Tolvaptan activates the Nrf2/HO-1 antioxidant pathway through PERK phosphorylation

Tolvaptan, a vasopressin type 2 receptor antagonist initially developed to increase free-water diuresis, has been approved for the treatment of autosomal dominant polycystic kidney disease in multiple countries. Furthermore, tolvaptan has been shown to improve the renal functions in rodent models of chronic kidney disease (CKD); however, the underlying molecular mechanisms remain unknown. CKD is characterized by increased levels of oxidative stress, and an antioxidant transcription factor—nuclear factor erythroid 2-related factor 2 (Nrf2)—has been gaining attention as a therapeutic target. Therefore, we investigated the effects of tolvaptan and a well-known Nrf2 activator, bardoxolone methyl (BARD) on Nrf2. To determine the role of tolvaptan, we used a renal cortical collecting duct (mpkCCD) cell line and mouse kidneys. Tolvaptan activated Nrf2 and increased mRNA and protein expression of antioxidant enzyme heme oxygenase-1 (HO-1) in mpkCCD cells and the outer medulla of mouse kidneys. In contrast to BARD, tolvaptan regulated the antioxidant systems via a unique mechanism. Tolvaptan activated the Nrf2/HO-1 antioxidant pathway through phosphorylation of protein kinase RNA-like endoplasmic reticulum kinase (PERK). As a result, tolvaptan and BARD could successfully generate synergistic activating effects on Nrf2/HO-1 antioxidant pathway, suggesting that this combination therapy can contribute to the treatment of CKD.


Tolvaptan induces Nrf2 nuclear translocation and HO-1 expression in mpkCCD cells. To eval-
uate the effect of tolvaptan on Nrf2 signaling in the kidney, we administered tolvaptan to mpkCCD cells and collected the nuclear extract to evaluate Nrf2 nuclear translocation. The effect of tolvaptan was compared with that of known Nrf2 activator, sulforaphane, as a positive control. Nrf2 nuclear translocation was significantly increased by tolvaptan in mpkCCD cells (Fig. 1a). Further, tolvaptan increased HO-1 mRNA and protein expression (Fig. 1b,c). Tolvaptan-induced HO-1 expression was inhibited by an Nrf2 inhibitor, ML385 (Fig. 1d) 18 . These results indicated that tolvaptan promoted Nrf2 nuclear translocation and activated the antioxidant systems in mpkCCD cells.

Tolvaptan activates the Nrf2/HO-1 antioxidant pathway through PERK phosphorylation.
Further, we investigated the key mediators of the tolvaptan/Nrf2/HO-1 signaling pathway. In previous reports, Nrf2 phosphorylation as well as oxidative stress disrupted Keap1-Nrf2 interaction and activated the antioxidant systems 10 . Nrf2 is directly phosphorylated by protein kinase RNA-like endoplasmic reticulum kinase (PERK) and  www.nature.com/scientificreports www.nature.com/scientificreports/ splicing of Xbp-1 mRNA and increased the expression of Grp78 mRNA, tolvaptan did not affect the IRE-1/XBP-1 pathway (Fig. 2e). These results indicated that tolvaptan could selectively activate the PERK signaling pathway without activation of IRE-1.

Tolvaptan induces Nrf2 nuclear translocation and HO-1 expression in vivo.
We verified the effect of tolvaptan on the Nrf2/HO-1 antioxidant pathway in vivo; to this end, we administered 0.5% tolvaptan via diet to male C57BL/6J mice for 24 h because 0.05%-0.5% tolvaptan in diet is required to attenuate renal damage 6,7 . Further, we measured the amount of water and feed intake and calculated the total 24 h dose of tolvaptan. Mean water intake was 10.5 ± 1.7 g in the control group and 35.6 ± 5.3 g in the tolvaptan-treated group. The average dose of tolvaptan was 23.8 ± 7.7 mg. On evaluating Nrf2 nuclear translocation and HO-1 protein expression in the kidneys using nuclear extracts (Table 1) and whole tissue lysates ( Table 2) of tolvaptan-treated mice, it was observed that although tolvaptan did not increase HO-1 protein expression in the renal cortex, it could successfully induce Nrf2 nuclear translocation and HO-1 protein expression in the renal outer medulla (Fig. 3a,b). However, no PERK phosphorylation was detected by western blotting and immunostaining analysis using commercially available antibodies.

Tolvaptan activates the Nrf2/HO-1 antioxidant pathway independently of cAMP signaling.
Considering that tolvaptan activates the Nrf2/HO-1 pathway, other V2R antagonists could probably exert the same effect. Therefore, we examined the effect of mozavaptan, which has a chemical structure quite similar to that of tolvaptan (Fig. 4a). The administered doses of tolvaptan and mozavaptan were sufficiently high to inhibit vasopressin/cAMP signaling and completely counteracted the effect of [deamino-Cys1, d-Arg8]-vasopressin (dDAVP) on aquaporin-2 (AQP2) phosphorylation at serine 269 ( Fig. 4b) 5,27 . Nevertheless, in contrast to tolvaptan, mozavaptan did not induce HO-1 protein expression in mpkCCD cells (Fig. 4c). These results indicate that the suppression of cAMP signaling is insufficient to induce HO-1 transcription.
Tolvaptan and BARD synergistically activate the Nrf2/HO-1 antioxidant pathway. Tolvaptan activated the Nrf2/HO-1 pathway via a different mechanism to that of BARD. BARD interacts with cysteine residues of Keap1 and inhibits the Keap1-Nrf2 binding, leading to Nrf2 nuclear translocation 28 . In contrast, tolvaptan activated Nrf2 through PERK phosphorylation. We examined whether tolvaptan and BARD additively or synergistically activate the Nrf2/HO-1 antioxidant pathway and found that BARD of concentration >25 nM activated the Nrf2/HO-1 antioxidant pathway in mpkCCD cells (Fig. 5a,b). Interestingly, 200 μM tolvaptan plus 25 nM BARD synergistically activated Nrf2 nuclear translocation and increased HO-1 mRNA and protein expression ( Fig. 5c-e). PERK phosphorylation was only induced by tolvaptan.

Water intake (g)
Dose of tolvaptan (mg)  Table 1. The amount of water intake and the dose of tolvaptan of mice analyzed as detailed in Fig. 3a. The dose of tolvaptan was calculated from the amount of feed intake. The average amount of water intake is 11.1 ± 1.0 g in control group and 39.2 ± 4.1 g in the tolvaptan-treated group. The average dose of tolvaptan is 28.5 ± 8.5 mg.
Water intake (g) Dose of tolvaptan (mg)  Table 2. The amount of water intake and the dose of tolvaptan of mice analyzed as detailed in Fig. 3b. The dose of tolvaptan was calculated from the amount of feed intake. The average amount of water intake is 9.95 ± 2.0 g in control group and 31.9 ± 3.5 g in the tolvaptan-treated group. The average dose of tolvaptan is 19.1 ± 1.7 mg.

Discussion
In the present study, we clarified that tolvaptan activated the Nrf2/HO-1 antioxidant pathway in mpkCCD cells and the outer medulla of mouse kidneys. To date, the V2R antagonist tolvaptan has provided clinical benefits in patients with heart failure, SIADH, and ADPKD by inhibiting intracellular cAMP production in the kidney 1-4 . We found novel pharmacological properties of tolvaptan that upregulated the Nrf2-antioxidant systems independently of cAMP signaling (Fig. 4a-c). Although inflammation and oxidative stress are prevalent in CKD, paradoxical Nrf2-dysregulation and unresponsiveness of antioxidant enzymes are observed 13,29,30 . To improve this situation, Nrf2 activators, such as BARD, have received much attention as the next-generation therapeutic target of CKD. Therefore, the activation of tolvaptan/PERK/Nrf2/HO-1 signaling pathway is a potential therapeutic target of CKD (Fig. 6).
Moreover, we found that tolvaptan and BARD synergistically activated the Nrf2/HO-1 antioxidant pathway ( Fig. 5c-e). In addition to the enhancement of their drug efficacies, tolvaptan offers the possibility of avoiding the undesirable side-effects of BARD. BARD increased cardiovascular diseases, particularly heart failure, in the phase-3 Bardoxolone Methyl Evaluation in Patients with Chronic Kidney Disease and Type 2 Diabetes Mellitus: the Occurrence of Renal Events (BEACON) trial, resulting in premature termination of the trial 31 . In the BARD-treated group, fluid retention may have caused hemodilution, thereby increasing blood pressure, and leading to a higher incidence of heart failure. On the contrary, the diuretic effect of tolvaptan decreases fluid overload without deterioration of the renal functions in patients with heart failure and CKD 32 . Consequently, the combined therapy of tolvaptan and BARD may improve BARD-induced positive fluid balance and contribute to the treatment of CKD via synergistic induction of the antioxidant defense systems.
Tolvaptan-induced Nrf2/HO-1 antioxidant pathway was mediated by ER-localized transmembrane kinase, PERK (Fig. 2b-d); however, the precise mechanism of PERK phosphorylation remains unclear. Interestingly, tolvaptan is known as a cell-permeable pharmacological chaperon that can directly bind to misfolded V2R mutants retained in the ER and thereby facilitate their proper folding and plasma membrane trafficking 33,34 . Both V2R mutants and wild-type V2R are constitutively misfolded and degraded in the ER. Protein folding efficiency of G-protein coupled receptors, including V2R, is only <50% 35 . Tolvaptan may directly bind to misfolded wild-type V2R in the ER and subsequently modulate PERK phosphorylation without activation of the ER stress transducer, IRE-1 (Figs 2e, 6). The significant difference in terms of HO-1 activation between tolvaptan and mozavaptan www.nature.com/scientificreports www.nature.com/scientificreports/ further supports our notion (Fig. 4c). Tolvaptan is a more potent pharmacological chaperon than mozavaptan, and only tolvaptan has been shown to increase membrane trafficking of wild-type V2R 34 . The ability of tolvaptan to bind to wild-type V2R in the ER suggests that it can activate the PERK/Nrf2/HO-1 signaling pathway in other V2R-expressing cells. Indeed, tolvaptan activated Nrf2-regulated antioxidant enzymes, such as HO-1 and NAD(P)H:quinone oxidoreductase-1 (NQO-1), in heart-derived H9C2 cells, which endogenously expressed V2R ( Supplementary Fig. 1a-c) 11,36 . Tolvaptan may activate the antioxidant systems in extra-renal organs as well as in kidneys. Conversely, tolvaptan was not effective in renal proximal tubule-derived HK2 cells, which did not endogenously express V2R ( Supplementary Fig. 2a-c) 37 .
The Nrf2/HO-1 antioxidant pathway was successfully activated in the renal outer medulla of tolvaptan-treated mice. Mean dose of tolvaptan for mice in the present study was approximately 1000 mg/kg/day (Tables 1 and 2). Based on the results of the area under the curve of tolvaptan, the exposure level of 1000 mg/kg/day tolvaptan in a mouse is equivalent to that of 250 mg/day in a human 38 . In a previous report, 0.05% tolvaptan in diet improved the renal function and histopathology in a rodent model of end-stage heart failure 7 . Moreover, 0.1% tolvaptan is protective against podocyte damage and proteinuria in a rodent model of puromycin aminonucleoside induced-nephrosis 6 . These beneficial effects of tolvaptan are presumably partly caused by the activation of the Nrf2/HO-1 antioxidant pathway. Previous evidence and our results propose that 25-250 mg/day tolvaptan in clinical use may exert renal protective effects. In addition, the combination therapy of tolvaptan and BARD is a promising strategy to reduce the dose of tolvaptan.
In the kidney, V2R is strongly expressed in the outer medulla 37 . In addition, tolvaptan antagonizes the binding of vasopressin to V2R especially in the medulla region of the kidney 39 . Based on the fact that tolvaptan induced antioxidant pathway in the outer medulla (Fig. 3b), tolvaptan may contribute to the treatment of acute kidney injury (AKI) as well as CKD. Due to the relatively larger consumption of O 2 , the renal medulla is vulnerable to hypoxia. Nrf2 activation is a promising therapeutic target to ameliorate tubular necrosis and apoptosis of the outer medulla caused by ischemic renal injury 40 . Tolvaptan is a potential therapeutic target of ischemic AKI which is a common clinical event and causes progression of CKD.

Animals. All experiments were performed in accordance with the guidelines for animal research of Tokyo
Medical and Dental University, and the study protocol was approved by The Animal Care and Use Committee of Tokyo Medical and Dental University (approval number: A2019-183C3). Male C57BL/6J mice (8-9-week-old) (CLEA JAPAN) were maintained under standard lightning conditions (12 h:12 h light-dark cycle). The mice were randomly divided into two groups: the control group (n = 8) and the tolvaptan-treated group (n = 10). Individual www.nature.com/scientificreports www.nature.com/scientificreports/ housing (one per cage) was provided for all mice, and they had free access to water and feed. The control group received a normal chow without tolvaptan, whereas the tolvaptan-treated group received a normal chow with 0.5% tolvaptan for 24 h. Following the administration of tolvaptan, the mice were euthanized and their kidneys were removed and separated into the cortex and outer medulla for protein extraction.
Reverse transcription-polymerase chain reaction (Rt-pCR) analysis. Total RNA was extracted using the Sepazol ® -RNA ISuper G (Nacarai Tesque), and cDNA was synthesized using the ReverTra ® Ace (Toyobo), according to the manufacturer's instruction. The forward and reverse Xbp-1 primers used were the same as previously described 44 . PCR amplification consisted of 35 cycles (95 °C for 10 s, 62 °C for 15 s, 72 °C for 30 s) after an initial denaturation step at 95 °C for 3 min. The PCR products were analyzed by electrophoresis on 2.0% agarose gel. Tolvaptan phosphorylated PERK independently of cAMP signaling, following which PERK directly phosphorylates Nrf2, leading to the dissociation of Nrf2 from Keap1 10,25 . Once released from Keap1, Nrf2 is not degraded by proteasome and translocates from the cytosol to nucleus. Tolvaptan increases the transcription of an antioxidant enzyme HO-1 in mpkCCD cells and the outer medulla of mouse kidneys. A PERK inhibitor, GSK2606414, counteracted the effect of tolvaptan on HO-1 expression. Although the precise mechanisms remain unclear, tolvaptan may bind to membrane-and ER-localized V2R and modulate PERK phosphorylation. Tolvaptan activated Nrf2 via a different mechanism to that of BARD. As a consequence, the combination therapy of tolvaptan and BARD could synergistically activate the Nrf2/HO-1 antioxidant pathway.