Severe acute dehydration in a desert rodent elicits a transcriptional response that effectively prevents kidney injury

Animal living in desert environments are forced to survive despite severe heat, intense solar radiation, and both acute and chronic dehydration. Indeed, these animals have evolved phenotypes that effectively address these environmental stressors. To begin to understand the ways in which the desert adapted rodent P. eremicus survives, we performed an experiment by which we subjected reproductively mature adults to profound acute dehydration, during which they lost on average 23% of their body weight. Animals react via a series of changes in the kidney, which include modulating expression of genes responsible for reducing the rate of transcription, and maintaining water and salt balance. Extracellular matrix turnover appears to be decreased, and apoptosis is limited. Serum Creatinine and other biomarkers of kidney injury are not elevated, which is different than the canonical human response, suggesting that transcriptional changes caused by acute dehydration effectively prohibit widespread kidney damage in the cactus mouse.


Introduction
Dehydration, whether caused by exposure to extreme environmental conditions, water deprivation, or by infection ( e.g., diarrheal illnesses), represents a significant threat to human life (1) . In spite of modern medicine, millions of people die every year from dehydration (2) .
Compounding issues of exposure and illness are public health issues regarding the delivery of safe drinking water. With global climate change, it is expected that these challenges will only become more severe (3) . As a result, research providing insight into the ecological genomics underlying renal homeostasis and physiologic resistance to acute and chronic dehydration is urgently needed. The response to severe acute dehydration in humans and traditional mammalian models is generally limited and may fail to prevent severe electrolyte imbalance, renal impairment or failure, or even death (4)(5)(6)(7) . In contrast, desert-living mammals have evolved phenotypes making them adept at surviving acute and chronic dehydration (8,9) .
In addition to the mechanisms described above, desert animals are thought to possess specialized machinery allowing for the efficient maintenance of water and solute balance, in spite of the obvious challenges presented to them as part of desert living. The aquaporins, a large protein family integral to the maintenance of water balance in the kidney (16)(17)(18) were the first genes implicated in desert rodents' remarkable abilities (19) , and immediately became canonized as one of the critical adaptation required by at least some desert rodents. To this end, several other studies provided evidence of the import of this gene family (20)(21)(22)(23) , which in brief, allows water to be resorbed from the urinary lumen back into circulation, thereby reducing water loss. With regards to solute balance, several studies have found members of another gene family -the Solute Carriers -to be differentially expressed or under lineage-specific positive selection (20,24) . Broadly speaking, these transport molecules are responsible for the maintenance of electrolyte levels requisite for the normal function of virtually all bodily processes (25) .
Dehydration and the resultant hyperosmolar state, the direct result of intense heat and aridity, is typically coupled with electrolyte derangement (26, 27) and therefore maintaining electrolyte gradients requisite for proper function may be particularly challenging for desert animals. Given these physiological challenges, specialized function of the Aquaporins and Solute Carriers may provide at least partial mechanisms thereby allowing for rodents to survive in desert-conditions.
The cell-level consequences of failure of these mechanisms ( e.g., salt and water imbalance) is well characterized, at least for non-desert adapted animals (27-29) . In brief, dehydration results in a hyperosmotic state, which, amongst other things, results in cell cycle arrest (30) , double strand DNA breaks (31) and disruption of repair mechanisms (32) , oxidative stress (33) , inhibition of transcription and translation (34) . Additionally, hyperosmolarity is known to inhibit protein folding in the Endoplasmic Reticulum (ER -1, 35) . Inhibited protein folding results in physical crowding of the ER, and invocation of the unfolded protein response (UPR), which provides mechanisms for either restoring homeostasis, or in cases where stress is too severe or prolonged, facilitating apoptosis (36) . Given this, the response to a hyperosmolar environment is profound, with disruptions to vital cellular processes. Here, we hypothesize that these responses will be less profound in an animal adapted to exceptionally dry conditions. In particular, we predict that while cellular stress will be apparent, signs of widespread apoptosis and tissue damage will not. To this end this study seeks to provide transcriptomic evidence to test this hypothesis.
To better understand the effects of dehydration in desert-adapted animals, and in particular the cellular effects, we conducted an experiment whereby we exposed a set of animals ( Peromyscus eremicus ) to experimental dehydration. The rodent P. eremicus (cactus mouse) represents an ideal model in which to study the genomic architecture of adaptive dehydration-resistance, and that of acute dehydration. These animals have evolved in the extremely hot and dry desert regions of the US Southwest (37) . They survive their entire lives without drinking water, and in many cases urinating. Though highly adapted to desert environments, P. eremicus retains many of the useful characteristics of more well established rodent models, including the facility with which they reproduce in the lab (38) and the availability of multiple sequenced genomes ( P. maniculatus , P. polionotus , P. californicus ). In addition, the genus Peromyscus -known as the Drosophila of North American Mammalogy (39) , has been the focus of ecological study for decades (24,38,(40)(41)(42)(43) , and has recently emerged as a powerful clade of organisms within which to study the genetics of adaptive phenotypes (43)(44)(45) .

Animal Care and Experimental Model
The cactus mice used for this study include only captive born individuals purchased from the Peromyscus Genetic Stock Center (Columbia, South Carolina). The animals, originally collected from a hot-desert location in Arizona, have been housed for several generations at the University of New Hampshire in conditions that mimic temperature and humidity levels in southwestern US deserts, as described previously (46) . Animals were randomly assigned to either a dehydration treatment group (n=19) or a control group (n=19). Mice that were assigned to the dehydration group were weighed and then water deprived for~72 hours directly prior to euthanasia. All mice were again weighed prior to sacrifice. Individuals in the study were collected at approximately noon (6 hours after lights on), and when exposed to 90°F for four hours, between September 2014 -April 2016.
Cactus mice were sacrificed via isoflurane overdose and decapitation in accordance with

Illumina Library Preparation and Sequencing
Tissues frozen in RNALater were thawed on ice in an RNAse-free work environment. Total RNA was extracted using a standard Trizol extraction protocol (Thermo Fisher Scientific, Waltham, MA

Sequence Quality Control and Assembly
Sequence read data were downloaded and quality checked using FastQC version 0.11.5 (48) . A de novo transcriptome for the cactus mouse kidney was assembled following the Oyster River Protocol for Transcriptome Assembly (49) . In brief, 59 million paired end reads from two individuals (1 control and 1 dehydrated) were error corrected using RCorrector version 1.0.2 (50) . Adapters, as well as bases with a Phred score <2 were trimmed using Skewer 0.

Mapping and Global Analysis of Differential Gene Expression
After quality and adapter trimming to a Phred score =2, reads were quasimapped to the cactus mouse kidney transcriptome after an index was prepared using Salmon 0.7.2 (57) . Kidney transcript IDs were mapped to genes from Mus musculus proteome version GRCm38.p5, using BLAST (58) . All data were then imported into the R statistical package (version 3.3.0) (59) using tximport (60) for gene level evaluation of gene expression, which was calculated using edgeR (version 3.1.4) (61) following TMM normalization and correction for multiple hypothesis tests by setting the false discovery rate (FDR) to 1%. Gene ontology enrichment analysis was carried out using the Kolmogorov-Smirnov test (62) for significance in the R package, topGO (63) .

Coexpression network analysis
TMM normalized count data for each individual, as well as physiology data consisting of individual water intake, weight loss and electrolyte data (described in [ (64) ], and available as Supplementary dataset 1, name = phys4.csv on GitHub) were imported into the package WCGNA (65) . Gene co-expression modules were described, then a heatmap that describes the relationship between co-expression modules and phenotype was produced. Gene ontology terms for genes residing in specific modules were generated.
Accordingly, serum electrolytes (Sodium, Calcium, Bicarb) were elevated in dehydrated animals, as well BUN. Interestingly, a serum marker of kidney damage, Creatinine, is not.

Sequence Read Data & Code Availability
In total, 38 kidneys (19 dehydrated, 19 control) were sequenced. Each sample was sequenced with between 2.3 million and 60.6 million read pairs (mean=16.8M, SD=1.3M). All read data are available using the project ID PRJEBXXX . Code used for the analysis of these data are available at https://git.io/vMN6l .

Transcriptome Assembly and Evaluation
The kidney transcriptome assembly consists of 79,660 transcripts. This assembly, along with the annotations contained in gff3 format, is available at (dryad on acceptance

Sequence Read Mapping and Estimation of Gene Expression.
Raw sequencing reads corresponding to individual kidney samples were mapped to the reference kidney transcriptome using Salmon, which resulted in an average mapping rate of 85% (sd=4%). These mapping data were imported into R and summarized into gene-level counts using tximport, after which, edgeR was used to generate normalized estimates of gene expression.

Global Analysis of Differential Gene Expression
Analysis of global patterns of gene expression, aimed at uncovering previously unknown differences in renal gene expression related to water deprivation by examining the entire transcriptome, was conducted using edgeR. After normalizing the count data using the TMM method (69) , which is done by finding a set of scaling factors for the library sizes that minimize the log-fold changes between the samples for most genes, we controlled for over 13,600 multiple comparisons to yield a robust statistical analysis of differential gene expression. This  Each dot is a gene, with red dots indicating significantly different gene expression (FDR < 0.01).
Gene ontology analysis of the 252 genes more highly expressed in acute dehydration (Table 3) suggests that acute dehydration is linked to first, the cellular starvation response, which occurs   Gene ontology analysis of the 213 genes lowly expressed in acute dehydration (Table 4) suggests that acute dehydration is linked to lower expression in genes related to cholesterol biosynthetic processes. Genes related to this pathway include LSS (Lanosterol synthase), HMGCS1 (Hydroxymethylglutaryl-CoA synthase, cytoplasmic) and FDFT1 (Squalene synthase).
Second, genes expressed significantly lower in the cactus mouse kidney during acute dehydration cluster around the GO term extracellular matrix organization. Genes involved in this pathway, and differentially expressed, include FN1 (Fibronectin), COL4A1, COL4A6, COL3A1, COL2A1, COL1A1 and COL1A2 (Collagen alpha chain genes) as well as LUM (Lumican).
With regards to Fibronectin and Collagen, these proteins have been commonly associated with extracellular matrix turnover and renal interstitial fibrosis (7,77) . Renal interstitial fibrosis, a hallmark of kidney injury, has been causally linked to renal functional impairment (78) , with fibrosis leading to a reduction in glomerular filtration rate (GFR) and overall function. That these genes are lowly expressed in the kidney of acutely dehydrated cactus mice provides strong evidence for the hypothesis that dehydration is not related to widespread kidney damage, and further, that extracellular matrix turnover is lowered.

Critical Process Analysis
To better understand the response to acute dehydration in desert adapted animals, we analyzed patterns of differential expression between control animals and those exposed to acute dehydration for specific processes proposed to be critical to desert survival. To identify genes related to specific processes of interest (water balance and sodium regulation), we selected relevant gene ontology terms then identified genes in Mus that correspond to that term. Expression of the Peromyscus orthologs of these genes was estimated.
Water --For desert animals, the regulation of water is obviously critical (8,14,15) . Here, the Aquaporin genes are thought to be critical to the maintenance of homeostasis, and as such have been the focus of study for decades (79,80) . How desert animals use these genes to regulate water is largely unstudied, though one study demonstrates that AQP4 is absent in at least one species of the Dipodomys rodents (19) . The transcriptomic mechanisms P. eremicus uses to regulate water appear to depends critically on INPP5K (Figure 3), which is known to be expressed in the collecting duct, and is functionally responsible for urine concentration (81) and is involved in insulin signaling (82) . Interestingly, this gene may also have a role in negatively regulating actin in the extracellular matrix (83) , which provides yet another indication of the general depression of turnover in the extracellular matrix, as does the lower expression of Fibronectin and the Collagens.
In addition to the INPP5K gene, Aquaporin 4 ( Figure 4) is significantly differentially expressed, being lower in acute dehydration relative to control animals. AQP4, expressed in the basolateral membrane of the collecting duct, is responsible for transport of water out of the cell, to the interstitium (84,85) . Defects in this non-vasopressin-responsive AQP may result in deficiencies in urine concentration (86) . The functional correlates of lower expression are currently unknown. On one hand, lower expression might suggest a reduced capacity for moving water from membrane cells to the interstitium while on the other, in severe acute dehydration, very little water is available for movement, so perhaps there is little need to manufacture such protein complexes. Of note, no other Aquaporin was significantly differentially expressed in acute dehydration. This is perhaps surprisingly, especially for AQP2, whose role is predominately in reabsorption of water from the urinary lumen into the cell (84) .
Such RNAseq studies of acute dehydration have not been previously conducted, and as such, the complexity of the response has not been previously fully characterized. Ongoing work in the lab aims to provide further functional characterization of the responses to acute dehydration.   2), and GALC (galactosylceramidase). Dehydration-linked weight loss of animals is has a significant negative relationship (higher gene expression related to less weight loss) with the 'grey' module, which contains GO-terms linked to monooxygenase activity, steroid metabolic process, and negative regulation of transport. The three described genes mostly tightly linked to this module include ITGA99 (integrin alpha 9), SGCG (sarcoglycan, gamma) and MAGBE4 (melanoma antigen, family B, 4). Broadly speaking, the integrins are receptors for extracellular matrix components (96) and are involved in the regulation in intracellular osmolarity (97) .
Expression of integrins, which could allow for sensing of osmotic stress and subsequent production of osmolytes, seem to have important links to dehydration-linked weight loss.

Biomarkers of kidney damage
A great deal of research has gone into identifying markers of acute kidney injury (1,(98)(99)(100) , and as a result, the measurement of kidney damage has moved well beyond assaying serum Creatinine, which is known to be relatively insensitive and slow to indicate damage (101) . In humans, dehydration and other clinical conditions that cause a pathological reduction in renal perfusion pressure are responsible for acute kidney injury (102) . If acute dehydration results in substantial tissue damage in the cactus mouse, this damage will be evident when looking at a panel of biomarkers specific for kidney injury. Despite these markers not having been validated in the cactus mouse, that a large number of statistically independent tests are concordant ( Figure 7) showing no differential expression, suggests that no significant tissue damage is occurring. Figure 7: Biomarkers of acute kidney injury. In all cases, the Y-axis is the log transformed TPM. Brown boxplot refers to the acute dehydration group (n=17) while blue refers to the fully hydrated control animals (n=18). Botplot titles are of the format Gene Name:Entrez ID.

Conclusion
Animal living in desert environments are forced to survive despite severe heat, intense solar radiation, and both acute and chronic dehydration. Indeed, these animals have evolved phenotypes that address these environmental stressors. To begin to understand the ways in which the desert adapted rodent P. eremicus survives, we performed an experiment by which we subjected reproductively mature adults to profound acute dehydration during which they lost, on average 23% of their body weight over the three day experiment. We modeled this loss after a scenario of a summertime rainfall event, followed by intense drought. While animals lost a substantial amount of weight, they were behaviorally intact. Animals react via a series of changes in the kidney, which include up-regulating genes responsible for 1. reducing the rate of transcription, and 2. Maintaining water and salt balance. Extracellular matrix turnover appears to be substantially decreased, and apoptosis appears to be limited. Serum Creatinine and other markers of kidney injury are not elevated over baseline, which is different than Mus exposed to less severe dehydration (7) and suggests that acute dehydration is not responsible for widespread kidney damage in the cactus mouse.
The work presented here are the results of experiment allowing us to peer deeply into the renal machinery of P. eremicus allowing for survival despite severe acute dehydration. In contrast, humans are exquisitely sensitive to alteration on water balance, with even minor dehydration resulting in physiological compromise (26) . The molecular toolkit employed by the cactus mouse -the genes identified in this study -have orthologues in human. One exciting potential outcome of this line of work is the ability to develop manipulative genetic or pharmacologic techniques enabling a more robust human response to dehydration. Indeed, such a response could be extremely valuable in a wide variety of situation where potable water is limited.