Renal Ischemia Tolerance Mediated by eIF5A Hypusination Inhibition Is Regulated by a Specific Modulation of the Endoplasmic Reticulum Stress

Through kidney transplantation, ischemia/reperfusion is known to induce tissular injury due to cell energy shortage, oxidative stress, and endoplasmic reticulum (ER) stress. ER stress stems from an accumulation of unfolded or misfolded proteins in the lumen of ER, resulting in the unfolded protein response (UPR). Adaptive UPR pathways can either restore protein homeostasis or can turn into a stress pathway leading to apoptosis. We have demonstrated that N1-guanyl-1,7-diamineoheptane (GC7), a specific inhibitor of eukaryotic Initiation Factor 5A (eIF5A) hypusination, confers an ischemic protection of kidney cells by tuning their metabolism and decreasing oxidative stress, but its role on ER stress was unknown. To explore this, we used kidney cells pretreated with GC7 and submitted to either warm or cold anoxia. GC7 pretreatment promoted cell survival in an anoxic environment concomitantly to an increase in xbp1 splicing and BiP level while eiF2α phosphorylation and ATF6 nuclear level decreased. These demonstrated a specific modulation of UPR pathways. Interestingly, the pharmacological inhibition of xbp1 splicing reversed the protective effect of GC7 against anoxia. Our results demonstrated that eIF5A hypusination inhibition modulates distinctive UPR pathways, a crucial mechanism for the protection against anoxia/reoxygenation.


Introduction
The endoplasmic reticulum (ER) is an intracellular organelle that plays an important role in synthesis, folding, modification and degradation, processing and trafficking of proteins [1]. Accumulation of unfolded or misfolded proteins in the lumen of ER results in a cellular stress response named ER stress or unfolded protein response (UPR). To face this ER stress, cells activate UPR adaptive pathways to restore protein homeostasis. Once this mechanism is saturated, unfolded proteins accumulate in the ER lumen and apoptotic pathways are activated. Thus, the balance between adaptative and apoptotic pathways plays a critical role in cell fate along ER stress [2,3]. Under physiological conditions, ER stress sensors (inositol-requiring protein 1 (IRE-1α), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6)) are inactivated by binding to the ER-resident chaperone BiP/GRP78 (immunoglobulin heavy chain binding protein/78 kDa glucose-regulated protein) on the side of the ER lumen [4,5]. In contrast, when cells are exposed to a stress generating proteins misfolding/unfolding, i.e., ER stress, BiP dissociates from these sensors to catch mis/unfolded proteins, hence, leading to the activation of the sensors downstream signaling pathways to tentatively solve this crisis [2]. induced CHOP expression during cold ischemia and reoxygenation, and their inhibitions allowed to increase cell survival [24].
Recently a new pharmacological approach identified the polyamine pathway and more particularly the eukaryotic initiation factor 5A (eIF5A) as a pharmacological target related to ischemia tolerance. eIF5A is activated by hypusination, a unique post-translational modification extremely well conserved across evolution [25]. This modification takes place on a specific lysine residue and is carried out from the polyamine spermidine by the successive action of deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). eIF5A has been originally characterized as a translation factor involved in protein synthesis initiation, but recently its described functions appear more diversified and complex as it promotes the translation of proteins with particular polyproline residues and participates to the nuclear exports of several mRNAs in cooperation with the exportin complex [26]. Of note, eIF5A exists under two isoforms, A1 ubiquitously expressed, and A2, extensively studied in cancer and displaying alternative functions [27]. We have previously demonstrated that GC7 (N1-guanyl-1,7-diamineoheptane), a spermidine analogue specifically inhibiting DHPS activity and thus hypusination of eIF5A, allows a protection from ischemic events at the cell and organ level [28][29][30]. Transposed to a preclinical model of renal transplantation in pig, GC7 pretreatment of the donor allowed a better functional recovery in the recipient [29,30]. From a metabolic point of view, GC7 decreases oxidative phosphorylation and reversibly reprograms the function and metabolism of kidney cells [31]. This metabolic shift drives cells toward the use of glucose as their single source of energy permitting cells therefore to be oxygen-independent through anaerobic glycolysis [30,31]. In addition, GC7 has been shown to decrease the oxidative stress, a phenomenon well-known to be associated to I/R both in vivo and in vitro, limiting I/R effects [29,30].
Herein we demonstrated that a GC7 preconditioning prevents kidney proximal tubule cell death induced by both cold and warm anoxia, both associated with a strong ER stress. This protective effect is triggered by IRE-1α/XBP1 pathway activation and associated to an inhibition of PERK/eIF2α pathway and an increased expression of the chief chaperone BIP.

Cell Survival Analysis
Cell viability was evaluated by using Calcein-AM 1 µM (Invitrogen, Thermofisher Scientific, Illkirch-Graffenstaden, France) and Ethidium homodimer 4 µM (Sigma-Aldrich) to stain live and dead cells respectively. Cells were incubated for 30 min at 37 • C in presence of both dyes and then washed two times with HBSS (w/o Phenol Red) before analysis. Fluorescent micrographs were recorded using an observer D1 microscope (Carl Zeiss, Le Pecq, France) and analyzed with imageJ software. Results are expressed as "survival rate" compared to corresponding normoxia condition.

Immunofluorescence
Cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100/BSA 3% for 10 min, and then sequentially incubated with primary antibody overnight at 4 • C (anti-CHOP, Cell Signaling technology #2895, dilution 1:100) and with the relevant secondary antibody coupled to Alexa-594 (Invitrogen, dilution 1:500) for 30min at RT. Cells were finally mounted and visualized with an inverted epifluorescence Axiovert microscope (Carl Zeiss, Le Pecq, France) under oil immersion and photographs were acquired and processed with Zen software (Carl Zeiss).

mRNA Analysis
Procedures follow MIQE recommendations [36]. Total RNA was extracted using TRIzol (Invitrogen, Thermofisher Scientific) according to the manufacturer's instructions. In addition, tissues were solubilized using Precellys tissue homogenizer in TRIzol reagent and using CK14 beads. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using M-MLV-RT (Promega, Charbonnières-les-Bains, France). SYBR qPCR premix Ex TaqII from Takara (Ozyme) was used for quantitative PCR (qPCR), and assays were run on a StepOne Plus ABI real-time PCR machine (PerkinElmer Life and Analytical Sciences, Boston, MA, USA). Expression of selected genes was normalized to that of gapdh and rplp0 housekeeping genes, and then quantified using the comparative-∆Ct method. Primer sequences are available upon request.

Statistical Analysis
Data were analyzed by GraphPad Prism 6 software and using ANOVA followed by Tukey's post hoc test to assess statistical differences between multiple experimental groups. Differences were considered statistically significant with p < 0.01. Data are displayed as scatter plot of independent values and group mean values ± SD.

GC7 Preconditioning Prevents Anoxia-and Tunicamycin-Induced Cell Death
We previously demonstrated that inhibition of eIF5A hypusination by GC7 prevented anoxia-induced cell death when applied for 24 h before stress. Here, we modified our pretreatment protocol to highlight the potential preconditioning effect of GC7 in a different time frame. For this, confluent kidney cells, originating from the Proximal Convoluted Tubule (PCT cells [33]), were treated with 30 µM GC7 for only 8 h, washed and maintained in culture medium without GC7 for an additional period of 24 h before being exposed to stressful conditions ( Figure 1A). We observed that this preconditioning protocol was sufficient to protect cells from warm anoxia-induced cell death (24 h, 37 • C, ≤0.1% O 2 ) as assessed by the live/dead fluorescent labelling assay ( Figure 1B). Indeed, up to 90 % of PCT cells died after 24 h of anoxia as previously described, while less than 20 % of cells died when they were pretreated with GC7 ( Figure 1C). To further mimic in vivo transplantation conditions (cold ischemia/reperfusion process), we exposed the cells to cold anoxia (16 h, 4 • C, ≤0.1% O 2 ) followed or not by a normothermic reoxygenation step (2 h, 37 • C, 20% O 2 ). We chose 16 h of anoxia because increasing the time of cold anoxia led to maximal death of untreated cells and thus did not allow to study the impact of reoxygenation. Untreated PCT cells showed a death rate of approximately 60% after cold anoxia and 80% after reoxygenation ( Figure 1C). Mortality was completely prevented when cells were pretreated with GC7 24 h before. groups. Differences were considered statistically significant with p < 0.01. Data are displayed as scatter plot of independent values and group mean values ± SD.

GC7 Preconditioning Prevents Anoxia-and Tunicamycin-Induced Cell Death
We previously demonstrated that inhibition of eIF5A hypusination by GC7 prevented anoxia-induced cell death when applied for 24 h before stress. Here, we modified our pretreatment protocol to highlight the potential preconditioning effect of GC7 in a different time frame. For this, confluent kidney cells, originating from the Proximal Convoluted Tubule (PCT cells [33]), were treated with 30 μM GC7 for only 8 h, washed and maintained in culture medium without GC7 for an additional period of 24 h before being exposed to stressful conditions ( Figure 1A). We observed that this preconditioning protocol was sufficient to protect cells from warm anoxia-induced cell death (24 h, 37 °C, ≤0.1% O2) as assessed by the live/dead fluorescent labelling assay ( Figure 1B). Indeed, up to 90 % of PCT cells died after 24 h of anoxia as previously described, while less than 20 % of cells died when they were pretreated with GC7 ( Figure 1C). To further mimic in vivo transplantation conditions (cold ischemia/reperfusion process), we exposed the cells to cold anoxia (16 h, 4 °C, ≤0.1% O2) followed or not by a normothermic reoxygenation step (2 h, 37 °C, 20% O2). We chose 16 h of anoxia because increasing the time of cold anoxia led to maximal death of untreated cells and thus did not allow to study the impact of reoxygenation. Untreated PCT cells showed a death rate of approximately 60% after cold anoxia and 80% after reoxygenation ( Figure 1C). Mortality was completely prevented when cells were pretreated with GC7 24 h before. later, exposed to either normoxia, warm anoxia (≤0.1% O2 at 37 °C for 24 h) or cold anoxia (≤0.1% O2 at 4 °C for 16 h) followed or not by a normothermic reoxygenation step ("Rep", 20% O2 at 37 °C for 2 h), or treated with tunicamycin 10 μg/ml. At the end of the experiment, cell viability was evaluated using a live/dead fluorescence assay. (B) Representative images of each condition with live (green, calcein-FITC) and dead (read, ethidium bromide homodimer) cells and (C) Corresponding survival rate (live/dead cells). Scatter plot representation of individual values along with mean ± SD for each condition, n = 5-7. *** = p ≤ 0.0001. GC7 treatment has been shown to induce a metabolic shift toward anaerobic glycolysis and limit oxidative stress generation, two effects protecting cells from O 2 deprivation [30,31]. Nevertheless, several studies demonstrated the key role of ER stress in the alteration of cell function and survival during ischemia/reperfusion [24]. To determine if GC7 was able to protect cells from cell death induced by UPR activation, we treated cells with 10 µg/mL of tunicamycin which is a well-known inductor of ER stress. As shown in Figure 1, cells pretreated with GC7 were protected from cell death induced by a 24 h tunicamycin treatment. To define the part of ER stress in cell death induced by warm and cold anoxia, we also analysed chop mRNA expression and CHOP nuclear translocation which is the ultimate event linking ER stress to cell death. Chop mRNA expression was increased after either warm or cold anoxia and reoxygenation in control cells but not in cells pretreated with GC7 ( Figure 2A). Concomitantly, PCT cells exposed to warm or cold anoxia and reoxygenation exhibited a strong positive nuclear staining for CHOP ( Figure 2B). This staining was absent in cells pretreated with GC7 prior the anoxic exposure. These interesting results suggested a new link between the modulation of ER stress and the protective effect of GC7. They also prompted us to thoroughly analyzed the expression and activation of the different ER stress pathways components in PCT cells preconditioned with GC7 and exposed to anoxic conditions. Cells 2023, 12, 409 6 of 15 GC7 treatment has been shown to induce a metabolic shift toward anaerobic glycolysis and limit oxidative stress generation, two effects protecting cells from O2 deprivation [30,31]. Nevertheless, several studies demonstrated the key role of ER stress in the alteration of cell function and survival during ischemia/reperfusion [24]. To determine if GC7 was able to protect cells from cell death induced by UPR activation, we treated cells with 10 μg/mL of tunicamycin which is a well-known inductor of ER stress. As shown in Figure  1, cells pretreated with GC7 were protected from cell death induced by a 24 h tunicamycin treatment. To define the part of ER stress in cell death induced by warm and cold anoxia, we also analysed chop mRNA expression and CHOP nuclear translocation which is the ultimate event linking ER stress to cell death. Chop mRNA expression was increased after either warm or cold anoxia and reoxygenation in control cells but not in cells pretreated with GC7 ( Figure 2A). Concomitantly, PCT cells exposed to warm or cold anoxia and reoxygenation exhibited a strong positive nuclear staining for CHOP ( Figure 2B). This staining was absent in cells pretreated with GC7 prior the anoxic exposure. These interesting results suggested a new link between the modulation of ER stress and the protective effect of GC7. They also prompted us to thoroughly analyzed the expression and activation of the different ER stress pathways components in PCT cells preconditioned with GC7 and exposed to anoxic conditions. GC7 pretreatment prevents the rise in CHOP expression and its nuclear translocation following anoxia and reoxygenation. PCT cells were pre-treated for 8 h either with a vehicle or with 30 μM GC7 and 24 hours later, exposed to either normoxia, warm anoxia (≤0.1% O2 at 37 °C for 24

GC7 Modulates Differentially ER Stress Pathway Activation during Anoxia
To investigate the activation of the different ER stress pathways, we first assessed xbp1 mRNA splicing and the expression of two of its target genes (bip and edem) as indicator of IRE-1α pathway activation. Warm anoxia, causing dramatic mortality after 24 h, induced a strong splicing of xbp1 correlated with increased expression of bip ( Figure 3A). GC7 pretreatment did not modify xbp1 splicing and bip mRNA expression under warm anoxia ( Figure 3A) but strongly increased BiP protein level ( Figure 3B). Otherwise, edem mRNA expression was not modified under warm anoxia independently of GC7 pretreatment ( Figure 3A). Interestingly, when cells were maintained under normoxia, pretreatment with GC7 induced a slight but significant increase in xbp1 splicing, in edem and bip mRNA levels as well as in BiP protein level ( Figure 3A,B). To analyze PERK pathway, we evaluated eIF2α phosphorylation which is due to PERK activation. Warm anoxia induced eIF2α phosphorylation, which was not the case when cells were pretreated with GC7 ( Figure 3B). Nuclear cleaved ATF6, which is an indicator of ATF6 pathway activity, was strongly decreased under warm anoxia independently of GC7 pretreatment ( Figure 3B). Finally, Protein Disulfide Isomerase (PDI) level, a chaperone protein rectifying incorrect disulfide bonding involved in ER stress resolution, were equivalent in untreated and GC7 treated cells both under normoxia and warm anoxia ( Figure 3B). h), or cold anoxia (≤0.1% O2 at 4 °C for 16 h) followed or not by a normothermic reoxygenation step ("Rep", 20% O2 at 37 °C for 2 h). At the end of the experiment cells were either (A) lysed for mRNA expression analysis or (B) fixed and processed for CHOP immunodetection. (A) Scatter plot representation of individual values along with mean ± SD for each condition of chop mRNA n = 3-5, * = p ≤ 0.01; ** = p ≤ 0.001; ***= p ≤ 0.0001. (B) Representative images of CHOP immunostaining (red) and nuclei counterstained with DAPI (blue).

GC7 Modulates Differentially ER Stress Pathway Activation during Anoxia
To investigate the activation of the different ER stress pathways, we first assessed xbp1 mRNA splicing and the expression of two of its target genes (bip and edem) as indicator of IRE-1α pathway activation. Warm anoxia, causing dramatic mortality after 24 h, induced a strong splicing of xbp1 correlated with increased expression of bip ( Figure 3A). GC7 pretreatment did not modify xbp1 splicing and bip mRNA expression under warm anoxia ( Figure 3A) but strongly increased BiP protein level ( Figure 3B). Otherwise, edem mRNA expression was not modified under warm anoxia independently of GC7 pretreatment ( Figure 3A). Interestingly, when cells were maintained under normoxia, pretreatment with GC7 induced a slight but significant increase in xbp1 splicing, in edem and bip mRNA levels as well as in BiP protein level ( Figure 3A,B). To analyze PERK pathway, we evaluated eIF2α phosphorylation which is due to PERK activation. Warm anoxia induced eIF2α phosphorylation, which was not the case when cells were pretreated with GC7 (Figure 3B). Nuclear cleaved ATF6, which is an indicator of ATF6 pathway activity, was strongly decreased under warm anoxia independently of GC7 pretreatment ( Figure 3B). Finally, Protein Disulfide Isomerase (PDI) level, a chaperone protein rectifying incorrect disulfide bonding involved in ER stress resolution, were equivalent in untreated and GC7 treated cells both under normoxia and warm anoxia ( Figure 3B).  To understand the protective mechanism provided by GC7 treatment, we decided to expose PCT cells to cold anoxia for 4 or 16 h followed or not by 2 h of warm reoxygenation. Of note, in contrast to 16 h of cold anoxia, 4 h under this condition did not induce cell death or CHOP nuclear translocation even after normothermic reoxygenation (data not shown). Under control condition, 4 h of anoxia followed or not by normothermic reoxygenation did not affect mRNA levels of ER stress pathways but induced ATF6 nuclear localization. Longer anoxia (16 h) increased xbp1 splicing and eIF2α phosphorylation ( Figure 4A,B). Interestingly, no clear modulation of ER stress pathways was found after 16 h of cold anoxia and 2 h of reoxygenation, suggesting normalization of protein folding and translation function in cells. As expected, PDI levels remained unchanged across all tested conditions ( Figure 4B). Moreover, following 4 and 16 h of anoxia, GC7 pre-treatment increased xbp1 splicing correlated with an increased expression of edem and bip mRNAs ( Figure 4A). These expressions were further increased after 2 h of reoxygenation, certainly due to sustained activity of the spliced XBP1 protein despite the decrease in spliced mRNA level. bip mRNA over-expression was confirmed at the protein level, with an increase in the level of BIP whatever the time of cold anoxia and reoxygenation ( Figure 4B), while the PDI level did not change after GC7 preconditioning. Nuclear ATF6 levels and eIF2α phosphorylation were decreased after 4 h of cold anoxia and after 16 h for eIF2α when cells have been pretreated with GC7 ( Figure 4B). translation function in cells. As expected, PDI levels remained unchanged across all tested conditions ( Figure 4B). Moreover, following 4 and 16 h of anoxia, GC7 pre-treatment increased xbp1 splicing correlated with an increased expression of edem and bip mRNAs ( Figure 4A). These expressions were further increased after 2 h of reoxygenation, certainly due to sustained activity of the spliced XBP1 protein despite the decrease in spliced mRNA level. bip mRNA over-expression was confirmed at the protein level, with an increase in the level of BIP whatever the time of cold anoxia and reoxygenation ( Figure 4B), while the PDI level did not change after GC7 preconditioning. Nuclear ATF6 levels and eIF2α phosphorylation were decreased after 4 h of cold anoxia and after 16 h for eIF2α when cells have been pretreated with GC7 ( Figure 4B).
Similar results were obtained when the cells were exposed to mild hypoxia (1% O2, 37°C) for 24 h. Indeed, GC7 preconditioning resulted in increased xbp1 splicing concomitantly with increased BIP mRNA and protein expressions ( Figure S2). It is interesting to note that, mild hypoxia alone does not lead to cell death, suggesting a potential broader/conserved protective mechanism.
Together, these results demonstrate that GC7 preconditioning increased IRE-1α pathway activity during warm and cold anoxia but inhibited PERK and ATF6 pathways during cold anoxia and normothermic reoxygenation.  Similar results were obtained when the cells were exposed to mild hypoxia (1% O 2 , 37 • C) for 24 h. Indeed, GC7 preconditioning resulted in increased xbp1 splicing concomitantly with increased BIP mRNA and protein expressions ( Figure S2). It is interesting to note that, mild hypoxia alone does not lead to cell death, suggesting a potential broader/conserved protective mechanism.
Together, these results demonstrate that GC7 preconditioning increased IRE-1α pathway activity during warm and cold anoxia but inhibited PERK and ATF6 pathways during cold anoxia and normothermic reoxygenation.

Involvement of IRE-1α Pathway in GC7 Protective Effect
To determine the importance of the IRE-1α pathway in the protection conferred by the GC7 preconditioning, we used 4µ8c (1 µM), a known inhibitor of IRE-1α to prevent xbp1 splicing ( Figure S3). 4µ8c treatment did not change control cells viability to either cold or warm anoxia but led to a partial inhibition of the protective effect of GC7 against warm anoxia ( Figure 5A) and a complete inhibition against cold anoxia ( Figure 5B). Interestingly, under normoxia, 4µ8c treatment did not show any toxic effect on PCT cells in presence or absence of GC7 ( Figure 5).

Involvement of IRE-1α Pathway in GC7 Protective Effect
To determine the importance of the IRE-1α pathway in the protection conferred by the GC7 preconditioning, we used 4μ8c (1 μM), a known inhibitor of IRE-1α to prevent xbp1 splicing ( Figure S3). 4μ8c treatment did not change control cells viability to either cold or warm anoxia but led to a partial inhibition of the protective effect of GC7 against warm anoxia ( Figure 5A) and a complete inhibition against cold anoxia ( Figure 5B). Interestingly, under normoxia, 4μ8c treatment did not show any toxic effect on PCT cells in presence or absence of GC7 ( Figure 5). An mRNA analysis demonstrated that 4μ8c treatment at the time of both warm and cold anoxia resulted in an expected inhibition of xbp1 splicing (Figure 6), particularly visible with the decrease in spliced xbp1 without affecting the level of unspliced xbp1 mRNA ( Figure S3). Concomitantly, as bip and edem expressions are partly controlled by spliced XBP1, we can observe a decrease in mRNA expressions, but only partial following 4μ8c treatment ( Figure 6). An mRNA analysis demonstrated that 4µ8c treatment at the time of both warm and cold anoxia resulted in an expected inhibition of xbp1 splicing (Figure 6), particularly visible with the decrease in spliced xbp1 without affecting the level of unspliced xbp1 mRNA ( Figure S3). Concomitantly, as bip and edem expressions are partly controlled by spliced XBP1, we can observe a decrease in mRNA expressions, but only partial following 4µ8c treatment ( Figure 6).
Inhibition of XBP1 splicing as well as the decrease in bip mRNA expression could be associated with the loss of the GC7 protective effect. Analysis of BiP protein level did not confirm this hypothesis. Indeed, while BiP level was increased in PCT cells pretreated with GC7, we did not observe a decrease of BiP when cells were treated with 4µ8c during anoxia (Figure 7). Furthermore, we have previously shown that GC7 pretreatment limited chop mRNA expression during warm and cold anoxia ( Figure 2A) and chop expression is partly controlled by spliced XBP1. Surprisingly, this inhibition of chop mRNA expression in GC7 pretreated cells was not prevented by 4µ8c treatment (Figure 6). Inhibition of XBP1 splicing as well as the decrease in bip mRNA expression could be associated with the loss of the GC7 protective effect. Analysis of BiP protein level did not confirm this hypothesis. Indeed, while BiP level was increased in PCT cells pretreated with GC7, we did not observe a decrease of BiP when cells were treated with 4μ8c during anoxia ( Figure 7). Furthermore, we have previously shown that GC7 pretreatment limited chop mRNA expression during warm and cold anoxia ( Figure 2A) and chop expression is partly controlled by spliced XBP1. Surprisingly, this inhibition of chop mRNA expression in GC7 pretreated cells was not prevented by 4μ8c treatment (Figure 6).   Inhibition of XBP1 splicing as well as the decrease in bip mRNA expression associated with the loss of the GC7 protective effect. Analysis of BiP protein leve confirm this hypothesis. Indeed, while BiP level was increased in PCT cells pr with GC7, we did not observe a decrease of BiP when cells were treated with 4μ8 anoxia (Figure 7). Furthermore, we have previously shown that GC7 pretreatmen chop mRNA expression during warm and cold anoxia ( Figure 2A) and chop expr partly controlled by spliced XBP1. Surprisingly, this inhibition of chop mRNA ex in GC7 pretreated cells was not prevented by 4μ8c treatment (Figure 6).

Impact of GC7 Preconditioning on ER Stress Pathway
A preconditioning treatment is thought to prepare cells to resist to future stress by inducing a small stress response, according to the hormesis concept. Thus, we analyzed PCT cells at the end of the 8 h of GC7 treatment and 16 h later. At the end of treatment, GC7 did not induce by itself an activation of ER stress pathways ( Figure 8A,B). In contrast, 16 h later PCT cells pretreated with GC7 displayed an increase in bip and edem mRNA levels which were related to a slight increase in xbp1 splicing ( Figure 8A). While phosphorylation of eIF2α decreased without reaching significance 16 h after GC7 pretreatment, a significant increase in the amount of BiP protein was found ( Figure 8B). Cells 2023, 12, 409 11 of 15 experiment cells were lysed for protein expression analysis. Representative western blots of BiP and β-tubulin protein levels. Scatter plot representation of individual values along with mean ± SD for each condition, n = 3-4. * = p ≤ 0.01; **= p ≤ 0.001.

Impact of GC7 Preconditioning on ER Stress Pathway
A preconditioning treatment is thought to prepare cells to resist to future stress by inducing a small stress response, according to the hormesis concept. Thus, we analyzed PCT cells at the end of the 8 h of GC7 treatment and 16 h later. At the end of treatment, GC7 did not induce by itself an activation of ER stress pathways ( Figure 8A,B). In contrast, 16 h later PCT cells pretreated with GC7 displayed an increase in bip and edem mRNA levels which were related to a slight increase in xbp1 splicing ( Figure 8A). While phosphorylation of eIF2α decreased without reaching significance 16 h after GC7 pretreatment, a significant increase in the amount of BiP protein was found ( Figure 8B).

Discussion
Warm and cold ischemia as well as reperfusion lead to various organ/cells damages [13,14] due to energy shortage, oxidative stress and ER stress [15,16]. Numerous studies have described the benefit of many compounds when used as a pre-or per-conditioning I/R treatment, notably GC7 [37]. This compound is a specific inhibitor of DHPS and thus of eIF5A hypusination, and has been characterized as a valuable in vivo protective agent against renal I/R in rat and pig [29,30]. From a mechanistic point of view, preconditioning with GC7 leads to metabolic changes as it significantly decreases mitochondrial oxidative phosphorylation and promotes anaerobic glycolysis, which is a mechanism of adaptation and response to oxygen deprivation. This allows cells to survive under anoxic conditions for more than 24 h, with the cells completely returning to their original metabolic status after a 48 h recovery period under normoxic conditions [30,31]. In addition, preconditioning with GC7 lowers oxidative stress under anoxic and reoxygenation conditions,

Discussion
Warm and cold ischemia as well as reperfusion lead to various organ/cells damages [13,14] due to energy shortage, oxidative stress and ER stress [15,16]. Numerous studies have described the benefit of many compounds when used as a pre-or per-conditioning I/R treatment, notably GC7 [37]. This compound is a specific inhibitor of DHPS and thus of eIF5A hypusination, and has been characterized as a valuable in vivo protective agent against renal I/R in rat and pig [29,30]. From a mechanistic point of view, preconditioning with GC7 leads to metabolic changes as it significantly decreases mitochondrial oxidative phosphorylation and promotes anaerobic glycolysis, which is a mechanism of adaptation and response to oxygen deprivation. This allows cells to survive under anoxic conditions for more than 24 h, with the cells completely returning to their original metabolic status after a 48 h recovery period under normoxic conditions [30,31]. In addition, preconditioning with GC7 lowers oxidative stress under anoxic and reoxygenation conditions, although it has not been characterized whether this is a cause or a consequence of the GC7-induced protection [30]. Similar results were found in an animal model of brain-dead donor pigs, in which preconditioning with GC7 decreased oxidative stress production in the graft [29]. In conclusion, GC7 preconditioning prevents energy shortage and limits oxidative stress in kidney submitted to I/R, which participates to improve graft function recovery. Nevertheless, GC7 preconditioning impact on ER stress remained to be defined as this stress has a pivotal role in graft outcome.
Kidney I/R in mice activates ER stress pathways, first as an adaptative response early after ischemia, and later when ischemia is sustained to a proapoptotic response [18]. These activations seem mainly localized in tubular epithelial cells, particularly within the corticomedullary junction which is a region highly sensitive to oxygen variation [16,23]. ER stress is highly deleterious and central in the sensitivity of kidney to I/R, and modulation of ER stress protected partly from I/R injury. Interestingly, several studies linked ER stress activation to the development of renal fibrosis after I/R [21,22]. Hence, we previously demonstrated that GC7 preconditioning of donor pigs limited drastically the development of renal fibrosis in recipients several months after the graft [29,30], potentially linking GC7 treatment to ER stress prevention. Herein, we demonstrated that PCT cells pre-treated with GC7 were able to resist to either warm or cold anoxia, and that resistance was linked to a decreased in CHOP nuclear translocation, known to induce an apoptotic program. Similarly, we found a decrease of PERK pathway activity characterized by a reduced phosphorylation of eIF2α. Nevertheless, GC7 did not inhibit all UPR pathways as we found an increased xbp1 splicing and BiP expression. This demonstrated a differential impact of GC7 treatment on UPR stress pathways, perhaps highlighting a positive effect on the adaptive response concomitantly with an inhibition of proapoptotic ER stress pathways.
It is well-known that ER stress pathways activation is not simultaneous and can lead to adaptative or proapoptotic responses. This is especially true for endothelial cells during I/R episodes, as they display a sequential activation of UPR pathways depending on the duration of anoxia and leading first to adaptative response and then to cell death [24]. Using the same approaches on PCT cells, we showed that, while IRE-1α is activated early and sustained along cold anoxia, ATF6 was only activated in the first hours while PERK pathway was only activated during sustained anoxia. As expected, CHOP nuclear translocation happened after 16 h of cold anoxia but not for shorter exposure times (4 h anoxia, not shown), correlating anoxia duration and cell death rate. This response to anoxia of tubular epithelial cells is sequential but different from that observed in endothelial cells [24], demonstrating the multiplicity and the complexity of the UPR response in different cells/tissues facing adverse conditions. GC7 preconditioning led to an increase of xbp1 splicing and BiP expression, the first certainly leading to the second. We assume that increased BiP levels in GC7 pretreated cells allow a higher capacity to chaperone proteins for their folding. This could increase the cell's tolerance threshold to excessive folding demand at the time of ER stress, here induced by anoxia, and consequently limiting the activation of UPR downstream pathways, PERK-eIF2α-ATF4 and ATF6. As no nuclear ATF6 was found after 16 h (cold) or 24 h (warm) of anoxia, we can postulate that the PERK pathway controls CHOP expression in PCT cells. Thus, the survival level obtained in GC7 pretreated cells could be due ultimately to the inhibition of this pathway characterized in our work by the inhibition of eIF2α phosphorylation under anoxia. Altogether, these results suggest that GC7 treatment induces xbp1 splicing which in turn increases BiP levels allowing to maintain inactive the PERK pathway at the time of stress. Increased splicing of xbp1 was a necessary step to trigger the GC7 protective effect, as splicing inhibition by the IRE-1α inhibitor 4µ8c reversed GC7 mediated protection. Intriguingly, while we correlated this to reduce bip mRNA levels, we did not confirm this decrease at the protein level. As 4µ8c was added on cells only during anoxia, we can suppose that the increase in BiP protein levels found under anoxia was more related to the effect of GC7 preconditioning before stress which potentially enhances the tolerance of cells to stress. In contrast, during anoxia the increase in XBP1 splicing seemed related to another stress tolerance pathway independent of BiP and involving one of the other actors of UPR not identified in our work.
We did not link the loss of GC7 protective effect under 4µ8c treatment to an increase in chop expression. This was not completely surprising because chop mRNA expression is mainly controlled by ATF4 and ATF6 pathways instead of by IRE-1α but it raises the question about the loss of the protective effect which could be unrelated to the CHOP pathway. Interestingly, in insulinoma cells, GC7 treatment protected cells from ER stressinduced death by decreasing CHOP level and caspase-3 cleavage, without affecting chop expression. In this situation, it was proposed that eIF5A hypusination was required for CHOP translation and thus that GC7 inhibited it [38]. As CHOP does not contain polyproline residue and has not been identified as a translational product of hypusinated eIF5A [39][40][41][42], we can suspect another role. Here, using a preconditioning treatment, we report a similar protective effect as observed in insulinoma cells, therefore excluding a direct and acute effect of GC7 on CHOP translation. Therefore, the underlying mechanism of the protective effect against ER stress-induced cell death conferred by GC7 treatment remains to be discovered.
Nevertheless, we clearly demonstrate that GC7 preconditioning delayed the activation of UPR when cells were exposed to a stress. This could be associated with an alteration of protein translation due to eIF5A hypusination inhibition by GC7 as hypusinated eIF5A is globally involved in translation, from translation initiation to the termination of multiple proteins [43]. Thus, several hypotheses are still relevant to explain the effect of GC7 on ER stress: (1) a decrease in protein synthesis which could limit ER stress amplitude; (2) an inhibition of translation of specific proteins involved in or modulating UPR; (3) an indirect effect involving the role of eIF5A in the translation of proteins involved in mitochondria activity and oxidative stress, both involved in ER stress activation [39].
A deeper analysis of the multiple pathways involved in ER stress and/or cell apoptosis is required to clearly characterize the protective effect of eIF5A hypusination inhibition under anoxia. Nevertheless, we demonstrated clearly that GC7 treatment modulates UPR by activating IRE-1α/XBP1 pathway while inhibiting PERK/eIF2α, concomitantly to preventing CHOP nuclear translocation. Together these results demonstrated a new protective effect conferred by GC7 against ER stress-induced cell death under anoxic environment. Knowing that ER stress has been linked to the development of renal fibrosis [19][20][21][22], the in vitro effect described here can be related to the anti-fibrotic action of GC7 during kidney transplantation [29,30]. Also, we must now transpose these results in more physiological situations, such as in an in vivo kidney model of warm and cold ischemia followed by reperfusion.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells12030409/s1, Figure S1: Time course of O 2 level decrease after switching to a 100% N2 atmosphere; Figure S2: GC7 increases XBP1 splicing and BIP expression under mild hypoxia; Figure S3: Impact of 4µ8c treatment on the splicing of XBP1.