Administration of mesenchymal stromal cells before renal ischemia/reperfusion attenuates kidney injury and may modulate renal lipid metabolism in rats

Mesenchymal stromal cells (MSC) have been demonstrated to attenuate renal ischemia/reperfusion (I/R) damage in rodent models. The mechanisms of such nephro-protection remain largely unknown. Furthermore, the optimal timing of MSC administration has been poorly investigated. Here, we compare the impact of MSC injection 7 days before (MSCD − 7) versus 1 day after (MSCD + 1) renal I/R in rats. Control groups received equivalent volumes of saline at similar time-points (SD − 7 and SD + 1). Right nephrectomy was performed, and left renal ischemia lasted 45 min. After 48-hour reperfusion, we observed significantly improved renal function parameters, reduced apoptotic index and neutrophil/macrophage infiltration in kidney parenchyma, and lower expression of tubular damage markers and pro-inflammatory cytokines in MSCD − 7 in comparison to MSCD + 1 and saline control groups. Next, comparative high-throughput RNA sequencing of MSCD − 7 vs. SD − 7 non-ischemic right kidneys highlighted significant down-regulation of fatty acid biosynthesis and up-regulation of PPAR-α pathway. Such a preferential regulation towards lipid catabolism was associated with decreased levels of lipid peroxidation products, i.e. malondialdehyde and 4-hydroxy-2-nonenal, in MSCD − 7 versus SD − 7 ischemic kidneys. Our findings suggest that MSC pretreatment may exert protective effects against renal I/R by modulating lipid metabolism in rats.

vitro and in vivo studies 5, 10-12 . Moreover, MSC exert tissue repair function in damaged organ by reducing inflammation and stimulating vascular supply 4 . Their beneficial effect predominantly involves paracrine and endocrine pathways rather than trandifferentiation 13 . MSC-derived microvesicles may also allow horizontal transfers of mRNA, microRNA and proteins to their neighboring cells 14,15 .
A number of experimental studies have provided promising data using MSC therapy in various models of I/R-related AKI 4 , and clinical trials are ongoing 16,17 . Hence, MSC administration either immediately or 24 h after renal ischemia significantly improved renal function with higher proliferative and lower apoptotic indexes in anesthetized rats exposed to I/R injury 13 . In strong contrast, Perico N. et al. 18,19 reported on an engraftment syndrome characterized by AKI following MSC infusion in both rodent models and human clinical trials of kidney transplantation (KTx). KTx necessarily conveys renal I/R. These authors demonstrated that pre-transplant administration of MSC 7 days before KTx reduces neutrophil infiltration into kidney parenchyma and prolongs allograft survival in mice 19 . Outcome differences in pre-versus post-transplant administration of MSC may be explained by the differential homing location of MSC into the spleen and lymphoid organs versus the ischemic organ, respectively. Similarly, Merino A. et al. demonstrated, in a rat renal allograft model, that the optimal time schedule for MSC infusion to prevent acute rejection was 7 days before KTx 20 . MSC could thus exert contrasting effects depending on the timing of administration related to the injury, which, in turn, conditions the microenvironment 21,22 .
In the field I/R-related AKI, the administration of MSC prior to the ischemic insult may mimic ischemic preconditioning (IPC) 23 . IPC is thought to exert its nephro-protection in 2 consecutive, immediate and delayed, phases 24 . The delayed phase of IPC initiates a complex genomic and proteomic response, and is considered as the most efficient one in terms of nephro-protection 24 . The present study aims at (i) determining the impact of the timing of MSC infusion, i.e. before versus after I/R, on structural and functional parameters of kidney injury in rats, and (ii) identifying the cellular pathways implicated in MSC-induced IPC, including their impact on kidney metabolism.
In comparison to saline infusion, MSC administration 7 days before renal I/R reduces neutrophil and macrophage infiltration, apoptosis and cell proliferation, while MSC administration 1 day after I/R increases apoptosis and cell proliferation. Following renal I/R, the quantification of tubular cells expressing proliferating cell nuclear antigen (PCNA) and heat-shock protein 70 kDa (HSP70) is classically used to assess the severity of acute tubular necrosis 25 . Apoptosis was measured using TUNEL assay. Here, the administration of MSC at D − 7 was associated with a significantly reduced number of HSP70-positive (p ≤ 0.01), PCNA-positive (p ≤ 0.05) and apoptotic cells (p ≤ 0.001) along renal tubules (Fig. 2a), as well as a lower number of myeloperoxidase (MPO)-positive polymorphonuclear neutrophils infiltrating kidney parenchyma (p ≤ 0.05), in comparison to the control SD − 7 group. The number of F4/80-positive macrophages was higher in SD − 7 versus MSCD − 7 ischemic kidneys. Conversely, CD163-positive M2 macrophages were more numerous in ischemic kidneys exposed to MSCD − 7 compared to saline exposure (SD − 7) (Fig. 2a). No significant difference in neutrophil and macrophage recruitment was found between MSC D + 1 and SD + 1 groups (Fig. 2b). By contrast, HSP70-expressing (p ≤ 0.01) and PCNA-immunoreactive (p ≤ 0.01) epithelial cells were more numerous in MSCD + 1 than in SD + 1 kidneys. The number of TUNEL-positive cells was significantly increased in MSCD + 1 than SD + 1 groups (p ≤ 0.05) (Fig. 2b).
SCIENTIFIC REPoRts | 7: 8687 | DOI:10.1038/s41598-017-08726-z Transcriptomics indicate a down-regulation of fatty acid biosynthetic pathways at day 7 post-administration of MSC. High-throughput RNA sequencing technology was used to probe the differential transcriptomic renal profiles of rats infused with MSC compared to saline (Fig. 3a,b). From the methodological point of view, messenger RNAs were extracted from the right kidneys of rats exposed (MSCD − 7, n = 6) or not-exposed (SD − 7, n = 6) to MSC 7 days before sampling. These kidneys were not exposed to ischemia, and were harvested before the 45-min ischemia of the contralateral kidneys. After (i) mapping the reads onto the rat genome (rn5), (ii) transcriptome reconstruction and (iii) abundance calculation, differentially expressed gene were identified using Cufflinks. A total of 25908 genes were assessed for differential expression calculation The mRNA expression levels were standardized using Gapdh as housekeeping gene. Significant differences are indicated, *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001.
between MSCD − 7 and SD − 7 non-ischemic groups. Among these genes, 748 genes were found to be significantly differentially expressed (False Discovery Rate, FDR < 0.05): 493 and 255 genes were down-and up-regulated in MSCD − 7 group, respectively (Fig. 3a). To allow the identification of relevant groups of related genes sharing One-way analysis of variance was performed among groups. Significant differences are indicated, *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001. biological functions or pathways, functional enrichment analysis was performed using Database for Annotation, Visualization and Integrated Discovery (DAVID) and WEB-based GEne SeT AnaLysis Toolkit 26,27 . Using gene ontology analysis via WebGestalt 28 , 457 identified genes with unambiguous gene symbol were allocated to ontology categories depending on their biological functions (Fig. 3b). Using Overrepresentation Enrichment analysis based on Wikipathway Enrichment Categories, we found that the metabolic pathways mostly affected by MSC pre-infusion are implicated in adipogenesis, insulin signaling, fatty acid (FA) biosynthesis, IL-6 signaling, B-cell receptor signaling, IL-3 pathway, proteasome degradation, and nuclear receptors involved in lipid metabolism ( Table 1). Because of previous reports suggesting renal lipotoxicity as a key mechanism in I/R AKI 29 , we selected 6 downregulated genes (Stearoyl-CoA desaturase (Scd), Serum-and glucocorticoid-inducible kinase 1 (Sgk1), Fatty acid synthase (Fasn), Acetyl-CoA carboxylase (Acaca), ATP citrate lyase (Acly), GRB2-associated binding protein (Gab)) and 1 upregulated gene (Peroxisome proliferator-activated receptor alpha (Ppara)) to validate the  RNA-Sequencing differential data between MSCD − 7 versus SD − 7 groups using real-time (RT)-qPCR. The mRNA expression level of Ppara was significantly increased in MSCD − 7 group compared to SD − 7 group, whereas the mRNA expression levels of Acly, Gab, Fasn, and Scd were significantly decreased in MSCD − 7 group, as suggested by the high-throughput RNA sequencing (Fig. 4a,b).

In comparison to saline infusion, MSC administration induces the expression and activation of
PPARα and FAT/CD36, and attenuates I/R-associated lipid peroxidation. Using immunoblotting, we found that PPARα and phosphorylated PPARα were significantly increased in MSC-exposed (MSCD − 7) kidneys in comparison to saline-infused controls, in both non-ischemic (p ≤ 0.001) and ischemic (p ≤ 0.05) conditions. Additionally, we observed an induced expression of FAT/CD36 in kidneys of MSC-pre-infused animals, significantly contrasting with controls (p ≤ 0.01) (Fig. 4c,d). Immunohistochemistry revealed a preferential localization of FAT/CD36 in the cytoplasm of the epithelial cells lining the proximal tubules (Fig. 4e). Finally, we found that MSC administration 7 days prior to I/R significantly attenuated lipid peroxidation in kidney parenchyma, as quantified by malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) modified proteins (Fig. 4f-h).

Discussion
Cell-based therapy has emerged as a promising strategy for the treatment of various conditions, including AKI.
Preclinical and pilot clinical studies support that MSC may attenuate AKI and accelerate recovery 4,5 . Still, controversies remain concerning the modalities of MSC infusion 8,30 . Here, we show that infusing rats with 1.5 × 10 6 MSC 7 days before renal I/R improves renal function, decreases inflammation and apoptosis in kidney parenchyma, and may modulate FA biosynthesis. Conversely, injecting MSC after renal I/R is deleterious in our model, with worsened kidney function and higher scores of inflammation and apoptosis. These observations further emphasize the impact of the timing of MSC administration with respect to organ injury 10,22 . Furthermore, exposure to MSC may participate to renal conditioning, with activation of the PPARα/CD36 pathway. Our model is limited to 48-hour reperfusion, and may not fully reflect the complexity of I/R-associated AKI observed in humans. Additionally, lipid metabolism is different between rats and humans, particularly concerning de novo lipogenesis 31 . Finally, MSC used in the present study were suspended in saline at the time of the i.v. delivery in order to avoid infusing rats with culture medium (enriched with multiple and various solutes and metabolites, including fetal bovine serum and antibiotics). Saline infusion at the time of renal I/R may alter renal perfusion because of its supraphysiological concentration of chloride leading to vasoconstriction of glomerular arterioles 32,33 . This methodological particularity may partly explain the discrepancy between our observations and previous reports 4 . The influence of MSC infusion timing has been poorly investigated, although in vitro and in vivo data have demonstrated that the environment strongly influences MSC phenotype and properties 22,34 . Indeed, MSC-associated immunomodulation may be due to a stepwise activation induced by soluble factors not constitutively expressed by MSC but triggered by inflammation, including IFN-γ, TNF-α, IL1α and IL1β 22,35 . The in vitro addition of MSC to CD4 lymphocytes exerts different consequences upon the status of target T-cells. Late addition of MSC after T-cell induction only suppresses Th1 cell lineage, subsequently expanding pro-inflammatory Th17 cells 36 . Casiraghi F. and colleagues demonstrated in vivo in a sensitized mouse model of KTx that pre-transplant administration of MSC prolonged allograft survival by promoting the expansion of donor-specific regulator T-lymphocytes, whereas post-transplant administration of MSC was associated with early graft dysfunction characterized by increased renal recruitment of neutrophils and in situ complement activation 19 . Similar observations of "engraftment syndrome" were made in pilot clinical trials including kidney transplant recipients 18,37 . The authors hypothesized that KTx triggers graft inflammation, which in turn causes the recruitment of MSC to the transplant and favors MSC differentiation towards a pro-inflammatory phenotype.
The nephro-protection induced by MSC infusion before renal I/R may mimic "delayed remote conditioning" 24 . Indeed, MSC do not transdifferentiate into mature tissue, but rather act via the secretion of endocrine or paracrine factors 38 . Such humoral alert may activate signal transduction pathways implicated in cell resistance to I/R and in cell recovery. Interestingly, we observed a significant impact of MSC on FA biosynthesis and lipid peroxidation in kidney parenchyma. Lipotoxicity has been well documented in various models of renal I/R injury 39,40 . Particularly, I/R injury-associated accumulation of triglycerides (TG) depends on (i) FA mobilization from membrane phospholipids by phospholipase A2 (PLA2) 40,41 , (ii) inhibition of TG degradation 42 , and (iii) acceleration of TG synthesis from free FA 42 . Exposure of proximal tubule cells to the mitochondrial-blocking agent, antimycin A (as an in vitro model of ischemia), upregulates TG formation, namely via fatty acid synthase (FAS) 42 . In our model, in vivo administration of MSC caused a down-regulation of key enzymes in FA biosynthesis, including FAS, as suggested by high-throughput RNA sequencing and confirmed by RT-qPCR. Additionally, MSC infusion activates PPARα pathway. PPARα is a member of the nuclear receptor superfamily of ligand-activated transcription factors, regulating lipid homeostasis, inflammation and vascular integrity 43 . PPARα is highly expressed in metabolically active tissues, including renal proximal tubules 44 . In case of renal I/R, PPARα expression decreases 45 , along with the rapid inhibition of microsomal and peroxisomal FA oxidation 46 . Conversely, pharmacological stimulation of PPARα by fibrates has been shown to attenuate I/R-associated AKI and accelerate kidney recovery 45,[47][48][49] . In rats, administration of PPARα agonist 5 days prior to renal I/R beneficially modulates the genes involved in FA oxidation, thereby preserving kidney structure and function 50 . Likewise, MSC infusion 7 days prior to renal I/R appears to up-regulate PPARα and attenuates AKI severity. In line with our observations, MSC have been previously shown in vitro to prevent lipotoxicity and improve cell viability and regeneration in high palmitic conditions 51 . MSC-induced nephro-protection may thus be linked to reduced lipotoxicity and lipid peroxidation at the time of renal I/R injury, as supported by a significant reduction in the abundance of MDA and 4-HNE modified proteins observed in our model. Besides modulating FA oxidation, PPARα also regulates transmembrane import of FA in a tissue-specific manner [52][53][54] . FAT/CD36 is a class B scavenger receptor broadly expressed, including in monocytes/macrophages and smooth muscle cells. This receptor has been implicated in several biological processes, and may respond to various ligands, such as thrombospondin-1, modified LDL and long-chain fatty acids. FAT/CD36 participates to the regulation of innate immunity, FA transport and angiogenesis 55 . Focusing on lipid metabolism, FAT/CD36 may be involved in mitochondrial FA oxidation, both at rest and in cases of metabolic challenges. In kidneys, FAT/ CD36 is mostly expressed in proximal tubular cells and podocytes, where it could contribute to glomerulosclerosis and albuminuria in diabetic nephropathy 56 . Palmitic acid-driven upregulation and translocation of CD36 from the cytosol to the plasma membrane lead to an increase in lipid uptake, ROS production and apoptosis in podocytes of patients with diabetic nephropathy and hyperlipidemia. In the field of I/R-related injury, controversies remain concerning the role of FAT/CD36. In mouse, the loss of FAT/CD36 results in impaired FA oxidation and reduced levels of glycogen, triglycerides and ATP in the heart. Consequently, CD36-deficient hearts are more susceptible to I/R injury because of lower energy storages before I/R and defective energy regeneration after I/R 57 . In strong contrast, hyperlipidemia exacerbates I/R injury in brain by promoting CD36-mediated inflammation in ApoE knock-out mice under high-fat diet 58 . The role of PPARα in governing the expression of FAT/CD36 in kidney has been poorly investigated to the best of our knowledge. In our study, MSC infusion is associated with both the activation of PPARα and the upregulation FAT/CD36 in both non-ischemic and ischemic conditions in comparison to saline infusion.
In addition to renal lipid metabolism, signaling pathways involved in inflammation modulation are also influenced by MSC pre-infusion, as suggested by our observations. Both innate and adaptive immune responses crucially contribute to the pathophysiology of renal I/R 5 . Activation of Toll-like receptors through the release of endogenous danger-associated molecular patterns (DAMPs) by ischemic renal cells leads to the initiation of a pro-inflammatory response. Hence, HMGB1, a ubiquitous nuclear protein which is actively released by stimulation of the innate immune system and passively released by ischemic tissues, may trigger TLR4. Waterman et al. corroborated the paradigm for MSC immune properties in emphasizing the particular role of TLRs exposure 34 . They observed that TLR3 stimulation of MSC supports their immunosuppressive effects while TLR4 activation provides a pro-inflammatory phenotype, characterized in particular by their dissimilar secretions of cytokines and chemokines 34 . TLR4 priming results in upregulation of pro-inflammatory cytokines, such as IL6 or IL8, while TLR3 priming results in production of anti-inflammatory molecules, such as IL4, IDO, or PGE2. Here, we found we found a significant upregulation of mRNA expression of HMGB1 in MSCD + 1 versus MSCD − 7 ischemic kidneys (Supplementary Figure 1). Furthermore, chemotactic cytokines, including MCP1, are produced by injured renal tubular epithelial cells, subsequently leading to the attraction of inflammatory cells 2 . MCP1 expression was found to be down-regulated in MSCD − 7 group in comparison to control group. Infiltration of kidney parenchyma by inflammatory cells, including MPO-positive neutrophils and F4/80-positive macrophages, is a typical feature of I/R injury 59 . The release of proteases, myeloperoxidases and cytokines by neutrophils, as well as the local production of ROS, further aggravate kidney injury via increased vascular permeability and reduced cell integrity 2 . In our study, MSC administration 7 days prior renal I/R was associated with a lower infiltration of neutrophils and macrophages in comparison to saline infusion. Furthermore, macrophage phenotype appears orientated towards M2 immunoregulatory subtype in case of a priori exposure to MSC. M2 macrophages are characterized by a low ability to secrete inflammatory cytokines and a high ability to phagocyte apoptotic cells 38 . In line with these observations, IL-6 signaling pathway was found to be down-regulated in MSCD − 7 kidneys compared to control SD − 7 group. The deleterious role of IL-6 in I/R-related AKI has been suggested in murine models: IL-6-knockout mice are less susceptible to I/R AKI, whereas transfer of IL-6-sufficient macrophages by transplantation of wild-type bone marrow into IL-6-deficient mice restore the susceptibility to the ischemic damage 60 .
As a whole, our data suggest that MSC-induced nephro-protective conditioning prior to I/R may involve critical modifications of lipid metabolism, including (i) down-regulated FA biosynthesis, (ii) activated PPARα pathway, (iii) prioritization of FA as source of energy in renal proximal tubule cells, and (iv) decreased availability of free FA, which in turn prevent lipid peroxidation and attenuate renal I/R damage. Additional in vitro and in vivo studies, including the comparative use of inhibitors of PPARα or CD36, are needed to further decipher the impact of MSC on FA oxidation in renal epithelial cells.

Materials and Methods
All methods were carried out in accordance with the relevant guidelines and regulations.

Isolation of MSC from bone marrow. Bone marrow was flushed from both femurs and tibias of male
10-week-old inbred Lewis rats (Charles River laboratories). Lewis rats are regarded as inbred for several congenic strains. After homogenization in Phosphate-Buffered Saline (PBS, Lonza) +2% fetal bovine serum (FBS, Lonza), the suspension was filtered and centrifuged at 100 g for 10 min. Cells were resuspended in DMEM medium (Lonza) and gently sieved through Ficoll (Healthcare Life Sciences). After an additional centrifugation at 200 g for 45 min at room temperature (RT), mononuclear cells were removed from the gradient interface and suspended in DMEM solution before a new centrifugation of 10 min at 100 g. The cells were then plated in 75 cm 2 culture flask (Falcon) containing DMEM supplemented with 10% FBS, 1% L-Glutamine (Lonza) and 1% penicillin (Lonza). Cells were then cryopreserved at early (<P3) passages. Following thawing in a water-bath at 37 °C, cells were centrifuged and re-suspended in pre-heated DMEM culture medium. MSC from 3 donors were pooled and expanded together. Several studies have demonstrated that MSC proliferation potential, phenotypic characteristics and ability to differentiate are largely preserved throughout the cryopreservation process 61 . MSC were only used before passage P5.
Supplemented DMEM was changed twice a week. When cells reached 80% of confluency, they were split in two 75 cm 2 culture flasks. On the basis of the conventional criteria 7, 8 , MSC were phenotyped twice, i.e. (i) before their cryopreservation and (ii) before each i.v. injection. Accordingly, the MSC used in the present project adhered to plastic supports and presented a spindle-shaped morphology. MSC were positive for MSC markers as evidenced by flow cytometry, using AlexaFluor-conjugated anti-rat CD29 antibody (BD Pharmingen) and APC-conjugated anti-rat CD90 (BD Pharmingen) antibody. MSC were negative for PE-conjugated anti-CD79a (abcam) antibody, V450-conjugated anti-rat CD45 (BD Horizon) antibody and FITC-conjugated anti-rat CD11b (BD Pharmingen). Cell fluorescence was evaluated by flow cytometry on a FACS Calibur flow cytometer. Data were analysed using FACS Diva softwares. Potential to differentiate into osteoblast, adipocyte, and chondroblast lineages was demonstrated by positive staining for Alizarin Red, Oil Red O and toluidine blue staining, respectively, as previously described 62 . These data concerning MSC quality are summarized in Supplementary Figure 2. Rat model of renal ischemia/reperfusion. The Institutional Animal Care and Use Committee of the University of Liege approved the present protocol (#1651). A total of 35 (including 3 dead rats, i.e. 1 in MSCD − 7 group (preoperative) and 2 in MSCD + 1 group (perioperative)) Lewis male rats aged of 8-10 weeks were randomly assigned to 4 groups (Fig. 5): MSC injection 7 days before renal I/R (MSCD − 7, n = 11), saline injection 7 days before renal I/R (S D − 7, n = 6), MSC injection 1 day after renal I/R (MSCD + 1, n = 9), saline injection 1 day after renal I/R (S D + 1, n = 6). Rats were anesthetized with pentobarbital (60 mg/kg). Analgesia was performed preoperatively using buprenorphine (0.05 mg/kg). Median laparotomy was performed on heating pads, and a vascular clamp was applied for 45 min on the left renal pedicle. The right kidney was nephrectomized, half-cut and fixed in paraformaldehyde or snap-frozen in liquid nitrogen. During the 45-minute period, the laparotomy area was covered with moistened gauze. Saline (0.5 mL/100 g) was infused intraperitoneally, as well as antibiotics (Enrofloxacin 2.5%, 0.1 mL/rat SC). After surgery, rats were monitored, with ad libitum access to food and water. Forty-eight hours after renal I/R, rats were anesthetized. Blood was collected by puncture of the inferior vena cava, and centrifuged at 100 g for 5 min at 4 °C. Serum levels of BUN and SCr were measured by enzymatic methods (Roche/Hitachi Cobas). The left kidney was excised, half-cut and fixed in paraformaldehyde or snap-frozen in liquid nitrogen. Snap-frozen (right and left) half-kidneys were grinded into homogeneous powder using B.Braun Mikro-Dismembrator before protein or mRNA extractions for further analyses.
Administration of MSC. MSC were detached from culture flasks by Trypsin-EDTA, and centrifuged at 200 g for 5 min in DMEM. Cells were counted in a Thoma chamber, and 1.5 × 10 6 cells were suspended in 1 mL of sterile saline (in order to avoid infusing rats with culture medium). The dose of 1.5 × 10 6 cells per rat was chosen on the basis of previous preclinical investigations 4 . Cell suspensions were slowly injected into the tail vein 7 days before (MSCD − 7) or 1 day after (MSCD + 1) renal I/R (Fig. 5). MSC were i.v. injected in parallel in all groups over a 2-day period. Quality of MSC administered in MSCD − 7 and MSCD + 1 groups can thus be technically regarded as identical. Control rats were infused with an equal volume of saline at the same time-points.

RNA sequencing and real-time quantitative polymerase chain reaction (RT-qPCR). Messenger
RNAs were extracted from the right kidneys of rats exposed (MSCD − 7) or not-exposed (SD − 7) to MSC 7 days before sampling. These kidneys did not suffer from I/R, and were harvested before the 45-min ischemia of the contralateral kidneys. After homogenization in 1 mL Tripure solution (Roche) and 200 μL of chloroform, lysates were centrifuged at 200 g at 4 °C for 15 min. The upper aqueous phase was diluted with 500 μL of isopropyl alcohol. The mixtures were centrifuged at 200 g at 4 °C for 10 min, and pellets were suspended in 500 μL of 70% ethyl-alcohol before centrifugation at 100 g at 4 °C for 5 min. Pellets were finally dissolved in 100 μL RNase-free water. RNA concentration and purity were assessed using NanoDrop Lite spectrophotometer (Thermo Scientific). All RNA samples had an absorbance [260 nm/280 nm] ratio above 1.8. Libraries were prepared for each sample using Truseq mRNA stranded kit from Illumina and sequenced on a NextSeq. 500 sequencer producing an average of 20 million 2 × 75-bp reads per library. BaseSpace Sequence Hub Illumina was used for the evaluation of the sequencing data. Reads were mapped onto the rat reference genome (rn5) using TopHat. Quality control metrics meet the expectations for this type of libraries, with especially (i) a percentage of reads that align to the selected reference genome >93% for each sample, (ii) a median coefficient of variation of coverage of the 1000 most highly expressed transcripts lower than 0.4 for each sample (as a measure of the uniformity of coverage across transcripts) and (iii) a percentage of reads that align to the correct strand (compared to reference genome annotation) higher than 99.4% for each sample (Supplementary Table 1). Library quality were also confirmed by Picard analysis showing the expected coverage for mRNA transcripts (Supplementary Figure 3a) and the absence of degradation (Supplementary Figure 3b). After running TopHat, resulting data were transferred to Cufflinks and Cuffmerge to generate a transcriptome assembly. Identification of genes differentially regulated was then performed with Cuffdiff 26 . BaseSpace Sequence Hub Illumina was used for the evaluation of the sequencing data. To interpret the gene lists derived from RNA sequencing, functional enrichment analysis was performed using Database for Annotation, Visualization and Integrated Discovery (DAVID) 27 and WEB-based GEne SeT AnaLysis Toolkit (WebGestalt) 28 . Significantly differentially expressed genes were classified into gene ontology categories with WebGestalt (2015, October). Relevant pathways were further detected using an Over Representation Analysis with WebGestalt, based on the proportion of differential expressed genes within a given pathway surpassing the proportion of genes that could be randomly expected (WikiPathways as enrichment category; GeneSymbol as ID type) 65 . Afterwards, cDNAs were generated using Reverse Transcription Kit (Promega) according to manufacturer's instruction. Primers used for RT-qPCR are listed in Table 2. Semi-quantitative mRNA expression levels were calculated using threshold cycle (Ct) values following the classical 2 −ΔCT equation. The housekeeping gene used for RT-qPCR was GAPDH.