cre increased with time (Table 2). 2
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Fig. 1. Body weight changes after bilateral nephrectomy (Nx) or sham operation (sham) (A). Effects of bilateral nephrectomy (Nx) or sham operation (sham) on cumulative food intake in conscious rats (B-a). Effects of bilateral nephrectomy (Nx) or sham operation (sham) on cumulative water intake in conscious rats (B-b). Data for cumulative food intake and water intake are expressed as means ± SEM (each group at surgery, n = 6). Data are presented as means ± SEM (each group at each time point, n = 6). †P < 0.05, ††P < 0.01 compared with the untreated group. *P < 0.05, **P < 0.01 compared with the sham-operated group.
3.4. Gene expressions of hypothalamic neuropeptides controlling feeding behaviors
Table 2 Effects of i.p. bilateral nephrectomy (Nx) and of sham operation (Sham) on plasma blood urea nitrogen (BUN) and creatinine (Cre) levels at 3, 12, and 20 h after treatment. Data are presented as means ± SEM (each group at each time point, n = 6). *P < 0.05, **P < 0.01 compared with Sham. Plasma BUN and Cre levels in untreated rats (n = 6) are 18.8 ± 0.5 mg/dl and 0.25 ± 0.01 mg/dl, respectively.
Time after treatments (h) Sham Nx Time after treatments (h) Sham Nx
Plasma concentration of 6 12 42.2 ± 2.7 29.9 62.8 ± 1.7** 89.1 Plasma concentration of 6 12 0.29 ± 0.02 0.23 1.24 ± 0.06** 2.11
Representative images obtained by in situhybridization of hypothalamic anorexigenic neuropeptides (CRH, POMC, and CART) at 6, 12, and 24 h after bilateral nephrectomy or sham operation are shown in Fig. 2. Similarly, representative images of orexigenic neuropeptides (AgRP, NPY, MCH, and Orexin) are shown in Fig. 3. All gene expression levels of hypothalamic neuropeptides controlling feeding behaviors in this study were calculated using the untreated group values as the standard values (Fig. 4). Gene expression of CRH in PVN was markedly higher in bilateral nephrectomized rats than in sham-operated rats (Fig. 4A-a).Gene expression of POMC and CART in Arc were significantly higher in bilateral nephrectomized rats than in sham-operated rats (Fig. 4A-b, c). The AgRP and NPY levels were significantly lower in bilateral nephrectomized rats than in sham-operated rats (Fig. 4B-a, b). The MCH and Orexin levels were significantly lower in bilateral nephrectomized rats than in sham-operated rats (Fig. 4B-c, d). In summary, bilateral nephrectomy upregulated the hypothalamic anorexic neuropeptide levels (Fig. 4A) and downregulated the hypothalamic orexigenic neuropeptide levels (Fig. 4B).
BUN (mg/dl) ± 2.2 ± 1.6**
24 19.1 ± 0.7 155.1 ± 6.1**
Cre (mg/dl) ± 0.01 ± 0.09**
24 0.22 ± 0.01 2.92 ± 0.14**
3.3. Water and food intake Cumulative water intake and food intake were measured every 6 h for 24 h in all groups (Fig. 1B-a, b). There were no differences in the water intake of the sham-operated group and the untreated group at all time points. The water intake of bilateral nephrectomized rats was significantly lower than of sham-operated and untreated rats (Fig. 1Ba). The food intake of sham-operated rats was significantly lower than that of untreated rats. The food intake of bilateral nephrectomized rats was further reduced compared with that of sham-operated rats. The decrease in food intake of bilateral nephrectomized rats was more pronounced than the decrease in water intake, resulting in the rats eating very little.
3.5. Plasma leptin levels The plasma leptin levels at 24 h after bilateral nephrectomy were significantly increased compared with those of sham-operated rats (Fig. 5). 4. Discussion Anorexia, a common clinical symptom in patients with acute and chronic kidney dysfunction, could have a major impact on patients’ prognosis and quality of life by causing malnutrition. Therefore, it is 3
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Fig. 2. Representative autoradiographs of sections hybridized with a 35S-labeled oligodeoxynucleotide probe complementary to the hypothalamic neuropeptides that promote feeding behaviors 6, 12, and 24 h after bilateral nephrectomy (Nx) or sham operation (Sham). CRH in the PVN (A), CART in the Arc (B), and POMC in the Arc (C) at 3, 12, and 20 h are shown. Black scale bar =1 mm. Signal intensity ranges from high (black) to low (white).
hypothalamic neuropeptides controlling feeding behaviors. These results lead to the conclusion that changes in hypothalamic neuropeptides after bilateral nephrectomy decreased food consumption. However, there is concern that bilateral nephrectomized rats could not eat due to surgical invasion or a prolonged anesthetic effect induced by kidney dysfunction. Bilateral nephrectomized rats have not only reduced food intake but also reduced water intake compared with sham-operated rats and untreated rats. The food intake of sham-operated rats decreased compared with that of untreated rats. These results may be due to anesthesia or surgical invasion. However, bilateral nephrectomized rats could drink some amount of water, but they could hardly eat. In other words, there was a qualitative difference in the water intake reduction and the food intake reduction in bilateral nephrectomized rats.
important to elucidate the pathophysiology of anorexia in kidney dysfunction. In the present study, the hypothalamic neuropeptides controlling feeding behaviors were shown to be affected by bilateral nephrectomy. The hypothalamus has a central role in the regulation of feeding behaviors and energy metabolism. Our team has shown the changes of gene expressions of hypothalamic neuropeptides controlling feeding behaviors under various physiological stresses or after administration of some drugs [16,19,20]. However, there have been few reports on the involvement of central mechanisms in appetite decreases in patients with kidney dysfunction. The food consumption of bilateral nephrectomized rats decreased significantly compared with sham-operated and untreated rats in the present study. Bilateral nephrectomy changed the levels of
Fig. 3. Representative autoradiographs of sections hybridized with a 35S-labeled oligodeoxynucleotide probe complementary to the hypothalamic neuropeptides that suppress feeding behaviors 6, 12, and 24 h after bilateral nephrectomy (Nx) or sham operation (Sham). AgRP in the Arc (A), NPY in the Arc (B), MCH in the LHA (C), and Orexin in the LHA (D) at 3, 12, and 20 h are shown. Black scale bar =1 mm. Signal intensity ranges from high (black) to low (white). 4
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Fig. 4. Gene expressions of the hypothalamic neuropeptides that promote (A) or suppress (B) feeding behaviors 6, 12, and 24 h after bilateral nephrectomy (Nx) or sham operation (Sham). CRH (A-a), CART (A-b), POMC (A-c), AgRP (B-a), NPY (B-b), MCH (B-c), and Orexin (B-d) are shown. Data are presented as means ± SEM (each group at each time point, n = 6). †P < 0.05, ††P < 0.01 compared with the untreated group. *P < 0.05, **P < 0.01 compared with the sham-operated group.
untreated rats. Kidney dysfunction is a serious stressor for the body, because the kidney is an important organ for maintaining homeostasis in the body. CRH plays an important role in the biological response for stress by secreting adrenocorticotropic hormone (ACTH) from the anterior pituitary, and it also has an inhibitory effect on food intake [5,22]. Recently, we showed that synthesis of CRH and vasopressin, which are stress hormones, increased in the hypothalamus after bilateral nephrectomy [8]. There are many previous reports that stress and appetite are closely related, and it is generally considered that acute stress suppresses feeding behaviors [23,24]. In the present experiment, results consistent with the previous report were obtained. Leptin, a peptide hormone secreted from adipocytes, is known to have a major effect on feeding behaviors and energy metabolism. Previous studies reported that plasma leptin levels were several times higher in patients with chronic kidney dysfunction than in healthy subjects, and plasma leptin levels in dialysis patients were inversely correlated with nutritional status and serum albumin levels [25,26]. Based on these reports, it appears that elevated leptin levels in patients with kidney dysfunction are involved in anorexia and nutritional disorders. In the present study, the plasma leptin levels in bilateral nephrectomized rats increased significantly compared with those in shamoperated rats. Leptin is excreted mainly from the kidneys, and thus the plasma leptin level is reported to increase as kidney function decreases [27]. Elevated leptin levels in this experiment may also have been triggered by impaired excretion associated with acute kidney dysfunction. Previous studies have reported that leptin promotes the synthesis of POMC and CART, which are neuropeptides that inhibit feeding behaviors and, conversely, inhibit the synthesis of AgRP, NPY, and orexin, which are neuropeptides that enhance feeding behaviors [28–31]. Therefore, the increasing plasma leptin level may have contributed to changes in hypothalamic neuropeptides controlling feeding behaviors
Fig. 5. The changes of plasma leptin levels 12 and 24 h after bilateral nephrectomy (Nx) or sham operation (Sham). Data are presented as means ± SEM (each group at each time point, n = 6). *P < 0.05, **P < 0.01 compared with the sham-operated group.
Furthermore, if the amount of food intake were decreased due to such physiological dysfunction induced by anesthesia or surgical invasion, the body would become starved, and the gene expressions of hypothalamic neuropeptides should change to promote feeding behaviors. On the other hand, the gene expressions of hypothalamic neuropeptides after bilateral nephrectomy changed to suppress feeding behaviors in the present study. Based on these results, the hypothalamic neuropeptide changes appear to have been located upstream of feeding behaviors in this experiment. In addition, previous reports have shown that food intake and water intake are interrelated [21]. Accompanied by an extreme reduction in food intake, water intake in bilateral nephrectomized rats may have decreased compared to sham-operated and 5
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Science (JSPS).
after bilateral nephrectomy. However, it should be noted that plasma concentrations of ghrelin, which antagonizes leptin and promotes feeding behaviors, have also been reported to be elevated in patients with kidney dysfunction [32]. Ghrelin is also excreted by the kidney, and it is reported that its blood concentration increases with decreasing kidney function. Further experiments are needed to prove this hypothesis. We assume that the decline in feeding behaviors in kidney dysfunction is a major problem when it lasts for a long time. However, bilateral nephrectomized rats cannot survive for a long time due to lethal arrhythmias and uremic syndrome. This point is the biggest limitation in this research. In the experimental design stage, an experiment using ischemic reperfusion injury instead of bilateral nephrectomy as a model of acute renal dysfunction in rats was also planned. This is because renal ischemia-reperfusion injury in rats is considered to be a more physiological experimental model of acute kidney dysfunction than bilateral nephrectomy. However, the renal ischemia reperfusion model rarely causes severe acute kidney dysfunction that could induce anorexia. In this experiment, the changes in gene expression of hypothalamic neuropeptides that control the feeding behavior of bilateral nephrectomized rats were validated, keeping in mind the limitations of bilateral nephrectomy. In order to address this issue, we are currently preparing a plan to investigate hypothalamic feeding neuropeptides in a more physiological animal model with kidney dysfunction that could be observed over a long period.
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5. Conclusion Anorexia is a serious complication that contributes to malnutrition in patients with advanced kidney dysfunction. However, the mechanism of anorexia is poorly understood. To the best of our knowledge, this report is the first to focus on the relationships of anorexia with kidney dysfunction and hypothalamic neuropeptides controlling feeding behaviors. In conclusion, the changes in gene expressions of hypothalamic neuropeptides controlling feeding behaviors were demonstrated, and a significant reduction of food intake was found after bilateral nephrectomy. This report is the first step to elucidating the physiological mechanism of anorexia in patients with kidney dysfunction. We hope that elucidation of the pathophysiology of anorexia in kidney dysfunction will contribute to improving the prognosis and quality of life of patients with kidney dysfunction. Authors’ contributions Research design: H.U., T.M., Y.O., and Y.U. Carried out the research: H.U., K.S., K. N., K.T., H.N., K. N., S.S., M.Y., and T.M. Analysis of the data: H.U., M.T., R.S., and Y.O. Drafting of the manuscript: H.U. Critical revision of the article: T.M. and Y.U. All authors approved the final version of the manuscript and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy are answered. All authors designated as authors qualify for authorship, and all those who qualify for authorship are listed. Declaration of Competing Interest The authors declare no conflict of interest associated with this manuscript. Acknowledgments The authors wish to express their appreciation to Ms. Yuki Nonaka for her technical assistance. This work was supported by a Grant-in-Aid for Scientific Research (B) (No. 17H04027), (C) (No. 17K08582), and (C) (No. 18K11010) from the Japan Society for the Promotion of 6
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Changes in gene expressions of hypothalamic neuropeptides controlling feeding behaviors in bilateral nephrectomized rats
T
Hiromichi Uenoa,b, Tetsu Miyamotob, Kenya Sanadab, Kazutoshi Nakazonob, Kentaro Tanakaa, Haruki Nishimuraa, Kazuaki Nishimuraa, Satomi Sonodaa, Mitsuhiro Yoshimuraa, ⁎ Takashi Maruyamaa, Ryota Serinoc, Yutaka Otsujib, Yoichi Uetaa, a
Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan The Second Department of Internal Medicine, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan c Department of Nephrology, Yoshino Hospital, Kitakyushu 808-0034, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Neuropeptides controlling feeding behaviors Anorexia Kidney dysfunction In situ hybridization Hypothalamus
Anorexia is one of the most widespread eating disorders that appears to contribute to malnutrition in patients with advanced kidney dysfunction. The changes of neuropeptides controlling feeding behaviors synthesized in the hypothalamus under several physiological condition could induce anorexia. While several mechanisms underlying uremic anorexia have been proposed, the changes of hypothalamic neuropeptides controlling feeding behaviors of uremic patients are poorly understood. The gene expressions of hypothalamic neuropeptides controlling feeding behaviors were evaluated after bilateral nephrectomy, which is a model of acute kidney dysfunction, by in situ hybridization histochemistry. Food consumption decreased markedly in bilateral nephrectomized rats. The mRNA levels of corticotrophin-releasing hormone, proopiomelanocortin, cocaine- and amphetamine-regulated transcript, which suppress feeding behavior, were significantly higher in bilateral nephrectomized rats than in sham-operated rats. On the other hand, the mRNA levels of Agouti-related peptide, neuropeptide Y, melanin-concentrating hormone, and orexin, which promote feeding behavior, were significantly lower in bilateral nephrectomized rats than in sham-operated rats. In addition, the plasma level of leptin, which has an anorexic effect, increased after bilateral nephrectomy. The results suggest that hypothalamic neuropeptides controlling feeding behaviors may be involved in the development of anorexia in bilateral nephrectomized rats. This report is the first step to elucidating the physiological mechanisms of anorexia in patients with kidney dysfunction.
1. Introduction It is well known that anorexia is a typical clinical manifestation of both acute and chronic kidney dysfunction. Anorexia is one of the most common disorders that contribute to malnutrition in patients with advanced kidney dysfunction. Accumulated evidence suggests that malnutrition is common and strongly associated with substantially increased morbidity and mortality in patients with kidney dysfunction, especially dialysis patients [1–3]. Although several strategies, including appetite stimulants, for prevention and treatment have been reported to improve nutritional status in patients with kidney dysfunction, there
are no nutritional interventional strategies that have shown effectiveness in large, randomized, clinical trials [4]. Feeding behaviors are controlled by complicated interactions of circulatory signals to maintain energy homeostasis or to satisfy the emotional desire for eating pleasure and are needed to acquire and consume food [5–7]. It is known that these complicated signals in feeding behaviors are integrated by the hypothalamus, which is an essential center for the endocrine system of the brain. The hypothalamus is an important area in which various neuropeptides controlling feeding behaviors are synthesized, and food intake is greatly affected by the expression levels of these neuropeptides. Representative hypothalamic neuropeptides that attenuate
Abbreviations: CRH, corticotrophin-releasing hormone; POMC, proopiomelanocortin; CART, cocaine- and amphetamine-regulated transcript; AgRP, agouti-related peptide (AgRP); NPY, neuropeptide Y; MCH, melanin-concentrating hormone; PVN, paraventricular nucleus (PVN); Arc, arcuate nucleus; LHA, lateral hypothalamic area ⁎ Corresponding author at: Department of Physiology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. E-mail address: [email protected] (Y. Ueta). https://doi.org/10.1016/j.neulet.2019.134426 Received 21 May 2019; Received in revised form 31 July 2019; Accepted 8 August 2019 Available online 08 August 2019 0304-3940/ © 2019 Elsevier B.V. All rights reserved.
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Table 1 The sequences of the probes used for in situ hybridization. Probe
sequence
CRH POMC CART AgRP NPY MCH Orexin
5’-CAG TTT CCT GTT GCT GTGAGC TTG CTG AGC TAA CTG CTC TGC CCT GGC-3’ 5’-TGG CTG CTC TCC AGG CAC CAG CTC CAC ACA TCT ATGGAG G-3’ 5’-TCC TTC TCG TGG GAC GCA TCA TCC ACG GCA GAG TAG ATG TCC AGG-3’ 5’-CGA CGC GGA GAA CGA GAC TCGCGG TTC TGT GGA TCT AGC ACC TCT GCC-3’ 5’-CAA ATG GAT GAT TGG TCA TTT CAA CAT AGA GTT GGG GGC TTGCT-3’ 5’-CCA ACA GGG TCG GTA GAC TCG TCC CAG CAT-3’ 5’-TCC TCA TAG TCT GGA GGC AGG TGG AAG GGTTCC CCA CTG CTA GTG-3’
2.4. In situ hybridization histochemistry for hypothalamic neuropeptides controlling feeding behaviors
feeding behaviors are corticotrophin-releasing hormone (CRH) in the paraventricular nucleus (PVN), proopiomelanocortin (POMC), and cocaine- and amphetamine-regulated transcript (CART) in the arcuate nucleus (Arc). On the other hand, representative hypothalamic neuropeptides that promote feeding behaviors are Agouti-related peptide (AgRP) and neuropeptide Y (NPY) in the Arc, and melanin-concentrating hormone (MCH) and orexin in the lateral hypothalamic area (LHA). We hypothesized that kidney dysfunction affected the synthesis of the hypothalamic neuropeptides controlling feeding behaviors, which could induce anorexia. To the best of our knowledge, these changes in hypothalamic neuropeptides in kidney dysfunction are poorly understood. In this study, the effects of bilateral nephrectomy, which is one of the classical animal models of acute kidney dysfunction [8–14], on food consumption and gene expressions of the hypothalamic neuropeptides were investigated. Furthermore, the change of plasma leptin levels after bilateral nephrectomy was examined.
To analyze the changes of hypothalamic neuropeptides controlling feeding behaviors by in situ hybridization histochemistry, rats were decapitated at 6, 12, and 24 h after treatment at 11:00-13:00 (n = 6 in each group at each time point). The procedure for in situ hybridization is described briefly, since the details of the procedure were provided in our previous reports [16,17].The brains were carefully removed, frozen on crushed dry, ice and stored at −80 °C. Blood was collected for measurement of plasma concentrations of blood urea nitrogen (BUN), creatinine (Cre), and leptin at decapitation. After centrifugation of the collected blood, plasma was extracted, and it was sent to a company (SRL, Tokyo, Japan) for analysis of plasma levels of BUN and Cre. Plasma leptin levels after treatment were quantified using a sandwich enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Morinaga Rat Leptin ELISA Kit; Morinaga, Yokohama, Japan). The frozen brains were cut to a thickness of 12 μm using a cryostat at −20 °C. The observation area was determined with reference to an atlas [18]. The probe sequences used for in situhybridization are shown in Table 1. The probe used in this experiment has been used before, and we believe that its reliability is guaranteed [16,19]. The hybridized sliced sections were exposed to autoradiography films (Amersham Hyperfilm, Buckinghamshire, UK) for 5 days for CRH, and 1 week for POMC, CART, AgRP, NPY, MCH, and orexin. Semi-quantitative analyses of gene expression were performed on the autoradiographic images using the Image J system (National Institutes of Health, Baltimore, MD, USA).
2. Materials and methods 2.1. Animals Adult male Wistar rats weighing 200–300 g at 7 weeks of age were used for this experiment. They were bred three per cage and were free to eat meals and drink tap water. The light and dark periods were alternated every 12 h (light on at 7:15 a.m.), and the room was kept at 23–25 °C. All experiments were carried out in accordance with the guidelines on the use and management of experimental animals of the Japan Physiological Society and were approved by the Animal Ethics Committee of the University of Occupational and Environmental Health (Kitakyushu, Japan).
2.5. Statistical analysis 2.2. Bilateral nephrectomy
Statistical analysis was performed using one-way analysis with the Bonferroni post hoc test for multiple comparisons. P < 0.05 was considered significant. The data are expressed as means ± standard error of the mean (SEM).
Wistar rats that underwent bilateral nephrectomy or sham surgery were sedated by a mixture of three anesthetic agents, medetomidine (0.3 mg/kg), midazolam (4.0 mg/kg), and butorphanol (5.0 mg/kg), during the operation [15]. Bilateral kidneys were removed after dorsal incision. The surgery was performed carefully so as not to disturb the adrenal glands and other intra-abdominal cavity organs.
3. Results 3.1. Changes in body weight
2.3. Measurements of body weight, water intake, and food intake
There were no changes in body weight before surgery in all groups (Fig. 1A). The untreated group then tended to have increased body weight, and the sham-operated and bilateral nephrectomized groups tended to have decreased body weight after surgery (Fig. 1A). However, there were no significant differences in weight change before and after surgery in all groups (Fig. 1A).
All rats were randomly divided into three groups, the bilateral nephrectomized group (n = 6), the sham-operated group (n = 6), and the untreated group (n = 6). All rats were put into a metabolic cage for measurements and adapted to the environment for 3 days before the experiment. All rats were able to consume food and tap water at any time during the experiment. Body weight was measured immediately before surgery and 24 h after surgery in the bilateral nephrectomized and sham-operated rats. In the untreated group, body weight was measured immediately before and 24 h after the start of measurement. Surgeries were performed from 11:00 am to 01:00 pm, and food and water intake were measured 0, 6, 12, and 24 h after bilateral nephrectomy or sham surgery.
3.2. Plasma levels of BUN and Cre After bilateral nephrectomy, the plasma levels of BUN and Cre increased with time (Table 2). 2
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Fig. 1. Body weight changes after bilateral nephrectomy (Nx) or sham operation (sham) (A). Effects of bilateral nephrectomy (Nx) or sham operation (sham) on cumulative food intake in conscious rats (B-a). Effects of bilateral nephrectomy (Nx) or sham operation (sham) on cumulative water intake in conscious rats (B-b). Data for cumulative food intake and water intake are expressed as means ± SEM (each group at surgery, n = 6). Data are presented as means ± SEM (each group at each time point, n = 6). †P < 0.05, ††P < 0.01 compared with the untreated group. *P < 0.05, **P < 0.01 compared with the sham-operated group.
3.4. Gene expressions of hypothalamic neuropeptides controlling feeding behaviors
Table 2 Effects of i.p. bilateral nephrectomy (Nx) and of sham operation (Sham) on plasma blood urea nitrogen (BUN) and creatinine (Cre) levels at 3, 12, and 20 h after treatment. Data are presented as means ± SEM (each group at each time point, n = 6). *P < 0.05, **P < 0.01 compared with Sham. Plasma BUN and Cre levels in untreated rats (n = 6) are 18.8 ± 0.5 mg/dl and 0.25 ± 0.01 mg/dl, respectively.
Time after treatments (h) Sham Nx Time after treatments (h) Sham Nx
Plasma concentration of 6 12 42.2 ± 2.7 29.9 62.8 ± 1.7** 89.1 Plasma concentration of 6 12 0.29 ± 0.02 0.23 1.24 ± 0.06** 2.11
Representative images obtained by in situhybridization of hypothalamic anorexigenic neuropeptides (CRH, POMC, and CART) at 6, 12, and 24 h after bilateral nephrectomy or sham operation are shown in Fig. 2. Similarly, representative images of orexigenic neuropeptides (AgRP, NPY, MCH, and Orexin) are shown in Fig. 3. All gene expression levels of hypothalamic neuropeptides controlling feeding behaviors in this study were calculated using the untreated group values as the standard values (Fig. 4). Gene expression of CRH in PVN was markedly higher in bilateral nephrectomized rats than in sham-operated rats (Fig. 4A-a).Gene expression of POMC and CART in Arc were significantly higher in bilateral nephrectomized rats than in sham-operated rats (Fig. 4A-b, c). The AgRP and NPY levels were significantly lower in bilateral nephrectomized rats than in sham-operated rats (Fig. 4B-a, b). The MCH and Orexin levels were significantly lower in bilateral nephrectomized rats than in sham-operated rats (Fig. 4B-c, d). In summary, bilateral nephrectomy upregulated the hypothalamic anorexic neuropeptide levels (Fig. 4A) and downregulated the hypothalamic orexigenic neuropeptide levels (Fig. 4B).
BUN (mg/dl) ± 2.2 ± 1.6**
24 19.1 ± 0.7 155.1 ± 6.1**
Cre (mg/dl) ± 0.01 ± 0.09**
24 0.22 ± 0.01 2.92 ± 0.14**
3.3. Water and food intake Cumulative water intake and food intake were measured every 6 h for 24 h in all groups (Fig. 1B-a, b). There were no differences in the water intake of the sham-operated group and the untreated group at all time points. The water intake of bilateral nephrectomized rats was significantly lower than of sham-operated and untreated rats (Fig. 1Ba). The food intake of sham-operated rats was significantly lower than that of untreated rats. The food intake of bilateral nephrectomized rats was further reduced compared with that of sham-operated rats. The decrease in food intake of bilateral nephrectomized rats was more pronounced than the decrease in water intake, resulting in the rats eating very little.
3.5. Plasma leptin levels The plasma leptin levels at 24 h after bilateral nephrectomy were significantly increased compared with those of sham-operated rats (Fig. 5). 4. Discussion Anorexia, a common clinical symptom in patients with acute and chronic kidney dysfunction, could have a major impact on patients’ prognosis and quality of life by causing malnutrition. Therefore, it is 3
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Fig. 2. Representative autoradiographs of sections hybridized with a 35S-labeled oligodeoxynucleotide probe complementary to the hypothalamic neuropeptides that promote feeding behaviors 6, 12, and 24 h after bilateral nephrectomy (Nx) or sham operation (Sham). CRH in the PVN (A), CART in the Arc (B), and POMC in the Arc (C) at 3, 12, and 20 h are shown. Black scale bar =1 mm. Signal intensity ranges from high (black) to low (white).
hypothalamic neuropeptides controlling feeding behaviors. These results lead to the conclusion that changes in hypothalamic neuropeptides after bilateral nephrectomy decreased food consumption. However, there is concern that bilateral nephrectomized rats could not eat due to surgical invasion or a prolonged anesthetic effect induced by kidney dysfunction. Bilateral nephrectomized rats have not only reduced food intake but also reduced water intake compared with sham-operated rats and untreated rats. The food intake of sham-operated rats decreased compared with that of untreated rats. These results may be due to anesthesia or surgical invasion. However, bilateral nephrectomized rats could drink some amount of water, but they could hardly eat. In other words, there was a qualitative difference in the water intake reduction and the food intake reduction in bilateral nephrectomized rats.
important to elucidate the pathophysiology of anorexia in kidney dysfunction. In the present study, the hypothalamic neuropeptides controlling feeding behaviors were shown to be affected by bilateral nephrectomy. The hypothalamus has a central role in the regulation of feeding behaviors and energy metabolism. Our team has shown the changes of gene expressions of hypothalamic neuropeptides controlling feeding behaviors under various physiological stresses or after administration of some drugs [16,19,20]. However, there have been few reports on the involvement of central mechanisms in appetite decreases in patients with kidney dysfunction. The food consumption of bilateral nephrectomized rats decreased significantly compared with sham-operated and untreated rats in the present study. Bilateral nephrectomy changed the levels of
Fig. 3. Representative autoradiographs of sections hybridized with a 35S-labeled oligodeoxynucleotide probe complementary to the hypothalamic neuropeptides that suppress feeding behaviors 6, 12, and 24 h after bilateral nephrectomy (Nx) or sham operation (Sham). AgRP in the Arc (A), NPY in the Arc (B), MCH in the LHA (C), and Orexin in the LHA (D) at 3, 12, and 20 h are shown. Black scale bar =1 mm. Signal intensity ranges from high (black) to low (white). 4
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Fig. 4. Gene expressions of the hypothalamic neuropeptides that promote (A) or suppress (B) feeding behaviors 6, 12, and 24 h after bilateral nephrectomy (Nx) or sham operation (Sham). CRH (A-a), CART (A-b), POMC (A-c), AgRP (B-a), NPY (B-b), MCH (B-c), and Orexin (B-d) are shown. Data are presented as means ± SEM (each group at each time point, n = 6). †P < 0.05, ††P < 0.01 compared with the untreated group. *P < 0.05, **P < 0.01 compared with the sham-operated group.
untreated rats. Kidney dysfunction is a serious stressor for the body, because the kidney is an important organ for maintaining homeostasis in the body. CRH plays an important role in the biological response for stress by secreting adrenocorticotropic hormone (ACTH) from the anterior pituitary, and it also has an inhibitory effect on food intake [5,22]. Recently, we showed that synthesis of CRH and vasopressin, which are stress hormones, increased in the hypothalamus after bilateral nephrectomy [8]. There are many previous reports that stress and appetite are closely related, and it is generally considered that acute stress suppresses feeding behaviors [23,24]. In the present experiment, results consistent with the previous report were obtained. Leptin, a peptide hormone secreted from adipocytes, is known to have a major effect on feeding behaviors and energy metabolism. Previous studies reported that plasma leptin levels were several times higher in patients with chronic kidney dysfunction than in healthy subjects, and plasma leptin levels in dialysis patients were inversely correlated with nutritional status and serum albumin levels [25,26]. Based on these reports, it appears that elevated leptin levels in patients with kidney dysfunction are involved in anorexia and nutritional disorders. In the present study, the plasma leptin levels in bilateral nephrectomized rats increased significantly compared with those in shamoperated rats. Leptin is excreted mainly from the kidneys, and thus the plasma leptin level is reported to increase as kidney function decreases [27]. Elevated leptin levels in this experiment may also have been triggered by impaired excretion associated with acute kidney dysfunction. Previous studies have reported that leptin promotes the synthesis of POMC and CART, which are neuropeptides that inhibit feeding behaviors and, conversely, inhibit the synthesis of AgRP, NPY, and orexin, which are neuropeptides that enhance feeding behaviors [28–31]. Therefore, the increasing plasma leptin level may have contributed to changes in hypothalamic neuropeptides controlling feeding behaviors
Fig. 5. The changes of plasma leptin levels 12 and 24 h after bilateral nephrectomy (Nx) or sham operation (Sham). Data are presented as means ± SEM (each group at each time point, n = 6). *P < 0.05, **P < 0.01 compared with the sham-operated group.
Furthermore, if the amount of food intake were decreased due to such physiological dysfunction induced by anesthesia or surgical invasion, the body would become starved, and the gene expressions of hypothalamic neuropeptides should change to promote feeding behaviors. On the other hand, the gene expressions of hypothalamic neuropeptides after bilateral nephrectomy changed to suppress feeding behaviors in the present study. Based on these results, the hypothalamic neuropeptide changes appear to have been located upstream of feeding behaviors in this experiment. In addition, previous reports have shown that food intake and water intake are interrelated [21]. Accompanied by an extreme reduction in food intake, water intake in bilateral nephrectomized rats may have decreased compared to sham-operated and 5
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Science (JSPS).
after bilateral nephrectomy. However, it should be noted that plasma concentrations of ghrelin, which antagonizes leptin and promotes feeding behaviors, have also been reported to be elevated in patients with kidney dysfunction [32]. Ghrelin is also excreted by the kidney, and it is reported that its blood concentration increases with decreasing kidney function. Further experiments are needed to prove this hypothesis. We assume that the decline in feeding behaviors in kidney dysfunction is a major problem when it lasts for a long time. However, bilateral nephrectomized rats cannot survive for a long time due to lethal arrhythmias and uremic syndrome. This point is the biggest limitation in this research. In the experimental design stage, an experiment using ischemic reperfusion injury instead of bilateral nephrectomy as a model of acute renal dysfunction in rats was also planned. This is because renal ischemia-reperfusion injury in rats is considered to be a more physiological experimental model of acute kidney dysfunction than bilateral nephrectomy. However, the renal ischemia reperfusion model rarely causes severe acute kidney dysfunction that could induce anorexia. In this experiment, the changes in gene expression of hypothalamic neuropeptides that control the feeding behavior of bilateral nephrectomized rats were validated, keeping in mind the limitations of bilateral nephrectomy. In order to address this issue, we are currently preparing a plan to investigate hypothalamic feeding neuropeptides in a more physiological animal model with kidney dysfunction that could be observed over a long period.
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5. Conclusion Anorexia is a serious complication that contributes to malnutrition in patients with advanced kidney dysfunction. However, the mechanism of anorexia is poorly understood. To the best of our knowledge, this report is the first to focus on the relationships of anorexia with kidney dysfunction and hypothalamic neuropeptides controlling feeding behaviors. In conclusion, the changes in gene expressions of hypothalamic neuropeptides controlling feeding behaviors were demonstrated, and a significant reduction of food intake was found after bilateral nephrectomy. This report is the first step to elucidating the physiological mechanism of anorexia in patients with kidney dysfunction. We hope that elucidation of the pathophysiology of anorexia in kidney dysfunction will contribute to improving the prognosis and quality of life of patients with kidney dysfunction. Authors’ contributions Research design: H.U., T.M., Y.O., and Y.U. Carried out the research: H.U., K.S., K. N., K.T., H.N., K. N., S.S., M.Y., and T.M. Analysis of the data: H.U., M.T., R.S., and Y.O. Drafting of the manuscript: H.U. Critical revision of the article: T.M. and Y.U. All authors approved the final version of the manuscript and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy are answered. All authors designated as authors qualify for authorship, and all those who qualify for authorship are listed. Declaration of Competing Interest The authors declare no conflict of interest associated with this manuscript. Acknowledgments The authors wish to express their appreciation to Ms. Yuki Nonaka for her technical assistance. This work was supported by a Grant-in-Aid for Scientific Research (B) (No. 17H04027), (C) (No. 17K08582), and (C) (No. 18K11010) from the Japan Society for the Promotion of 6
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