**1. 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., HgCl2) [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

**Citation:** Blas-Valdivia, V.;

Rojas-Franco, P.; Serrano-Contreras, J.I.; Sfriso, A.A.; Garcia-Hernandez, C.; Franco-Colín, M.; Cano-Europa, E. C-phycoerythrin from *Phormidium persicinum* Prevents Acute Kidney Injury by Attenuating Oxidative and Endoplasmic Reticulum Stress. *Mar. Drugs* **2021**, *19*, 589. https://doi.org/ 10.3390/md19110589

Academic Editors: Donatella Degl Innocenti and Marzia Vasarri

Received: 4 September 2021 Accepted: 16 October 2021 Published: 20 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

environment. The increase in oxidants is not counteracted by the antioxidant system and therefore leads to oxidative stress [5,6] and endoplasmic reticulum (ER) stress [3].

ER stress disrupts proteostasis in this organelle, causing the accumulation of unfolded and misfolded proteins. To maintain ER function, the unfolded protein response is activated through the protein kinase RNA-like ER kinase (PERK), activating transcription factor 6α (ATF6α), and inositol-requiring enzyme 1α (IRE1α) pathways.

The PERK pathway, crucial in regulating the unfolded protein response, reduces transcription through phosphorylation of the eukaryotic translation initiation factor-2α (eIF2α). If ER stress is controlled, protein folding can resume, and the phosphorylated eIF2α dephosphorylates. In the event that ER stress is sustained, the activating transcription factor 4 (ATF4)/growth arrest and DNA damage-inducible gene 153 (GADD153, also called CHOP) pathway activates the expression of genes that participate in redox homeostasis, autophagy, and/or apoptosis. The particular genes involved depend on the level of ER stress [7,8].

In parallel, the ATF6α pathway diminishes ER stress by regulating genes that encode ER chaperones and enzymes responsible for promoting folding, maturation, secretion, or degradation of misfolded proteins. When ER stress is sustained, the cell activates autophagy and apoptosis by upregulating the generation of reactive oxygen species (ROS) and activating ER membrane-associated caspase 12 through the ATF4/GADD153 pathway [7,8].

Additionally, IRE1α contributes to adaptation or apoptosis under chronic ER stress. The adaptation response of IRE1α is activated by selective cleavage of X-box binding protein 1 (XBP1) mRNA to produce spliced isoforms of XBP1, which enhance the transcription of chaperones, foldases, and components of the ER-associated protein degradation (ERAD) response to restore proteostasis. In case ER stress is still uncontrolled, IRE1α activates c-Jun N-terminal kinases 1 (JNK1) to promote the translocation of B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) into the mitochondrial membrane, which triggers the release of cytochrome c and the second mitochondrial-activated factor (Smac), leading to the activation of caspases 3 and 9 [9].

Some research groups have been developing eco-friendly therapeutic strategies for AKI from microalgae pigments such as phycobiliproteins, toroidal light-harvesting proteins in cyanobacteria, and the photosynthetic apparatus in algae. The most studied phycobiliprotein with nephroprotective activity is C-phycocyanin (C-PC). It impedes kidney failure by decreasing oxidative stress and ER stress in mice intoxicated with mercury [6,10,11]. Moreover, other phycobiliproteins, including C-phycoerythrin (C-PE), have nutraceutical activity against metabolic and toxic injury that affects certain organs (e.g., the liver) in animal models [12,13].

C-PE, an oligomeric chromoprotein of cyanobacteria, is composed of monomers αβ and prosthetic covalently linked open-chain tetrapyrrole moieties denominated Cphycoerythrobilin. In *Phormidium* sp., the monomer units oligomerize to form trimers (αβ)3 and then stack as hexamers [(αβ)3]2 [14]. C-PE is widely used in the food and cosmetic industries, as well as in diagnosis and research. There are reports on its nutraceutical properties, which stem from scavenging and antioxidant activity [15]. Our research group demonstrated that a C-PE-rich protein extract from *Pseudanabaena tenuis* has nephroprotective activity [16], although its mechanism is still not completely understood. The aim of the current contribution was to determine whether the nephroprotective activity of C-PE (purified from *Phormidium persicinum*) is related to a reduction in oxidative stress and ER stress, and consequently an attenuation of the alterations in the levels of nephrin and podocin normally caused by HgCl2-induced AKI.

#### **2. Results**

#### *2.1. Characterization of C-PE from Phormidium persicinum*

The absorbance spectra from various steps of purification (Figure 1) show an absorbance peak at 562 nm. The A562/A280 ratio increased with each purification step, thus being the greatest (4.35) for the final product of purified C-PE.

**Figure 1.** The absorbance spectra for the process of purification of C-phycoerythrin (C-PE) from *Phormidium persicinum* taken after the following events: the centrifugation cycles (**A**), Sephadex column chromatography (**B**), (NH4)2SO4 precipitation (**C**), and dialysis and concentration (**D**).

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).

**Figure 2.** Representative native- and sodium dodecyl sulfate (SDS)-polyacrylamide gels (PAGE) during the process of purification of C-phycoerythrin (C-PE) from *Phormidium persicinum*, taken after the following events: the centrifugation cycles (A), Sephadex column chromatography (B), (NH4)2SO4 precipitation (C), and dialysis and concentration (D).

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.

**Figure 3.** 3D spectrum of the excitation-emission matrix (EEM) of C-phycoerythrin (C-PE), with the emission and excitation regions in the range of 550–640 and 300–600 nm, respectively (**A**). Expansion of the EEM for emission (555–595 nm) and excitation (510–577 nm) (**B**).

#### *2.2. 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 GSH2/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 GSH2/GSSG ratio, while ameliorating the elevated level of nitrites (from 171% to 139%).

**Figure 4.** Effect of C-phycoerythrin (C-PE) on HgCl2-induced oxidative stress and alterations in the redox environment of the kidney. Oxidative stress markers (**A**–**C**). Redox environment markers (**D**–**F**). Data are expressed as the mean ± SEM (*n* = 6 mice/group). One-way ANOVA and the Student-Newman-Keuls post hoc test. RFU, relative fluorescence units. \* *p* < 0.05 vs. the control group. \*\* *p* < 0.05 vs. the HgCl2 group.

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).

**Figure 5.** Effect of C-phycoerythrin (C-PE) on the decreased expression of nephrin (**A**) and podocin (**B**) in the kidneys that results from the exposure of mice to HgCl2. (**C**) Representative Western blots of each experimental group. Data are expressed as the mean ± SEM (*n* = 6 mice/group). OD, optical density. One-way ANOVA and the Student-Newman-Keuls post hoc test. \* *p* < 0.05 vs. the control group. \*\* *p* < 0.05 vs. the HgCl2 group.

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.

**Figure 6.** Representative photomicrographs of the renal cortex of animals intoxicated with HgCl2 and treated with C-phycoerythrin (C-PE). HgCl2 causes cell atrophy, hyperchromatic nuclei, and edema. Histological alterations were ameliorated in groups treated with C-PE. The tissue was stained with hematoxylin-eosin. The lower right bar represents 250 μm.

> 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 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.

**Figure 7.** The effect of C-phycoerythrin (C-PE) on the activity of caspases 3 (**A**) and 9 (**B**) and the kidney damage score (**C**) in mice with HgCl2-induced AKI. In (**A**) and (**B**), data are expressed as the mean ± SEM (*n* = 6 mice/group). Data were analyzed with one-way ANOVA and the Student-Newman-Keuls post hoc test. \* *p* < 0.05 vs. the control group. \*\* *p* < 0.05 vs. the HgCl2 group. In (**C**), each box represents the median ± interquartile space. Data were examined with the Kruskal–Wallis test and Dunn post hoc test. \* *p* < 0.05 vs. the control group. \*\* *p* < 0.05 vs. the HgCl2 group.
