**4. Materials and Methods**

#### *4.1. 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 HgCl2 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 HgCl2 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 HgCl2 ip and 100 mg/kg/day C-PE og).

C-PE or the vehicle was administered 30 min before the injection of HgCl2 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.

#### *4.2. Cultivation, Purification, and Characterization of C-PE from Phormidium persicinum*

*P. persicinum* was obtained from the culture collection of the Centro de Investigaciones Biológicas del Noroeste, S. C. (CIB 84). It was grown in a synthetic medium (denominated NM), created and optimized by our group (composition: 29 g/L of commercial sea salt, 0.8 g/L NaHCO3, 0.05 g/L K2HPO4, 2.16 g/L NaNO3, 5 mg/L MgSO4, 1 mg/L FeSO4, and 1 mL of a micronutrient solution containing 0.2 mM EDTA, 46.2 mM H3BO3, 9.3 mM MnCl2, 0.95 mM ZnSO4, 2.03 mM Na2MoO4, 0.49 mM Ca(NO3)2, and 0.77 mM CuSO4). Incubation was carried out at 21 ± 2 ◦C with constant aeration provided by an air pump, under green LED illumination (24 W, 3000 Lx) and a 12/12 h light/dark cycle (lights on at 8:00 AM).

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 (NH4)2SO4 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:

$$\text{CPE}\left(\frac{m\chi}{dL}\right) = \frac{[Absorhana\varepsilon\_{562\text{ }nm} - 0.1374]}{0.3540}; r^2 = 0.9899; r = 0.9949; \dots$$

The purity index was calculated as the ratio of the maximum absorbance peak to the absorbance peak of the proteins (A562/A280) [38].
