The one-humped Arabian camel (Camelus dromedarius) is the most important livestock animal in arid and semi-arid regions and continues to provide basic necessities to millions of people. In the current context of global warming, there is renewed interest in the adaptive mechanisms that enable camelids to survive in arid conditions. Recent investigations described genomic signatures that revealed evolutionary adaptations to desert environments. We now present a comprehensive catalogue of the transcriptomes and proteomes of the dromedary kidney and describe how the gene expression profiles of Differentially Expressed Genes (DEGs) are modulated as a consequence of chronic dehydration and subsequent acute rehydration. We performed RNAseq and quantification of peptides in samples from 15 dromedaries (5 controls, 5 dehydrated and 5 rehydrated). Gene Ontology analyses suggested an enrichment of the cholesterol biosynthetic process and an overrepresentation of categories related to ion transmembrane transport in the camel kidney, and RTN analyses confirmed alterations in the transcriptional machinery involved in cholesterol synthesis. These data were validated by RT-qPCR. Based on our hypothesis of a role for cholesterol during dehydration, we identified DEGs with roles in the countercurrent multiplication process which are affected by changes in the level of cholesterol. Thus, we further validated differentially expressed genes with known roles in water conservation which are affected by changes in cholesterol levels. Our datasets suggest that suppression of cholesterol biosynthesis may facilitate water retention in the kidney by indirectly facilitating the AQP2-mediated water reabsorption.
Overall design: Nineteen male dromedary camels aged 4-5 years, body weight range 276-416 kg, were used in the present study. The camels were supplied with alfalfa hay as feed and ranch-housed in the United Arab Emirates. Veterinary supervision was provided throughout the experimental period and no signs of distress or illness were identified. After a short adaptive period, the camels were divided into 3 groups, control (n=5), dehydrated (n=8) and rehydrated (n=6). The control group had free access to food and water during the entire experimental period. The dehydrated group was water-deprived but had ad libitum access to food for 20 days. Meanwhile the rehydrated group was subjected to the same protocol as the dehydrated animals followed by ad libitum water supply for 3 additional days. After the experimental period, the camels were sacrificed in the local central abattoir for human consumption in April 2016. Kidney samples were harvested within 1 hour after killing the animals, immediately frozen in liquid nitrogen and kept at -80°C for later physiological, histological, morphological and molecular analysis. Samples were shipped frozen on dry ice to the University of Bristol under the auspices of a DEFRA Import Licence (TARP/2016/063). This study was approved by the Animal Ethics Committee of the United Arab Emirates University (approval ID: AE/15/38) and the University of Bristol Animal Welfare and Ethical Review Board. Total RNA was extracted from fresh frozen kidney medulla and cortex of 5 control, 5 dehydrated and 5 rehydrated camels (for a total of 30 samples). These 15 animals were randomly selected out of the 19 animals included in the experimental set up while the remaining 4 were used for preliminary analyses only. Frozen tissue was added to a pre-cooled mortar containing a small volume of liquid nitrogen. Tissue was then grounded to a fine powder using a pestle and the nitrogen was allowed to evaporate. The powdered tissue was placed into pre-chilled tubes on dry ice and stored at -80°C. For RNA extraction, 1ml of Qiazol (79306, Qiagen) was added to 100mg of powdered tissue, then immediately vortexed for 2 minutes, then incubated on ice for 10 minutes. Lysed samples were spun at 12000xg for 10 minutes at 4°C and the supernatant was transferred to a new tube. 1/5 volume Chloroform (22711.244, VWR) was added to each sample and vortexed for 15 seconds. Samples were then spun at 12000xg for 15 minutes at 4°C. 350ml of the upper phase was removed and mixed with an equal volume of absolute ethanol. RNA was extracted using ZymoTM Direct-Zol RNA miniprep (Zymo Research) as per the manufacturer’s instructions. Total RNA concentration and 260/280 ratios were measured using a Nanodrop 2000c (Thermo Scientific). Illumina Sequencing was performed by Bristol Genomics Facility, University of Bristol, using the poly-A selection method for library preparation and the Illumina NextSeq500 system. RIN were 8.1 (SD=0.5), 7.7 (SD=0.2) and 7.6 (SD=0.6) for control, dehydrated and rehydrated cortex samples, and 8.5 (SD=0.5), 8.8 (SD=0.2) and 8.7 (SD=0.4) for control, dehydrated and rehydrated medulla samples, respectively.
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