*2.3. Evaluation of ER Stress*

The effects of C-PE on the PERK/p-eIF2α (Ser52)/ATF4 and PERK/p-eIF2α (Ser52)/ ATF6α signaling pathways is portrayed in Figure 8. HgCl2-induced AKI was manifested as an overexpression of PERK (A), p-eIF2α (Ser 52) (B), ATF4 (C), GADD153 (D), GADD34 (E), and ATF6α (F). The C-PE treatment did not prevent the alteration in the expression of these proteins in both pathways. A representative Western blot of the marker for the PERK/eIF2α/ATF4 and PERK/eIF2α/ATF6α signaling pathways is shown in Figure 9.

Figure 10 shows the effect of C-PE on the IRE1α pathway and the proteins associated with cellular damage. HgCl2 exposure generated an overexpression of IRE1α (panel A), XBP1 (panel B), caspase 12 (panel C), Bax (panel D), p-p53 (Thr 155) (panel G), and p53 (panel H). It also increased the Bax/Bcl2 and p-p53 (Thr 155)/p53 ratios (panels F and I, respectively) and reduced the expression of Bcl2 (panel E). With C-PE treatment, there was no alteration in the level of any of the proteins evaluated, which is observed in the corresponding Western blot depicted in Figure 11.

**Figure 8.** Effect of C-phycoerythrin (C-PE) on HgCl2-induced endoplasmic reticulum stress through the PERK/p-eIF2α (Ser 52)/ATF4-GADD153 and PERK/p-eIF2α (Ser 52)/ATF6α/GADD153 pathways in the kidney. An evaluation was made of the expression of PERK (**A**), p-eIF2α (Ser 52) (**B**), ATF4 (**C**), GADD153 (**D**), GADD34 (**E**), and ATF6α (**F**). Data are expressed as the mean ± SEM (*n* = 3 mice/group). OD, optical density. \* *p* < 0.05 vs. the control group.

**Figure 9.** Representative Western blot of the effect of C-phycoerythrin (C-PE) on HgCl2-induced endoplasmic reticulum stress through the PERK/p-eIF2α (Ser 52)/ATF4/GADD153 and PERK/p-eIF2α (Ser 52)/ATF6α/GADD153 pathways.

**Figure 10.** Effect of C-phycoerythrin (C-PE) on HgCl2-induced endoplasmic reticulum stress and cell death. Protein expression was evaluated for IRE1α (**A**), XBP1 (**B**), caspase 12 (**C**), Bax (**D**), Bcl2 (**E**), the Bax/Bcl2 ratio (**F**), p53 (**G**), p-p53 (Thr 155) (**H**), and the p53/p-p53 (Thr 155) ratio (**I**). Data are expressed as the mean ± SEM (*n* = 3 mice/group). OD, optical density. \* *p* < 0.05 vs. the control group. \*\* *p* < 0.05 vs. the HgCl2 group.

**Figure 11.** Representative Western blot of the effect of C-phycoerythrin (C-PE) on HgCl2-induced endoplasmic reticulum stress through the IRE1α pathway and the attenuation of cell death.

#### **3. Discussion**

C-PE is reported to have nutraceutical activity against the damage resulting from cell insult [12,13]. Our group has demonstrated that treatment with a protein extract rich in C-PE prevented oxidative stress and cellular damage in an animal model of HgCl2-induced AKI [16]. This model was chosen because mercury produces ER stress, which leads to renal damage. However, the aforementioned study only associated the nutraceutical properties of C-PE with scavenging and antioxidant activity. Thus, the aim of the current contribution was to explore the molecular mechanism of action of C-PE (purified from *P. persicinum*) by examining its nephroprotective activity against HgCl2-induced ER stress, oxidative stress, and alterations in the redox environment in the same animal model.

HgCl2 produces oxidative stress and alterations in the redox environment by three mechanisms: Fenton and Haber-Weiss reactions that generate free radicals and ROS [17], the activation of ER stress [3], and the binding of Hg2+ with intracellular sulfhydrylcontaining proteins and low-molecular-weight compounds (e.g., GSH) capable of affecting the redox environment and protein function [18]. As a consequence of these reactions, nephrin and podocin are downregulated, and the slit diaphragm is injured, which is observed as HgCl2-induced AKI. The resulting inflammatory process participates in the progression of AKI [19].

In recent years, the use of nutraceuticals from cyanobacteria and their metabolites has proven effective against renal damage (e.g., AKI) stemming from toxicants or chronic kidney disease [16,20–22]. Purified C-PE presently demonstrated nephroprotective activity when tested against HgCl2-induced AKI, as evidenced by the reduction found in oxidative stress and ER stress.

C-PE, a protein with a molecular weight of ~240 KDa, has nutraceutical properties in vitro as an ROS scavenger [23]. Moreover, it prevents oxidative stress and cellular damage in vivo [12,13]. All reports on C-PE suggest that it is a potent antioxidant. By scavenging ROS, it avoids alterations in the redox environment and therefore impedes cellular damage [12,13,24]. However, animal studies have not yet completely defined the nutraceutical protection mechanism.

C-PE may act as a prodrug that leads to the release of the phycoerythrobilin moiety into the gastrointestinal tract, as previously demonstrated by our group for C-PC and phycocyanobilin [22]. C-PC is known to break down into chromo-peptides that contain phycocyanobilin, followed by the apparent absorption of linear tetrapyrrole compounds facilitated by the action of intestinal peptidases [24,25]. Once in serum, phycoerythrobilin could bind to albumin due to its low water solubility, which would extend its therapeutic activity into the entire organism [26].

The protective effect of C-PE against HgCl2-induced AKI is associated with antioxidant, anti-inflammatory, and chelation mechanisms. C-PE acts as an antioxidant because it contains PEB. In addition, the chemical structure of phycoerythrobilin acts as a nucleophilic compound, neutralizing free radicals and ROS [24]. According to an in vitro model, the chelation of Hg2+ by PEB suppresses the degranulation of RBL-2H3 mast cells and decreases the intracellular concentration of Ca2+ [27], giving rise to anti-inflammatory and nephroprotective effects. Hg2+ binds to PEB thioether bridges in C-PE, which assume a cyclic helical form capable of chelation [28]. The antioxidant and chelating activity of C-PE can avoid Fenton and Haber-Weiss reactions and consequently ameliorate the production of free radicals, the generation of oxidative stress, and the alteration of the redox environment in kidney cells. All the aforementioned mechanisms of C-PE are related to the maintenance of the redox environment and therefore prevent the dysfunction of organelles such as the ER.

In the current evaluation of proteostasis, HgCl2-induced ER stress was found to activate the IRE1α pathway and promote cell death. At the same time, mercury activated the PERK pathway, which restored proteostasis through PERK/eIF2α/ATF-4/GADD153. When the cell was incapable of compensating for imbalances in proteostasis, the activation of ATF4 and GADD153 in the same pathway led to the expression of proapoptotic proteins and the triggering of cell death. As can be appreciated, PERK and IRE1α have a synergic effect in prompting kidney cell death by increasing the Bax/Bcl-2 ratio and the level of caspases 3, 8, 9, and 12 [3,10]. Hence, HgCl2 was capable of generating AKI in the present study by fomenting oxidative stress, an alteration in the redox environment, and ER stress. The resulting histological damage was considerable (grade 4), affecting over 75% of tubular and glomerular cells.

C-PE treatment enhanced the canonical ER response through the PERK/p-eIF2α (ser 52)/ATF-4/GADD153 pathway, involving ER-associated degradation (ERAD), known to process misfolded and unfolded proteins. The phosphorylation of eIF2α (ser 52) is able to suppress the overall translation of mRNA, thus reducing protein stress in the ER. Furthermore, the moderate increment in ATF6α upregulates several genes that participate in the adaptative phase of the unfolded protein response [29]. C-PE treatment is herein proposed to have activated the PERK and ATF6 signaling pathways, maintaining proteostasis by avoiding oxidative stress and alterations in the redox environment and by activating the unfolded protein response [30,31].

The response elicited by C-PE is distinct from that of other phycobiliproteins. For instance, C-PC averts the overexpression of GADD34 by activating GADD153, which is related to the inhibition of apoptosis [11,32]. On the other hand, both C-PC and C-PE maintain proteostasis. The differences between these two responses should be explored in depth in future research.

C-PE and C-PC have a similar effect on the IREα pathway, decreasing cell death mediated by caspases 3, 9, and 12 as well as reducing the disruption in p53 activation and the alteration of the Bax/Bcl2 ratio [10,11]. This idea is supported by neurotoxicological models, where C-PE prevents ER stress linked to calcium deregulation and mitochondrial dysfunction [33].

In the control group, interestingly, C-PE per se increased the phosphorylation of p53 (Thr 155), which is a genome gatekeeper because it is a master transcriptional factor that induces cellular senescence and suppresses cell growth and tumor formation. Exposure to various cellular stressors, however, causes p53 to be overexpressed and phosphorylated in several regions, leading to cell cycle arrest or apoptosis. Accordingly, p53 is phosphorylated by the C-Jun activation domain-binding protein-1 (Jab1) in Thr 155, promoting its translocation into the cytoplasm to favor interaction with the COP9 signalosome complex. These nuclear export mechanisms of p53 provide a practical future approach to a possible C-PE-induced activation of anti-cancer therapy by p53 [34], as evidenced by the lack of histological irregularities in the C-PE control group as well as the capacity of C-PE treatment of AKI mice to prevent oxidative stress, ER stress, and alterations in the redox environment and cell death markers.
