C-phycoerythrin from Phormidium persicinum Prevents Acute Kidney Injury by Attenuating Oxidative and Endoplasmic Reticulum Stress

C-phycoerythrin (C-PE) is a phycobiliprotein that prevents oxidative stress and cell damage. The aim of this study was to evaluate whether C-PE also counteracts endoplasmic reticulum (ER) stress as a mechanism contributing to its nephroprotective activity. After C-PE was purified from Phormidium persicinum by using size exclusion chromatography, it was characterized by spectrometry and fluorometry. A mouse model of HgCl2-induced acute kidney injury (AKI) was used to assess the effect of C-PE treatment (at 25, 50, or 100 mg/kg of body weight) on oxidative stress, the redox environment, and renal damage. ER stress was examined with the same model and C-PE treatment at 100 mg/kg. C-PE diminished oxidative stress and cell damage in a dose-dependent manner by impeding the decrease in expression of nephrin and podocin normally caused by mercury intoxication. It reduced ER stress by preventing the activation of the inositol-requiring enzyme-1α (IRE1α) pathway and avoiding caspase-mediated cell death, while leaving the expression of protein kinase RNA-like ER kinase (PERK) and activating transcription factor 6α (ATF6α) pathways unmodified. Hence, C-PE exhibited a nephroprotective effect on HgCl2-induced AKI by reducing oxidative stress and ER stress.


Introduction
Acute kidney injury (AKI), a syndrome engendered by sepsis, cardiorenal syndrome, urinary tract obstruction, and nephrotoxins, is known to increase the level of serum creatinine and/or decrease urine output. It is an important public health issue because of being a serious complication for 10-15% of hospitalized patients and~50% of those in intensive care [1].
Animal models of AKI are induced by administering a drug or toxicant (e.g., HgCl 2 ) [2,3]. Mercury targets the kidney by binding to thiol-containing proteins in the tubular and glomerular nephron portion, disrupting the tubular transport mechanism related to Na +/ K + -ATPase [4]. It also alters the intracellular calcium current and consequently the redox The images of native-and SDS-PAGE at each step of the purification process show that the α and β C-PE subunits correspond to ~19 and ~21 KDa, respectively ( Figure 2). The excitation-emission matrix (EEM) spectrum corresponding to the 3D fluorescence fingerprint of purified C-PE is shown in Figure 3 (panel A). The expansion of the same EEM displays the emission and excitation regions in the range of 555-595 and 510-570 nm, respectively (panel B). The fingerprint of C-PE exhibits a sharp fluorescence peak at Eex/Eem 563/574 nm (corresponding to fluorochrome) next to Rayleigh-Tyndall's scattered light lines. The 3D spectrum of EEM features three principal Ex/Em peaks at 563/574, 545/574, and 530/574, and a small Ex/Em peak at 385/575. Two shoulders are present on the lower part of the main peak, the first at Eex/Eem 545/574 nm and the second at Eex/Eem 530/574 nm. Another weak peak can be observed at Eex/Eem 385/575 nm. The excitation-emission matrix (EEM) spectrum corresponding to the 3D fluorescence fingerprint of purified C-PE is shown in Figure 3 (panel A). The expansion of the same EEM displays the emission and excitation regions in the range of 555-595 and 510-570 nm, respectively (panel B). The fingerprint of C-PE exhibits a sharp fluorescence peak at E ex /E em 563/574 nm (corresponding to fluorochrome) next to Rayleigh-Tyndall's scattered light lines. The 3D spectrum of EEM features three principal Ex/Em peaks at 563/574, 545/574, and 530/574, and a small Ex/Em peak at 385/575. Two shoulders are present on the lower part of the main peak, the first at E ex /E em 545/574 nm and the second at E ex /E em 530/574 nm. Another weak peak can be observed at E ex /E em 385/575 nm.

Evaluation of Oxidative Stress, the Redox Environment, the Activity of Effector Caspases 3 and 9, the Expression of Nephrin and Podocin, and Renal Damage
The effect of C-PE on HgCl2-induced oxidative stress and alterations in the redox environment is illustrated in Figure 4 (panels A-C and D-E, respectively). Animals intoxicated with HgCl2 showed higher renal oxidative stress, indicated by the corresponding increase in lipid peroxidation (panel A, ~374%), ROS (panel B, ~211%), and nitrites (panel C, ~171%). Mercury intoxication also caused a lower GSH 2 /GSSG ratio (panel F, ~66%) and greater GSSG content (panel E, ~269%). On the other hand, all doses of C-PE treatment prevented the HgCl2-induced increase in lipid peroxidation, ROS, and GSSG, and the alteration in the GSH 2 /GSSG ratio, while ameliorating the elevated level of nitrites (from 171% to 139%).

Evaluation of Oxidative Stress, the Redox Environment, the Activity of Effector Caspases 3 and 9, the Expression of Nephrin and Podocin, and Renal Damage
The effect of C-PE on HgCl 2 -induced oxidative stress and alterations in the redox environment is illustrated in Figure 4 (panels A-C and D-E, respectively). Animals intoxicated with HgCl 2 showed higher renal oxidative stress, indicated by the corresponding increase in lipid peroxidation (panel A,~374%), ROS (panel B,~211%), and nitrites (panel C,~171%). Mercury intoxication also caused a lower GSH 2 /GSSG ratio (panel F,~66%) and greater GSSG content (panel E,~269%). On the other hand, all doses of C-PE treatment prevented the HgCl 2 -induced increase in lipid peroxidation, ROS, and GSSG, and the alteration in the GSH 2 /GSSG ratio, while ameliorating the elevated level of nitrites (from 171% to 139%). Regarding the proteins associated with glomerular damage ( Figure 5), mercury decreased the expression of nephrin (A) and podocin (B) by ~65% and ~71%, respectively. Treatment with C-PE partially reduced, by ~36% and ~48%, the downregulation of nephrin and podocin, respectively. These changes can be appreciated by the corresponding Western blots ( Figure 5C). Regarding the proteins associated with glomerular damage ( Figure 5), mercury decreased the expression of nephrin (A) and podocin (B) by~65% and~71%, respectively. Treatment with C-PE partially reduced, by~36% and~48%, the downregulation of nephrin and podocin, respectively. These changes can be appreciated by the corresponding Western blots ( Figure 5C).  According to typical photomicrographs of the renal cortex stained with hematoxylineosin (H&E) (Figure 6), the control (vehicle only) and C-PE only groups had normal cytoarchitecture, which is characterized by glomeruli and the surrounding tubules with cuboidal epithelium. The photomicrographs of the group treated with mercury only display edema, cellular atrophy of distal and proximal tubules, distortion of cellular continuity, loss of the cell nucleus, hyperchromatic nuclei, and glomerulosclerosis. The AKI mice According to typical photomicrographs of the renal cortex stained with hematoxylineosin (H&E) (Figure 6), the control (vehicle only) and C-PE only groups had normal cytoarchitecture, which is characterized by glomeruli and the surrounding tubules with cuboidal epithelium. The photomicrographs of the group treated with mercury only display edema, cellular atrophy of distal and proximal tubules, distortion of cellular continuity, loss of the cell nucleus, hyperchromatic nuclei, and glomerulosclerosis. The AKI mice treated with C-PE exhibited a dose-dependent nutraceutical effect capable of preventing cellular damage. treated with C-PE exhibited a dose-dependent nutraceutical effect capable of preventing cellular damage. The effect of C-PE on the activity of caspases 3 and 9 is shown in Figure 7 (panels A and B, respectively). HgCl2 generated an increase of ~511% and ~347% in the level of caspases 3 and 9, respectively. These results indicate grade 4 histological damage (panel C), affecting over 75% of the tubules and glomerulus. C-PE diminished damage in a dosedependent manner (panel C). The highest C-PE dose (100 mg/kg/day) led to grade 1-2 kidney damage, affecting 25-50% of the tubules and glomerulus. The effect of C-PE on the activity of caspases 3 and 9 is shown in Figure 7 (panels A and B, respectively). HgCl 2 generated an increase of~511% and~347% in the level of caspases 3 and 9, respectively. These results indicate grade 4 histological damage (panel C), affecting over 75% of the tubules and glomerulus. C-PE diminished damage in a dose-dependent manner (panel C). The highest C-PE dose (100 mg/kg/day) led to grade 1-2 kidney damage, affecting 25-50% of the tubules and glomerulus. Mar. Drugs 2021, 19, x 9 of 19

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. HgCl 2 -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. HgCl 2 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.

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.   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.

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 Hg 2+ with intracellular sulfhydryl-containing 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][21][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

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 HgCl 2 -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 HgCl 2 -induced ER stress, oxidative stress, and alterations in the redox environment in the same animal model. HgCl 2 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 Hg 2+ 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 HgCl 2 -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][21][22]. Purified C-PE presently demonstrated nephroprotective activity when tested against HgCl 2 -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 HgCl 2 -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 Hg 2+ by PEB suppresses the degranulation of RBL-2H3 mast cells and decreases the intracellular concentration of Ca 2+ [27], giving rise to anti-inflammatory and nephroprotective effects. Hg 2+ 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, HgCl 2 -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, HgCl 2 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 his-tological 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.

Animals
Forty-eight male albino NIH Swiss mice (25-30 g) were kept in a cool room (21 ± 2 • C) with 40-60% relative humidity under a 12/12 h light/dark cycle (lights on at 8 AM). Food and water were provided ad libitum. The experimental procedures were in accordance with the Official Mexican Norm (NOM-062-ZOO-1999, technical specifications for the production, care, and use of laboratory animals) [35]. The protocol was approved by the institutional Internal Bioethics Committee (ZOO-013-2021).
The animals were divided into two lots to carry out distinct protocols, one to assess oxidative stress and kidney damage and another to analyze ER stress. For the evaluation of oxidative stress and kidney damage, 36 mice were randomly allocated to 6 groups (n = 6). Three were control groups: (1) the vehicle (negative control), with 100 mM of phosphate buffer (PB, at pH 7.4) administered by oral gavage (og) + 0.9% of saline solution (SS) applied intraperitoneally (ip), (2) AKI induced by a single application of 5 mg/kg HgCl 2 ip + the vehicle (PB) og, and (3) C-PE treatment, consisting of 100 mg/kg/day C-PE og + 0.9% SS ip. The other three groups received a single application of HgCl 2 ip as well as 25, 50, or 100 mg/kg/day C-PE og. For the analysis of ER stress, twelve mice were randomly allocated to four groups with the following treatments (n = 3): (1) the control (vehicle), (2) mercury-induced AKI, (3) the C-PE treatment, and (4) the AKI + C-PE treatment (a single application of HgCl 2 ip and 100 mg/kg/day C-PE og).
C-PE or the vehicle was administered 30 min before the injection of HgCl 2 or 0.9% of SS. C-PE was administered once daily for five days (the first protocol) or for three days (the second protocol) at the same time (12:00 AM) each day. Whereas the mice assigned to the evaluation of oxidative stress and renal damage were euthanized 5 days after mercury intoxication, those employed for assessing ER stress were euthanized 3 days after the same event. The right kidneys were frozen at −70 • C to await examination of the markers of oxidative stress and the redox environment by Western blot, while the left kidneys were put into paraformaldehyde in PBS (4% v/v) to appraise cell damage. Regarding the purification of C-PE, the cyanobacterial biomass was centrifuged at 10,000× g for 1 min and 5-10 g of the resulting cell pellet was re-suspended in 20 mL of distilled water. Subsequently, three freeze-thaw cycles were performed, freezing at −20 • C and thawing at 4 • C during 24 h. The resulting slurry was centrifuged in 4 cycles at 21,400× g for 10 min at 4 • C to remove the cell debris. An aliquot of 20 mL of the phycobiliprotein-rich extract was injected into a column (33 cm long × 4.7 cm in diameter) containing Sephadex G-250 gel previously equilibrated with 10 mM of PB (pH 7.4). The pink fractions were obtained and precipitated with a saturated solution of (NH 4 ) 2 SO 4 at 4 • C for 24 h in the dark. This mixture was centrifuged at 21,400× g for 2 min at 4 • C, and the resulting pellet was resuspended in 100 mM of PB at pH 7.4. The membrane was then dialyzed with PB for 24 h, after which time an aliquot of C-PE was immediately lyophilized to construct a calibration curve, obtain an absorption spectrum, and characterize the extract fluorometrically with an EEM. The C-PE extract was solubilized in PB and 5 mM of sucrose and frozen at −20 • C to await administration to the animals [36].
The EEM was recorded by scanning excitation and emission simultaneously in a Luminescent Spectrometer (Perkin Elmer LS 55) equipped with a Xenon discharge lamp and an excitation/emission slit 5/5. The scans were processed by 3D View Perkin Elmer software to produce 3D fingerprint contour maps by using fluorescence lines (with emission plotted on the X-axis and excitation on the Y-axis), as previously reported [37].
The calibration curve of 0.6-6 mg/mL of C-PE solubilized in PB was calculated as follows: The purity index was calculated as the ratio of the maximum absorbance peak to the absorbance peak of the proteins (A 562 /A 280 ) [38].

Evaluation of Oxidative Stress, the Redox Environment, and the Activity of Effector Caspases 3 and 9
Kidneys were homogenized in 3.5 mL of 10 mM PB for all assays. The quantification of oxidative stress, the redox environment, and the activity of effector caspases 3 and 9 was performed with a previously described method [3,22].
The lipid peroxidation technique employed an aliquot of 500 µL of homogenate, which was added to 4 mL of chloroform-methanol (2:1, v/v). The mixture was agitated and kept at 4 • C for 30 min (protected from light) to allow for the separation of the polar and nonpolar phases. Afterwards, the aqueous phase was aspirated and discarded. With an aliquot of 2 mL of the organic phase (chloroform), fluorescence was determined at 370 nm (excitation) and 430 nm (emission). The results were expressed as relative fluorescence units (RFU) per mg of protein.
The level of ROS was quantified by the formation of 2,7-dichlorofluorescein (DCF), and 10 µL of the homogenate was added to 1945 µL of TRIS-HEPES (18:1 v/v) and incubated in the presence of 50 µL of 2,7-dichlorofluorescin diacetate (DCFH-DA) at 37 • C for 1 h. The reaction was stopped by freezing, and the fluorescence was measured at 488 nm (excitation) and 525 nm (emission).
Nitrites were assessed as indirect markers of nitrergic stress. An aliquot of 500 µL of homogenate was added to 500 µL of concentrated chlorohydric acid and 500 µL of 20% zinc suspension. The mixture was stirred and incubated at 37 • C for 1 h, followed by centrifugation at 4000× g for 2 min. The supernatant (50 µL) was added to a 96-well polystyrene plate containing 50 µL of 0.6% sulfanilamide and 0.12% N-(naftyl)-ethylenediamine, and then incubated for 15 min at room temperature. The absorbance was measured at 530 nm in a Multiscan Go ® plate spectrophotometer.
A determination was made of two redox environment markers, GSH and GSSG, in a sample of 300 µL, treated with 500 µL of 30% phosphoric acid and centrifuged at 10,000× g for 30 min at 4 • C. To analyze GSH, an aliquot of 30 µL of the supernatant was diluted in 1.9 mL of FEDTA (1:10, 100 mM phosphate and 5 mM EDTA), and the mixture was reacted with 100 µL of o-phthaldialdehyde. To assess GSSG, 130 µL of the supernatant was added to 60 µL of N-ethylmaleimide and left for 30 min. Subsequently, an aliquot of 60 µL of the mixture was combined with 1.84 mL of FEDTA and 100 µL of o-phthaldialdehyde. The two chemical species were measured at 350 nm (excitation) and 420 nm (emission).
The activity of caspases 3 and 9 was evaluated using a commercial colorimetric assay kit as specified in the manufacturer's instructions (Millipore, APT165 and APT173, respectively). Accordingly, p-nitroaniline (pNA) was cleaved from the substrate N-Acetyl-Asp-Glu-Val-Asp p-nitroaniline (DEVD-pNA, caspase 3) or N-Acetyl-Leu-Glu-His-Asp

Conclusions
The nutraceutical effect of C-PE on HgCl 2 -induced AKI stems from its antioxidant activity, which reduces the level of oxidative stress markers and maintains the redox environment. Additionally, C-PE modulates intracellular signaling pathways involved in proteostasis, avoiding the disruption of podocytes and damage to glomerular and tubular cells. Hence, the nephroprotective activity of C-PE is related to the prevention of oxidative stress and ER stress in the kidney of animals intoxicated with mercury. The nutraceutical effect may also be related to anti-inflammatory activity, possibly triggering autophagy as a survival pathway linked to the unfolded protein response. This mechanism is worthy of greater attention in future research.

Institutional Review Board Statement:
The experimental procedures were in accordance with the Official Mexican Norm (NOM-062-ZOO-1999, technical specifications for the production, care, and use of laboratory animals) [35]. The protocol was approved by the institutional Internal Bioethics Committee (ZOO-013-2021).

Data Availability Statement:
Publicly available datasets were analyzed in this study. This data can be found here: [https://drive.google.com/file/d/15HqGDpXfEdC6_lv9RdAO3cq8glJocEbF/view? usp=sharing, accessed on 2 October 2021].