Chronic renal insufficiency does not induce behavioral and cognitive alteration in rats

Physiology & Behavior 138 (2015) 133–140

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Chronic renal insufficiency does not induce behavioral and cognitive alteration in rats Ľubomíra Tóthová a,b,⁎, Janka Bábíčková a,b, Veronika Borbélyová a, Barbora Filová a, Katarína Šebeková a, Július Hodosy a,b,c a b c

Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia Center for Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovakia Institute of Physiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• A comprehensive evaluation of behavior was performed in 5/6 nephrectomy rat model of chronic kidney disease • General locomotion, depression and spatial memory did not change with 5/6 nephrectomy after 9 months • Anxiety level was more alleviated in nephrectomized rats after nine months • Psychosocioeconomical traits should be taken into account when assessing behavioral changes in chronic kidney disease patients

a r t i c l e

i n f o

Article history: Received 6 September 2014 Received in revised form 13 October 2014 Accepted 23 October 2014 Available online 31 October 2014 Keywords: Renal insufficiency Locomotor activity Anxiety Depression Memory 5/6 Nephrectomy

a b s t r a c t In humans, chronic kidney disease (CKD) is associated with cognitive decline, increase in anxiety, or depression. The underlying mechanisms of these changes remain unclear. The aim of this study was to elucidate whether and how experimentally induced long-term CKD affects cognitive functions in rats. Thirty male Wistar rats underwent 5/6 nephrectomy (5/6 Nx), an established model of CKD, or sham surgery. Development of CKD was monitored using biochemical analyses and confirmed by renal histology. Behavioral tests of anxiety, depression and spatial behavior were performed before, and at 3 and 9 months after the surgery. CKD in 5/6 Nx rats was characterized by significant decrease of renal function, e.g., glomerular filtration rate, and progressive glomerulosclerosis, tubular atrophy, and interstitial fibrosis; and increased plasma uremic toxins. Mortality was higher in 5/6 Nx rats in comparison with controls. Compared to control group, the surviving 5/6 Nx rats presented similar general locomotor activity, depression traits, and spatial abilities (p = 0.43, p = 0.84, p = 0.71, respectively). At 9 months, lower anxiety in the light–dark box test was observed in 5/6 Nx rats if compared with the control group (p = 0.02). Despite the development of progressive CKD in 5/6 Nx rats, no expected behavioral changes were observed. Further experimental studies associating behavioral responses to severity of CKD are definitely needed to confirm the solely psychosocial aspect background of CKD-associated cognitive impairment in humans. © 2014 Published by Elsevier Inc.

⁎ Corresponding author at: Institute of Molecular Biomedicine, Comenius University, Sasinkova 4, 811 08 Bratislava, Slovakia. Tel.: +421 2 59357371; fax: +421 2 59357631. E-mail address: [email protected] (Ľ Tóthová).

http://dx.doi.org/10.1016/j.physbeh.2014.10.027 0031-9384/© 2014 Published by Elsevier Inc.

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1. Introduction Chronic kidney disease (CKD) is defined as kidney damage resulting in glomerular filtration rate lower than 60 ml/min/1.73 m2 for 3 months or more, irrespective of cause [1]. Its severity is classified according to glomerular filtration rate (GFR) into 5 stages, those graded IV and V representing the most severe cases. CKD represents a serious public health problem worldwide: increasing incidence and prevalence of CKD imposes burden on the utilization of medical care for CKD itself as well as increase in costs of treating complications from CKD [2]. In humans, one of the most sensitive complications, severely decreasing the quality of life, is the cognitive decline [3,4]. Several studies examining the link between the CKD and cognitive outcomes revealed higher rates of dementia and cognitive dysfunction when compared to those in general population [5,6]. Although the patients with most severe CKD stages are most seriously afflicted, symptoms may manifest also in mild or moderate stages of CKD, and even in subjects with albuminuria without reduced GFR [7,8]. The potential pathogenic factors involved in the development of cognitive disorders observed in humans include increased concentrations of circulating uremic toxins (particularly neurotoxins), pro-inflammatory cytokines, and reactive oxygen species partially released from damaged kidneys [9–11], and changes in serotoninergic and adrenergic neurotransmission [12,13]. Due to the systemic stress, overactivation of sympathetic nervous system may also contribute to the pathogenesis of cognitive development in CKD patients [14]. Cognitive impairment associated with albuminuria may be a consequence of endothelial dysfunction and sub-clinical cerebral microvessel disease [47]. However, the underlying pathological mechanism of cognitive disorders is not very well understood. Rats in initial stage of CKD are characterized by insignificant decline of creatinine clearance, 3 to 4-fold rise in proteinuria, and mild accumulation of uremic toxins present alterations in activation of neural cells in several brain regions with different functional properties [15]. Observed central sympathetic overactivity, activation of dorsal raphe serotoninexpressing neurons, histamine-expressing neurons in the hypothalamic tuberomamillary nucleus, those in the prefrontal and anterior cingulate cortex and stress-related brain areas and nuclei might be implicated in

affecting (among others) attention, memory processing, cognition, learning and synaptic plasticity. However, current experimental studies regarding behavioral changes in CKD show some controversies [16]. For example, in adenine-induced CKD model in rats decrease in motor activity and increase in depression-like behavior were reported [17]. In 5/6 Nx model of CKD, mice exhibited alterations in working memory performance [16]. A different study showed significantly impaired cognitive functions without differences in anxiety between CKD and control mice [18]. Alterations in short-term memory without changes in long-term memory were described in rats with severe CKD induced by subtotal nephrectomy [19]. Different approaches to CKD induction, duration of experiments, and selection of limited number of different tests to study behavioral characteristics of CKD animals do not allow a straightforward comparison of results obtained in different studies. Herein we aimed to determine the effects of long-term CKD induced in rats by subtotal nephrectomy on a variety of behavioral traits such as locomotor activity, anxiety and depression-like behavior, and memory impairment in a single study. 2. Material and methods 2.1. Animals Thirty 12-weeks-old male Wistar rats were obtained from Anlab (Prague, Czech Republic). Animals were maintained in 12:12 h light/dark cycle and had ad libitum access to water and food throughout the experiments. After arrival, rats were left 2 weeks for acclimatization and handled daily. The room temperature was maintained at 22 ± 1 °C throughout the whole study. All animal experiments were carried out according to Slovak legislation after approval by the institutional ethics committee. After acclimatization, the animals were randomly assigned into subtotally nephrectomized (5/6 Nx, n = 15) and sham operated (CTRL, n = 15) groups. The animals in the 5/6 Nx group underwent subtotal (5/6) nephrectomy in two surgical steps as reported previously [20]. Briefly, animals were anesthetized by ketamine/xylazin administered intraperitoneally (100 mg/kg and 10 mg/kg respectively, Richterpharma, Wels, Austria). An incision was made approximately

Fig. 1. Physiological panel. (A) Glomerular filtration rate, (B) proteinuria, (C) body weight and (D) survival after nephrectomy measured at baseline and 3 and 9 months after 5/6 nephrectomy. * denotes p b 0.05, ** p b 0.01 and *** p b 0.001 vs. corresponding CTRL group. Data are presented as mean ± SEM.

Ľ Tóthová et al. / Physiology & Behavior 138 (2015) 133–140 Table 1 Blood nitrogen urea, creatinine, AOPP concentrations in plasma at 9 months.

Blood urea nitrogen [mmol/l] Creatinine [μmol/l] AOPP [μmol/g protein]

CTRL

5/6 Nx

9.66 ± 0.87 57.84 ± 6.76 4.6 ± 0.3

28.15 ± 12.26 ** 197.7 ± 72.97 ** 6.1 ± 0.4 *

Data presented as mean ± SEM. * denotes p b 0.05, ** denotes p b 0.01, both vs. CTRL group.

1 cm beneath the last rib on the left side and the left kidney was exposed. The kidney was decapsulated and upper and lower kidney poles were excised. Bleeding was stopped by Gelaspon (Chauvin Ankerpharm GmbH, Rudolstadt, Germany) and the incision was sutured in two layers by Chirlac 4-0 (Chirmax, Modrany, Czech Republic). Fourteen days later, a similar incision was made at the right flank, the renal vessels were ligated and the decapsulated kidney was excised. Rats from each group were sacrificed at the end of the experiment.

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2.3. AOPP measurement Protein oxidation was determined by the measurement of advanced oxidation protein products [22]. Two hundred microliters of plasma samples were mixed with 20 μl of glacial acetic acid. The absorbance at 340 nm was measured. Chloramine T with potassium iodine was used to construct the calibration curve (intra-assay variability 3–7% and inter-assay variability 2–10%). 2.4. Behavioral tests All animals were tested for behavioral traits before, 3 months and 9 months after the surgery, except water maze task, which was performed after 9 months only. Because of high inter-individual variability, the initial testing before the surgery served as a control for correct random assignment into the groups, i.e. no behavioral differences between groups at the beginning. Behavioral tests were recorded and analyzed by automated tracking software Ethovision XT 10.1 (Noldus, Wageningen, Netherlands).

2.2. Kidney function Blood samples for standard biochemistry were collected at 3 different time points (0, 3 and 9 months after surgery). The blood was obtained either from the tail vein (0 and 3 months) or from the abdominal aorta at the end of the experiment. All biochemical markers were measured at certified biochemical laboratory (Institute of Biochemistry, Saint Michael Hospital, Bratislava, Slovakia). Creatinine in serum and urine was determined using analyzer Olympus AU400 (Beckman Coulter, California, USA). Proteinuria was analyzed on COBAS 6000 (Roche, Basel, Switzerland). Glomerular filtration rate was calculated from creatinine clearance as follows:

ccrGFR ¼

UCr  24 h volume PCr  24 h  60 min

where, ccrGFR creatinine clearance glomerular filtration rate [ml/min], UCr creatinine concentration in urine [mg/ml], 24 h volume – volume of urine in 24 h [ml], and PCr – creatinine concentration in plasma [mg/ml]. The ccrGFR result was subsequently corrected for 100 g of body weight [21].

2.4.1. Open field Open field test serves as a test for general locomotor activity and as habituation for other testing. The maze arena is made of 100 × 100 × 40 cm washable plastic square. The maze was situated in the dimly lit room. The maze was virtually divided into the central zone and border zone. Animals were put in the middle of the arena and they were allowed 5 min to freely explore. The total distance moved as well as the speed inside the arena were determined to evaluate the locomotor activity. The time that rats spent in the center zone was assessed as an index of anxiety. The longer time the rat spent in the center zone of maze was considered as lower anxiety level, as described elsewhere [20]. 2.4.2. Simple novelty recognition test Rats were tested in the same arena as used for open field test for simple novelty recognition which refers to how easily the rats are willing to adapt and explore new objects in known environment. The maze was enriched by a new object – a ceramic cup placed into the maze. The total time spent interacting with the new object was recorded within for 5 min. Touching or sniffing the object with nose was classified as interaction. The longer interaction with the object was regarded as lower anxiety and higher exploration behavior [23,24].

Fig. 2. Histological features of the kidneys. Control, sham-operated rats (A) had normal glomeruli (a) and interstitium (b), but showed mild age-related findings in the renal parenchyma (c), i.e. – perivascular infiltration of lymphocytes (arrow) and thickening of the tubular basement membranes (arrowhead). In Sirius red staining (C), collagen positive area (red) was strictly localized to the Bowman's capsules, vascular beds, capillary and tubular basement membranes. Nephrectomized rats (B), showed progressive renal fibrosis, including glomerulosclerosis (d), proteinuric tubular casts, expansion of the interstitial cells (lightning) and interstitial deposition of the extracellular matrix (asterisk; e) and tubular dilation and atrophy (f). In Sirius red staining, massive expansion of the collagen deposition was observed in both glomerular mesangium, as well as tubulointerstitium (D). Scale bars represent 100 μm (magnification 100×).

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Fig. 3. Baseline behavior. No significant results of rats before nephrectomy were found in (A) average speed, (B) distance moved, and (C) time in border zone of open field describing general locomotor activity. No significant changes were found in (D) time interacting with object, (E) time spent in light zone and (F) immobility time. Data are presented as mean ± SEM.

2.4.3. Light/Dark box The light/dark box apparatus consisted of 60 cm × 40 cm rectangular plastic box. The box was divided into closed (dark) – covered with a lid and opened (light) – brightly illuminated parts. Rats were placed in the light part and were allowed 5 min to move freely. The time spent in the light part served as an indicator of anti-anxiety behavior [25]. 2.4.4. Elevated plus maze Elevated plus maze testing was performed in an apparatus with four arms located 50 cm above the floor. The length of each arm was 50 cm and width 10 cm. Two of the arms were opened and 2 were closed by 30 cm high walls. The rats were placed at the central plate and were observed for 5 min. Time spent in the open arms was assessed as an index of anxiety behavior. Rats were considered to be in the open arms only if all 4 paws were in the open arm of the maze [20]. 2.4.5. Forced swim test Animals were individually placed into a plastic cylinder (height 46 cm, 35 cm in diameter) filled with water (25 ± 1 °C). Their behavior was observed for 5 min: the specific movements were manually recorded and immobility time was calculated. The time spent immobile was considered to reflect depression-like behavior [26,27]. After the session, the rats were removed from the pool, dried with a towel and returned into their cages. 2.4.6. Sucrose preference test (SPT) During this test, animals were offered two bottles for 24 h, one containing regular tap water and the other containing 2% of sucrose solution. To prevent the possible effect of side preference, the position of the bottles was swapped after 12 h, equally. The consumption of both solutions was measured by weighing the bottles. The sucrose preference was calculated as the amount of sucrose solution consumed and divided with the total amount of liquid drunk. The lower percentage of sucrose consumption is interpreted as a lack of interest in rewarding stimuli, thus serves as a marker of depressive behavior [28]. 2.4.7. Modified Morris water maze The modified water maze that consisted of a dark plastic circular pool (125 cm in diameter, 60 cm in height) filled with water (water

temperature 25 ± 1 °C) was used. The maze was virtually divided into four quadrants, and on the wall of each quadrant one geometrical figure was placed for orientation as an external cue. A circular platform 10 cm in diameter was placed in one of the quadrants 0.5 cm beneath the water surface. Due to the high learning ability of rats, the water maze was performed only once, at the end of the 9 months period. The memory and spatial ability testing was performed during 4 consecutive days. Each day, the rats received one block of the acquisition phases, with four trials in a block. The platform remained hidden 0.5 cm beneath the surface of the water in a constant quadrant. A different starting quadrant was used each trial. A trial consisted of a swim followed by a 30-second rest on the platform. Any rat that did not find the platform within 60 s was gently guided to it by an experimenter. Latency times (time to find the platform) reflecting status of a short-term working memory were assessed for each day and group. On day 5, one probe trial was performed after removing the platform. The duration of the trial was 60 s and time spent in the platform quadrant was recorded. During this probe the quadrant opposite the platform served as starting position for all animals. This trial reflects long-term reference memory status [29].

2.5. Histopathological analysis At sacrifice the remnant kidneys from 5/6 Nx rats and left kidneys from CTRL rats were harvested, immersion-fixed in methyl Carnoy's solution and embedded in paraffin. Sections (1 μm thick) were stained with Periodic acid Schiff (PAS) reagent and Sirius red stain. The tissue injury was analyzed in a single blind manner by a skilled pathologist.

2.6. Statistical analysis Data are presented as mean ± standard error of the mean. A two-way ANOVA (one factor being the time, the second being the assigned group) with subsequent Sidak post-hoc test were used to compare the results of biochemistry and behavioral tasks. The survival rate was determined by Kaplan–Meier survival curves. The p values less than 0.05 were considered significant. The data were analyzed using the Graphpad Software, version 6.1 (Graphpad Software, Inc., CA, USA).

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mortality rate in the 5/6 Nx group when compared to the control group (Fig. 1D; p b 0.01) with odds ratio of 28 (95% confidence interval 2.8 to 278.1). Since only 6 rats were available at the 9 month period in the 5/6 Nx group, only these were further behaviorally analyzed. PAS stained kidney sections of CTRL rats (Fig. 2A) showed normal architecture of the glomeruli and interstitium with only mild age-related histological alterations presented as focal segmental glomerulosclerosis, perivascular infiltration of lymphocytes and thickening of the tubular basement membranes. 5/6 Nx rats (Fig. 2B), showed progressive renal fibrosis and a histological picture of advanced chronic renal disease with massive fibrosis (Fig. 2D). The baseline behavioral tests performed before surgery showed no differences in general locomotor activity and exploration, anxiety or depression level in the performed tests (Fig. 3A–F). Similarly, the behavioral tests performed at 3 months showed no significant changes (data not shown). After 9 months, test in open field showed no significant differences in general locomotor activity expressed as average speed and distance moved in the arena (Fig. 4A and B, respectively). No difference in open field anxiety level was found expressed as time in border zone (Fig. 4C). However, 5/6 Nx rats spent more time in the light part of the light/dark box when compared to the CTRL group (Fig. 5A; t = 2.59; p b 0.05). Other behavioral tests analyzing anxiety showed no significant differences between the groups (Fig. 5B and C). In the forced swim test, both 5/6 Nx and CTRL rats spent comparable time as immobile (Fig. 5D). Similarly, the anhedony test confirmed that nephrectomy did not induce depressive like behavior, since no difference in preference between sweetened or normal water was observed (Fig. 6). In water maze task, 5/6 Nx and CTRL rats performed similarly. In working memory, the rats gradually decreased the latency times, reaching significant improvement on the 3rd and 4th day for both groups (Fig. 7A; F = 19.29; p b 0.01) when compared to the first day. For reference memory, there was no significant difference between the 5/6 Nx and CTRL rats (Fig. 7B). 4. Discussion

Fig. 4. General locomotor activity. Results for open field and general locomotor activity after 9 months of nephrectomy. No significant differences were found in (A) speed, (B) distance moved, and (C) time spent in border zone. Data are presented as mean ± SEM.

3. Results Rats with 5/6 Nx had significantly lower ccrGFR at 3 months by 67% and after 9 months by 79% if compared to the CTRL group (Fig. 1A; F = 46.63; p = 0.012 and p = 0.010 respectively). No significant decrease of ccrGFR in CTRL group was observed. Significant effect of time and subtotal nephrectomy was observed to play a role in the development of proteinuria. Proteinuria was significantly higher, 3-fold, in the 5/6 Nx group when compared to the CTRL at 9 months (Fig. 1B; F = 32.38; p b 0.001) and 10-fold when compared to the 5/6 Nx baseline and at 3 months (p b 0.001). CTRL group had also significantly higher proteinuria at 9 months when compared to the corresponding baseline and at 3 months (Fig. 1B; F = 86.35; p b 0.001). Blood urea nitrogen (BUN; mean 9.66, range 7.94 to 15.53 for CTRL group vs. mean 28.15, range 13.6 to 64.8 mmol/l for 5/6 Nx group) and plasma creatinine (mean 57.84, range 32.2 to 96.4 μmol/l for CTRL group vs. mean 197.7, range 137.05 to 539.75 μmol/l for 5/6 Nx group) were both significantly increased in the 5/6 Nx group only at 9 months when compared to control. AOPPs were significantly higher after 9 months in the 5/6 Nx group (p b 0.05; Table 1). No differences in body weight between the two groups were observed, however, there was a main effect of time observed, where both groups doubled their weight during the experiment (Fig. 1C; F = 117.2; p b 0.001). Additionally, there was a higher

CKD is a risk factor for development of cognitive impairment which might be manifested already in patients with early-stage of CKD [30]. A prevalence of cognitive impairment associated with CKD is estimated to be 30%–60%, i.e. at least twice higher as that observed in age-matched controls [31]. Presence of cognitive impairment further decreases the quality of life of the CKD patient, enhances existing social and economic burden, but also results in higher risk for mortality [32]. Causes of CKD-associated cognitive impairment in humans are multifactorial (including cerebrovascular disease, renal anemia, secondary hyperparathyroidism, dialysis disequilibrium, and uremic toxins accumulation [32], and mechanisms of induction of cognitive impairment not fully understood yet). Rodent models of CKD are important experimental tools used to investigate potential pathophysiological pathways of human disease. Since mechanisms underlying CKD-associated behavioral changes and cognitive impairment in humans are not very well understood yet, experimental studies might help with their elucidation. However, results of behavioral testing in CKD animals are scarce and controversial. Discrepancies may arise from use of different methods for CKD induction, e.g., surgery [19] vs. chemical induction potentially imposing additional toxicity [17], different time points of behavioral testing reflecting early or advanced stages of CKD [18,33], as well as variety of used behavioral tests. A comprehensive assessment of the behavioral changes during CKD in experimental animals is missing. Thus the aim of our experiment was to describe comprehensively the changes in rat behavior 3 and 9 months after induction of CKD by 5/6 nephrectomy employing battery of behavioral tests concurrently assessing the general locomotion, anxiety, depression-like, exploratory and memory components. Our animals presented decreased renal function with proteinuria,

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Fig. 5. Anxiety and depression behavior. Results for (A) light/dark box, where nephrectomized rats spent more time in light zone when compared to CTRL group, (B) elevated plus maze with no significant differences, (C) simple novelty test and (D) forced swim test. No significant differences were found. Data are presented as mean ± SEM. * denotes p b 0.05 vs. CTRL group.

and histologically confirmed CKD, as well as increased plasma concentration of determined uremic toxins findings corresponding with those in humans with CKD [34]. In contrast to our hypothesis, our multifaceted testing approach did not reveal substantial changes in behavior of rats either in early or advanced stage of CKD. We assessed the general locomotor activity by open field test, while the anxiety level was evaluated by light/dark box and elevated plus maze. Open field evaluates the locomotor activity by determination of overall distance walked, and speed. Moreover, an open field test enables anxiety trait assessment by detection of time an animal spends in the center part of the maze [35]. Light/dark box and elevated plus maze enable to evaluate two distinct aversion related properties of anxiety: in the light/dark box the animals demonstrate natural aversion from light preferring to stay in the dark [36], while elevated plus maze enables testing of aversion from high and open grounds, that rodents express as spending more time in the closed arms of the maze when allowed to choose [37]. We observed no changes in general locomotor activity and anxiety level as assessed by open field and elevated plus maze. In the light–dark box, 5/6 Nx rats spent more time in the light zone, possibly reflecting decrease in anxiety in comparison with the control group. These results are in contrast to the clinical studies demonstrating higher anxiety level in both moderate and severe CKD patients [38]. Moreover, these findings contradict some animal studies describing decreased locomotor activity, exploratory and emotional-like types of behavior in CKD of rodents [17,33]. On the other hand, Fujisaki et al. [16] did not reveal decrease in physical performance of CKD rats. In different study, CKD mice manifested decreased anxiety level presented as longer time spent in the light part of light/dark box [18]. Reasons leading to disparate results in different studies remain unclear, but might be on the account of different CKD stages of tested animals, which make them difficult to compare due to different approaches of determination of CKD severity. The CKD stage was assessed by the level of plasma creatinine and urea, while in our study ccrGFR and presence of proteinuria. We did not observe any significant increase in BUN or plasma creatinine after 3 months as did other research groups,

although creatinine clearance glomerular filtration rate was already significantly decreased. It is probable, that during this time there might have been ongoing adaptive processes in central nervous system after nephrectomy during initial stages of CKD [39], which could in turn affect the behavioral results. If compared with the control rats, our CKD animals did not show difference in depression-like behavior monitored as immobility time in the forced swim task. The result of Porsolt depressive test was also confirmed in our experiment by unchanged preference for the sucrose solution. This preference did not change throughout the time and nephrectomy did not affect this type of behavior. This is in direct contrast to observations in humans, where patients with CKD show higher incidence of depression symptoms [40,41]. It is also in contrast to another animal experiment [17]. Rats with adenine-induced CKD presented increased immobility time in forced swim test at 28 days. Severe anemia [42] and hyperparathyreoidism [43] induced by feeding of adenine could contribute to observed behavioral changes in this model of CKD. Direct toxic effects of adenine administration were also

Fig. 6. Anhedonia. Results of sucrose preference test. Although slight decrease in sucrose preference was observed after 3 and 9 months, this was not significant. Neither time, nor treatment had effect on sucrose preference. Data are presented as mean ± SEM.

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severe stage of CKD with broad range of plasma creatinine and BUN, but low number of remaining animals did not allow for performance of relevant subgroup analysis. Nevertheless, our data suggests that CKDassociated cognitive impairment manifested in humans could be underlined more by the psychosocial factors such as low self-esteem, feelings of uselessness, impaired physical activity possibilities and the overall distraction of the body image. Such factors are irrelevant in rats. To conclude, this study did not reveal any cognitive decline in rats after induction of chronic kidney disease by 5/6 Nx. Locomotion, anxiety, depression-like behavior as well as memory were well preserved after 9 months of CKD in surviving animals. However, further experimental studies focusing on CKD-associated behavioral responses are needed to confirm possible psychosocial aspects of cognitive decline in humans. Disclosure No conflict of interest to declare. Acknowledgment This study was funded by the Slovak Ministry of Education, Science, Research and Sport – grant number VEGA 1/0222/14. The authors would like to thank MSc. Jana Bellova, PhD., for her assistance with figures preparation. References Fig. 7. Spatial memory. Results from water maze and (A) short-term working memory and (B) long-term reference memory. No significant effect of the treatment on the observed data. Data are presented as mean ± SEM. ** denotes p b 0.01 vs. 1st and 2nd day for both groups.

reflected by significant decrease in body weight [17]. In our study body weight of 5/6Nx rats remained similar to that of the control group throughout the experiment. Moreover, we did not gather behavioral data as soon as 28 days after the induction of CKD. These findings might suggest that the serotonin system, which plays a role in mediating depression-like behavior and the mesolimbic dopamine system, which plays a role in the mediation of reward stimuli, could have been adapted after 9 months, being more complex in influencing the behavior. In contrast to human studies [44], we did not reveal any decline either in short-term working memory or in long-term reference memory. In the model of 5/6 nephrectomy, long-term memory impairment was detected after 24 weeks only in rats with severe CKD [19]. The severity of the CKD was classified as 3-fold rise of serum creatinine vs. controls, the severe group presenting mean creatinine concentration of 1.19 mg/dl (105 μmol/l). In our study, CCr was used to classify CKD severity. However, at 9 months, mean creatininemia of 5/6 Nx rats was 3.5-fold higher in comparison with the controls, ranged widely according to GFR. When CKD severity would be staged according to the plasma creatinine as proposed by [19], it would be possible to divide the rats into the 3 rats with higher plasma creatinine and 3 with the lower plasma creatinine. However, all 6 rats would be still classified as rats with severe CKD. Additionally, all 6 surviving rats had CKD linked with significant proteinuria at 9 months. To the best of our knowledge, this is the first comprehensive experimental study describing complexly different aspects of behavior in CKD including general locomotion, anxiety, depression and memory. Indeed, our study did not come without some limitations. The major limitation is the mortality rate in the 5/6 Nx group which was 60% after 9 months, leaving only 6 animals to test at the end of the experiment. Although this rate is comparable to other studies [45,46], no relevant behavioral data are available for animals that died prior to 90 or 270 day testing in our study. Therefore we can only hypothesize that animals that died earlier could be more susceptible to cognitive decline than surviving rats. Additionally, surviving animals displayed

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