Rapamycin Does Not Act as a Dietary Restriction Mimetic in the Protection against Ischemia Reperfusion Injury

Introduction: Short-term fasting protects against renal ischemia reperfusion injury (IRI). mTOR signaling is downregulated and may be involved in its protective effect. Rapamycin is considered a possible mimetic as it inhibits the mTOR pathway. This study examines the effect of rapamycin on renal IRI. Material and Methods: Mice were divided into four groups: ad libitum (AL), fasted (F), AL treated with rapamycin (AL+R), and F treated with rapamycin (F+R). Rapamycin was administered intraperitoneally 24 h before bilateral renal IRI was induced. Survival was monitored for 7 days. Renal cell death, regeneration, and mTOR activity were determined 48 h after reperfusion. Oxidative stress resistance of human renal proximal tubular and human primary tubular epithelial cells after rapamycin treatment was determined. Results: All F and F+R mice survived the experiment. Although rapamycin substantially downregulated mTOR activity, survival in the AL+R group was similar to AL (10%). Renal regeneration was significantly reduced in AL+R but not in F+R. After IRI (48 h), pS6K/S6K ratio was lower in F, F+R, and AL+R groups compared to AL fed animals (p = 0.02). In vitro, rapamycin also significantly downregulated mTOR activity (p < 0.001) but did not protect against oxidative stress. Conclusion: Rapamycin pretreatment does not protect against renal IRI. Thus, protection against renal IRI by fasting is not exclusively mediated through inhibition of mTOR activity but may involve preservation of regenerative mechanisms despite mTOR downregulation. Therefore, rapamycin cannot be used as a dietary mimetic to protect against renal IRI.


Introduction
Dietary restriction (DR), defined as reduced food intake without causing malnutrition, can be applied by short-term fasting or calorie restriction for a longer period. DR has been reported to extend lifespan in several organisms, from rodents [1] to other life forms including nonhuman primates [2]. Furthermore, animals undergoing DR have increased resistance to different forms of stress [3,4]. In humans, the effects of DR on health and lifespan are difficult to determine, although long-term DR seems to have a favorable impact on age-related morbidity including cardiovascular fitness, BMI, and insulin sensitivity [5][6][7]. Ischemia reperfusion injury (IRI) is still an inevitable problem during kidney transplantation, karger@karger.com www.karger.com/esr resulting in oxidative damage by production of reactive oxygen species. Especially proximal tubular cells are prone to oxidative stress. Previously, we showed that both short-term fasting (water only diet) and 14 days of 30% calorie restriction protect against renal IRI in C57BL/6 mice, improving both kidney function and survival [8]. In humans, we showed that DR improved outcome after kidney donation and transplantation and may improve the therapeutic window of toxic anticancer agents [9,10]. Although we showed that DR can attenuate renal IRI, it remains to be elucidated which underlying mechanisms cause these protective effects. Furthermore, applying fasting or any form of DR to humans before surgery is still considered unwanted. A DR mimetic, with similar effects as DR, would be an alternative method to attenuate renal IRI in kidney transplantation. The target of rapamycin (TOR) pathway is a nutrisensing pathway which is known for its longevity effects in C. elegans and Drosophila when suppressed [9,10]. Rapamycin, an mTOR inhibitor, has been shown to have similar effects as DR in inducing increased lifespan in yeast and in mice [11][12][13]. Since downregulation of mTOR activity is a molecular hallmark of fasting and is believed to be involved in its protective effect, rapamycin could act as a DR mimetic and may provide protection against IRI by downregulating mTOR signaling. This study was designed to assess the effects of rapamycin on in vitroinduced oxidative stress and to examine its potential as a DR mimetic to attenuate renal IRI in mice.

Materials and Methods
In vivo Male C57BL/6 mice (~25 g) were obtained from Harlan (Horst, The Netherlands). On arrival, mice were placed with 3-4 mice in individually ventilated cages and housed under standard circumstances. All mice had free access to water. All mice acclimated for 7 days before start of the experiment. Fasted mice had free access to water but no access to food for 3 days prior to IRI. Animal experiments were approved by the University Animal Experiments Committee (Protocol No. 105-12-13) under the Dutch National Experiments on Animals Act, compiled with Directive 86/609/EC (1986) of the Council of Europe.
Rapamycin 24 h before IRI, all mice received an intraperitoneal injection of 0.2 mL with either 5 mg/kg rapamycin (LC Laboratories) dissolved in dimethylsulfoxide (Sigma-Aldrich) and diluted in phosphatebuffered saline (PBS) or vehicle of dimethylsulfoxide diluted with PBS.
Surgical Procedure Animals were anaesthetized with isoflurane 4% (Pharmachemie BV) in O 2 and placed on heated plates. Through midline incision, both kidneys were localized and renal pedicles were dissected. A vascular clamp was placed for 37 min to induce bilateral renal ischemia. After the clamps were removed, reperfusion of the kidneys was visually assessed and the abdomen was closed with 5/0 Vicryl sutures. Animals were monitored until they regained consciousness before moving them from the operating room to the stable. At 48 h and 7 days after reperfusion, animals were exsanguinated through heart puncture under anesthesia. Kidneys were collected and immediately stored in either liquid nitrogen or 4% formaldehyde.
Apoptosis. To determine apoptosis, the protein levels of PARP/ cleaved PARP (Cell Signaling) were determined using 6% SDS-PAGE gel and the primary antibody PARP (1:1,000).

Western Blot
The protein expression levels of mTOR were determined by Western blot. 15 × 10 6 cells were used and blotted as described before.
Cell Death Cells were stressed using H 2 O 2 as described above. 120,000 cells were plated/well in a 6-well plate. 10 µL trypan blue (Sigma-Aldrich) was added to 10 µL cell suspension, and cell death was determined using a Bürker counter.

Apoptosis
For the in vitro apoptotic assay, 1 × 10 6 HK-2 cells were plated in a 96-well plate. Cells were stressed with H 2 O 2 as described before. Cells were stained with the PE Annexin V Apoptosis Detection Kit I (BD Pharmingen TM ). Cells were washed with PBS and resuspended in 1X binding buffer. 100 µL of the solution was transferred to a 5-mL tube. Then, 5 µL PE annexin V and 5 µL 7-AAD were added and incubated for 15 min at room temperature in the dark. After incubation, 400 µL of binding buffer was added and after, cells were acquired on FACS LSRII TM flow cytometer (BD Biosciences). A minimum of 20,000 live events were acquired based on forward and side scatter. Data analysis was performed using FlowJo TM (Tree Star) with PE channel on x-axis and PE-Cy5 on y-axis. The plots were dissected into three populations by placing a quadrant. Cells negative for both annexin V and 7-AAD were considered live nonapoptotic cells. Cells which were positive for annexin V and negative for 7-AAD were the cells undergoing apoptosis (early apoptosis), while the cells positive for both annexin-V and 7-AAD were considered late apoptotic or necrotic cells.

Statistical Analysis
All data are expressed as means ± SEM. Significance was tested with Student's t test using SPSS, version 20.0 (IBM Corp.). p values <0.05 were considered significant.

In vivo
Rapamycin Pretreatment Does Not Protect against Renal IRI As expected, survival after renal IRI in fasted animals was 100% at postoperative day 7. Fasting + rapamycin gave a similar survival rate (100%). There was no difference in survival between vehicle-treated controls and rapamycin-treated animals. Ninety percent of the animals died or had to be killed because of morbidity associated with irreversible kidney failure within 5 days after IRI (Fig. 1a).
mTOR Activity Decreases after Rapamycin Treatment Twenty-four hours after intraperitoneal injection with rapamycin, pS6K/S6K ratio was measured to determine mTOR activity. In AL mice treated with rapamycin and fasted animals treated with rapamycin, the pS6K/S6K ratio was significantly lower compared to vehicle-treated mice (p = 0.01 and p = 0.048, respectively). Fasted mice showed a similar decrease in mTOR activity. Forty-eight hours after reperfusion, pS6K/S6K ratios were significantly higher in AL mice compared to all other groups (p = 0.03, p = 0.01, p = 0.002, rapamycin-treated animals, fasted, and fasted with rapamycin treatment, respectively) (Fig. 1b).
Cellular proliferation is inhibited after renal IRI in mice treated with rapamycin. Proliferation is needed after renal IRI to replace apoptotic cells by healthy cells. PCNA is used as a marker for proliferation and cell repair. Fortyeight hours after reperfusion, PCNA levels in kidney tissue lysates were significantly higher in fasted animals (p = <0.001) compared to rapamycin-treated and control animals (Fig. 1c).
Rapamycin Does Not Reduce Apoptosis after IRI PARP is cleaved during the apoptotic cascade and is therefore used as a biomarker for apoptosis. At t = 0, all fasted animals (both with and without rapamycin) had significantly higher cleaved PARP/PARP ratio (p < 0.05). At 48 h after reperfusion, this ratio was significantly lower in fasted animals (p < 0.001, p < 0.001, and p = 0.04 compared to AL, F+, and AL+R, respectively). In fasted mice treated with rapamycin, this effect was diminished, resulting in significantly higher cleaved PARP/PARP ratio, compared to fasted animals. In AL mice, there was no difference between rapamycin-or vehicle-treated mice and no difference with fasted and rapamycin-treated mice (Fig. 1d).

In vitro
In mice, rapamycin did not protect against IRI. The pathophysiology of IRI, however, is complex, involving oxidative stress and a subsequent immunological response, which could cause even more cell damage. Therefore, we investigated the effect of rapamycin on oxidative stress resistance per se in two renal cell lines. Rapamycin Does Not Protect against Oxidative Stress in vitro Rapamycin-treated cells had similar growth curves as control cells, although rapamycin seemed to delay proliferation. H 2 O 2 is a robust stressor in both HK-2 cells and PTECs, resulting in reduced proliferation up to 72 h. In PTECs, regeneration was seen after 48 h, when cells started to proliferate again. Hypoxia/reoxygenation had a similar, although less distinctive, effect (Fig. 2). When cells were incubated with rapamycin before oxidative stress, a significant reduction in proliferation was seen compared to controls. Rapamycin did not improve proliferation after oxidative stress, compared to controls.
In HK-2 cells, no difference in cell death was seen between rapamycin and controls. Hypoxia/reoxygenation resulted in significantly more cell death at 24 h but not at 48 and 72 h (Fig. 3). Oxidative stress in rapamycin-treated cells resulted in significantly more cell death compared to controls and to rapamycin-treated cells without stress.
Cell death was equal between the two stressed groups (Fig. 3). In both HK-2 and PTEC, there was no difference in the percentage of dead cells after 1 h of incubation with H 2 O 2 between rapamycin-treated cells and control cells (Fig. 3). After staining with annexin V, no significant differences were found in percentage of apoptotic cells or necrotic cells when stressed HK-2 cells were compared to stressed HK-2 cells pretreated with rapamycin (Fig. 4).

Discussion
Both DR and mTOR inhibition by rapamycin are associated with prolonged longevity in multiple species [1,2,11,12]. DR, like short-term fasting, induces robust protection against renal IRI. Although preoperative overnight fasting is practiced to prevent aspiration of stomach content, the period is not sufficient to induce a protective response as seen after 3 days of fasting in mice. Our hypothesis was that rapamycin could act as a DR mimetic, inducing similar protection against IRI. Unfortunately, our study failed to prove this hypothesis. Rapamycin targets mTOR, an atypical serine/threonine protein kinase which forms two distinct complexes (mTORC1 and mTORC2) by interaction with several proteins. mTORC2 is downregulated after continuous administration of rapamycin [14]. One dose of rapamycin, as we administered, is therefore likely to only downregulate mTORC1 [15]. Most of the functions of mTOR are attributed to mTORC1, and less is known about mTORC2. mTORC1 activity is regulated through multiple cellular processes, like cell growth, cellular stress, energy status, oxygen consumption, and amino acid metabolism [14]. mTORC1 activation leads to protein and lipid synthesis in proliferating cells and activates metabolism and ATP production [14,16]. Likewise, inhibition of mTOR activity by DR decreases cellular proliferation and metabolism. Furthermore, mTOR inhibition stimulates autophagy, a process where cells are broken down and damaged organelles can be recycled, which is needed during nutritional deprivation [17]. Autophagy is implied as an obligatory event conferring resistance to oxidative stress [18]. Nevertheless, after IRI, proliferation is needed to regenerate the damaged renal cells. In earlier murine experiments, we showed that fasted mice resume normal food intake immediately after surgery and show significant weight gain [16]. This restoration to normal diet activates mTOR and promotes cell proliferation. mTOR activity in our study remained suppressed up to 48 h after IRI. Perhaps due to this extended mTOR inhibition, damaged cells cannot be regenerated, resulting in irreversible kidney failure. This suggests that the timing of mTOR inhibition determines the difference between attenuating or ameliorating IRI. Another explanation for differences in results in rapamycin studies is that the administration of rapamycin differs. Longevity experiments administered rapamycin in microencapsulated form mixed with food, while others report intraperitoneal injections [19]. Difference in pharmacokinetics and bioavailability, caused by different administration techniques, may induce different effects. We administered a single dose of rapamycin intraperitoneally 24 h before IRI. Both the dosages, as well as the route, timing, and number of administrations, may influence the outcome. In addition to our in vivo study, our in vitro experiments did not show any protective effects of rapamycin against oxidative stress. Conclusion mTOR inhibition by rapamycin before the induction of renal IRI does not mimic the protective effect against IRI induced by DR. The difference in kinetics of mTOR inhibition and activation between DR and refeeding and