Quiescence-inducing neurons-induced hypometabolism ameliorates acute kidney injury in a mouse model mimicking cardiovascular surgery requiring circulatory arrest

Objectives Acute kidney injury is a serious complication after cardiovascular surgery requiring circulatory arrest. It is reported that mice can be induced into a hibernation-like hypometabolic state by stimulating a specific neuron located at the hypothalamus (quiescence-inducing neurons-induced hypometabolism [QIH]). Here, we investigated the efficacy of QIH for the amelioration of acute kidney injury in an experimental circulatory arrest using a transgenic mouse model. Methods We genetically prepared mice in which QIH can be conditionally induced (QIH-ready mice). Mice were divided into 4 groups (n = 6 for each): QIH-ready normothermia (QN), QIH-ready hypothermia (QH), control normothermia (CN), and control hypothermia (CH). After induction of QIH, left thoracotomy and descending aorta crossclamping were conducted. After reperfusion, we collected kidneys and evaluated histologic changes and serum biochemical markers, specifically neutrophil gelatinase-associated lipocalin and cystatin C, indicating early kidney injury. Results Normothermia showed higher tubular injury scores than those in hypothermia (QN vs QH [P = .0021] and CN vs CH [P < .001]). QN exhibited lower neutrophil gelatinase-associated lipocalin and cystatin C levels than those in CN (neutrophil gelatinase-associated lipocalin: CN vs QN: 1.51 ± 0.71 vs 0.82 ± 0.32; P = .0414 and cystatin C: 1.48 ± 0.39 vs 0.71 ± 0.26; P = .0015). There was no significant difference between QN and QH. Conclusions QIH partly ameliorated acute kidney injury in a mouse ischemia model even in normothermia. QIH might be a promising approach to achieving sufficient kidney protection without hypothermic circulatory arrest in the future.

Q neurons-induced hypometabolism ameliorates acute kidney injury mediated by ischemia even in normothermia in a mice model mimicking cardiovascular surgery requiring circulatory arrest. PERSPECTIVE We have experimentally validated that Q neuronsinduced hypometabolism (QIH) can protect the kidneys from acute kidney injury (AKI) mediated by ischemia in cardiovascular surgeries requiring circulatory arrest even in normothermia. QIH may contribute to establishing a new strategy to prevent AKI in cardiovascular surgeries requiring circulatory arrest without deep hypothermia in the future.
Video clip is available online.
Acute kidney injury (AKI) is a major complication after cardiovascular surgery requiring circulatory arrest and is associated with increased short-term and long-term mortality. The incidence of AKI after aortic surgery requiring circulatory arrest is reported to be approximately 5% to 50%, which is higher than those of coronary artery bypass surgeries or valve surgeries [1][2][3] ; indicating that circulatory arrest and associated kidney ischemia is a cause of the AKI.
It is well recognized that tissue oxygen consumption decreases in accordance with the reduction of body temperature, and the advantages of the reduction of body temperature in resuscitative medicine in cases of cardiopulmonary arrest or brain death are numerously reported since the 1950s. [4][5][6] In the field of cardiovascular surgery requiring circulatory arrest, induction of hypometabolism by deep hypothermia below 20 C is broadly applied since the 1970s for organ protection during the circulatory arrest. 7 However, due to the tendency of postoperative coagulopathy and platelet dysfunction mediated by hypothermia, 8 deep hypothermic circulatory arrest (DHCA) is associated with increased postoperative bleeding and blood transfusion requirement. 9 Furthermore, DHCA is a cause of prolonged surgical duration, which may increase the risk of postoperative infectious complications, 10,11 which encouraged surgeons and researchers to find an alternative to achieve sufficient organ protection during cardiovascular surgeries requiring circulatory arrest.
It is well known that some mammals, such as squirrels, can undergo a state of decreased physiological activity associated with reduced body temperature and metabolic activities called hibernation or torpor. 12,13 The hibernators are capable of living in low body temperature and metabolic state under extremely reduced food intake and energy production. 12,14 The fundamental of hibernation is the resistance to the hypometabolic state, or the mechanism allowing the animals to stay healthy even with a lower oxygen supply. Mice are recognized as a nonhibernator; however, our recent research revealed that mice can enter a hibernation-like state by stimulating pyroglutamylated RFamide peptide (QRFP)-containing neurons located at the hypothalamus (quiescence-inducing neurons [Q neurons]). 15 The physiological function of Q neurons is not fully documented, yet, we found that excitation of Q neurons induces Q neurons-induced hypometabolism (QIH), a multiday hypometabolic state resembling hibernation. Moreover, we found that QIH mice exhibit decreased heart and respiratory rates associated with an extremely reduced rate of oxygen consumption (VO 2 ). 15 This discovery indicates that nonhibernators, including humans, can enter a hibernation-like hypometabolic state. Because hypometabolism resistance is the key function of torpor, QIH may possibly contribute to kidney protection under ischemic conditions. To validate this hypothesis, we investigated the efficiency of kidney protection during experimental circulatory arrest in normal and reduced body temperature conditions, using a transgenic mice model in which hypometabolism is induced by Q neurons activation.

MATERIALS AND METHODS Animals
We prepared 36 male C57BL/6J background mice in which Q neurons specifically express codon-improved Cre recombinase (iCre), a Cre recombinase (Qrfp iCre heterozygous mice), aged 16 to 23 weeks and of 24.4 to 36.2 g body weight as previously reported. 15 The Qrfp iCre heterozygous mice were generously provided by Dr Takeshi Sakurai, University of Tsukuba and bred at RIKEN Center for Biosystems Dynamics Research. We prepared QIH-ready mice as previously reported. 15 In brief, the Qrfp iCre heterozygous mice were anesthetized with isoflurane and positioned in a stereotaxic frame (David Kopf Instruments) to inject adeno-associated virus (AAV) at the hypothalamus to introduce designer receptors exclusively activated by designer drugs. AAV8-hSyn-DIO-hM3D (Gq)-mCherry (Addgene; 44361-AAV8, Lot #v78582, 2.1 3 10 13 genome copies/mL) 0.3 mL was injected at the position of anterior-posterior (þ0.38 mm), medial-lateral (AE0.30 mm), and dorsal-ventral (-5.25 mm). Virus was injected at a controlled rate of 0.1 mL/min using a Hamilton needle syringe. When this virus infects the cells expressing iCre, the cell will start expressing hM3Dq, which can excite the neurons at the existence of clozapine-Noxide (CNO) (designer receptors exclusively activated by designer drugs system). In this case, intraperitoneal injection of CNO will induce QIH through excitation of Q neurons. 15,16 For the control, we injected AAV8-hSyn-DIO-mCherry (Addgene, 50459-AAV8, Lot #v48443, 2.3 3 10 13

Abbreviations and Acronyms
AAV8 ¼ the rate of oxygen consumption genome copies/mL) to express mCherry to the Q neurons. Because mCherry cannot respond to CNO, administrating CNO will not induce QIH in the control animals ( Figure 1, A). Both QIH-ready and control mice are produced from the same strain Qrfp iCre heterozygous mice, which has the C57BL/6J background. After at least 3 weeks from the virus injection, all mice were intraperitoneally injected with CNO to test the capability of QIH. Mice that showed QIH were used as QIH-ready for further ischemia induction surgery. Control mice were also intraperitoneally injected with CNO after the AAV injection. Mice were divided into 4 groups: QIH-ready normothermia (QN) (n ¼ 8), QIH-ready hypothermia (QH) (n ¼ 8), control normothermia (CN) (n ¼ 10), and control hypothermia (CH) (n ¼ 10) (Figure 1, B

Evaluation of the Rate of VO 2 in Mice Under QIH
The body temperature of mice under QIH decreases because of induced hypometabolism. 15 To evaluate whether the metabolic condition of mice under QIH is affected by external control of body temperature maintained at normothermia, the rate of VO 2 in QIH mice was evaluated in accordance with the change of body temperature (T B ) regulated by the ambient temperature (T A ) (Figure 1, C). We prepared 5 QIH-ready mice aged from 17 to 22 weeks and housed in a temperature-controlled chamber (LP-400P-AR, Nippon Medical & Chemical Instruments). We recorded T B using a telemetry temperature sensor (TA11TA-F10; DSI) and VO 2 using a respiratory-gas analyzer (ARCO-2000 mass spectrometer; ARCO System). We kept the animals at T A 20 C (0 hours) and induced QIH by intraperitoneal injection of CNO 2 hours later (2 hours). After 3 hours from the initiation of QIH, we changed T A to 36 C for 3 hours (5-8 hours), then decreased T A to 20 C until 48 hours. We repeated the experiment in the same animals 2 times in a month interval (10 experiments in total).

Surgical Procedure
QIH-ready and control mice were transferred to Kyoto University and acclimated for 1 to 7 days with free access to food and water. The experimental protocol is indicated in Figure 1, B. On the day of surgery, mice were injected with CNO (1 mg/kg) intraperitoneally 3 hours before anesthesia. For tracheal intubation, mice were anesthetized with 3% to 5% isoflurane, followed by a ventral midline incision on the trachea and then tracheal intubation with a 22-gauge intravenous catheter (Terumo SR-OT2225C). Both lungs were mechanically ventilated with a tidal volume of 0.6 mL and a rate of 120 breaths per minute in room air. Vecuronium (0.1 mg/kg) and butorphanol (5 mg/kg) were intraperitoneally injected and inhalation of isoflurane ceased simultaneously. A thermometer probe was inserted into the abdominal cavity, and a custom-made temperature control device (Alice Co Ltd) controlled the body temperature modified from a previous report. 17 In the normothermic groups, the intraperitoneal temperature was maintained at 35.0 AE 1.3 C, whereas in the hypothermic groups, the intraperitoneal temperature was lowered until it reached 21.5 AE 1.3 C and was maintained at a low level by the temperature control device (Figure 2 was finally maintained at approximately 35 C. After 3 hours, all survived mice were humanely put to death. Whole blood was collected from the heart and unilateral kidney was excised. The surgical procedure is shown in Video 1.

Measurement of Blood Flow
For the measurement of blood flow before, during and after aortic clamp, we used a nonpreheating, noninvasive blood pressure, and heart rate monitor (MK-2000ST; Muromachi Kikai Co. Ltd). Using a cuff pulse sensor attached to tail arteries, we detected and measured the blood flow pattern using photoplethysmography. We measured the blood flow in 6 rats (CN:1, QN:2, CH:2, CN:1) from the induction of anesthesia to the time of death (Figure 2, B).

Measurement of Serum Biochemical Parameters
Blood samples were collected and centrifuged at 10,000 rpm for 10 minutes to obtain serum. The concentration of serum neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C were measured using the Mouse NGAL ELISA Kit (R&D Systems) and Mouse Cystatin C ELISA Kit (Bio Vendor R&D) according to the manufacturer's instruction.

Histologic Examination
The excised kidneys were fixed in 10 w/v% neutral-buffered formalin, sectioned, stained with periodic acid-Schiff and photographed using a digital microscope (Biorevo BZ-9000; Keyence). Tubular injury was defined as the tubular epithelial brush border loss, lucency, flattening, loss of nuclei, and intraluminal debris/cast. Injury was scored with a semiquantitative scale; 0 ¼ no tubular injury; 1 ¼ 10% or less; 2 ¼ 10% to 25%; 3 ¼ 26% to 50%; 4 ¼ 51% to 75%; and 5 ¼ more than 75% of tubules injured at the cortex. 18,19 Statistical Analysis The values are presented as mean AE SD. Multiple comparisons between groups were performed by 1-way analysis of variance followed by Tukey as post hoc. Statistical analyses were performed with JMP Pro 12 (SAS Institute Inc).

Evaluation of the Rate of VO 2 in Mice Under QIH
As shown in Figure 1, C, T B and VO 2 decreased after the initiation of QIH (5 hours). The T B recovered to normal 3 hours after changing T A from 20 to 36 C (8 hours). However, the VO 2 at 8 hours did not recover to the level at the initiation of QIH (2 hours). These results indicate that QIH decreases the oxygen demand of organs independent of body temperature.

Mouse Ischemia Model
In the present study, we performed ischemia induction of the lower body of mice by clamping the descending thoracic aorta, mimicking clinical aortic surgery as previously reported 20,21 (Figure 2, A). As preliminary experiments, we tested several clamping duration times and found that the survival rate of the mice was extremely reduced when the clamping time was longer than 20 minutes (survival rate ¼ 44% (4 out of 9) with a 30-minute clamp; 50% (1 out of 2) with a 20-minute clamp); therefore, we fixed our protocol to a 15-minute aortic clamping time. The survival rate was 78% (28 out of 36; 2 in CN, 3 in CH, 2 in QN, and 1 in QH died during surgery). Four animals are excluded from the experiment due to technical failure of the procedure (failure in aortic clamping) (2 in CN, 1 in CH, and 1 in QH). Finally, we evaluated 24 mice (n ¼ 6 for each group).
We checked the blood flow of the tails of each mouse to evaluate the lower body perfusion during the procedure (Figure 2, B). The blood flow almost disappeared during the clamp of the aorta, which immediately recovered after de-clamping. The changes in T B in all the mice are indicated in Figure 2, C. T B in QIH mice were lower than those in non-QIH mice before aortic crossclamping, indicating successful QIH.

Histopathologic Assessment for Renal Tubular Injury
The degree of renal tubular injury investigated by histologic analyses is shown in Figure 3. Severe tubular injury, indicated by tubular epithelial brush border loss, flattening,  (Figure 3, B). These results indicate that hypothermia protected the kidney from sublethal renal tubular damage.

DISCUSSION
In the present study, we revealed that QIH ameliorates AKI and dysfunction induced by the ischemia-reperfusion even in normothermic mice. QIH reduced kidney damage that was confirmed by histologic evaluations and the reduction of serum biomarkers levels indicating early kidney damage ( Figure 5).
In the histopathology evaluations, the extent of the injury of proximal convoluted tubule was lower in hypothermic groups compared with those in normothermic groups, which may indicate the effect of kidney protection by the reduction of body temperature. On the other hand, biomarkers for early kidney damage were almost equivalent in QIH groups regardless of the body temperature, indicating that QIH, a hibernation-like hypometabolism, may confer an equivalent level of kidney protection in the situation of circulatory arrest as that mediated by hypothermia. Hibernation is a hypothermic, hypometabolic, adaptive response engaged by animals known as hibernators to reduce metabolic demand. 22 There are several reports that have investigated the ischemiareperfusion injury of hibernators. Lindell and colleagues 23 evaluated liver ischemia-reperfusion injury in a liver transplantation model using squirrels as a hibernator and rats as a nonhibernator and found that the squirrel model showed significantly less elevation of serum level of lactate dehydrogenase compared with the rat model; indicating that the induction of hypometabolism in hibernators would be advantageous for organ protection. It is possible that the hibernation-like energy demand reduction of QIH realized the amelioration of ischemic kidney damage in the present study.
Passive organ protection and hypometabolism induced by hypothermia have been applied broadly and traditionally not  only in the field of cardiovascular surgery, but also in various fields of medicine such as ambulatory medicine and organ transplantation, with research reports that date to the 1940s. 4,24 QIH is a brand-new, promising approach to achieving active hypometabolism induced by the activation of Q neurons located in the hypothalamus. 15 In a transgenic mouse model ready for QIH, the hibernation-like state can be maintained for several days. The QIH reduces body temperature by approximately 10 C associated with massively reduced tissue oxygen consumption, heart rate, respiratory rate, and activities, resembling the passive hypometabolism induced by hypothermia. However, a hibernation-like state in the QIH is an active induction of hypometabolism, which may confer additional effects in organ protection from that in hypothermia and can be applied in not only the field of cardiovascular surgery but also in various medical fields. It would be ideal for cardiovascular surgeries requiring circulatory arrest if sufficient organ protection is achieved even with normal body temperature by the QIH, which may avoid bleeding tendency mediated by hypothermia, 8,9 and possible kidney injury mediated by DHCA, which is reported in a rabbit cardiopulmonary bypass model. 25 A method to induce hibernation in humans is expected for cardiovascular surgery requiring circulatory arrest, and extrapolating mouse QIH to humans is highly anticipated. Manipulation of specific neurons at the hypothalamus is required for QIH. Stimulating neurons directly in the brain may be a choice, although such invasive approaches should be avoided in patients receiving anticoagulation therapy who are undergoing cardiovascular surgery. Pharmacologic excitation of Q neurons is yet another approach to inducing human QIH. However, Q neurons-specific molecular targets, such as membrane receptors or channels, are unknown. We think that reproducing QIH is not the only answer to achieving human hibernation because brain is merely the center of torpor but not the frontline of hypometabolism. The peripheral tissues or organs during torpor should have gained resistance to hypometabolism. Therefore, among our research goals at the top of the list would be to induce torpor in humans pharmacologically by targeting the peripheral organs but not the brain.
For the evaluation of renal ischemia in mice, it is common to clamp renal arteries and veins en bloc. 26 Although our model, in which we clamped thoracic descending aorta, would be more invasive than renal vessels clamp models, we selected this method as a model relevant to human aortic surgeries requiring the circulatory arrest of lower body. 20,21 In the present study, we anesthetized mice with muscle relaxant and analgesic reagent under the approval of the institutional review board, which would have attenuated the effect of the reduction of body temperature and metabolic activities mediated by general anesthesia itself and enabled us to evaluate the effect of QIH on the reduction of metabolic activities more directly.
There are several limitations in the present study. First, we might have not completely eliminated the effect of anesthesia on the reduction of metabolic activities, although we had arranged the methods of anesthesia. Further, larger animals would be less susceptible to passive T B change influenced by T A under general anesthesia. Second, it would be preferable if we could evaluate kidney injury at a longer duration of reperfusion after ischemia considering that the majority of the research evaluated kidney injury with a longer duration of reperfusion. 27,28 In the present study, we conducted intraoperative management using a tracheostomy that prohibited us from returning the mice after procedures for longer observation after the surgery. Third, we could not evaluate physiological parameters indicating AKI such as urine output because of relatively short observation period of renal reperfusion. Physiological end point data are anticipated in future preclinical experiments with larger animals and longer observation period, and subsequent clinical studies.

CONCLUSIONS
In the present study, we found that QIH partly ameliorated AKI in a mouse ischemia model even in normothermia. QIH might be a promising approach to achieving sufficient kidney protection without DHCA in the future.