Denervation Aggravates Renal Ischemia Reperfusion Injury via BMAL1-mediated Nrf2/ARE pathway

Denervation Aggravates Renal Ischemia Reperfusion Injury via BMAL1-mediated Nrf2/ARE pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Denervation Aggravates Renal Ischemia Reperfusion Injury via BMAL1-mediated Nrf2/ARE pathway qian sun, YIFEI WANG, QIAO TANG, CHENG ZENG, LI DU, CHONG DONG This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2017001/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Denervation is an inevitable pathological situation of renal graft. This study was to explore the change of clock gene rhythm under renal denervation (RDN) and its effect on renal function and oxidative stress during renal ischemia-reperfusion (IR) injury. Method C57/BL6 mice were randomly divided into 4 groups at daytime 7AM (zeitgeber time [ZT] 0) or at nighttime 7PM (ZT12) in respectively: Sham (S) group, RDN group, IR group and RDN+ IR (DIR) group. Renal pathological and functional changes were assessed by H&E staining, and serum creatinine, urea nitrogen and neutrophil gelatinase-associated lipocalin levels. Renal oxidative stress was detected by SOD and MDA levels, and renal inflammation was measured by IL-6, IL-17AF and TNF-ɑ levels. BMAL1, CLOCK, Nrf2 and HO-1 mRNA and protein expressions were tested by qPCR and Western Blot. Result Compared with S groups, the rhythm of BMAL1 and CLOCK genes in the kidney was disordered in RDN groups, while renal pathological and functional indexes did not change significantly. Compared with IR groups, renal pathological and functional indexes were significantly higher in the DIR groups, as well as oxidative stress and inflammation in renal tissues. The protein expressions of BMAL1 and Nrf2 were upregulated by RDN at ZT12 timepoint. In DIR groups, renal injury was aggravated after the Brusatol treatment, but there was no significant improvement after the t-BHQ treatment, which might be consistent with the changes of Nrf2 and HO-1 protein expressions. Knockdown BMAL1 gene alone didn’t reverse the aggravation of renal IR injury at nighttime caused by RDN, but it could recover the protective effect of activating Nrf2/ARE pathway by t-BHQ. Conclusion RDN lead to the disruption of BMAL1-mediatedNrf2 rhythm accumulation in the kidney, which reduced the renal ability to resist oxidative stress and inflammation, due to the impaired effect of activating Nrf2/ARE pathway in renal IR injury at nighttime. circadian rhythm nuclear factor E2 related factor 2 renal ischemia-reperfusion injury renal denervation tert-butylhydroquinone Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Background In recent years, with the increasing incidence of diabetes and hypertension, a growing number of patients with end-stage renal failure (ESRF) need renal transplantation, which is the only effective treatment (Hariharan, et al., 2021). Although the success rate of renal transplantation has improved enormously, ischemia-reperfusion injury (IRI), as an inevitable pathophysiological process in transplantation, is one of the leading causes of delayed graft function. IRI affects the long-term survival of grafts, and increases the incidence of cardiovascular disease and mortality of renal transplant recipients (Granata, et al., 2022 ). Therefore, it is an imperative lesson to deeply clarify the main mechanism of transplanted renal IRI and put forward corresponding early effective regulation strategies. Nuclear factor E2 related factor 2 (Nrf2) has been recognized as the core transcription factor of antioxidant stress induced by IR (Sadrkhanloo, et al., 2022). Our previous studies have confirmed that Nrf2/ARE pathway played an important role in renal injury induced by IR (Zeng, et al., 2020) (Sun, et al., 2013) (Sun, et al., 2012) (Dong, et al., 2021). Surprisingly, we also found that the expressions of Nrf2 and its downstream antioxidant genes were markedly variable in renal IRI models established at different time-of-day along with the renal injury (Sun, et al., 2021). A large number of evidences have shown that the core clock genes such as circadian locomotor output cycles kaput (CLOCK) and brain and muscle ARN-t like protein 1 (BMAL1) can regulate the rhythmic expression of Nrf2 through E-box elements on the promoter in oxidative stress injury of multiple organs (Mezhnina, et al., 2022). Rhythmic accumulation is the hinge pattern for Nrf2 to give full scope to antioxidant stress. At the same time, some studies have shown that Nrf2 inhibits the homeostasis of CLOCK/BMAL1 by regulating Rev-Erbα, which is the downstream target gene of BMAL1 (Tamaru, et al., 2016) (Bevinakoppamath, et al., 2022). Therefore, Nrf2 and the clock genes form a chain reaction loop to integrate the cell redox signal into the rhythmic oscillation of circadian clock. As a peripheral organ, the kidney has an independent circadian clock system, which is involved in maintaining the circadian rhythm of renal physiological function such as blood pressure and water and sodium excretion. The renal circadian rhythm is synchronously regulated by the suprachiasmatic nucleus (SCN) of hypothalamus that is the control center of circadian clock, which is mainly completed by the peripheral nerve (Gumz, 2016). And the impairment of renal function is closely related to the abnormal rhythmic expressions of clock genes (Mohandas, et al., 2022). Clinically, patients with ERSF show a variety of rhythm disorders of physiological functions, such as low sleep quality, recurrence of hypertension, restless legs syndrome and so on, which can be aggravated by hemodialysis treatment (So, et al., 2020 ) (Firsov, et al., 2018). Unfortunately, renal transplantation can only temporarily improve a series of symptoms caused by circadian rhythm disorder, but the long-term syndromes still exist (Mendoza-Romo-Ramírez, et al., 2021 ) (Pisano, et al., 2021). Meanwhile, the latest cohort study with 10291 patients observed that compared to nocturnal de-clamping, day-time de-clamping was associated with a better prognosis of kidney transplantation despite a longer duration of cold ischemia (Montaigne, et al., 2021 ). So far, there is a lack of research on the changes of renal function and its regulatory mechanism in transplanted kidney due to renal denervation (RDN). This study found that RDN further aggravated the rhythmic oscillation disorder of expressions of BMAL1 and Nrf2 during renal IRI, and exacerbated the renal oxidative stress injury. RDN lead to the disruption of BMAL1-mediated Nrf2 rhythm accumulation in the kidney, which reduced the renal ability to resist oxidative stress and impaired the protective effect of activating Nrf2/ARE pathway in renal IR injury at nighttime. We also explored the effect of tert-butylhydroquinone (t-BHQ), an agonist of Nrf2, on oxidative stress injury induced by IR in the kidney following RDN. 2. Materials And Methods 2.1 Materials Male C57BL/6 mice (25 ± 3g; 6–8 weeks) were purchased from the Animal Center of Renmin Hospital of Wuhan University (Wuhan, China, nos. 2015-0027). Mice were housed in specific-pathogen-free (SPF) conditions at 22–24˚C, a relative humidity of 50 ± 15%, receiving standard laboratory chow and water. A total of ≥ 10 days prior to experimentation, the mice were housed in a strict 12-h light/dark cycle [The light time is 7 a.m.-7 p.m. (zeitgeber time (ZT) 0-ZT12), and the dark time is 7 p.m.-7 a.m. (ZT12-ZT24)]. The experimental protocol of the present study was approved by the Ethics Committee of Renmin Hospital of Wuhan University and in accordance with the principles of Laboratory Animal Care by the National Institutes of Health (permit no. 20210124). Antibodies for CLOCK, BMAL1, Nrf2 and HO-1 were purchased from Cell Signaling Technology, Inc. (cat. nos. 5157S, 14020S, 12721S, and 43966S, respectively), GADPH and Lamin B1 were from Proteintech (cat. nos. 60004-1-Ig and 66095-1-Ig), and tyrosine hydroxylase (TH) antibody was from ABclonal Technology Co., Ltd. (cat. nos. A12756). Blood urea nitrogen (BUN) and serum creatinine (Scr) were measured using an Olympus automatic analyzer, and norepinephrine (NE) and neutrophil gelatinase associated lipocalin (NGAL) levels were quantified using the corresponding enzyme-linked immunosorbent assay (ELISA) kit purchased from Wuhan USCN Business Co., Ltd. Superoxide dismutase (SOD) and malondialdehyde (MDA) assay kits were purchased from Nanjing Jiancheng Biochemicals Ltd. Interleukin-6 (IL-6), IL-17AF (heterodimer) and tumor necrosis factor (TNF-α) assay kits were purchased from Thermo Fisher Scientific. 2.2 Renal Denervation (RDN) model Animals were anesthetized using 5% chloral hydrate injected intraperitoneally at the dose of 10ml/kg. The RDN model was established by wrapping bilateral renal artery with 10% phenol for 15 minutes until the renal artery turned white (Xiao, et al., 2015). The decrease of NE content in renal tissue by more than 85% can be used as the standard to judge the effect of complete denervation. 2.3 Renal Ischemia Reperfusion (IR) model Animals were anesthetized using 5% chloral hydrate injected intraperitoneally at the dose of 10ml/kg. The IR model was established by bilateral renal pedicle occlusion for ischemia (30 min), followed by removal of the microvascular clip for 24 h reperfusion (Shiva, et al., 2020). The procedure was successful if the kidney turned from red to black after the pedicle occlusion, then black to red after gradual removal the clip. 2.4 Experimental protocol Mice were randomly assigned into one of 4 experimental groups in ZT0 or ZT12 individually (n = 6 per group) as follows: (1) Sham group (S group) that underwent laparotomy without RDN and occlusion for kidney; (2) RDN group; (3) IR group; (4) RDN + IR group (DIR group) that established IR model 7 days after the RDN model was successfully established. To examine the effects of Nrf2 pathway in renal IR injury following RDN, the specific inhibitor of Nrf2-Brusatol (Sigma-Aldrich, Shanghai, China) was dissolved in 1% of DMSO and intraperitoneally injected at a dose of 2 mg/kg every 2 days for 10 days before IR model established. The Nrf2 activator-tert-butylhydroquinone (t-BHQ, Sigma-Aldrich, Shanghai, China), was diluted in 1% of DMSO and intraperitoneally injected at a dose of 16.7 mg/kg every 8 hours for one day before IR model established. To examine the effects of BMAL1 gene knockdown in renal IR injury following RDN, we used recombinant adeno-associated virus serotype 9 (AAV9) vectors which carry a CMV promoter with GFP reporter (AAV9-BMAL1 shRNA-GFP) or AAV9-GFP NC which were produced by Obio Technology (Shanghai, China) to knock down BMAL1 gene expression or as control. The three pairs of shRNA sequence were in supplementary table1. AAV-BMAL1 was given via tail vein injection at a dose of 2 × 10 12 vg/kg once 2 weeks before RDN model established. 2.5 Histopathology of kidney tissue The left kidney was sectioned, then fixed with 4% formaldehyde for 24 h at room temperature, then embedded in paraffin, from which 4-µm sections were cut and stained with hematoxylin for 3 mins and eosin for 60 sec at room temperature. The slides were evaluated using light microscopy (original magnification, ×200; Olympus BX50; Olympus Corporation). Renal histological assessment was conducted using the Banff 97 working classification (Racusen, et al., 1999 ) and a semi-quantitative scale, as described (Spandou, et al., 2006): For each kidney, ≥ 100 cortical tubules from 10 different regions were scored. Higher scores represented more severe damage, scoring as follows: 0 = no damage; 1 = minimal damage (< 5% involvement of the cortex or outer medulla); 2 = mild damage (5–25% involvement of the cortex or outer medulla); 3 = moderate damage (25–75% involvement of the cortex or outer medulla); 4 = severe damage (> 75% involvement of the cortex or outer medulla). 2.6 Measurement of Scr, BUN and NGAL levels After the end of IR, 1 ml blood was collected from left ventricle apical area, centrifuged at 3,000 × g for 10 mins at 4˚C, and then serum was separated and stored at -20˚C. Scr and BUN were measured using an Olympus automatic analyzer (AU5400; Olympus Corporation), and NGAL level were measured using ELISA assay kit (cat. no. SEB388Mu; Wuhan USCN Business Co., Ltd) according to the manufacturer’s protocol. 2.7 Measurement of SOD activity, MDA, IL-6, IL-17AF (heterodimer) and TNF-ɑ levels in renal tissues Renal tissues were harvested and immediately homogenized on ice in 5 volumes of normal saline. The homogenates were centrifuged at 1,200 × g for 10 min at 4˚C. SOD activity (cat. no. A001-1) and MDA levels (cat. no. A003-1) were measured using chemical assay kits (Nanjing Jiancheng Biochemicals Ltd.), and IL-6 (cat. no. 88-7064-88), IL-17AF (heterodimer) (cat. no. 88-8711-88) and TNF-ɑ levels (cat. no. 88-7324-88) were measured using chemical assay kits (Thermo Fisher Scientific) in accordance with the manufacturer’s protocol. SOD activity was expressed as U/mg protein and 1 unit of enzyme is defined as the enzyme activity that inhibits the autoxidation of pyrogallol by 50%. The MDA content was expressed in nmol of MDA/ mg protein. The IL-6, IL-17AF (heterodimer) and TNF-ɑ levels was expressed in pg/ mg protein 2.8 Western blot analysis Cytoplasmic and nuclear proteins of the renal tissues were extracted using nuclear and cytoplasmic protein extraction kit (cat. no. P0028; Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. After measurement of the protein concentration using the bicinchoninic acid method, an equal quantity of 50 µg protein was separated by 12% SDS‑PAGE at 100 V for 3 h. After electrophoresis, proteins were transferred onto polyvinylidene difluoride membranes at 200 mA for 2 h. Each membrane was blocked with 5% nonfat milk for 2 h at room temperature, then incubated overnight at 4˚C with an appropriate primary antibody: CLOCK, BMAL1, Nrf2 and HO-1 (each at 1:1000 dilution). After repeated washing with Tween‑20 (TBS‑T) the membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. The immunoreactive bands were visualized by enhanced chemiluminescence (cat. nos. NCI5079; Thermo) and captured on BLT PHOTON TECHNOLOGY (Pro Ⅱ). The optical density of the bands was measured with ImageJ Software version 1.6 (National Institutes of Health, Bethesda, MD, USA). 2.9 RNA extraction and quantitative PCR (qPCR) According to the manufacturer's protocol, TRIzol reagent (Thermo Fisher Scientific) was used for the extraction of total RNA from the tissues and the BeyoRT™ First Strand cDNA synthesis kit (cat. no. D7166; Beyotime Institute of Biotechnology) was used for the preparation of cDNA. All qPCR reactions were performed in triplicates with iQ SYBR Green Supermix on an iCycler Real-Time Detection System (Bio-Rad Laboratories, Inc., USA). The thermocycling conditions were as follows: 95˚C for 10 min, followed by 40 cycles of 95˚C for 15 sec, 60˚C for 60 sec, and 72˚C for 60 sec. The expression levels of each targeted mRNA sequence were calculated by applying the 2 −∆∆ Ct method after being normalized to GADPH. Primers were in supplementary table1. 2.10 mRNA-sequencing and data processing 2 µg of total RNA was used to sequence mRNA libraries (30–40 million paired-end reads in each sample) through High Seq 2000 (Illumina, San Diego, CA, USA). The Fastp and FastQC were used to process the original image data for base calling, preliminary quality analysis and data filtering. Gene expression calculation was based on read counts from StringTie and fragments per kilo bases per million reads (FPKM) > 0.1 indicates meaningful expression. For samples with biological replicates, differentially expressed gene (DEG) clustering analysis was performed using the DESeq2 according to the criteria of |Fold Change| > 2 and p-adjust < 0.05. The Gene Ontology (GO, Gene Ontology database) functional enrichment analysis provided information on how the DEGs were related to certain biological functions using GOSeq. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis was based on KEGG pathway units and used a hypergeometric test to find the pathways of the DEGs. 2.11 Statistical analysis All outcome measurements were expressed as means ± SD values (n = 6) and analyzed using Graph Pad Prism 9.0 (GraphPad Software, Inc.). Statistical significance of differences among groups was determined by a one-way ANOVA with Tukey’s post-hoc test. Comparison of two groups was evaluated using a t-test. P < 0.05 was considered to indicate a statistically significant difference. 3. Results 3.1 Circadian rhythm disorder in the kidney following RDN After bilateral RDN model has been established 7 days, the decreases of NE and TH levels in the kidney was identified the success of RDN model with the disordered arrangement of renal nerve fibers, necrosis of a large number of nerve cells, vacuolization of cytoplasm and pyknosis of nuclei (Supplementary Figure S1). In order to explore the circadian rhythm disorder after RDN, qPCR was used to measure the mRNA levels of the CLOCK, BMAL1 and Nrf2 in the kidney that were collected every 4 h from ZT0, finding that they displayed a strong endogenous circadian rhythm in the S group, but the rhythmic oscillations of those genes were obviously disordered in the kidney following RDN (Fig. 1 A). Western blot analysis indicated that the CLOCK, BMAL1 and Nrf2 protein expression levels also exhibited the synchronous rhythm in the normal kidney, as ZT20-ZT0 was the expression peak phase and ZT8-ZT12 was the tough phase. However, the synchronous rhythm of CLOCK, BMAL1 and Nrf2 protein expressions were broken in the kidney following RDN, which were upregulated by RDN at ZT12 timepoint (Fig. 1 B and C). 3.2 RDN aggravated the diurnal variability of renal injury induced by IR Base on the above results, the renal IR model was established at the timepoints close to the peak or trough of Nrf2 protein expression in the normal kidney. In both ZT0 and ZT12 timepoint, there was no significant injury in the kidney by H&E staining and detection of BUN and Scr levels after RDN, indicated that RDN didn’t cause damage to the normal kidney (Supplementary Figure S2). Thus in Fig. 2 A, IR induced histological changes to renal tubules, including tubular epithelial edema and swelling, lumen dilation, epithelial simplification, nuclear necrosis, cytoplasmic translucency and vacuolation. Compared with ZT0, the histological injury was significantly aggravated when the IR model was established at ZT12 both in IR and DIR groups, while the histological injury score significantly increased after RDN treatment compared to IR groups at both ZT0 and ZT12 timepoint (Fig. 2 B). As the same, Scr, BUN and NAGL levels were higher in the DIR group compared with the IR group at both ZT0 and ZT12 timepoint (Fig. 2 C). Overall, the results indicated that renal injury induced by IR has diurnal variability, and RDN aggravated the diurnal variability of renal injury induced by IR, which might be related to the circadian rhythm disorder after RDN. 3.3 RDN increased oxidative stress and inflammation in the kidney following nocturnal IR As shown in Fig. 3 , compared with ZT0 timepoint, the establishment of IR model at ZT12 timepoint with or without RDN significantly decreased SOD activity and increased MDA content, IL-6, IL-17AF and TNF-ɑ levels in the renal tissues, which indicated that IR induced increased oxidative stress and inflammation in the kidney at nighttime. After bilateral RDN model has been established 7 days, SOD activity was lower and MDA content, IL-6, IL-17AF and TNF-ɑ levels were higher in the ZT12 DIR group than those in the IR ZT12 groups, which was not such remarkable between the DIR ZT0 and IR ZT0 groups. In addition, there was no significant changes in the indexes of oxidative stress and inflammation in the kidney in both ZT0 and ZT12 timepoint after RDN (Supplementary Figure S3). To this end, RDN increased oxidative stress and inflammation in the kidney following nocturnal IR, which was consistent with the results of renal morphological and functional injury. It’s suggested that RDN disordered the rhythm of Nrf2 might result in the decline of renal ability to resist oxidative stress and the aggravation of inflammatory response induced by IR. 3.4 Gene Comparison Analysis from kidneys following IR injury with/ without RDN Principal component analysis (PCA) and correlation heatmap of all expressed genes (Fig. S4) revealed that the transcriptomic profiles among different groups were distinct and well separated. Heatmap and ingenuity pathway analysis of differentially expressed (DE) genes (Supplementary Fig. S5) indicated that circadian rhythm of kidneys was significant different in S ZT0 and S ZT12 groups. Except circadian rhythm, IL-17 signaling pathway, cortisol and cholesterol metabolism and cytokine-cytokine receptor interaction were also different in RDN ZT0 and RDN ZT12 groups. Especially, PI3K-AKT pathway was strongly influenced by RDN treatment in ZT12, which was not reflected between S ZT0 and RDN ZT0 groups. As expected, IR effected catalytic activity, transmembrane transport and inflammatory response with or without RDN treatment, including TNF signaling pathway, cytokine-cytokine receptor interaction and PI3K-AKT signaling pathway (Supplementary Fig. S6). Compared with ZT0 groups, ingenuity pathway analysis revealed that the inflammatory and immune response signaling pathways were highly modulated in kidneys from ZT12 groups in both IR and DIR groups, including IL-17, cytokine-cytokine receptor interaction and TNF signaling pathways. Noteworthily, compared with IR ZT12 group, circadian rhythm, JAK-STAT, NF-kappa B and HIF-1 signaling pathways were emerged in DIR ZT12 group (Fig. 4 ). 3.5 RDN regulated clock genes and Nrf2/ARE pathway in the kidney following IR Next, we further verified the role of BMAL1/Nrf2/ARE pathway in RDN aggravating the diurnal variability of renal IRI. In the IR groups, the protein expressions of BMAL1, CLOCK, Nrf2 and HO-1 were all lower in the ZT12 timepoint than those of ZT0 timepoint. Thus, in the DIR groups, the protein expressions of BMAL1, CLOCK, Nrf2 and HO-1 were all higher in the ZT12 timepoint than those of ZT0 timepoint. In other words, the protein expressions of BMAL1, CLOCK, Nrf2 and HO-1 were upregulated in DIR ZT12 group compared to IR ZT12 group (Fig. 5 ). 3.6 RDN impaired the protective effect of activating Nrf2/ARE pathway in renal IR injury at nighttime As known that Nrf2/ARE pathway played a key role in renal IR injury, we further explored the effect of RDN on regulation of Nrf2 in renal IR injury at nighttime using the t-BHQ (Nrf2 activator) or Brusatol (Nrf2 antagonist) treatment. In IR groups without RDN, renal injury was significantly enhanced after the Brusatol treatment with the increase of histological injury score and the levels of BUN, Scr and NGAL, as well as the decrease of SOD activity and increase of the levels of MDA, IL-6, IL-17AF (heterodimer) and TNF-ɑ in the kidney, while renal IR injury was attenuated after the t-BHQ treatment (Fig. 6 A-J left). Thus, in DIR groups, there was only aggravated damage after the Brusatol treatment but no significant improvement after the t-BHQ treatment (Fig. 6 A-J right), which might be consistent with the changes of Nrf2 and HO-1 protein expressions. Compare with IR group, t-BHQ increased Nrf2 and HO-1 protein expressions, which were decreased by Brusatol. Nrf2 and HO-1 protein expressions were only decreased by Brusatol in DIR groups, but there was no significant increase induced by t-BHQ treatment in DIR groups. The above results suggested that RDN might interfere with the expression of Nrf2 protein in renal IR injury, resulting in impairing the protective effect of activating Nrf2/ARE pathway (Fig. 6 D). 3.7 Genetic knockdown of BMAL1 recovered the activation of Nrf2/ARE pathway after RDN in renal IR injury at nighttime To explore the role of BMAL1, which was disordered by RDN, in regulating the activation of the Nrf2/ARE pathway in renal IR injury at nighttime, the BMAL1 gene was knockdown by AAV9-BMAL1 shRNA injection. As shown in Supplementary Figure S7, AAV successfully transfected kidney cells by GFP detection, and BMAL1 protein was effectively downregulated in kidney by Western Blot analysis. After downregulation of BMAL1, t-BHQ treatment could significantly attenuate the renal IR injury in the DIR ZT12 group, with the decrease of histological injury score and the levels of BUN, Scr and NGAL, as well as the increase of the level of SOD and decrease of the level of MDA, IL-6, IL-17AF (heterodimer) and TNF-ɑ compared with AAV-NC group, which was not appeared in the AAV-BMAL1 + DIR group without t-BHQ treatment (Fig. 7 A-J). Furthermore, the protein expressions of Nrf2 and HO-1 were significantly increased in the AAV-BMAL1 + DIR + t-BHQ group compared with the AAV-NC + DIR + t-BHQ group (Fig. 7 K-M). To this end, it’s illustrated that knockdown BMAL1 gene alone cannot reverse the aggravation of renal IR injury at nighttime associated with RDN treatment, but it could recover the protective effect of activating Nrf2/ARE pathway by t-BHQ. 4. Discussion Our present study is the first time focusing on the impact of RDN on renal IR injury with the following novel findings. First, RDN disordered circadian rhythm of clock genes and Nrf2 in the kidney without the histological and functional injury. Second, RDN aggravated the diurnal variability of renal injury with increased oxidative stress and inflammation in the kidney following nocturnal IR. Third, RDN lead to the disruption of BMAL1-mediated Nrf2 rhythm accumulation in the kidney, which reduced the ability to resist oxidative stress and impaired the protective effect of activating Nrf2/ARE pathway in renal IR injury at nighttime. Moreover, knockdown BMAL1 gene alone cannot reverse the aggravation of renal IR injury at nighttime caused by RDN, but it could recover the protective effect of activating Nrf2/ARE pathway by t-BHQ. Above all, our results suggested that RDN aggravated renal IRI at nighttime associated with BMAL1-mediated Nrf2/ARE pathway. Clinical observations have shown that the rhythm disorder after renal transplant remained, such as sleep disturbance, fatigue, hypertension and metabolic impairment in ERSF patients, which indicates that circadian clock system involved in the graft’s function of renal transplant patient (Mendoza-Romo-Ramírez, et al., 2021 ) (Pisano, et al., 2021). The literature has demonstrated that nocturnal surgery might increase the morbidity and mortality with a higher risk of postoperative complications compared to daytime surgery in lung transplant and cardiac surgery and myocardial tolerance to IRI imposed daytime variation (Qiu, et al., 2018) (Montaigne, et al., 2018 ) (Cunningham, et al., 2019). Thus, the cohort studies referring to the impact of time-of-day on graft outcomes and complications in patients undergoing renal transplant is still controversial with limited numbers in recent years. Although a few studies did not observe the difference of time-of-day on outcomes, it may be due to the disorder of preoperative biological rhythm in patients with ESRF (Ville, et al., 2021 ) (Treacy, et al., 2022 ) (Sugünes, et al., 2019) (Guerrero, et al., 2021 ). Meanwhile, the latest cohort study with 10291 patients observed that compared to nocturnal declamping, day-time declamping was associated with a better prognosis of kidney transplantation despite a longer duration of cold ischemia (Montaigne, et al., 2021 ). Similarly, our previous studies have confirmed that bilateral occlusion IR model established at nighttime induced graver renal injury regarding that circadian rhythm of clock genes, and Nrf2 accumulation were a novel endogenous mechanism in renal IRI and repair processes (Sun, et al., 2021). It has also been confirmed in this study that renal IRI was aggravated at nighttime associated with BMAL1-mediated Nrf2/ARE pathway, which could be attenuated by Nrf2 activator t-BHQ. Moreover, this study has paid close attention to the impact of RDN on the diurnal variation of renal IR injury, which is inevitable in the process of graft acquisition. The newest study showed that renal IRI aggravated in bilateral sympathetic denervation rat model by superior cervical ganglionectomy via enhancing the inflammatory response (Zhang, et al., 2022 ). And numerous studies have shown that RDN mediated circadian blood pressure control in resistant hypertensive patients that might contribute to a significant improvement in dipping pattern and nocturnal systolic blood pressure (Becker, et al., 2019 ) (Osborn, et al., 2021). It’s likely that the peripheral nerves of kidneys play an important role in circadian variability of renal function, which is synchronously regulated by the suprachiasmatic nucleus (SCN) of hypothalamus that is the control center of circadian clock (Gumz, 2016) (Mohandas, et al., 2022). Similarly, our results indicated that RDN disordered circadian rhythm of core clock genes and Nrf2 in the kidney, which couldn’t induce any histological and functional injury in the kidney. Nevertheless, RDN aggravated the diurnal variability of renal injury induced by IR with increased oxidative stress and inflammation in the kidney, as the renal injury was grater following nocturnal IR. To explore the internal mechanism, we examined the expression levels of genes associated with circadian clock system and Nrf2/ARE pathway. Evidence from an increasing number of clinical and laboratory studies has indicated that there is obvious diurnal variation in IR injury of different organs including myocardial, renal and hepatic IR, as the circadian clock plays a vital role to protect the tissues and cells from oxidative injury induced by excessive ROS (Qiu, et al., 2021) (Lin, et al., 2020) (Correa-Costa, et al., 2018). As known, Nrf2 is recognized as the master transcription factor of the ROS homeostasis in renal IR injury (Zhuang, et al., 2022). Recent studies have found that the Nrf2/ARE pathway is regulated by the circadian clock that BMAL1 controls Nrf2 rhythm accumulation via E-BOX element (Mezhnina, et al., 2022) (Tamaru, et al., 2016). The activity of Nrf2 on circadian rhythm manner is vital to maintain ARE related target genes for protecting against oxidative injury in many pathological processes (Bevinakoppamath, et al., 2022). Similarly, in our previous and this study, we found that renal IR model established at nighttime with the trough expressions of BMAL1-mediated Nrf2 suffered the severe oxidative injury due to the weaken expressions of downstream antioxidant proteins. But far more than this, RDN disordered circadian rhythm of core clock genes and Nrf2 in the kidney, and the dysrhythmia of the Nrf2/ARE pathway reduced the rhythmic regulation to impair the sensitivity to resist oxidative stress induced by IR at nighttime. Moreover, knockdown BMAL1 gene cannot reverse the aggravation of renal IR injury at nighttime caused by RDN, which may be associated with that the circadian rhythm manner can’t be recovered by knockdown BMAL1 gene alone. In addition, our previous study has shown that IR-mediated oxidative stress injury in the kidney is ameliorated by treatment with t-BHQ, as an Nrf2 activator (Chen, et al., 2020 ). On the one hand, the canonical Nrf2 activation pathway is that electrophilic compounds such as t-BHQ help Nrf2 dissociate from Keap1, translocate into the nucleus and bind to the ARE resulting in transcription of the phase II genes. On the other hand, t-BHQ can increase Wilms tumor gene in chromosome X protein that decreases Nrf2 ubiquitination and activates Nrf2 transcriptional activity through non-canonical Nrf2 activation pathway (Kim and Jeon, 2022) (Silva-Islas and Maldonado, 2018). Unfortunately, the nocturnal IR injury couldn’t be attenuated by t-BHQ in the kidney following RDN in this study, while knockdown BMAL1 gene could recover the protective effect of activating Nrf2/ARE pathway by t-BHQ. Thus, we speculate that Nrf2 has been exhausted in the renal IR at nighttime following RDN, which can’t be continuously activated by t-BHQ. Meanwhile, decreased BMAL1 may alleviate the depletion of Nrf2 and restore the protective effect of t-BHQ to a certain extent. Of course, there are other possibilities. It’s reported that the role of t-BHQ on ROS-mediated dissociation of Nrf2-Keap1 may differ according to phase II enzyme and cell line investigated, in which the treatment with t-BHQ alone did not induce ROS (Gharavi, et al., 2007). Respecting to Nrf2 stability, t-BHQ only increases the level of Nrf2 protein, but not the Nrf2 mRNA level in HepG2 cells (Nguyen, et al., 2003). And t-BHQ does not inhibit the Nrf2 ubiquination directly, which merely stabilizes the ubiquinated Nrf2 (Li, et al., 2005). To this end, furthermore studies are required to elucidate the intrinsic mechanisms of t-BHQ or its metabolites of IR-induced oxidative stress injury in the kidney with the dysrhythmia of the Nrf2/ARE pathway. 5. Limitation And Future Study This is the first and pioneering study involved in the effect of RDN on renal IR injury and its internal mechanism, so there are still many limitations in this study. First, we established RDN model using renal artery cauterization wrapped by phenol classically with high success rate and low mortality, which can’t distinguish between afferent and efferent nerves. Studies have shown that renal afferent and efferent nerves have different regulatory roles in maintaining renal function (Zhang, et al., 2022 ) (Becker, et al., 2019 ) (Osborn, et al., 2021). Consequently, we can improve the establishment of RDN to explore the effects of afferent and efferent nerves (Qiu, et al., 2018) on renal IR injury associated with circadian rhythm respectively. Secondly, a few clinic observations referring to the impact of time-of-day on graft outcomes and complications in patients undergoing renal transplant haven’t observe the difference of time-of-day on outcomes, which may be due to the disorder of preoperative circadian rhythm in patients with ESRF (Treacy, et al., 2022 ) (Sugünes, et al., 2019) (Guerrero, et al., 2021 ). However, the EDSF model has not been used in this study, which is a certain gap with clinical renal transplant. Hence, the conclusions of this study pay special attention to the function and regulation of circadian rhythm in normal kidney. We are conducting in-depth research using the EDSF model, but the establishment of this complex animal model is facing great challenges. Thirdly, in order to synchronize the circadian rhythm of experimental mice, we have homogenized the genes, light, diet, modeling and other related confounders, but there are still individual differences unavoidably. Therefore, the time-of-day for IR modeling in this study is nothing but near the peak and tough timepoint of circadian rhythm oscillation, and this interference can only be diminished by expanding the sample size. Finally, up to now, the researches about the pharmacokinetic parameters of t-BHQ or its metabolism in human and mice are not sufficient (Gharavi, et al., 2007). This study has observed that t-BHQ can’t effectively reverse the nocturnal renal IR injury after RDN by activating Nrf2/ARE pathway, which is closely related to the expression of core clock gene BMAL1. Respecting to the advance researches on chronotherapy in recent years (Gharavi, et al., 2007) (Seeman, et al., 2022), more researches are required to optimize the treatment scheme based on the specific characteristics of circadian rhythm for patients following renal transplantation. Conclusion After RDN, the clock rhythm of kidney is disordered, resulting in the aggravation of renal IR injury at night, which is related to BMAL1-mediated Nrf2/ARE pathway. The treatment of t-BHQ can’t recover the nocturnal IR injury in kidney following RDN by activating Nrf2/ARE pathway, which can be repaired by BMAL1 knockdown. It is suggested that more researches are required to optimize the reasonable and individualized chronotherapy based on the specific characteristics of circadian rhythm, which will effectively improve the graft survival rate and the quality of life for renal transplant patients. Declarations Ethics approval and consent to participate: The experimental protocol of the present study was approved by the Ethics Committee of Renmin Hospital of Wuhan University and in accordance with the principles of Laboratory Animal Care by the National Institutes of Health (permit no. 20210124). Consent for publication: Not applicable. Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests: The authors declare that they have no competing interests. Funding: This study was supported National Natural Science Foundation of China (No. 82072140), Tianjin Science and Technology Fund Planning Project (No. 21JCYBJC01130) and China Scholarship Council (No. 202006275056). Authors' contributions: Design, writing draft and funding acquisition, QS; data collection and analysis, YW, QT and CZ; investigation and resources, LD; review, editing and funding acquisition, CD. All authors have read and agreed to the published version of the manuscript. Acknowledgements: Not applicable. References Sundaram Hariharan, Ajay K, Israni GD. Long-Term Survival after Kidney Transplantation. N Engl J Med. 2021 Aug;19(8):729–43. 385(. Granata S, Votrico V, Spadaccino F, Catalano V, Netti GS, Ranieri E, Stallone G, Zaza G. Oxidative Stress and Ischemia/Reperfusion Injury in Kidney Transplantation: Focus on Ferroptosis, Mitophagy and New Antioxidants. Antioxidants (Basel). 2022 Apr 12;11(4):769. doi: 10.3390/antiox11040769 . 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C) Representative H&E staining photomicrographs of renal nerves in different groups (original magnification, ×200). The arrow refers to the renal nerve. F2S.tif Figure S2 Renal diurnal variability following RDN. A) Representative H&E staining photomicrographs of kidney in different groups (original magnification, ×200). B) Renal histologic evaluation score in different groups. C) The blood urea nitrogen (BUN), D) serum creatinine (Scr) and E) serum neutrophil gelatinase associated lipocalin (NAGL) levels in different groups. Data are expressed as means± SD, n = 6 from each group. F3S.tif Figure S3 Oxidative stress and inflammation in the kidney following RDN. A) The superoxide dismutase (SOD) activity, B) malondialdehyde (MDA), C) Interleukin-6 (IL-6), D) IL-17AF (heterodimer) and E) tumor necrosis factor (TNF-ɑ) levels in renal tissues. Data are expressed as means± SD, n = 6 from each group. F4S.tif Figure S4. (A) Principal component analysis (PCA) and (B) Correlation heatmap of all expressed genes in different groups. The expressed genes were FPKM>0.1. F5S.tif Figure S5. The Cluster, GO term enrichment and enriched KEGG pathway analysis of DEGs between S ZT0 and S ZT12 groups (A), RDN ZT0 and RDN ZT12 groups (B), S ZT0 and RDN ZT0 groups (C), and S ZT12 and RDN ZT12 groups (D). The DEGs were |Fold Change| > 2 and p-adjust < 0.05. F6S.tif Figure S6. The Cluster, GO term enrichment and enriched KEGG pathway analysis of DEGs between S ZT0 and IR ZT0 groups (A), S ZT12 and IR ZT12 groups (B), RDN ZT0 and DIR ZT0 groups (C), and RDN ZT12 and DIR ZT12 groups (D). The DEGs were |Fold Change| > 2 and p-adjust < 0.05. F7S.tif Figure S7 The knockdown of BMAL1 gene by AAV9-BMAL1 shRNA injection. A) Representative photomicrographs of AAV transfection in different groups (original magnification, ×200). The GFP- green fluorescence represents positive transfection of AAV, and DAPI- blue fluorescence represents nucleus. B) The protein expressions of BMAL1 in the kidney. Protein expressions were relative to AAV normal control (NC) group. GADPH was used as the housekeeping control. Data are expressed as means± standard deviation (SD), n = 5 from each group, * P < 0.05 as NC group vs. AAV BMAL1 shRNA (BM) group. TabelS1.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2017001","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":143836348,"identity":"851c4224-5c0a-4d92-b22e-02a2e662519d","order_by":0,"name":"qian sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYNACAzDJ+ICxAUQnEK+F2YAELRDAJkGUFr4byQcfFxTYMMjPyD1WzbvjMAM/e44Bw88duLVI3khLNp5hkMZgcCMv7TbvmcMMkj1vDBh7z+DxxY0cM2keg8MMBhI5Zrdz2w6DRAyYGdvwacn//pvH4D/QYTlmxSAt9oS15LAx8xgcYGAAWscMtkWCgBbJM8+MgQ5LZjA488ZY+m9bOo/EmWcFB3vxaOE7nvzwM88fOwb59hzDjzPbrOX425M3PviJRwvDAQhV3wDl8yAJ4tcyCkbBKBgFowA3AADSF01G1r6q9gAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-8038-0706","institution":"Renmin Hospital of Wuhan University: Wuhan University Renmin Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"qian","middleName":"","lastName":"sun","suffix":""},{"id":143836349,"identity":"ddc0b7bb-7a55-4697-87b7-60864adc972b","order_by":1,"name":"YIFEI WANG","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University: Wuhan University Renmin Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"YIFEI","middleName":"","lastName":"WANG","suffix":""},{"id":143836350,"identity":"57cfd72b-294f-440f-84e7-e62b212c8d59","order_by":2,"name":"QIAO TANG","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University: Wuhan University Renmin Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"QIAO","middleName":"","lastName":"TANG","suffix":""},{"id":143836351,"identity":"5371160a-9260-4901-b690-eaf459893388","order_by":3,"name":"CHENG ZENG","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University: Wuhan University Renmin Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"CHENG","middleName":"","lastName":"ZENG","suffix":""},{"id":143836352,"identity":"aa03d682-d004-43b2-a28a-ec62f72fea60","order_by":4,"name":"LI DU","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University: Wuhan University Renmin Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"LI","middleName":"","lastName":"DU","suffix":""},{"id":143836353,"identity":"f0cf538e-e532-4a04-984b-1d0d266bd0e0","order_by":5,"name":"CHONG DONG","email":"","orcid":"https://orcid.org/0000-0003-4287-303X","institution":"Tianjin First Central Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"CHONG","middleName":"","lastName":"DONG","suffix":""}],"badges":[],"createdAt":"2022-08-31 08:49:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2017001/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2017001/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":27871024,"identity":"5d2ff266-afe7-4821-b19c-c275c511124d","added_by":"auto","created_at":"2022-10-17 14:52:42","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":409907,"visible":true,"origin":"","legend":"\u003cp\u003eCircadian rhythm disorder in the kidney following RDN. A) The mRNA levels of the CLOCK, BMAL1 and Nrf2 in the kidney that were collected every 4 h from ZT0. B) The protein expressions of CLOCK, BMAL1 and Nrf2 in the kidney. Protein expressions were relative to Sham group. GADPH or Lamin B1 was used as the housekeeping control. Data are expressed as means± SD, n = 5 from each group, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 as Sham vs. RDN. C) Representative Western Blot bands of CLOCK, BMAL1 and Nrf2 protein in different groups.\u003c/p\u003e","description":"","filename":"F10826.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2017001/v1/ea7f62055cd92acfc67c6b99.jpg"},{"id":27871759,"identity":"c5821dc6-d08b-47c8-99dd-a231e0537c33","added_by":"auto","created_at":"2022-10-17 14:57:42","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":673922,"visible":true,"origin":"","legend":"\u003cp\u003eDiurnal variability of renal injury induced by IR following RDN. A) Representative H\u0026amp;E staining photomicrographs of kidney in different groups (original magnification, ×200). B) Renal histologic evaluation score in different groups. C) The blood urea nitrogen (BUN), D) serum creatinine (Scr) and E) serum neutrophil gelatinase associated lipocalin (NAGL) levels in different groups. Data are expressed as means± SD, n = 6 from each group, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 as IR at ZT12 timepoint (IR12) vs. DIR at ZT12 timepoint (DIR12), \u0026amp;\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 as ZT0 vs. ZT12 in IR or DIR groups respectively.\u003c/p\u003e","description":"","filename":"F2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2017001/v1/1428d88c20070bcd4e039026.jpg"},{"id":27871760,"identity":"eddd5070-795d-4556-9c7a-3ee86c0a9492","added_by":"auto","created_at":"2022-10-17 14:57:42","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":239544,"visible":true,"origin":"","legend":"\u003cp\u003eDiurnal variability of oxidative stress and inflammation induced by IR following RDN. A) The superoxide dismutase (SOD) activity, B) malondialdehyde (MDA), C) Interleukin-6 (IL-6), D) IL-17AF and E) tumor necrosis factor (TNF-ɑ) levels in renal tissues. Data are expressed as means± SD, n = 6 from each group, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 as IR at ZT12 timepoint (IR12) vs. DIR at ZT12 timepoint (DIR12), \u0026amp;\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 as ZT0 vs. ZT12 in IR or DIR groups respectively.\u003c/p\u003e","description":"","filename":"F3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2017001/v1/306289f2347ef922b1d40eea.jpg"},{"id":27871028,"identity":"ee5f1492-5e8c-4719-b91c-5b6851074438","added_by":"auto","created_at":"2022-10-17 14:52:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":515581,"visible":true,"origin":"","legend":"\u003cp\u003eRNA-seq Comparison Analysis. The Cluster, GO term enrichment and enriched KEGG pathway analysis of DEGs between IR ZT0 and IR ZT12 groups (A), DIR ZT0 and DIR ZT12 groups (B), IR ZT0 and DIR ZT0 groups (C), and IR ZT12 and DIR ZT12 groups (D). The DEGs were |Fold Change| \u0026gt; 2 and p-adjust \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"F4.png","url":"https://assets-eu.researchsquare.com/files/rs-2017001/v1/f403b106ee1655594bb5cf37.png"},{"id":27872412,"identity":"9db1c8c8-ed5e-4774-970e-d182d43af1d9","added_by":"auto","created_at":"2022-10-17 15:02:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":252101,"visible":true,"origin":"","legend":"\u003cp\u003eDiurnal variability of clock genes and Nrf2/ARE pathway induced by IR following RDN. A) Representative Western Blot bands of CLOCK, BMAL1, Nrf2 and HO-1 protein in different groups. The protein expressions of B) CLOCK, C) BMAL1, D) Nrf2 and E) HO-1 in the kidney. Protein expressions were relative to Sham group. GADPH or Lamin B1 was used as the housekeeping control. Data are expressed as means± SD, n = 6 from each group, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 as IR at ZT12 timepoint (IR12) vs. DIR at ZT12 timepoint (DIR12), \u0026amp;\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 as ZT0 vs. ZT12 in IR or DIR groups respectively.\u003c/p\u003e","description":"","filename":"F5.png","url":"https://assets-eu.researchsquare.com/files/rs-2017001/v1/70db44d323bd4b08d88dbdcd.png"},{"id":27871762,"identity":"425d3ccf-9140-46a3-94e2-8f5d833bb666","added_by":"auto","created_at":"2022-10-17 14:57:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":897174,"visible":true,"origin":"","legend":"\u003cp\u003eNrf2/ARE pathway in nocturnal IR injury following RDN. A) Representative H\u0026amp;E staining photomicrographs of kidney in different groups (original magnification, ×200). B) Renal histologic evaluation score in different groups. C) The blood urea nitrogen (BUN), D) serum creatinine (Scr) and E) serum neutrophil gelatinase associated lipocalin (NAGL) levels in different groups. F) The superoxide dismutase (SOD) activity, G) malondialdehyde (MDA), F) Interleukin-6 (IL-6), I) IL-17AF (heterodimer) and J) tumor necrosis factor (TNF-ɑ) levels in renal tissues. K) Representative Western Blot bands of Nrf2 and HO-1 protein in different groups. The protein expressions of L) Nrf2 and M) HO-1 in the kidney. Protein expressions were relative to Sham group. GADPH or Lamin B1 was used as the housekeeping control. Data are expressed as means± SD, n = 6 from each group, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 as IR at ZT12 timepoint (IR12) vs. DIR at ZT12 timepoint (DIR12), \u0026amp;\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 as ZT0 vs. ZT12 in IR or DIR groups respectively.\u003c/p\u003e","description":"","filename":"F6.png","url":"https://assets-eu.researchsquare.com/files/rs-2017001/v1/a294a0e1595712ef6e32d884.png"},{"id":27871763,"identity":"e1db89f8-28f2-4ab6-8ecb-7ad3f44600cd","added_by":"auto","created_at":"2022-10-17 14:57:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":689860,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of genetic knockdown of BMAL1 on Nrf2/ARE pathway in nocturnal IR injury following RDN. A) Representative H\u0026amp;E staining photomicrographs of kidney in different groups (original magnification, ×200). B) Renal histologic evaluation score in different groups. C) The blood urea nitrogen (BUN), D) serum creatinine (Scr) and E) serum neutrophil gelatinase associated lipocalin (NAGL) levels in different groups. F) The superoxide dismutase (SOD) activity, G) malondialdehyde (MDA), F) Interleukin-6 (IL-6), I) IL-17AF (heterodimer) and J) tumor necrosis factor (TNF-ɑ) levels in renal tissues. The protein expressions of K) Nrf2 and L) HO-1 in the kidney. M) Representative Western Blot bands of Nrf2 and HO-1 protein in different groups. Protein expressions were relative to Sham group. GADPH or Lamin B1 was used as the housekeeping control. Data are expressed as means± SD, n = 6 from each group, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 as t-BHQ vs. 1% DMSO treatment in AAV BMAL1 shRNA (BM group) or AAV normal control (NC group) respectively.\u003c/p\u003e","description":"","filename":"F7.png","url":"https://assets-eu.researchsquare.com/files/rs-2017001/v1/3253d06a89735ceb5e58c879.png"},{"id":31903313,"identity":"2bab1739-d25b-4ea4-9b22-348130aceeb7","added_by":"auto","created_at":"2023-01-21 20:05:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3237368,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2017001/v1/d4689819-9d5a-4c7a-aee4-29b75d8db29e.pdf"},{"id":27871025,"identity":"480eb378-358a-43a6-bb0c-d2a711af5963","added_by":"auto","created_at":"2022-10-17 14:52:42","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":708728,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S1 The levels of A) norepinephrine (NE) and B) tyrosine hydroxylase (TH) in the kidney. Protein expressions of TH were relative to Sham group. GADPH was used as the housekeeping control. Data are expressed as means± SD, n = 6 from each group, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 as Sham vs. RDN. C) Representative H\u0026amp;E staining photomicrographs of renal nerves in different groups (original magnification, ×200). The arrow refers to the renal nerve.\u003c/p\u003e","description":"","filename":"F1S.tif","url":"https://assets-eu.researchsquare.com/files/rs-2017001/v1/29424577c14f848207899547.tif"},{"id":27871032,"identity":"a7e323e4-6c03-49f3-942d-68b20fffc755","added_by":"auto","created_at":"2022-10-17 14:52:43","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1176018,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S2 Renal diurnal variability following RDN. A) Representative H\u0026amp;E staining photomicrographs of kidney in different groups (original magnification, ×200). B) Renal histologic evaluation score in different groups. C) The blood urea nitrogen (BUN), D) serum creatinine (Scr) and E) serum neutrophil gelatinase associated lipocalin (NAGL) levels in different groups. Data are expressed as means± SD, n = 6 from each group.\u003c/p\u003e","description":"","filename":"F2S.tif","url":"https://assets-eu.researchsquare.com/files/rs-2017001/v1/083ae0c8b8df898d7b3891e6.tif"},{"id":27871031,"identity":"af3d45db-3b53-4082-8786-c086a00ef752","added_by":"auto","created_at":"2022-10-17 14:52:43","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":264216,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S3 Oxidative stress and inflammation in the kidney following RDN. A) The superoxide dismutase (SOD) activity, B) malondialdehyde (MDA), C) Interleukin-6 (IL-6), D) IL-17AF (heterodimer) and E) tumor necrosis factor (TNF-ɑ) levels in renal tissues. Data are expressed as means± SD, n = 6 from each group.\u003c/p\u003e","description":"","filename":"F3S.tif","url":"https://assets-eu.researchsquare.com/files/rs-2017001/v1/f69f9537b173e3377c39b1df.tif"},{"id":27871038,"identity":"4264762d-55cd-4f09-83ee-00befa09a61a","added_by":"auto","created_at":"2022-10-17 14:52:43","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1897920,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S4. (A) Principal component analysis (PCA) and (B) Correlation heatmap of all expressed genes in different groups. The expressed genes were FPKM\u0026gt;0.1.\u003c/p\u003e","description":"","filename":"F4S.tif","url":"https://assets-eu.researchsquare.com/files/rs-2017001/v1/c338dfabc1e145baf6b4a914.tif"},{"id":27871034,"identity":"bdf152e9-325b-46ff-ae1c-9ab2d05b932d","added_by":"auto","created_at":"2022-10-17 14:52:43","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1647344,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S5. The Cluster, GO term enrichment and enriched KEGG pathway analysis of DEGs between S ZT0 and S ZT12 groups (A), RDN ZT0 and RDN ZT12 groups (B), S ZT0 and RDN ZT0 groups (C), and S ZT12 and RDN ZT12 groups (D). The DEGs were |Fold Change| \u0026gt; 2 and p-adjust \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"F5S.tif","url":"https://assets-eu.researchsquare.com/files/rs-2017001/v1/04f9a7e5c28be1ec8b51af5c.tif"},{"id":27871037,"identity":"052cc2c2-5004-48bc-a17a-83c978f9f9f5","added_by":"auto","created_at":"2022-10-17 14:52:43","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1911472,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S6. The Cluster, GO term enrichment and enriched KEGG pathway analysis of DEGs between S ZT0 and IR ZT0 groups (A), S ZT12 and IR ZT12 groups (B), RDN ZT0 and DIR ZT0 groups (C), and RDN ZT12 and DIR ZT12 groups (D). The DEGs were |Fold Change| \u0026gt; 2 and p-adjust \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"F6S.tif","url":"https://assets-eu.researchsquare.com/files/rs-2017001/v1/66078c5937214487e6d9886f.tif"},{"id":27871764,"identity":"0185c5bc-2133-4aff-b1cb-29cb988b3660","added_by":"auto","created_at":"2022-10-17 14:57:43","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":601312,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S7 The knockdown of BMAL1 gene by AAV9-BMAL1 shRNA injection. A) Representative photomicrographs of AAV transfection in different groups (original magnification, ×200). The GFP- green fluorescence represents positive transfection of AAV, and DAPI- blue fluorescence represents nucleus. B) The protein expressions of BMAL1 in the kidney. Protein expressions were relative to AAV normal control (NC) group. GADPH was used as the housekeeping control. Data are expressed as means± standard deviation (SD), n = 5 from each group, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 as NC group vs. AAV BMAL1 shRNA (BM) group.\u003c/p\u003e","description":"","filename":"F7S.tif","url":"https://assets-eu.researchsquare.com/files/rs-2017001/v1/05b19ee3ee4d7d39cace756f.tif"},{"id":27871035,"identity":"554cb90d-0d61-4f3d-8b6e-e81bcbe1c8d7","added_by":"auto","created_at":"2022-10-17 14:52:43","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":9729,"visible":true,"origin":"","legend":"","description":"","filename":"TabelS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2017001/v1/d14794a42e3eecf5710d3c22.xlsx"}],"financialInterests":"","formattedTitle":"Denervation Aggravates Renal Ischemia Reperfusion Injury via BMAL1-mediated Nrf2/ARE pathway","fulltext":[{"header":"1. Background","content":"\u003cp\u003eIn recent years, with the increasing incidence of diabetes and hypertension, a growing number of patients with end-stage renal failure (ESRF) need renal transplantation, which is the only effective treatment (Hariharan, et al., 2021). Although the success rate of renal transplantation has improved enormously, ischemia-reperfusion injury (IRI), as an inevitable pathophysiological process in transplantation, is one of the leading causes of delayed graft function. IRI affects the long-term survival of grafts, and increases the incidence of cardiovascular disease and mortality of renal transplant recipients (Granata, et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, it is an imperative lesson to deeply clarify the main mechanism of transplanted renal IRI and put forward corresponding early effective regulation strategies.\u003c/p\u003e \u003cp\u003eNuclear factor E2 related factor 2 (Nrf2) has been recognized as the core transcription factor of antioxidant stress induced by IR (Sadrkhanloo, et al., 2022). Our previous studies have confirmed that Nrf2/ARE pathway played an important role in renal injury induced by IR (Zeng, et al., 2020) (Sun, et al., 2013) (Sun, et al., 2012) (Dong, et al., 2021). Surprisingly, we also found that the expressions of Nrf2 and its downstream antioxidant genes were markedly variable in renal IRI models established at different time-of-day along with the renal injury (Sun, et al., 2021). A large number of evidences have shown that the core clock genes such as circadian locomotor output cycles kaput (CLOCK) and brain and muscle ARN-t like protein 1 (BMAL1) can regulate the rhythmic expression of Nrf2 through E-box elements on the promoter in oxidative stress injury of multiple organs (Mezhnina, et al., 2022). Rhythmic accumulation is the hinge pattern for Nrf2 to give full scope to antioxidant stress. At the same time, some studies have shown that Nrf2 inhibits the homeostasis of CLOCK/BMAL1 by regulating Rev-Erbα, which is the downstream target gene of BMAL1 (Tamaru, et al., 2016) (Bevinakoppamath, et al., 2022). Therefore, Nrf2 and the clock genes form a chain reaction loop to integrate the cell redox signal into the rhythmic oscillation of circadian clock.\u003c/p\u003e \u003cp\u003eAs a peripheral organ, the kidney has an independent circadian clock system, which is involved in maintaining the circadian rhythm of renal physiological function such as blood pressure and water and sodium excretion. The renal circadian rhythm is synchronously regulated by the suprachiasmatic nucleus (SCN) of hypothalamus that is the control center of circadian clock, which is mainly completed by the peripheral nerve (Gumz, 2016). And the impairment of renal function is closely related to the abnormal rhythmic expressions of clock genes (Mohandas, et al., 2022). Clinically, patients with ERSF show a variety of rhythm disorders of physiological functions, such as low sleep quality, recurrence of hypertension, restless legs syndrome and so on, which can be aggravated by hemodialysis treatment (So, et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) (Firsov, et al., 2018). Unfortunately, renal transplantation can only temporarily improve a series of symptoms caused by circadian rhythm disorder, but the long-term syndromes still exist (Mendoza-Romo-Ram\u0026iacute;rez, et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) (Pisano, et al., 2021). Meanwhile, the latest cohort study with 10291 patients observed that compared to nocturnal de-clamping, day-time de-clamping was associated with a better prognosis of kidney transplantation despite a longer duration of cold ischemia (Montaigne, et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSo far, there is a lack of research on the changes of renal function and its regulatory mechanism in transplanted kidney due to renal denervation (RDN). This study found that RDN further aggravated the rhythmic oscillation disorder of expressions of BMAL1 and Nrf2 during renal IRI, and exacerbated the renal oxidative stress injury. RDN lead to the disruption of BMAL1-mediated Nrf2 rhythm accumulation in the kidney, which reduced the renal ability to resist oxidative stress and impaired the protective effect of activating Nrf2/ARE pathway in renal IR injury at nighttime. We also explored the effect of tert-butylhydroquinone (t-BHQ), an agonist of Nrf2, on oxidative stress injury induced by IR in the kidney following RDN.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eMale C57BL/6 mice (25\u0026thinsp;\u0026plusmn;\u0026thinsp;3g; 6\u0026ndash;8 weeks) were purchased from the Animal Center of Renmin Hospital of Wuhan University (Wuhan, China, nos. 2015-0027). Mice were housed in specific-pathogen-free (SPF) conditions at 22\u0026ndash;24˚C, a relative humidity of 50\u0026thinsp;\u0026plusmn;\u0026thinsp;15%, receiving standard laboratory chow and water. A total of \u0026ge;\u0026thinsp;10 days prior to experimentation, the mice were housed in a strict 12-h light/dark cycle [The light time is 7 a.m.-7 p.m. (zeitgeber time (ZT) 0-ZT12), and the dark time is 7 p.m.-7 a.m. (ZT12-ZT24)]. The experimental protocol of the present study was approved by the Ethics Committee of Renmin Hospital of Wuhan University and in accordance with the principles of Laboratory Animal Care by the National Institutes of Health (permit no. 20210124). Antibodies for CLOCK, BMAL1, Nrf2 and HO-1 were purchased from Cell Signaling Technology, Inc. (cat. nos. 5157S, 14020S, 12721S, and 43966S, respectively), GADPH and Lamin B1 were from Proteintech (cat. nos. 60004-1-Ig and 66095-1-Ig), and tyrosine hydroxylase (TH) antibody was from ABclonal Technology Co., Ltd. (cat. nos. A12756). Blood urea nitrogen (BUN) and serum creatinine (Scr) were measured using an Olympus automatic analyzer, and norepinephrine (NE) and neutrophil gelatinase associated lipocalin (NGAL) levels were quantified using the corresponding enzyme-linked immunosorbent assay (ELISA) kit purchased from Wuhan USCN Business Co., Ltd. Superoxide dismutase (SOD) and malondialdehyde (MDA) assay kits were purchased from Nanjing Jiancheng Biochemicals Ltd. Interleukin-6 (IL-6), IL-17AF (heterodimer) and tumor necrosis factor (TNF-α) assay kits were purchased from Thermo Fisher Scientific.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Renal Denervation (RDN) model\u003c/h2\u003e \u003cp\u003eAnimals were anesthetized using 5% chloral hydrate injected intraperitoneally at the dose of 10ml/kg. The RDN model was established by wrapping bilateral renal artery with 10% phenol for 15 minutes until the renal artery turned white (Xiao, et al., 2015). The decrease of NE content in renal tissue by more than 85% can be used as the standard to judge the effect of complete denervation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Renal Ischemia Reperfusion (IR) model\u003c/h2\u003e \u003cp\u003eAnimals were anesthetized using 5% chloral hydrate injected intraperitoneally at the dose of 10ml/kg. The IR model was established by bilateral renal pedicle occlusion for ischemia (30 min), followed by removal of the microvascular clip for 24 h reperfusion (Shiva, et al., 2020). The procedure was successful if the kidney turned from red to black after the pedicle occlusion, then black to red after gradual removal the clip.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Experimental protocol\u003c/h2\u003e \u003cp\u003eMice were randomly assigned into one of 4 experimental groups in ZT0 or ZT12 individually (n\u0026thinsp;=\u0026thinsp;6 per group) as follows: (1) Sham group (S group) that underwent laparotomy without RDN and occlusion for kidney; (2) RDN group; (3) IR group; (4) RDN\u0026thinsp;+\u0026thinsp;IR group (DIR group) that established IR model 7 days after the RDN model was successfully established. To examine the effects of Nrf2 pathway in renal IR injury following RDN, the specific inhibitor of Nrf2-Brusatol (Sigma-Aldrich, Shanghai, China) was dissolved in 1% of DMSO and intraperitoneally injected at a dose of 2 mg/kg every 2 days for 10 days before IR model established. The Nrf2 activator-tert-butylhydroquinone (t-BHQ, Sigma-Aldrich, Shanghai, China), was diluted in 1% of DMSO and intraperitoneally injected at a dose of 16.7 mg/kg every 8 hours for one day before IR model established. To examine the effects of BMAL1 gene knockdown in renal IR injury following RDN, we used recombinant adeno-associated virus serotype 9 (AAV9) vectors which carry a CMV promoter with GFP reporter (AAV9-BMAL1 shRNA-GFP) or AAV9-GFP NC which were produced by Obio Technology (Shanghai, China) to knock down BMAL1 gene expression or as control. The three pairs of shRNA sequence were in supplementary table1. AAV-BMAL1 was given via tail vein injection at a dose of 2 \u0026times; 10\u003csup\u003e12\u003c/sup\u003e vg/kg once 2 weeks before RDN model established.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Histopathology of kidney tissue\u003c/h2\u003e \u003cp\u003eThe left kidney was sectioned, then fixed with 4% formaldehyde for 24 h at room temperature, then embedded in paraffin, from which 4-\u0026micro;m sections were cut and stained with hematoxylin for 3 mins and eosin for 60 sec at room temperature. The slides were evaluated using light microscopy (original magnification, \u0026times;200; Olympus BX50; Olympus Corporation). Renal histological assessment was conducted using the Banff 97 working classification (Racusen, et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) and a semi-quantitative scale, as described (Spandou, et al., 2006): For each kidney, \u0026ge;\u0026thinsp;100 cortical tubules from 10 different regions were scored. Higher scores represented more severe damage, scoring as follows: 0\u0026thinsp;=\u0026thinsp;no damage; 1\u0026thinsp;=\u0026thinsp;minimal damage (\u0026lt;\u0026thinsp;5% involvement of the cortex or outer medulla); 2\u0026thinsp;=\u0026thinsp;mild damage (5\u0026ndash;25% involvement of the cortex or outer medulla); 3\u0026thinsp;=\u0026thinsp;moderate damage (25\u0026ndash;75% involvement of the cortex or outer medulla); 4\u0026thinsp;=\u0026thinsp;severe damage (\u0026gt;\u0026thinsp;75% involvement of the cortex or outer medulla).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Measurement of Scr, BUN and NGAL levels\u003c/h2\u003e \u003cp\u003eAfter the end of IR, 1 ml blood was collected from left ventricle apical area, centrifuged at 3,000 \u0026times; g for 10 mins at 4˚C, and then serum was separated and stored at -20˚C. Scr and BUN were measured using an Olympus automatic analyzer (AU5400; Olympus Corporation), and NGAL level were measured using ELISA assay kit (cat. no. SEB388Mu; Wuhan USCN Business Co., Ltd) according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Measurement of SOD activity, MDA, IL-6, IL-17AF (heterodimer) and TNF-ɑ levels in renal tissues\u003c/h2\u003e \u003cp\u003eRenal tissues were harvested and immediately homogenized on ice in 5 volumes of normal saline. The homogenates were centrifuged at 1,200 \u0026times; g for 10 min at 4˚C. SOD activity (cat. no. A001-1) and MDA levels (cat. no. A003-1) were measured using chemical assay kits (Nanjing Jiancheng Biochemicals Ltd.), and IL-6 (cat. no. 88-7064-88), IL-17AF (heterodimer) (cat. no. 88-8711-88) and TNF-ɑ levels (cat. no. 88-7324-88) were measured using chemical assay kits (Thermo Fisher Scientific) in accordance with the manufacturer\u0026rsquo;s protocol. SOD activity was expressed as U/mg protein and 1 unit of enzyme is defined as the enzyme activity that inhibits the autoxidation of pyrogallol by 50%. The MDA content was expressed in nmol of MDA/ mg protein. The IL-6, IL-17AF (heterodimer) and TNF-ɑ levels was expressed in pg/ mg protein\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Western blot analysis\u003c/h2\u003e \u003cp\u003eCytoplasmic and nuclear proteins of the renal tissues were extracted using nuclear and cytoplasmic protein extraction kit (cat. no. P0028; Beyotime Institute of Biotechnology) according to the manufacturer\u0026rsquo;s instructions. After measurement of the protein concentration using the bicinchoninic acid method, an equal quantity of 50 \u0026micro;g protein was separated by 12% SDS‑PAGE at 100 V for 3 h. After electrophoresis, proteins were transferred onto polyvinylidene difluoride membranes at 200 mA for 2 h. Each membrane was blocked with 5% nonfat milk for 2 h at room temperature, then incubated overnight at 4˚C with an appropriate primary antibody: CLOCK, BMAL1, Nrf2 and HO-1 (each at 1:1000 dilution). After repeated washing with Tween‑20 (TBS‑T) the membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. The immunoreactive bands were visualized by enhanced chemiluminescence (cat. nos. NCI5079; Thermo) and captured on BLT PHOTON TECHNOLOGY (Pro Ⅱ). The optical density of the bands was measured with ImageJ Software version 1.6 (National Institutes of Health, Bethesda, MD, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 RNA extraction and quantitative PCR (qPCR)\u003c/h2\u003e \u003cp\u003eAccording to the manufacturer's protocol, TRIzol reagent (Thermo Fisher Scientific) was used for the extraction of total RNA from the tissues and the BeyoRT\u0026trade; First Strand cDNA synthesis kit (cat. no. D7166; Beyotime Institute of Biotechnology) was used for the preparation of cDNA. All qPCR reactions were performed in triplicates with iQ SYBR Green Supermix on an iCycler Real-Time Detection System (Bio-Rad Laboratories, Inc., USA). The thermocycling conditions were as follows: 95˚C for 10 min, followed by 40 cycles of 95˚C for 15 sec, 60˚C for 60 sec, and 72˚C for 60 sec. The expression levels of each targeted mRNA sequence were calculated by applying the 2\u003csup\u003e\u0026minus;∆∆\u003c/sup\u003eCt method after being normalized to GADPH. Primers were in supplementary table1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 mRNA-sequencing and data processing\u003c/h2\u003e \u003cp\u003e2 \u0026micro;g of total RNA was used to sequence mRNA libraries (30\u0026ndash;40\u0026nbsp;million paired-end reads in each sample) through High Seq 2000 (Illumina, San Diego, CA, USA). The Fastp and FastQC were used to process the original image data for base calling, preliminary quality analysis and data filtering. Gene expression calculation was based on read counts from StringTie and fragments per kilo bases per million reads (FPKM)\u0026thinsp;\u0026gt;\u0026thinsp;0.1 indicates meaningful expression. For samples with biological replicates, differentially expressed gene (DEG) clustering analysis was performed using the DESeq2 according to the criteria of |Fold Change| \u0026gt; 2 and p-adjust\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The Gene Ontology (GO, Gene Ontology database) functional enrichment analysis provided information on how the DEGs were related to certain biological functions using GOSeq.\u0026nbsp;KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis was based on KEGG pathway units and used a hypergeometric test to find the pathways of the DEGs.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003e2.11 Statistical analysis\u003c/span\u003e \u003c/p\u003e \u003cp\u003eAll outcome measurements were expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD values (n\u0026thinsp;=\u0026thinsp;6) and analyzed using Graph Pad Prism 9.0 (GraphPad Software, Inc.). Statistical significance of differences among groups was determined by a one-way ANOVA with Tukey\u0026rsquo;s post-hoc test. Comparison of two groups was evaluated using a t-test. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate a statistically significant difference.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Circadian rhythm disorder in the kidney following RDN\u003c/h2\u003e \u003cp\u003eAfter bilateral RDN model has been established 7 days, the decreases of NE and TH levels in the kidney was identified the success of RDN model with the disordered arrangement of renal nerve fibers, necrosis of a large number of nerve cells, vacuolization of cytoplasm and pyknosis of nuclei (Supplementary Figure S1). In order to explore the circadian rhythm disorder after RDN, qPCR was used to measure the mRNA levels of the CLOCK, BMAL1 and Nrf2 in the kidney that were collected every 4 h from ZT0, finding that they displayed a strong endogenous circadian rhythm in the S group, but the rhythmic oscillations of those genes were obviously disordered in the kidney following RDN (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Western blot analysis indicated that the CLOCK, BMAL1 and Nrf2 protein expression levels also exhibited the synchronous rhythm in the normal kidney, as ZT20-ZT0 was the expression peak phase and ZT8-ZT12 was the tough phase. However, the synchronous rhythm of CLOCK, BMAL1 and Nrf2 protein expressions were broken in the kidney following RDN, which were upregulated by RDN at ZT12 timepoint (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 RDN aggravated the diurnal variability of renal injury induced by IR\u003c/h2\u003e \u003cp\u003eBase on the above results, the renal IR model was established at the timepoints close to the peak or trough of Nrf2 protein expression in the normal kidney. In both ZT0 and ZT12 timepoint, there was no significant injury in the kidney by H\u0026amp;E staining and detection of BUN and Scr levels after RDN, indicated that RDN didn\u0026rsquo;t cause damage to the normal kidney (Supplementary Figure S2). Thus in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, IR induced histological changes to renal tubules, including tubular epithelial edema and swelling, lumen dilation, epithelial simplification, nuclear necrosis, cytoplasmic translucency and vacuolation. Compared with ZT0, the histological injury was significantly aggravated when the IR model was established at ZT12 both in IR and DIR groups, while the histological injury score significantly increased after RDN treatment compared to IR groups at both ZT0 and ZT12 timepoint (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). As the same, Scr, BUN and NAGL levels were higher in the DIR group compared with the IR group at both ZT0 and ZT12 timepoint (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Overall, the results indicated that renal injury induced by IR has diurnal variability, and RDN aggravated the diurnal variability of renal injury induced by IR, which might be related to the circadian rhythm disorder after RDN.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 RDN increased oxidative stress and inflammation in the kidney following nocturnal IR\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, compared with ZT0 timepoint, the establishment of IR model at ZT12 timepoint with or without RDN significantly decreased SOD activity and increased MDA content, IL-6, IL-17AF and TNF-ɑ levels in the renal tissues, which indicated that IR induced increased oxidative stress and inflammation in the kidney at nighttime. After bilateral RDN model has been established 7 days, SOD activity was lower and MDA content, IL-6, IL-17AF and TNF-ɑ levels were higher in the ZT12 DIR group than those in the IR ZT12 groups, which was not such remarkable between the DIR ZT0 and IR ZT0 groups. In addition, there was no significant changes in the indexes of oxidative stress and inflammation in the kidney in both ZT0 and ZT12 timepoint after RDN (Supplementary Figure S3). To this end, RDN increased oxidative stress and inflammation in the kidney following nocturnal IR, which was consistent with the results of renal morphological and functional injury. It\u0026rsquo;s suggested that RDN disordered the rhythm of Nrf2 might result in the decline of renal ability to resist oxidative stress and the aggravation of inflammatory response induced by IR.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Gene Comparison Analysis from kidneys following IR injury with/ without RDN\u003c/h2\u003e \u003cp\u003ePrincipal component analysis (PCA) and correlation heatmap of all expressed genes (Fig. S4) revealed that the transcriptomic profiles among different groups were distinct and well separated. Heatmap and ingenuity pathway analysis of differentially expressed (DE) genes (Supplementary Fig. S5) indicated that circadian rhythm of kidneys was significant different in S ZT0 and S ZT12 groups. Except circadian rhythm, IL-17 signaling pathway, cortisol and cholesterol metabolism and cytokine-cytokine receptor interaction were also different in RDN ZT0 and RDN ZT12 groups. Especially, PI3K-AKT pathway was strongly influenced by RDN treatment in ZT12, which was not reflected between S ZT0 and RDN ZT0 groups. As expected, IR effected catalytic activity, transmembrane transport and inflammatory response with or without RDN treatment, including TNF signaling pathway, cytokine-cytokine receptor interaction and PI3K-AKT signaling pathway (Supplementary Fig. S6). Compared with ZT0 groups, ingenuity pathway analysis revealed that the inflammatory and immune response signaling pathways were highly modulated in kidneys from ZT12 groups in both IR and DIR groups, including IL-17, cytokine-cytokine receptor interaction and TNF signaling pathways. Noteworthily, compared with IR ZT12 group, circadian rhythm, JAK-STAT, NF-kappa B and HIF-1 signaling pathways were emerged in DIR ZT12 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5 RDN regulated clock genes and Nrf2/ARE pathway in the kidney following IR\u003c/h2\u003e \u003cp\u003eNext, we further verified the role of BMAL1/Nrf2/ARE pathway in RDN aggravating the diurnal variability of renal IRI. In the IR groups, the protein expressions of BMAL1, CLOCK, Nrf2 and HO-1 were all lower in the ZT12 timepoint than those of ZT0 timepoint. Thus, in the DIR groups, the protein expressions of BMAL1, CLOCK, Nrf2 and HO-1 were all higher in the ZT12 timepoint than those of ZT0 timepoint. In other words, the protein expressions of BMAL1, CLOCK, Nrf2 and HO-1 were upregulated in DIR ZT12 group compared to IR ZT12 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.6 RDN impaired the protective effect of activating Nrf2/ARE pathway in renal IR injury at nighttime\u003c/h2\u003e \u003cp\u003eAs known that Nrf2/ARE pathway played a key role in renal IR injury, we further explored the effect of RDN on regulation of Nrf2 in renal IR injury at nighttime using the t-BHQ (Nrf2 activator) or Brusatol (Nrf2 antagonist) treatment. In IR groups without RDN, renal injury was significantly enhanced after the Brusatol treatment with the increase of histological injury score and the levels of BUN, Scr and NGAL, as well as the decrease of SOD activity and increase of the levels of MDA, IL-6, IL-17AF (heterodimer) and TNF-ɑ in the kidney, while renal IR injury was attenuated after the t-BHQ treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-J left). Thus, in DIR groups, there was only aggravated damage after the Brusatol treatment but no significant improvement after the t-BHQ treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-J right), which might be consistent with the changes of Nrf2 and HO-1 protein expressions. Compare with IR group, t-BHQ increased Nrf2 and HO-1 protein expressions, which were decreased by Brusatol. Nrf2 and HO-1 protein expressions were only decreased by Brusatol in DIR groups, but there was no significant increase induced by t-BHQ treatment in DIR groups. The above results suggested that RDN might interfere with the expression of Nrf2 protein in renal IR injury, resulting in impairing the protective effect of activating Nrf2/ARE pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003e3.7 Genetic knockdown of BMAL1 recovered the activation of Nrf2/ARE pathway after RDN in renal IR injury at nighttime\u003c/span\u003e \u003c/p\u003e \u003cp\u003eTo explore the role of BMAL1, which was disordered by RDN, in regulating the activation of the Nrf2/ARE pathway in renal IR injury at nighttime, the BMAL1 gene was knockdown by AAV9-BMAL1 shRNA injection. As shown in Supplementary Figure S7, AAV successfully transfected kidney cells by GFP detection, and BMAL1 protein was effectively downregulated in kidney by Western Blot analysis. After downregulation of BMAL1, t-BHQ treatment could significantly attenuate the renal IR injury in the DIR ZT12 group, with the decrease of histological injury score and the levels of BUN, Scr and NGAL, as well as the increase of the level of SOD and decrease of the level of MDA, IL-6, IL-17AF (heterodimer) and TNF-ɑ compared with AAV-NC group, which was not appeared in the AAV-BMAL1\u0026thinsp;+\u0026thinsp;DIR group without t-BHQ treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-J). Furthermore, the protein expressions of Nrf2 and HO-1 were significantly increased in the AAV-BMAL1\u0026thinsp;+\u0026thinsp;DIR\u0026thinsp;+\u0026thinsp;t-BHQ group compared with the AAV-NC\u0026thinsp;+\u0026thinsp;DIR\u0026thinsp;+\u0026thinsp;t-BHQ group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK-M). To this end, it\u0026rsquo;s illustrated that knockdown BMAL1 gene alone cannot reverse the aggravation of renal IR injury at nighttime associated with RDN treatment, but it could recover the protective effect of activating Nrf2/ARE pathway by t-BHQ.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur present study is the first time focusing on the impact of RDN on renal IR injury with the following novel findings. First, RDN disordered circadian rhythm of clock genes and Nrf2 in the kidney without the histological and functional injury. Second, RDN aggravated the diurnal variability of renal injury with increased oxidative stress and inflammation in the kidney following nocturnal IR. Third, RDN lead to the disruption of BMAL1-mediated Nrf2 rhythm accumulation in the kidney, which reduced the ability to resist oxidative stress and impaired the protective effect of activating Nrf2/ARE pathway in renal IR injury at nighttime. Moreover, knockdown BMAL1 gene alone cannot reverse the aggravation of renal IR injury at nighttime caused by RDN, but it could recover the protective effect of activating Nrf2/ARE pathway by t-BHQ. Above all, our results suggested that RDN aggravated renal IRI at nighttime associated with BMAL1-mediated Nrf2/ARE pathway.\u003c/p\u003e \u003cp\u003eClinical observations have shown that the rhythm disorder after renal transplant remained, such as sleep disturbance, fatigue, hypertension and metabolic impairment in ERSF patients, which indicates that circadian clock system involved in the graft\u0026rsquo;s function of renal transplant patient (Mendoza-Romo-Ram\u0026iacute;rez, et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) (Pisano, et al., 2021). The literature has demonstrated that nocturnal surgery might increase the morbidity and mortality with a higher risk of postoperative complications compared to daytime surgery in lung transplant and cardiac surgery and myocardial tolerance to IRI imposed daytime variation (Qiu, et al., 2018) (Montaigne, et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) (Cunningham, et al., 2019). Thus, the cohort studies referring to the impact of time-of-day on graft outcomes and complications in patients undergoing renal transplant is still controversial with limited numbers in recent years. Although a few studies did not observe the difference of time-of-day on outcomes, it may be due to the disorder of preoperative biological rhythm in patients with ESRF (Ville, et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) (Treacy, et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) (Sug\u0026uuml;nes, et al., 2019) (Guerrero, et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Meanwhile, the latest cohort study with 10291 patients observed that compared to nocturnal declamping, day-time declamping was associated with a better prognosis of kidney transplantation despite a longer duration of cold ischemia (Montaigne, et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similarly, our previous studies have confirmed that bilateral occlusion IR model established at nighttime induced graver renal injury regarding that circadian rhythm of clock genes, and Nrf2 accumulation were a novel endogenous mechanism in renal IRI and repair processes (Sun, et al., 2021). It has also been confirmed in this study that renal IRI was aggravated at nighttime associated with BMAL1-mediated Nrf2/ARE pathway, which could be attenuated by Nrf2 activator t-BHQ.\u003c/p\u003e \u003cp\u003eMoreover, this study has paid close attention to the impact of RDN on the diurnal variation of renal IR injury, which is inevitable in the process of graft acquisition. The newest study showed that renal IRI aggravated in bilateral sympathetic denervation rat model by superior cervical ganglionectomy via enhancing the inflammatory response (Zhang, et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). And numerous studies have shown that RDN mediated circadian blood pressure control in resistant hypertensive patients that might contribute to a significant improvement in dipping pattern and nocturnal systolic blood pressure (Becker, et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) (Osborn, et al., 2021). It\u0026rsquo;s likely that the peripheral nerves of kidneys play an important role in circadian variability of renal function, which is synchronously regulated by the suprachiasmatic nucleus (SCN) of hypothalamus that is the control center of circadian clock (Gumz, 2016) (Mohandas, et al., 2022). Similarly, our results indicated that RDN disordered circadian rhythm of core clock genes and Nrf2 in the kidney, which couldn\u0026rsquo;t induce any histological and functional injury in the kidney. Nevertheless, RDN aggravated the diurnal variability of renal injury induced by IR with increased oxidative stress and inflammation in the kidney, as the renal injury was grater following nocturnal IR.\u003c/p\u003e \u003cp\u003eTo explore the internal mechanism, we examined the expression levels of genes associated with circadian clock system and Nrf2/ARE pathway. Evidence from an increasing number of clinical and laboratory studies has indicated that there is obvious diurnal variation in IR injury of different organs including myocardial, renal and hepatic IR, as the circadian clock plays a vital role to protect the tissues and cells from oxidative injury induced by excessive ROS (Qiu, et al., 2021) (Lin, et al., 2020) (Correa-Costa, et al., 2018). As known, Nrf2 is recognized as the master transcription factor of the ROS homeostasis in renal IR injury (Zhuang, et al., 2022). Recent studies have found that the Nrf2/ARE pathway is regulated by the circadian clock that BMAL1 controls Nrf2 rhythm accumulation via E-BOX element (Mezhnina, et al., 2022) (Tamaru, et al., 2016). The activity of Nrf2 on circadian rhythm manner is vital to maintain ARE related target genes for protecting against oxidative injury in many pathological processes (Bevinakoppamath, et al., 2022). Similarly, in our previous and this study, we found that renal IR model established at nighttime with the trough expressions of BMAL1-mediated Nrf2 suffered the severe oxidative injury due to the weaken expressions of downstream antioxidant proteins. But far more than this, RDN disordered circadian rhythm of core clock genes and Nrf2 in the kidney, and the dysrhythmia of the Nrf2/ARE pathway reduced the rhythmic regulation to impair the sensitivity to resist oxidative stress induced by IR at nighttime. Moreover, knockdown BMAL1 gene cannot reverse the aggravation of renal IR injury at nighttime caused by RDN, which may be associated with that the circadian rhythm manner can\u0026rsquo;t be recovered by knockdown BMAL1 gene alone.\u003c/p\u003e \u003cp\u003eIn addition, our previous study has shown that IR-mediated oxidative stress injury in the kidney is ameliorated by treatment with t-BHQ, as an Nrf2 activator (Chen, et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). On the one hand, the canonical Nrf2 activation pathway is that electrophilic compounds such as t-BHQ help Nrf2 dissociate from Keap1, translocate into the nucleus and bind to the ARE resulting in transcription of the phase II genes. On the other hand, t-BHQ can increase Wilms tumor gene in chromosome X protein that decreases Nrf2 ubiquitination and activates Nrf2 transcriptional activity through non-canonical Nrf2 activation pathway (Kim and Jeon, 2022) (Silva-Islas and Maldonado, 2018). Unfortunately, the nocturnal IR injury couldn\u0026rsquo;t be attenuated by t-BHQ in the kidney following RDN in this study, while knockdown BMAL1 gene could recover the protective effect of activating Nrf2/ARE pathway by t-BHQ. Thus, we speculate that Nrf2 has been exhausted in the renal IR at nighttime following RDN, which can\u0026rsquo;t be continuously activated by t-BHQ. Meanwhile, decreased BMAL1 may alleviate the depletion of Nrf2 and restore the protective effect of t-BHQ to a certain extent. Of course, there are other possibilities. It\u0026rsquo;s reported that the role of t-BHQ on ROS-mediated dissociation of Nrf2-Keap1 may differ according to phase II enzyme and cell line investigated, in which the treatment with t-BHQ alone did not induce ROS (Gharavi, et al., 2007). Respecting to Nrf2 stability, t-BHQ only increases the level of Nrf2 protein, but not the Nrf2 mRNA level in HepG2 cells (Nguyen, et al., 2003). And t-BHQ does not inhibit the Nrf2 ubiquination directly, which merely stabilizes the ubiquinated Nrf2 (Li, et al., 2005). To this end, furthermore studies are required to elucidate the intrinsic mechanisms of t-BHQ or its metabolites of IR-induced oxidative stress injury in the kidney with the dysrhythmia of the Nrf2/ARE pathway.\u003c/p\u003e"},{"header":"5. Limitation And Future Study","content":"\u003cp\u003eThis is the first and pioneering study involved in the effect of RDN on renal IR injury and its internal mechanism, so there are still many limitations in this study. First, we established RDN model using renal artery cauterization wrapped by phenol classically with high success rate and low mortality, which can\u0026rsquo;t distinguish between afferent and efferent nerves. Studies have shown that renal afferent and efferent nerves have different regulatory roles in maintaining renal function (Zhang, et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) (Becker, et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) (Osborn, et al., 2021). Consequently, we can improve the establishment of RDN to explore the effects of afferent and efferent nerves (Qiu, et al., 2018) on renal IR injury associated with circadian rhythm respectively. Secondly, a few clinic observations referring to the impact of time-of-day on graft outcomes and complications in patients undergoing renal transplant haven\u0026rsquo;t observe the difference of time-of-day on outcomes, which may be due to the disorder of preoperative circadian rhythm in patients with ESRF (Treacy, et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) (Sug\u0026uuml;nes, et al., 2019) (Guerrero, et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the EDSF model has not been used in this study, which is a certain gap with clinical renal transplant. Hence, the conclusions of this study pay special attention to the function and regulation of circadian rhythm in normal kidney. We are conducting in-depth research using the EDSF model, but the establishment of this complex animal model is facing great challenges. Thirdly, in order to synchronize the circadian rhythm of experimental mice, we have homogenized the genes, light, diet, modeling and other related confounders, but there are still individual differences unavoidably. Therefore, the time-of-day for IR modeling in this study is nothing but near the peak and tough timepoint of circadian rhythm oscillation, and this interference can only be diminished by expanding the sample size. Finally, up to now, the researches about the pharmacokinetic parameters of t-BHQ or its metabolism in human and mice are not sufficient (Gharavi, et al., 2007). This study has observed that t-BHQ can\u0026rsquo;t effectively reverse the nocturnal renal IR injury after RDN by activating Nrf2/ARE pathway, which is closely related to the expression of core clock gene BMAL1. Respecting to the advance researches on chronotherapy in recent years (Gharavi, et al., 2007) (Seeman, et al., 2022), more researches are required to optimize the treatment scheme based on the specific characteristics of circadian rhythm for patients following renal transplantation.\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eAfter RDN, the clock rhythm of kidney is disordered, resulting in the aggravation of renal IR injury at night, which is related to BMAL1-mediated Nrf2/ARE pathway. The treatment of t-BHQ can\u0026rsquo;t recover the nocturnal IR injury in kidney following RDN by activating Nrf2/ARE pathway, which can be repaired by BMAL1 knockdown. It is suggested that more researches are required to optimize the reasonable and individualized chronotherapy based on the specific characteristics of circadian rhythm, which will effectively improve the graft survival rate and the quality of life for renal transplant patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eThe experimental protocol of the present study was approved by the Ethics Committee of Renmin Hospital of Wuhan University and in accordance with the principles of Laboratory Animal Care by the National Institutes of Health (permit no. 20210124).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This study was supported National Natural Science Foundation of China (No. 82072140), Tianjin Science and Technology Fund Planning Project (No. 21JCYBJC01130) and China Scholarship Council (No. 202006275056).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions:\u003c/strong\u003e Design, writing draft and funding acquisition, QS; data collection and analysis, YW, QT and CZ; investigation and resources, LD; review, editing and funding acquisition, CD. 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Pediatr Transpl. 2022 Mar;26(2):e14192. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/petr.14192\u003c/span\u003e\u003cspan address=\"10.1111/petr.14192\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"circadian rhythm, nuclear factor E2 related factor 2, renal ischemia-reperfusion injury, renal denervation, tert-butylhydroquinone ","lastPublishedDoi":"10.21203/rs.3.rs-2017001/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2017001/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e Denervation is an inevitable pathological situation of renal graft. This study was to explore the change of clock gene rhythm under renal denervation (RDN) and its effect on renal function and oxidative stress during renal ischemia-reperfusion (IR) injury.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod\u003c/strong\u003e C57/BL6 mice were randomly divided into 4 groups at daytime 7AM (zeitgeber time [ZT] 0) or at nighttime 7PM (ZT12) in respectively: Sham (S) group, RDN group, IR group and RDN+ IR (DIR) group. Renal pathological and functional changes were assessed by H\u0026amp;E staining, and serum creatinine, urea nitrogen and neutrophil gelatinase-associated lipocalin levels. Renal oxidative stress was detected by SOD and MDA levels, and renal inflammation was measured by IL-6, IL-17AF and TNF-ɑ levels. BMAL1, CLOCK, Nrf2 and HO-1 mRNA and protein expressions were tested by qPCR and Western Blot. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResult\u003c/strong\u003e Compared with S groups, the rhythm of BMAL1 and CLOCK genes in the kidney was disordered in RDN groups, while renal pathological and functional indexes did not change significantly. Compared with IR groups, renal pathological and functional indexes were significantly higher in the DIR groups, as well as oxidative stress and inflammation in renal tissues. The protein expressions of BMAL1 and Nrf2 were upregulated by RDN at ZT12 timepoint. In DIR groups, renal injury was aggravated after the Brusatol treatment, but there was no significant improvement after the t-BHQ treatment, which might be consistent with the changes of Nrf2 and HO-1 protein expressions. Knockdown BMAL1 gene alone didn’t reverse the aggravation of renal IR injury at nighttime caused by RDN, but it could recover the protective effect of activating Nrf2/ARE pathway by t-BHQ.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e RDN lead to the disruption of BMAL1-mediatedNrf2 rhythm accumulation in the kidney, which reduced the renal ability to resist oxidative stress and inflammation, due to the impaired effect of activating Nrf2/ARE pathway in renal IR injury at nighttime.\u003c/p\u003e","manuscriptTitle":"Denervation Aggravates Renal Ischemia Reperfusion Injury via BMAL1-mediated Nrf2/ARE pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-10-17 14:52:40","doi":"10.21203/rs.3.rs-2017001/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"11793e18-1ec1-4a51-94a4-1bb858f5c4e7","owner":[],"postedDate":"October 17th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-01-21T20:05:46+00:00","versionOfRecord":[],"versionCreatedAt":"2022-10-17 14:52:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2017001","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2017001","identity":"rs-2017001","version":["v1"]},"buildId":"ehx78VzkSd0WSzXnipQa-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}