Water deprivation up-regulates urine osmolality and renal aquaporin 2 in Mongolian gerbils (Meriones unguiculatus)

Water deprivation up-regulates urine osmolality and renal aquaporin 2 in Mongolian gerbils (Meriones unguiculatus)

    Water deprivation up-regulates urine osmolality and renal aquaporin 2 in Mongolian gerbils (Meriones unguiculatus) Meng-Meng Xu, De-H...

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    Water deprivation up-regulates urine osmolality and renal aquaporin 2 in Mongolian gerbils (Meriones unguiculatus) Meng-Meng Xu, De-Hua Wang PII: DOI: Reference:

S1095-6433(16)30003-4 doi: 10.1016/j.cbpa.2016.01.015 CBA 10002

To appear in:

Comparative Biochemistry and Physiology, Part A

Received date: Revised date: Accepted date:

1 November 2015 16 January 2016 16 January 2016

Please cite this article as: Xu, Meng-Meng, Wang, De-Hua, Water deprivation up-regulates urine osmolality and renal aquaporin 2 in Mongolian gerbils (Meriones unguiculatus), Comparative Biochemistry and Physiology, Part A (2016), doi: 10.1016/j.cbpa.2016.01.015

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ACCEPTED MANUSCRIPT Water deprivation up-regulates urine osmolality and renal aquaporin 2 in Mongolian gerbils (Meriones unguiculatus)

State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of

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Zoology, Chinese Academy of Sciences, Beijing 100101, China b

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Meng-Meng Xu a,b, De-Hua Wang a,*

Graduate University of the Chinese Academy of Sciences, Beijing 100049, China

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* Corresponding author:

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ms. has 24 pages, 7 figures, 1 table

Dr. De-Hua Wang

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Address: State Key Laboratory of Integrated Management of Pest Insects and

Chaoyang District,

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100101 Beijing, China

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Rodents, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road,

Tel: + 8610 64807073; Fax: 8610 64807099

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E-mail: [email protected]

Abstract To better understand how desert rodents adapt to water scarcity, we examined urine osmolality, renal distribution and expression of aquaporins (AQPs) in Mongolian gerbils (Meriones unguiculatus) during 7 days of water deprivation (WD). Urine osmolality of the gerbils during WD averaged 7503mOsm kg-1. Renal distributions of AQP1, AQP2, and AQP3 were similar to that described in other rodents. After the 7 day WD, renal AQP2 was up-regulated, while resting metabolic rate and total evaporative water loss decreased by 43% and 36%, respectively. Our data demonstrated that Mongolian gerbils showed high urine concentration, renal 1

ACCEPTED MANUSCRIPT AQPs expression and body water conservation to cope with limited water availability, which may be critical for their survival during dry seasons in cold deserts. Key words: aquaporins, AQPs; urine osmolality; total evaporative water loss,

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TEWL; Mongolian gerbils (Meriones unguiculatus).

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1. Introduction

Some desert rodents could survive on a diet of dry seeds without drinking by

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their ability to conserve body water stringently (Schmidt-Nielsen, 1964; Degen, 1997; Walsberg, 2000; Bozinovic and Gallardo, 2006). These rodents are

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characterized by a low basal metabolic rate (BMR), low evaporative water loss (EWL), low fecal water content, and large relative medullary thickness (RMT) (Degen, 1997; McNab, 2002; Bozinovic and Contreras, 1990; Schwimmer and Haim,

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2009). They can produce extremely concentrated urine, which could be 20-30 times

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that of plasma (Schmidt-Nielsen, 1964; Corte´s et al., 2000; Palgi and Haim, 2003). For instance, the spinifex hopping mouse (Notomys alexis) could concentrate its

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urine up to 9370 mOsm kg-1 (MacMillen and Lee, 1967), one of the highest values reported among mammals.

Aquaporins (AQPs), a family of membrane proteins which function as water

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channels, play a crucial role in water reabsorption along the nephron in mammals (Nielsen et al., 2002; King et al., 2004). Among the 8 AQPs distributed in kidney, the knockout of AQP1, AQP2, or AQP3 generates a severe urine-concentrating defect in rats and mice (Verkman, 2009; Kortenoeven and Fenton, 2014). The degus (Octodon degus) and the leaf-eared mouse (Phyllotis darwini) from South America deserts regulated renal AQP2 protein abundance to cope with variation in water availability (Bozinovic et al., 2003; Gallardo et al., 2005). There has been little investigation into the function and regulation of renal AQPs in desert rodents (Bozinovic and Gallardo, 2006), and, consequently, studies on a wider variety of species would further our understanding of this urine-concentrating mechanism (Pannabecker, 2013). Mongolian gerbils (Meriones unguiculatus) are distributed along a broad aridity 2

ACCEPTED MANUSCRIPT gradient in Inner Mongolian semi-deserts and grasslands of China, the Baikal region of Russia, and Mongolia (Walker 1968; Mallon, 1985; Wang et al., 2003). The habitats are characterized by severe climate and extreme seasonal ambient

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temperature fluctuations (Weiner and Górecki, 1981; Wang et al., 2000). In winter, Mongolian gerbils feed mainly on plant seeds stored in late autumn, while in late

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spring and summer they also feed on some leaves and stems of seasonal plants such as Salsola collina and Artemisia sieversiana (Qin, 1984; Zhou et al., 1985). The gerbils have a relatively wide thermal neutral zone (TNZ, 26.5–38.9) (Robinson,

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1959; Wang et al., 2000; Pan et al., 2014), and are capable of altering their thermogenesis, metabolism and mitochondrial membrane lipidome in response to a

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wide range of ambient temperatures (Steffen and Roberts, 1977; Luebbert et al., 1979; Zhang and Wang, 2007; Pan et al., 2014). Furthermore, the EWL of

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Mongolian gerbils shows a rather pronounced variation with habitat, ambient

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temperature, relative humidity, and body weight (Xiao and Sun, 1988; Shi et al., 2015). Mongolian gerbils were not able to maintain body mass on a diet of soy beans

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and sea water (Winkelmann and Getz, 1962), but were able to maintain water balance on a high protein (43.3%) diet and 2% saline (Edwards et al., 1983). The physiological mechanism of how Mongolian gerbils cope with water stress

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remains unclear. It has been established that the up-regulation of AQP2 expression is a water-sparing mechanism at the molecular level which occurs under conditions of negative water balance. This leads to an increased urine concentrating capacity at the organismal level (Nielsen et al., 1993; Yang et al., 1999; Nielsen et al., 2002; Bozinovic and Gallardo, 2006). The objective of our study was to examine urine osmolality, renal distribution and expression of AQPs in Mongolian gerbils during 7 days of water deprivation. We hypothesized that Mongolian gerbils would alter urine osmolality and renal AQP2 abundance to cope with water scarcity. We predicted that renal AQP2 protein abundance in Mongolian gerbils would be up-regulated for producing highly concentrated urine. We also determined associated parameters such as RMR, TEWL, serum osmolality, food intake, urine volume, fecal water content, 3

ACCEPTED MANUSCRIPT and body fat mass.

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2. Material and methods

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2.1 Animals

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All animal procedures were approved by the Animal Care and Use Committee of the Institute of Zoology, the Chinese Academy of Sciences. Mongolian gerbils in this study were from our laboratory colony established in 1999. The gerbils were housed

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in plastic cages (30 cm×15 cm× 20 cm) with sawdust as bedding at 23 ± 1 oC on a 16h: 8h light: dark cycle (lights on at 04:00). They were offered a commercial standard rat

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pellet (Beijing Huafukang Feed Co.) containing 21 % crude protein, 5 % crude fat, and 53% carbohydrate. Food and water were supplied ad libitum. Eighteen adult Mongolian gerbils of both sexes, weighing 55–75 g and aged 150–180 days were

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single metabolic cages.

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selected randomly. They were raised individually for 2 weeks before being placed into

2.2 Metabolic cage experiment

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The gerbils were transferred into transparent and gnaw-proof polycarbonate commercial metabolic cages with stainless steel grid floors, 30 cm in diameter and 20 cm high (Techniplast, MTB 0210, USA). A collection funnel (22.5 cm diameter) with

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a separating cone ensured immediate, complete separation of feces and urine. Each urine collection tube was covered by a small funnel to minimize evaporation (Dicker and Nunn, 1957).

After 3 days in the metabolic cages, the gerbils were divided into two groups: a control (C) group with food and drinking water ad libitum and a water deprived (WD) group with only food. Gerbils of both groups were kept in metabolic cages for 7 days, during which time, body mass (±0.1 g), food intake (±0.1 g), urine volume, and fresh fecal mass were measured each day between 0900 h and 1100h. Urine samples were collected in tubes and stored at − 80°C, while feces and food residues were collected, oven-dried to constant mass and separated. Then the fecal water content was calculated as: (fresh fecal mass - dried fecal mass)*100/ fresh fecal mass. 4

ACCEPTED MANUSCRIPT 2.3 RMR and TEWL measurement After 7 days WD, RMR and TEWL of the Mongolian gerbils were measured simultaneously using an open flow respirometry system (Sable, TurboFOX Complete

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Field System, including a humidity module RH-100, USA). Gerbils were placed in a transparent plastic chamber (2.7L) for 3h between 1200 h and 21:00h. An incubator

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(Yiheng Model LRH-250, Shanghai, China) was used to maintain the chamber at a Ta of 23±0.5oC. Fresh air was dried with Drierite desiccants (W. A. Hammond Drierite Co., USA) and warmed by passage through a copper tube, before entering the

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chamber at a flow rate of 600–700 mL min-1. The excurrent air flow from the chamber was subsampled and directed at approximately 100 mL min-1 to the humidity module

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for water vapor density measurement. Subsequently the sampled gas was dried using a non-chemical gas drier (ND-2, Sable Systems International Inc., USA) and then

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analyzed for oxygen and carbon dioxide. Data outputs of oxygen and carbon dioxide

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concentration, as well as water vapor density were recorded every 15 s. A baseline was recorded to correct for the fluctuation of analyzers before and after each trial.

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RMR and TEWL were calculated as: RMR = FR × ((FiO2 - FeO2) –FeO2 × (FeCO2- FiCO2)) / (1-FeO2) Where, FR = flow rate (mL/min), Fi = input fractional concentration (%), and Fe =

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excurrent fractional concentration (%; Li et al., 2010). TEWL = (Ve ρout – Vi ρin)×60 Ve = flow rate of air exiting the chamber (ml/min), Vi = flow rate of air entering the chamber (ml/min), ρin = water vapor density (mgH2O/ml) of the air entering chamber, ρout = water vapor density (mgH2O/ml) of the air exiting the chamber (Shi et al., 2015). RMR and TEWL were estimated from the average of the 20 lowest, stable consecutive data points (5 min) for each gerbil when steady-state was maintained for at least 30 minutes. Body mass (± 0.1g) was recorded before and after each metabolic trial and the average body mass was used for correcting RMR and TEWL (Li et al., 2010; Chi and Wang, 2011; Shi et al., 2015). 5

ACCEPTED MANUSCRIPT 2.4 Measurement of morphological and physiological parameters after WD Animals were sacrificed using CO2 overdose. Serum samples from the carotid artery were collected by centrifugation and stored at − 80°C, and kidneys, heart and

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liver were removed and weighed (±1 mg). The height (H), depth (D), and width (W) of the kidney and the length (L) of the medulla were measured to calculate the relative 0.33

(Sperber, 1944).Then, the

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medullary thickness (RMT): RMT = L/ (H*D*W)

kidney was cooled in liquid nitrogen and stored at − 80°C, while the other kidney was fixed wholly in Bouin’s solution for immunocytochemistry.

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The carcass was weighed (±1 mg) and oven-dried to determine body water and fat

content. Body fat was measured by a Soxtec Fat Extraction Systems (Soxtex Avanti

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2050, FOSS, Sweden). Urine and serum osmolalities were measured using a freezing-point osmometer (SMC-30B, Tian-he medical instruments, Tianjin, China).

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2.5 Immunolocalization of AQPs

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The kidneys fixed in Bouin's solution, were paraffin-embedded (7mm thick). Localization of AQP1, AQP2, and AQP3 was done by immunocytochemistry

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following the procedures of Gallardo et al., (2005). The polyclonal primary antibodies were rabbit anti-AQP1 (diluted 1: 2000, AB3272-50UL, Millipore, USA), anti-AQP3 (diluted 1:750, AB3276-50UL Millipore, USA), and goat anti AQP2 (diluted 1:1000,

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SC-9882, Santa Cruz, USA). Immunoreactivity was identified using biotinylated goat anti-rabbit and rabbit anti-goat immune globulin G (diluted 1:500, Jackson ImmunoResearch, USA). Sections were then incubated with ABC reagents (PK-6100, Vector Labs, USA) for 60 min. Immunoreactive sites were subsequently identified by the 3,3 diaminobenzidine (DAB substrate kit; Vector Labs, USA), and tissue sections were photographed using a Nikon optical microscope (Nikon H600L). Double immunofluorescence was used to examine the coexistence of AQP2 and AQP3 in collecting ducts; the secondary antibodies used were donkey anti-rabbit (Alxa Flour 488, green, 1:500; Jackson ImmunoResearch, USA) and donkey anti-goat (Alexa Flour 594, red, 1:500; Jackson ImmunoResearch, USA) immune globulin G respectively. 6

ACCEPTED MANUSCRIPT 2.6 Western blot analysis of AQPs AQP1 and AQP2 protein levels in the kidney were estimated as described by Gallardo et al., (2005). The kidney was lysed in buffer of 10 mM triethanolamine,

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1mM phenylmethylsulfonyl fluoride (PMSF), 250 mM sucrose, and protease inhibitor cocktail (P8340, Sigma , USA). Protein concentration was determined using the Folin

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phenol method (Lowry et al., 1951).Proteins (80μg) were added to each lane separated by 12% SDS-PAGE gels, and blotted into polyvinylidene difluoride (PVDF) (Xing et al., 2015).Then the PVDF membranes were incubated with the primary antibody

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(AQP1 and AQP2, diluted 1:1000; mouse anti-β-tubulin, diluted 1:3000, E7, DSHB, USA) and horseradish peroxidase conjugated secondary antibody (diluted 1:3000,

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ZSGB-BIO Co., China). The immunoblot was revealed by ECL (Beyotime, China) and densitometric analysis was done using Quantity One software (version 4.4.0,

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BioRad, Hercules, CA). AQPs level was expressed as relative units (RU), contents of

2.7 Statistical analysis

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AQPs were normalized with tubulin levels.

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Data were analyzed using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). Normality and homogeneity of variance of the data were examined using Shapiro-Wilk and Levene tests, respectively. Differences in body composition, RMR and TEWL

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between groups were analyzed by one-way ANCOVA with body mass as covariate followed by Tukey’s post hoc tests. Differences between groups in other parameters (final body mass, wet carcass mass, dry carcass mass, fat-free dry carcass mass, serum osmolality, RMT, AQP1and AQP2 protein content) were analyzed by independent-samples t-test. Body mass, food intake, fecal mass, fecal water content, and urine osmolality and volume were analyzed by repeated measures ANOVA followed by Tukey’s post hoc tests. Results are presented as means ± SE and the level of statistical significance was set at P < 0.05.

3. Results 3.1 Body composition After the 7 day WD period, body mass (group effect, P < 0.05; time effect, P < 7

ACCEPTED MANUSCRIPT 0.001; group × time effect, P < 0.001; repeated measures ANOVA, Fig. 1A)decreased by 12.8g, with a concomitant decrease in wet carcass mass (t = -5.934, df =16, P < 0.001), dry carcass mass (t = -3.045, df = 16, P < 0.01), fat-free dry

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carcass mass (t = -2.425, df = 16, P < 0.05), fat mass (F1, 15=6.481, P < 0.05) and heart mass (F1, 15 = 6.618, P < 0.05). There was no significant difference in kidney

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and liver mass, body water mass, and RMT between WD and control groups (Table 1). 3.2 Food intake and fecal mass

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After the 7 day WD period, food intake (group effect, P < 0.001; time effect, P < 0.001; group × time effect, P < 0.01; repeated measures ANOVA, Fig. 1B) and

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fecal mass (group effect, P < 0.001; time effect, P < 0.05; group × time effect, P < 0.01; repeated measures ANOVA, Fig. 1C) decreased by 3.4g and 1.0 g, respectively.

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3.3 Body water loss and RMR

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Fecal water content (group effect, P < 0.05; time effect, P < 0.01; group × time effect, P < 0.05; repeated measures ANOVA, Fig. 2A) and urine volume (group

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effect, P < 0.05; time effect, P = 0.055; group × time effect, P = 0.086; repeated measures ANOVA, Fig. 2B) of WD gerbils decreased by 4.5% and 0.6ml, respectively. After WD, TEWL (72.6 ± 4.6 mg H2O/h) decreased by 43% in WD

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gerbils compared to the control group (125.8 ± 3.4 ml O2/h; 126.7 ± 6.4 mg H2O/h) (F1,15 = 8.038, P = 0.013; Fig. 2C and Fig. 2D). After WD, RMR (80.8 ± 3.4 mL O2/h) was 36% lower in WD gerbils than in the control group (125.8 ± 3.4 ml O2/h; 126.7 ± 6.4 mg H2O/h) (F1,15 = 26.203, P = 0.000; Fig. 2C and Fig. 2D). 3.4 Urine and serum osmolalities During the 7 day WD period, urine osmolality increased significantly in WD gerbils from day 1, with the peak at day 2 (group effect, P < 0.01; time effect, P = 0.05; group × time effect, P = 0.61, repeated measures ANOVA, Fig. 3A). The average urine osmolality of the gerbils during WD was 7503 mOsm kg-1. After WD, serum osmolality was significantly higher in the WD group (348.4 ± 12.4 mOsm 8

ACCEPTED MANUSCRIPT kg-1) than in the control group (330.6 ± 5.5 mOsm kg-1) (t = -3.045, df = 16, P < 0.01, Fig. 3B). 3.5 Immunolocalization and abundance of AQPs

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AQP1 is present in the proximal tubules (Fig. 4A and 4B) in the cortex and the descending thin limbs in the medulla (Fig.5A and 5B).Neither immunoreactivity nor

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AQP1 abundance (t = -0.009, df = 16, P = 0.993; Fig. 7A)differed between groups. AQP2 was expressed in the apical plasma membrane of connecting tubule cells and the cortical, outer, and inner medullary collecting duct cells (Fig. 4C and 4D, Fig. 5C

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and 5D). Unlike AQP1, AQP2 labeling was more abundant in WD gerbils than control gerbils, and it was correlated by a twice increase in AQP2 abundance in WD

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gerbils revealed by densitometric analysis (t = -2.075, df = 16, P < 0.05, Fig. 7B). AQP3 is localized at the basolateral membranes of connecting tubule cells and the

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cortical, outer, and inner medullary collecting duct cells, which were the same cells

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labeled for AQP2 (Fig. 4E and 4F, Fig. 5E and 5F, Fig. 6A and 6B). Immunostain of AQP1, 2, 3 was much lighter in the cortex than the medulla (Fig. 4 and Fig. 5).

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4. Discussion

The present study showed that Mongolian gerbils increased renal AQP2 protein content and serum and urine osmolalities, but reduced RMR, TEWL, body mass, and

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body fat when exposed to a prolonged period of WD. The decrease of body mass and food intake in Mongolian gerbils was similar to other desert rodents (Cortes et al., 1988; Bozinovic et al., 2007; Tirado et al., 2008). It is believed that desert rodents mobilize their nutrients stored in the body to acquire water when they encounter water shortage to compensate for obligatory water loss (Schmidt-Nielsen, 1964; Walsberg, 2000; Takei et al., 2012). One gram fat oxidation could produce more metabolic water (1.017g) than starch (0.556g) and protein (0.396g) (Schmidt-Nielsen, 1964; Frank, 1988), so the body fat loss in Mongolian gerbils may help to produce some metabolic water and compensate the need of body water during WD. Desert rodents possess lower RMR and TEWL than the same species from mesic habitats (Degen, 1997; Tracy and Walsberg, 2001; Bozinovic et al., 2009; 9

ACCEPTED MANUSCRIPT Bozinovic et al., 2011; Shi et al., 2015). In our study, body water loss (including TEWL) and RMR of Mongolian gerbils were significantly reduced after the 7 day WD. Similarly, Northeast African spiny mice (Acomys cahirinus) reduced EWL

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(Daily and Haines, 1981) after water restriction and bushy tailed gerbils (Sekeetamys calurus) (Palgi and Haim, 2003) and golden spiny mice (Acomys russatus) (Ron and

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Haim, 2001) reduced RMR after drinking salty water under laboratory conditions. It was suggested that reduced metabolic rates not only helped to conserve energy but also body water via reducing gas exchange (Weissenberg and Shkolnik, 1994; Palgi

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and Haim, 2003).

Rodents from arid areas possess larger RMT than rodent from mesic areas

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(Degen, 1997; Mohammed et al., 2004). The RMT of Mongolian gerbils was 9.94 and average urine osmolality in WD gerbils was 7503mOsm kg-1, values which are

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comparable to the kangaroo rats (Dipodomys merriami) (which are 8.5 and 6382

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mOsm/kg-1; Sperber, 1944; Carpenter, 1966; respectively), a rodent well adapted to deserts. Our result is consistent with the previous study by Edwards et al., (1983),

conservation.

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suggest that the kidney of Mongolian gerbils is well adapted for body water

AQP1 is the constitutive water-specific channel, and AQP2 is present on the

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apical membrane in collecting duct cells, where the final fine-tuning of water and urea reabsorption is achieved. AQP3 is present on the basolateral membrane in the same cells labeled for AQP2; it helps AQP2 with the water reabsorption in the distal nephron (Nielsen et al., 2002; Kortenoeven and Fenton, 2014). Renal distribution of AQP1, AQP2, and AQP3 in the gerbils was similar to laboratory mice and rats (Nielsen et al., 2002), as well as the leaf-eared mouse (Gallardo et al., 2005), degus (Bozinovic et al., 2003), and kangaroo rats (Huang et al., 2001). However, patterns of renal AQPs expression in desert rodents do not always follow patterns of expression in rats and mice (Pannabecker, 2015). For instance, AQP4 is expressed in rat kidney, yet absent in kidney of kangaroo rats (Huang et al., 2001). In addition, kangaroo rats possess remarkably longer AQP1-positive descending thin limbs of 10

ACCEPTED MANUSCRIPT Henle’s loop and greater basal-to-apical AQP2 ratio in the inner medulla compared to Munich-Wistar rat (Urity et al., 2012; Espineira and Pannabecker, 2014). Similar to other rodents, renal AQP1 abundance of Mongolian gerbils was

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unaltered, suggesting that it plays a constitutive role in water absorption (Nielsen et al., 2002). The up-regulation of renal AQP2 in WD gerbils is consistent with the

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leaf-eared mouse following 5 days of WD (Gallardo et al., 2005), free-living degus during summer (Bozinovic et al., 2003), and rats and mice after short periods of water deprivation (Nielsen et al., 1993; Yang et al., 1999). It seems that desert

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rodents show a generality of AQP1 and AQP2 responses to water deprivations similar to rats and mice, and AQP2 plays an important role under conditions of

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negative water balance.

In conclusion, our results demonstrated that Mongolian gerbils showed high

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flexibility of renal AQP expression, osmoregulation, and body water conservation

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when water deprived. These efficient water conservation mechanisms could be potentially critical for their survival in their habitats, which are characterized by

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seasonal and spatial fluctuations of water availability.

Acknowledgments

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Thanks to Qi Chen, Zhong-Hong Cao of Embryo Biology Group, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences for their help in method of AQP3 immunofluorescence. We are also grateful to all the members of Animal Physiological Ecology Group for their helpful assistance and discussion. We thank the three anonymous reviewers for their helpful and constructive comments on this manuscript. This work was supported by the National Natural Scientific Foundation of China (No. 31472006 and 31272312), and the Chinese Academy of Sciences (KSCX-EW-N-005) to DHW.

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Fig.1 Time-course changes of body mass, food intake, and fecal mass in control (C) and water deprived (WD) Mongolian gerbils. Body mass (A), food intake (B), and fecal mass (C) *

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were significantly lower in the WD group than the C group. Data are means ± SE. P < 0.05.

Fig.2 Fecal water content, urine volume, total evaporative water loss (TEWL), and resting

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metabolic rate (RMR) in control (C) and water deprived (WD) Mongolian gerbils. Fecal water content (A), urine volume (B), TEWL (C), and RMR (D) were significantly lower in WD *

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P < 0.001

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group than the C group. Data are means ± SE. P < 0.05,

Fig.3 Urine and serum osmolalities in control (C) and water deprived (WD) Mongolian

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gerbils. Urine osmolality was significantly lower in WD gerbils (A), while serum osmolality was *

significantly higher in WD gerbils than the C group (B). P < 0.05.

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Fig.4 Immunolocalization of aquaporins (AQPs) in the renal cortex of control (C) and water deprivation (WD) Mongolian gerbils. A-B, AQP1 labeling in the proximal tubules; C-D, AQP2 labeling in the apical membrane of connecting tubule cells and the cortical collecting duct cells; E-F, AQP3 labeling in the basolateral membranes of connecting tubule cells and the cortical collecting duct cells.

Fig.5 Immunolocalization of aquaporins (AQPs) in the renal medulla of the control (C) and water deprived (WD) Mongolian gerbils. A-B, AQP1 labeling in the descending thin limbs; C-D, AQP2 labeling in the apical membrane of collecting duct cells; E-F, AQP3 labeling in the basolateral membranes of collecting duct cells .

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ACCEPTED MANUSCRIPT Fig.6 Immunolocalization of aquaporin 2 (AQP2) and aquaporin 3 (AQP3) in the same cells of renal collecting duct cells in Mongolian gerbils. A, outer medulla; B, inner medulla.

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Fig.7. Effects of water deprivation on aquaporins abundance in the kidney in Mongolian gerbils. No significant difference of AQP1 protein abundance was detected between WD (water

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deprived) and C (control) group (A). Gerbils in WD group had higher levels of AQP2 protein *

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abundance compared with the C group (B). Data are means ± SE. P < 0.05.

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AQP2 (red) and AQP3 (green)

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ACCEPTED MANUSCRIPT Table 1. Body composition and relative medullary thickness (RMT) of Mongolian gerbils in control (C) and water deprived (WD) group. Control

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P value

51.0 ± 1.4

41.4 ± 0.9

Dry carcass mass (g)

19.0 ± 1.0

15.7 ± 0.5

Fat-free dry carcass mass (g)

14.0 ± 0.3

13.0 ± 0.2

0.028

Body water mass (g)

32.1± 0.7

25.7 ± 0.6

0.096

Body fat mass (g)

5.0± 0.8

2.7±0.4

0.022

Kidney mass (g)

0.574 ± 0.013

0.539 ± 0.008

0.351

Liver mass (g)

2.087 ± 0.091

1.407 ± 0.077

0.161

Heart mass (g)

0.262 ± 0.005

0.208 ± 0.006

0.022

9.74 ± 0.18

0.429

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RMT

9.94±0.17

0.000

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Wet carcass mass (g)

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Body composition

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Values are means ± SE. Kidney mass, liver mass, heart mass, body water mass, and body fat mass

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were analyzed by one-way ANCOVA with final body mass as covariate followed by Tukey’s

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post-hoc tests. Final body mass, wet carcass mass, fat free body mass and RMT were determined

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by independent-samples t-test.

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