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.