Physiology & Behavior 138 (2015) 133–140

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Physiology & Behavior

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Chronic renal insufficiency does not induce behavioral and cognitivealteration 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 Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Bratislava, Slovakiab Center for Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovakiac 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 behaviorwas performed in 5/6 nephrectomy ratmodel of chronic kidney disease

• General locomotion, depression andspatial memory did not change with5/6 nephrectomy after 9 months

• Anxiety level was more alleviated innephrectomized rats after nine months

• Psychosocioeconomical traits shouldbe taken into account when assessingbehavioral changes in chronic kidneydisease patients

⁎ Corresponding author at: Institute of Molecular BiomE-mail address: tothova.lubomira@gmail.com (Ľ Tótho

http://dx.doi.org/10.1016/j.physbeh.2014.10.0270031-9384/© 2014 Published by Elsevier Inc.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 6 September 2014Received in revised form 13 October 2014Accepted 23 October 2014Available online 31 October 2014

Keywords:Renal insufficiencyLocomotor activityAnxietyDepressionMemory5/6 Nephrectomy

In humans, chronic kidney disease (CKD) is associated with cognitive decline, increase in anxiety, or depression.The underlyingmechanisms of these changes remain unclear. The aim of this studywas to elucidatewhether andhow 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. Behavioraltests 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, andprogressive 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 5/6Nx 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 ifcompared 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 CKDare definitely needed to confirmthe solely psychosocial aspect background of CKD-associated cognitive impairment in humans.

© 2014 Published by Elsevier Inc.

edicine, Comenius University, Sasinkova 4, 811 08 Bratislava, Slovakia. Tel.: +421 2 59357371; fax: +421 2 59357631.vá).

134 Ľ Tóthová et al. / Physiology & Behavior 138 (2015) 133–140

1. Introduction

Chronic kidney disease (CKD) is defined as kidney damage resultingin glomerular filtration rate lower than 60ml/min/1.73m2 for 3 monthsor more, irrespective of cause [1]. Its severity is classified according toglomerular filtration rate (GFR) into 5 stages, those graded IV and Vrepresenting the most severe cases. CKD represents a serious publichealth problem worldwide: increasing incidence and prevalence ofCKD imposes burden on the utilization of medical care for CKD itself aswell as increase in costs of treating complications from CKD [2]. Inhumans, one of the most sensitive complications, severely decreasingthe quality of life, is the cognitive decline [3,4]. Several studies examiningthe link between the CKD and cognitive outcomes revealed higher ratesof dementia and cognitive dysfunction when compared to those ingeneral population [5,6]. Although the patients with most severe CKDstages are most seriously afflicted, symptoms may manifest also inmild or moderate stages of CKD, and even in subjects with albuminuriawithout reduced GFR [7,8].

The potential pathogenic factors involved in the development ofcognitive disorders observed in humans include increased concentrationsof circulating uremic toxins (particularly neurotoxins), pro-inflammatorycytokines, and reactive oxygen species partially released from damagedkidneys [9–11], and changes in serotoninergic and adrenergic neuro-transmission [12,13]. Due to the systemic stress, overactivation ofsympathetic nervous system may also contribute to the pathogenesis ofcognitive development in CKD patients [14]. Cognitive impairment asso-ciatedwith albuminuriamaybe a consequence of endothelial dysfunctionand sub-clinical cerebral microvessel disease [47]. However, the underly-ing pathological mechanism of cognitive disorders is not very wellunderstood.

Rats in initial stage of CKD are characterized by insignificant declineof creatinine clearance, 3 to 4-fold rise in proteinuria, andmild accumu-lation of uremic toxins present alterations in activation of neural cells inseveral brain regionswith different functional properties [15]. Observedcentral sympathetic overactivity, activation of dorsal raphe serotonin-expressing neurons, histamine-expressing neurons in the hypothalamictuberomamillary nucleus, those in the prefrontal and anterior cingulatecortex and stress-related brain areas and nuclei might be implicated in

Fig. 1. Physiological panel. (A) Glomerular filtration rate, (B) proteinuria, (C) body weight anephrectomy. * denotes p b 0.05, ** p b 0.01 and *** p b 0.001 vs. corresponding CTRL gro

affecting (among others) attention, memory processing, cognition,learning and synaptic plasticity. However, current experimental studiesregarding behavioral changes in CKD show some controversies [16]. Forexample, in adenine-induced CKD model in rats decrease in motoractivity and increase in depression-like behavior were reported [17].In 5/6 Nx model of CKD, mice exhibited alterations in working memoryperformance [16]. A different study showed significantly impairedcognitive functions without differences in anxiety between CKD andcontrol mice [18]. Alterations in short-term memory without changesin long-term memory were described in rats with severe CKD inducedby subtotal nephrectomy [19]. Different approaches to CKD induction,duration of experiments, and selection of limited number of differenttests to study behavioral characteristics of CKD animals do not allow astraightforward comparison of results obtained in different studies.

Hereinwe aimed to determine the effects of long-term CKD inducedin rats by subtotal nephrectomy on a variety of behavioral traits such aslocomotor activity, anxiety and depression-like behavior, and memoryimpairment 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 hlight/dark cycle and had ad libitum access towater and food throughoutthe experiments. After arrival, rats were left 2 weeks for acclimatizationand handled daily. The room temperature was maintained at 22± 1 °Cthroughout the whole study. All animal experiments were carried outaccording to Slovak legislation after approval by the institutional ethicscommittee.

After acclimatization, the animals were randomly assigned intosubtotally nephrectomized (5/6 Nx, n = 15) and sham operated(CTRL, n=15) groups. The animals in the 5/6Nx group underwent sub-total (5/6) nephrectomy in two surgical steps as reported previously[20]. Briefly, animals were anesthetized by ketamine/xylazin admin-istered intraperitoneally (100 mg/kg and 10 mg/kg respectively,Richterpharma, Wels, Austria). An incision was made approximately

nd (D) survival after nephrectomy measured at baseline and 3 and 9 months after 5/6up. Data are presented as mean ± SEM.

Table 1Blood nitrogen urea, creatinine, AOPP concentrations in plasma at 9 months.

CTRL 5/6 Nx

Blood urea nitrogen [mmol/l] 9.66 ± 0.87 28.15 ± 12.26 **Creatinine [μmol/l] 57.84 ± 6.76 197.7 ± 72.97 **AOPP [μmol/g protein] 4.6 ± 0.3 6.1 ± 0.4 *

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

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1 cmbeneath the last rib on the left side and the left kidneywas exposed.The kidney was decapsulated and upper and lower kidney poles wereexcised. Bleeding was stopped by Gelaspon (Chauvin AnkerpharmGmbH, Rudolstadt, Germany) and the incisionwas sutured in two layersby Chirlac 4-0 (Chirmax, Modrany, Czech Republic). Fourteen days later,a similar incision was made at the right flank, the renal vessels wereligated and the decapsulated kidney was excised. Rats from each groupwere sacrificed at the end of the experiment.

2.2. Kidney function

Blood samples for standard biochemistrywere collected at 3 differenttime points (0, 3 and 9 months after surgery). The blood was obtainedeither from the tail vein (0 and 3 months) or from the abdominal aortaat the end of the experiment. All biochemical markers were measuredat certified biochemical laboratory (Institute of Biochemistry, SaintMichael Hospital, Bratislava, Slovakia). Creatinine in serum and urinewas determined using analyzer Olympus AU400 (Beckman Coulter,California, USA). Proteinuria was analyzed on COBAS 6000 (Roche,Basel, Switzerland). Glomerular filtration rate was calculated fromcreatinine clearance as follows:

ccrGFR ¼ UCr � 24hvolumePCr � 24h � 60 min

where, ccrGFR creatinine clearance glomerular filtration rate[ml/min], UCr creatinine concentration in urine [mg/ml], 24h volume– volume of urine in 24 h [ml], and PCr – creatinine concentration inplasma [mg/ml]. The ccrGFR result was subsequently corrected for100 g of body weight [21].

Fig. 2.Histological features of the kidneys. Control, sham-operated rats (A) had normal glomerulii.e. – perivascular infiltration of lymphocytes (arrow) and thickening of the tubular basement mlocalized to the Bowman's capsules, vascular beds, capillary and tubular basement membranes(d), proteinuric tubular casts, expansion of the interstitial cells (lightning) and interstitial depositstaining, massive expansion of the collagen deposition was observed in both glomerular mesangi

2.3. AOPP measurement

Protein oxidation was determined by themeasurement of advancedoxidation protein products [22]. Two hundred microliters of plasmasamples were mixed with 20 μl of glacial acetic acid. The absorbanceat 340 nm was measured. Chloramine T with potassium iodine wasused 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 and9 months after the surgery, except water maze task, which wasperformed after 9 months only. Because of high inter-individualvariability, the initial testing before the surgery served as a control forcorrect randomassignment into the groups, i.e. no behavioral differencesbetween groups at the beginning. Behavioral tests were recorded andanalyzed by automated tracking software Ethovision XT 10.1 (Noldus,Wageningen, Netherlands).

2.4.1. Open fieldOpenfield test serves as a test for general locomotor activity andas ha-

bituation for other testing. Themaze arena is made of 100 × 100 × 40 cmwashable plastic square. Themazewas situated in the dimly lit room. Themazewas virtually divided into the central zone andborder zone. Animalswere put in themiddle of the arena and theywere allowed 5min to freelyexplore. The total distance moved as well as the speed inside the arenawere determined to evaluate the locomotor activity. The time that ratsspent in the center zone was assessed as an index of anxiety. The longertime the rat spent in the center zone of maze was considered as loweranxiety level, as described elsewhere [20].

2.4.2. Simple novelty recognition testRats were tested in the same arena as used for open field test for

simple novelty recognition which refers to how easily the rats arewilling to adapt and explore new objects in known environment. Themaze was enriched by a new object – a ceramic cup placed into themaze. The total time spent interactingwith the new object was recordedwithin for 5min. Touching or sniffing the object with nosewas classifiedas interaction. The longer interaction with the object was regarded aslower anxiety and higher exploration behavior [23,24].

(a) and interstitium (b), but showedmild age-related findings in the renal parenchyma (c),embranes (arrowhead). In Sirius red staining (C), collagen positive area (red) was strictly. Nephrectomized rats (B), showed progressive renal fibrosis, including glomerulosclerosision of the extracellular matrix (asterisk; e) and tubular dilation and atrophy (f). In Sirius redum, as well as tubulointerstitium (D). Scale bars represent 100 μm (magnification 100×).

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 generallocomotor 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.

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2.4.3. Light/Dark boxThe light/dark box apparatus consisted of 60 cm×40 cm rectangular

plastic box. The box was divided into closed (dark) – covered with a lidand opened (light) – brightly illuminated parts. Rats were placed in thelight part and were allowed 5min tomove freely. The time spent in thelight part served as an indicator of anti-anxiety behavior [25].

2.4.4. Elevated plus mazeElevated plus maze testingwas performed in an apparatus with four

arms located 50 cm above the floor. The length of each arm was 50 cmand width 10 cm. Two of the arms were opened and 2 were closed by30 cm high walls. The rats were placed at the central plate and wereobserved for 5 min. Time spent in the open arms was assessed as anindex of anxiety behavior. Rats were considered to be in the openarms only if all 4 paws were in the open arm of the maze [20].

2.4.5. Forced swim testAnimals were individually placed into a plastic cylinder (height

46 cm, 35 cm in diameter) filled with water (25± 1 °C). Their behaviorwas observed for 5min: the specificmovementsweremanually recordedand immobility time was calculated. The time spent immobile wasconsidered to reflect depression-like behavior [26,27]. After the session,the rats were removed from the pool, dried with a towel and returnedinto 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 sucrosesolution. To prevent the possible effect of side preference, the positionof the bottles was swapped after 12 h, equally. The consumption ofboth solutions was measured by weighing the bottles. The sucrosepreference was calculated as the amount of sucrose solution consumedand dividedwith the total amount of liquid drunk. The lower percentageof sucrose consumption is interpreted as a lack of interest in rewardingstimuli, thus serves as a marker of depressive behavior [28].

2.4.7. Modified Morris water mazeThe 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 dividedinto four quadrants, and on the wall of each quadrant one geometricalfigure was placed for orientation as an external cue. A circular platform10 cm in diameter was placed in one of the quadrants 0.5 cm beneaththe water surface. Due to the high learning ability of rats, the watermaze was performed only once, at the end of the 9 months period.

The memory and spatial ability testing was performed during 4consecutive days. Each day, the rats received one block of the acquisitionphases, with four trials in a block. The platform remained hidden 0.5 cmbeneath the surface of the water in a constant quadrant. A differentstarting quadrantwas used each trial. A trial consisted of a swim followedby a 30-second rest on the platform. Any rat that did notfind the platformwithin 60 s was gently guided to it by an experimenter. Latency times(time to find the platform) reflecting status of a short-term workingmemory were assessed for each day and group. On day 5, one probetrial was performed after removing the platform. The duration of thetrialwas 60 s and time spent in the platformquadrantwas recorded. Dur-ing this probe the quadrant opposite the platform served as starting po-sition for all animals. This trial reflects long-term reference memorystatus [29].

2.5. Histopathological analysis

At sacrifice the remnant kidneys from 5/6 Nx rats and left kidneysfrom CTRL rats were harvested, immersion-fixed in methyl Carnoy'ssolution and embedded in paraffin. Sections (1 μm thick) were stainedwith Periodic acid Schiff (PAS) reagent and Sirius red stain. The tissueinjury was analyzed in a single blind manner by a skilled pathologist.

2.6. Statistical analysis

Data are presented asmean± standard error of themean. A two-wayANOVA (one factor being the time, the second being the assigned group)with subsequent Sidak post-hoc test were used to compare the results ofbiochemistry and behavioral tasks. The survival rate was determined byKaplan–Meier survival curves. The p values less than 0.05 wereconsidered significant. The data were analyzed using the GraphpadSoftware, version 6.1 (Graphpad Software, Inc., CA, USA).

Fig. 4. General locomotor activity. Results for open field and general locomotor activity after9 months of nephrectomy. No significant differences were found in (A) speed, (B) distancemoved, and (C) time spent in border zone. Data are presented as mean ± SEM.

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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 decreaseof ccrGFR in CTRL group was observed. Significant effect of time andsubtotal nephrectomy was observed to play a role in thedevelopment 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-foldwhen compared to the 5/6Nx baselineand at 3 months (p b 0.001). CTRL group had also significantly higherproteinuria at 9 months when compared to the corresponding baselineand at 3 months (Fig. 1B; F = 86.35; p b 0.001). Blood ureanitrogen (BUN; mean 9.66, range 7.94 to 15.53 for CTRL group vs. mean28.15, range 13.6 to 64.8 mmol/l for 5/6Nx group) andplasma 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 significantlyincreased in the 5/6 Nx group only at 9 months when compared tocontrol. AOPPs were significantly higher after 9 months in the 5/6 Nxgroup (p b 0.05; Table 1). No differences in body weight between thetwo groups were observed, however, there was a main effect of timeobserved, where both groups doubled their weight during the experi-ment (Fig. 1C; F = 117.2; p b 0.001). Additionally, there was a higher

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 2.8to 278.1). Since only 6 rats were available at the 9 month period in the5/6 Nx group, only these were further behaviorally analyzed.

PAS stained kidney sections of CTRL rats (Fig. 2A) showed normal ar-chitecture of the glomeruli and interstitium with only mild age-relatedhistological alterations presented as focal segmental glomerulosclerosis,perivascular infiltration of lymphocytes and thickening of the tubularbasement membranes. 5/6 Nx rats (Fig. 2B), showed progressive renalfibrosis and a histological picture of advanced chronic renal diseasewith massive fibrosis (Fig. 2D).

The baseline behavioral tests performed before surgery showed nodifferences in general locomotor activity and exploration, anxiety ordepression level in the performed tests (Fig. 3A–F). Similarly, thebehavioral tests performed at 3 months showed no significant changes(data not shown).

After 9 months, test in open field showed no significant differencesin general locomotor activity expressed as average speed and distancemoved in the arena (Fig. 4A and B, respectively). No difference in openfield 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 thelight/dark box when compared to the CTRL group (Fig. 5A; t = 2.59;p b 0.05). Other behavioral tests analyzing anxiety showed no significantdifferences 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 didnot induce depressive like behavior, since no difference in preferencebetween sweetened or normal water was observed (Fig. 6).

In water maze task, 5/6 Nx and CTRL rats performed similarly. Inworking memory, the rats gradually decreased the latency times,reaching significant improvement on the 3rd and 4th day for bothgroups (Fig. 7A; F = 19.29; p b 0.01) when compared to the first day.For reference memory, there was no significant difference betweenthe 5/6 Nx and CTRL rats (Fig. 7B).

4. Discussion

CKD is a risk factor for development of cognitive impairment whichmight be manifested already in patients with early-stage of CKD [30]. Aprevalence of cognitive impairment associatedwith CKD is estimated tobe 30%–60%, i.e. at least twice higher as that observed in age-matchedcontrols [31]. Presence of cognitive impairment further decreases thequality of life of the CKD patient, enhances existing social and economicburden, but also results in higher risk for mortality [32]. Causes ofCKD-associated cognitive impairment in humans are multifactorial(including cerebrovascular disease, renal anemia, secondary hyperpara-thyroidism, dialysis disequilibrium, and uremic toxins accumulation[32], and mechanisms of induction of cognitive impairment not fullyunderstood yet).

Rodent models of CKD are important experimental tools used toinvestigate potential pathophysiological pathways of human disease.Since mechanisms underlying CKD-associated behavioral changes andcognitive impairment in humans are not very well understood yet,experimental studies might help with their elucidation. However,results of behavioral testing in CKDanimals are scarce and controversial.Discrepanciesmay arise fromuse of differentmethods for CKD induction,e.g., surgery [19] vs. chemical induction potentially imposing additionaltoxicity [17], different time points of behavioral testing reflecting earlyor advanced stages of CKD [18,33], as well as variety of used behavioraltests. A comprehensive assessment of the behavioral changes duringCKD in experimental animals is missing. Thus the aim of our experimentwas to describe comprehensively the changes in rat behavior 3 and9 months after induction of CKD by 5/6 nephrectomy employing batteryof behavioral tests concurrently assessing the general locomotion,anxiety, depression-like, exploratory and memory components.Our animals presented decreased renal function with proteinuria,

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 mazewith 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. CTRLgroup.

Fig. 6. Anhedonia. Results of sucrose preference test. Although slight decrease in sucrosepreference 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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and histologically confirmed CKD, as well as increased plasmaconcentration of determined uremic toxins findings correspondingwith those in humans with CKD [34].

In contrast to our hypothesis, our multifaceted testing approach didnot reveal substantial changes in behavior of rats either in early oradvanced stage of CKD. We assessed the general locomotor activity byopen field test, while the anxiety level was evaluated by light/darkbox and elevated plusmaze. Open field evaluates the locomotor activityby determination of overall distance walked, and speed. Moreover, anopen field test enables anxiety trait assessment by detection of timean animal spends in the center part of the maze [35]. Light/dark boxand elevated plus maze enable to evaluate two distinct aversion relatedproperties of anxiety: in the light/dark box the animals demonstratenatural aversion from light preferring to stay in the dark [36], whileelevated plus maze enables testing of aversion from high and opengrounds, that rodents express as spending more time in the closedarms of the maze when allowed to choose [37]. We observed no changesin general locomotor activity and anxiety level as assessed by open fieldand elevated plus maze. In the light–dark box, 5/6 Nx rats spent moretime in the light zone, possibly reflecting decrease in anxiety in compari-son with the control group. These results are in contrast to the clinicalstudies demonstrating higher anxiety level in both moderate and severeCKD patients [38]. Moreover, these findings contradict some animalstudies describing decreased locomotor activity, exploratory andemotional-like types of behavior in CKD of rodents [17,33]. On the otherhand, Fujisaki et al. [16] did not reveal decrease in physical performanceof CKD rats. In different study, CKD mice manifested decreased anxietylevel presented as longer time spent in the light part of light/darkbox [18]. Reasons leading to disparate results in different studies remainunclear, but might be on the account of different CKD stages of testedanimals, which make them difficult to compare due to differentapproaches of determination of CKD severity. The CKD stagewas assessedby the level of plasma creatinine and urea, while in our study ccrGFR andpresence of proteinuria. We did not observe any significant increase inBUN or plasma creatinine after 3 months as did other research groups,

although creatinine clearance glomerular filtration rate was already sig-nificantly decreased. It is probable, that during this time there mighthave been ongoing adaptive processes in central nervous system after ne-phrectomy during initial stages of CKD [39], which could in turn affect thebehavioral results.

If compared with the control rats, our CKD animals did not showdifference in depression-like behavior monitored as immobility timein the forced swim task. The result of Porsolt depressive test was alsoconfirmed in our experiment by unchanged preference for the sucrosesolution. This preference did not change throughout the time andnephrectomy did not affect this type of behavior. This is in directcontrast to observations in humans, where patients with CKD showhigher incidence of depression symptoms [40,41]. It is also in contrastto another animal experiment [17]. Rats with adenine-induced CKDpresented increased immobility time in forced swim test at 28 days.Severe anemia [42] and hyperparathyreoidism [43] induced by feedingof adenine could contribute to observed behavioral changes in thismodel of CKD. Direct toxic effects of adenine administration were also

Fig. 7. Spatialmemory. Results fromwatermaze and (A) short-termworkingmemory and(B) long-term reference memory. No significant effect of the treatment on the observeddata. Data are presented as mean ± SEM. ** denotes p b 0.01 vs. 1st and 2nd day forboth groups.

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reflected by significant decrease in body weight [17]. In our study bodyweight of 5/6Nx rats remained similar to that of the control groupthroughout the experiment. Moreover, we did not gather behavioraldata as soon as 28 days after the induction of CKD. These findingsmight suggest that the serotonin system,which plays a role inmediatingdepression-like behavior and the mesolimbic dopamine system, whichplays a role in themediation of reward stimuli, could have been adaptedafter 9 months, being more complex in influencing the behavior.

In contrast to human studies [44],we did not reveal any decline eitherin short-termworkingmemory or in long-term referencememory. In themodel of 5/6 nephrectomy, long-termmemory impairmentwas detectedafter 24 weeks only in rats with severe CKD [19]. The severity of theCKD was classified as 3-fold rise of serum creatinine vs. controls, thesevere 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-foldhigher in comparison with the controls, ranged widely according toGFR. When CKD severity would be staged according to the plasmacreatinine as proposed by [19], it would be possible to divide the ratsinto the 3 rats with higher plasma creatinine and 3 with the lowerplasma creatinine. However, all 6 rats would be still classified as ratswith severe CKD. Additionally, all 6 surviving rats had CKD linkedwith significant proteinuria at 9 months.

To the best of our knowledge, this is the first comprehensiveexperimental study describing complexly different aspects of behaviorin CKD including general locomotion, anxiety, depression and memory.Indeed, our study did not come without some limitations. The majorlimitation is the mortality rate in the 5/6 Nx group which was60% after 9 months, leaving only 6 animals to test at the end of theexperiment. Although this rate is comparable to other studies [45,46],no relevant behavioral data are available for animals that died prior to90 or 270 day testing in our study. Therefore we can only hypothesizethat animals that died earlier could be more susceptible to cognitivedecline than surviving rats. Additionally, surviving animals displayed

severe stage of CKD with broad range of plasma creatinine and BUN,but low number of remaining animals did not allow for performance ofrelevant subgroup analysis. Nevertheless, our data suggests that CKD-associated cognitive impairment manifested in humans could beunderlined more by the psychosocial factors such as low self-esteem,feelings of uselessness, impaired physical activity possibilities and theoverall distraction of the body image. Such factors are irrelevant in rats.

To conclude, this study did not reveal any cognitive decline in ratsafter induction of chronic kidney disease by 5/6 Nx. Locomotion, anxiety,depression-like behavior as well as memory were well preserved after9 months of CKD in surviving animals. However, further experimentalstudies focusing on CKD-associated behavioral responses are needed toconfirm 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 authorswould like to thank MSc. Jana Bellova, PhD., for her assistance with fig-ures preparation.

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