*4.3. 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 (*p*NA) was cleaved from the substrate *N*-Acetyl-Asp-Glu-Val-Asp *p*-nitroaniline (DEVD-*p*NA, caspase 3) or *N*-Acetyl-Leu-Glu-His-Asp

*p*-nitroaniline (LEHD-*p*NA, caspase 9), and then the samples were measured spectrophotometrically at 405 nm.

#### *4.4. Examination of Kidney Damage*

Histopathological analysis was performed to appraise kidney damage. The kidneys were fixed for 48 h in 4% paraformaldehyde in PBS. Afterwards, they were embedded in paraffin to obtain 5 μm slices on a standard microtome. Each section was stained with H&E, dehydrated, and mounted in resin. The presence or absence of kidney cell damage was evaluated with a histopathological graded scale [6,20,22] as follows: 0, undamaged (indistinguishable from the controls); 1, minimal (affecting ≤25% of the tubules and glomerulus); 2, mild (affecting >25% and ≤50% of the tubules and glomerulus); 3, moderate (affecting >50% and ≤75% of the tubules and glomerulus); 4, severe (affecting >75% of the tubules and glomerulus).

#### *4.5. Western Blot Analysis for Nephrin, Podocin, and ER Stress Markers*

The expression of proteins was determined with Western blot assays. Briefly, the samples were prepared with 100 μL of the homogenate mixed with 50 μL of a complete protease inhibitor cocktail® (MilliporeSigma, Burlington, MA, USA) in lysis buffer, and then 150 μL of the 2× Laemmli sample buffer (Biorad, Hercules, CA, USA, 161-0737) was added. The samples were homogenized by vortex, placed in a boiling water bath for 3 min, and then kept at −20 ◦C to await processing. Fifty μg of protein samples were loaded in 15% polyacrylamide gel with sodium dodecyl sulfate (SDS-PAGE) and separated by electrophoresis (90 V for 60 min). Subsequently, the proteins were electrotransferred from the gels to PVDF membranes in a Trans-Blot Turbo system (Biorad) at 25 V and 2.05 A for 7 min. Upon completing this time, the membranes were blocked for 1 h under constant stirring in PBST (PBS with 0.05% Tween 20 and 5% low-fat milkSvelty®), followed by incubation overnight at 4 ◦C in a blocking buffer containing the primary antibodies. The primary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA), diluted 1:1000, were PERK (sc-377400), p-eIF2α (Ser 52, sc-12412), ATF-4 (sc-200), GADD153 (sc-56107), ATF-6α (sc-166659), IRE-1α (sc-390960), p-p53 (Thr 155, sc-377567), XBP1 (sc-7160), Bax (sc-20067), and Bcl2 (sc-7382). Podocin (orb337389), nephrin (orb11107), GADD34 (orb13417), and p53 (orb14498) were acquired from Byorbit (Cambridgeshire, Cambridge, UK) and diluted 1:500. After incubation, membranes were washed three times with fresh PBST (20 min/wash) and then incubated in a secondary antibody diluted 1:1500 (HPR-conjugated goat antirabbit; Life Technologies, Rockford, IL, USA, 65-6120) at room temperature for 1 h under constant stirring. Membranes were washed three times with fresh PBST. Finally, the protein bands were revealed on photographic plates (JUAMA, Mexico City, Mexico) by chemiluminescence, using Luminata TM Forte® (MilliporeSigma, Burlington, MA, USA). β-Actin protein expression served as the loading control and constitutive protein (Santa Cruz Biotechnology; sc-1615, dilution 1:4000). The optical density (OD) of all bands was quantified by the Image J program (NIH, Bethesda, MD, USA) and described as the protein/β-actin ratio.

#### *4.6. Statistical Analysis*

All data are expressed as the mean ± standard error, except for the kidney damage score. The latter is described as the median ± interquartile spaces, with the values analyzed with the Kruskal–Wallis method. The variables in the first protocol (to evaluate oxidative stress and the redox environment) were examined by one-way analysis of variance (ANOVA). Two-way ANOVA was utilized to assess ER stress, considering the treatment and absence/presence of AKI as factors. ANOVA was followed by the Student-Newman-Keuls post hoc test. Statistical significance was considered at *p* < 0.05.
