Introduction

Chronic kidney disease (CKD) is defined as structural and functional impairment of renal function, including a decrease in the glomerular filtration rate (GFR), glomerular sclerosis, tubular atrophy, interstitial fibrosis, and severe kidney inflammation [1, 2]. Nephrons that remain functional after CKD development cannot maintain the impairment in GFR, generating the remodeling of vascular endothelium and activating the renin-angiotensin system (RAS). The RAS is an essential regulator of cardiovascular homeostasis, and its major peptide, angiotensin II (Ang II), is the most well-characterized vasoconstrictor of the RAS [3]. Ang II induces blood pressure elevation through the AT1 receptor activation, producing vasoconstriction, oxidative stress, inflammation, and fibrosis [4]. A counter-regulatory peptide of the RAS is angiotensin 1–7 [Ang-(1–7)], which opposes the action of Ang II through activating endothelial NO synthase (eNOS) by Mas receptor [5]. Under homeostatic conditions, vascular endothelium regulates vascular tone and blood flow. However, chronic insults such as oxidative stress and inflammation alter vasoreactivity, producing advanced glycation end-products and accumulating endogenous inhibitors of eNOS [6]. These processes reduce NO bioavailability triggering endothelial dysfunction [7]. Thus, vascular endothelium damage occurs early in CKD, developing complications such as SAH, left ventricular hypertrophy, and coronary artery disease [8].

In addition, pharmacological therapies for systemic arterial hypertension (SAH) in CKD focus on maintaining blood pressure control using the minimum amount of medication and with minor adverse effects [9]. Therefore, developing therapies that retard endothelial dysfunction and control the SAH can delay CKD-induced cardiovascular complications. Nutraceuticals have attracted the attention of researchers and clinical physicians in the last few years because they reduce cardiovascular risk [10]. Spirulina (Arthrospira sp) is one of the most studied nutraceuticals with antihypertensive action [11]. It has been proposed that c-phycocyanin (CPC), a primary metabolite in the photosynthetic apparatus, is responsible for its nutraceutical antihypertensive actions [12, 13]. However, unknown is whether CPC treatment avoids CKD-altered vascular response to Ang II and Ang-(1–7). Thus, this study aims to determine if CPC prevents changes in the vascular response to Ang II and Ang-(1–7) and AT1, AT2, and Mas receptor expression in the aortic rings of rats with CKD.

Material and methods

Animals

Twenty-four male Wistar rats (250–280 g) were kept in a room with controlled conditions: constant temperate (21 ± 2 °C), relative humidity of 40–60%, and a 12/12 h light/dark cycle (lights on at 8 AM). Food and water were provided ad libitum. The experimental procedures in this research follow the guidelines of the Laws and Codes established in the Official Mexican Norm (NOM-062-ZOO- 1999) [14]. Furthermore, the protocol was approved by the institutional Internal Bioethics Committee (ZOO-018-2018).

Experimental protocol

Rats were randomly allocated into four groups (n = 6): (1) sham + 4 mL/kg/d vehicle (100 mM of phosphate buffer pH 7.4 (PBS) administered by oral gavage (og), (2) sham + 100 mg/kg/d CPC solubilized in PBS og, (3) CKD + 4 mL/kg/d vehicle og, (4) CKD + 100 mg/kg/d of CPC og. The animals were habituated to non-invasive blood pressure evaluation two weeks before surgery. Normotensive animals were used in the surgical procedure. CKD was induced by 5/6 nephrectomy, for that rats were anesthetized with ketamin-xylazine (40 and 2 mg/kg intramuscular (i.m.), respectively). In aseptic conditions, a ventral laparotomy was performed. Two branches of the left kidney artery were occluded with 4-0 black silk sutures to induce a renal infarction. Then, the right kidney was removed. After surgery, tramadol (10 mg/kg og) and enrofloxacin (10 mg/kg i.m.) were administered over two days to alleviate pain and prevent infection [13, 15]. CPC treatment began one week after surgery and continued for four weeks. The animals of the sham group were manipulated as CKD groups and treated with tramadol and enrofloxacin, except for left renal artery ligation and the right nephrectomy.

Arthrospira maxima (Spirulina) cultivation and CPC purification using for the treatment. As previously reported [13, 15]. A. maxima was cultivated in the Zarrouk medium. Incubation was carried out at 21 ± 2 °C with constant aeration provided by an air pump, under white LED illumination (24 W, 3000 Lx) and a 12/12 h light/dark cycle (lights on at 8:00 AM). For CPC purification, cyanobacterial biomass was obtained under a vacuum filtration system. Then the semi-dry biomass (5–10 g) was resuspended in 30 mL of distilled water. Subsequently, eight freeze-thaw cycles were performed, freezing at −80 °C and thawing at 4 °C. The resulting slurry was centrifuged in 10 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-100 previously equilibrated with 10 mM of PBS (pH 7.4). The bluish 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 PBS at pH 7.4. The membrane was then dialyzed with PBS for 24 h and quantified. The CPC was conservated at −20 °C with 5 mM of sucrose to await administration to the animals.

Hemodynamic variables and serum markers of renal function evaluation

The day before euthanasia, each animal was placed in metabolic cages (Tecniplast™ Metabolic Cage Systems for Rodents, Thermo Fisher Scientific) to collect 24 h urine. Systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), and heart rate were determined in conscious rats before the surgery and at the end of treatment by a non-invasive method coupled to the tail of the rat using a CODA system (Kent Scientific). Then, animals were anesthetized with isoflurane (3%) and euthanized by decapitation. Blood samples were taken to measure serum markers of renal function as blood urea nitrogen (BUN), uric acid, creatinine, and proteinuria using colorimetric assay kits (Spinreact).

Vascular response

Vascular reactivity was evaluated at the end of the CPC treatment. After euthanasia, the descending aorta was located, extracted, and immediately placed in a Petri dish with Krebs-Henseleit solution (119.5 mM NaCl, 12 mM dextrose, 24.9 mM NaHCO3, 4.74 mM KCl, 1.18 mM MgSO4·7H2O, 1.18 mM KH2PO4, 2.5 mM CaCl2·H2O, and 0.026 mM EDTA), constant carbon dioxide oxygenation (5% CO2 and 95% O2) at a temperature of 37 ± 0.2 °C and pH 7.4 ± 0.2. Aorta was carefully cleaned from the surrounding connective tissue and cut into rings approximately 3 mm wide. One section of the dissected aorta was stored at −70 °C for biochemical and molecular processing. Meanwhile, the rest of the aortic rings were mounted on two stainless steel hooks in an “L” shape and placed by duplicate in isolated organ chambers containing 2.5 mL of Krebs solution. The mechanical activity of the aortic rings was recorded isometrically using a tension transducer (FTO3, Grass Instrument Co., USA) connected to a data acquisition unit (MP 100; Biopac Systems, Inc., USA). Each ring was stabilized for 30 min under a basal tension of 2.5 g. Then, the evaluation of vascular reactivity was carried out. Concentration-response curves were performed using Ang-(1–7) to assess relaxing responses and Ang II to assess contractile responses.

The relaxing response was evaluated in aortic rings precontracted with submaximal concentrations of norepinephrine (NA, 1 × 10−7 M); after 10 min, cumulative concentrations of Ang-(1–7) (1 × 10−4 M–1 × 10−2 M) were added to the chambers, and the relaxing response was recorded. Then, the contractile responses caused by the addition of accumulated concentrations of Ang II (1 × 10−4 M - 1 × 10−2 M) were determined.

The AT1, AT2, and Mas protein expressions were determined by Western blot. Briefly, the aorta was homogenized in 200 μl of lysis buffer (Tris HCl pH 7.5, 5 M NaCl, 0.2 M NaF, 10 mM Na3VO4, NP40 1%) containing protease inhibitor (Santa Cruz Biotechnology, Dallas, Texas, USA). Subsequently, the concentration of proteins was determined using the Bradford technique. Samples were adjusted in 200 μl vials at a final concentration of 2 μg/μL with 2× loading buffer (0.125 M Tris-HCl, SDS. 4%, glycerol 20%, mercaptoethanol 10% pH 6.8); then, were placed in a boiling water bath for 3 min and kept at −20 °C to await processing. The proteins were separated by electrophoresis (100 V for 60 min) in 10% SDS-PAGE gels, to which 50 μg of protein was loaded. Subsequently, separated proteins were electrotransferred to PVDF membranes for 120 min at 70 V. Upon completing this time, the membranes were blocked for 80 min under constant stirring in PBST (PBS with 0.05% Tween 20 and 5% low-fat milkSvelty®). After three washes whit fresh PBST, membranes were cropped according to the molecular weight of the protein of interest and incubated separately overnight at 4 °C in a blocking buffer containing the corresponding primary antibodies: AT1 (1:1000, sc-515884), AT2 (1:100 sc-518054), and Mas (1:1000, sc-390453) from Santa Cruz Biotechnology. After incubation, membranes were washed three times with fresh PBST (20 min/wash) and then incubated with a secondary antibody diluted 1:1500 (goat anti-mouse-HPR from Santa Cruz Biotechnology) at room temperature one h under constant stirring. Membranes were washed three times with fresh PBST. Finally, protein signals were detected by chemiluminescence (Immobilon Western Chemiluminescent HRP Substrate; Millipore, Billerica, MA) and bands were visualized using a ChemiDocTM XRS + Imaging System (Bio-Rad, Hercules, CA) and quantified by densitometry using an image analysis software (Image Lab Software Version 5.2.1, Bio-Rad, Hercules, CA). Membranes were striped using a stripping buffer (Glycine, 200 mM; SDS, 0.1%; Tween 20, 1%; pH 2.2) and re-incubated to determine β-actin protein expression served as the loading control and constitutive protein (Santa Cruz Biotechnology; sc-1615, dilution 1:4000).

Statistical analysis

The hemodynamic variables, serum markers of renal function, and protein expression by Western blot were analyzed by two-way analysis of variance (ANOVA) followed by the Tukey post hoc test. The two factors evaluated were disease condition and CPC treatment. All data are expressed as the mean ± standard error. Statistical significance was considered at P < 0.05.

Vascular-dependent relaxation to Ang-(1–7) was expressed as a percentage inhibition of the contraction to norepinephrine. The negative logarithm of the effective concentration 50 (pEC50) represents the Ang-(1–7) concentration that induces fifty percent inhibition of the contraction to norepinephrine. Meanwhile, for the contractile vascular response to Ang II, pEC50 represents a negative logarithm of Ang II concentration that induces fifty percent of the contractile response. The pEC50 and Emax (maximum efficacy response) were calculated by the sigmoidal (4-parameter) non-linear regression curve fit using GraphPad Prism 8 (GraphPad Software, San Diego, CA). Also, the data were analyzed by three-way analysis of variance (ANOVA) followed by the Tukey post hoc test. The three factors evaluated were disease condition, CPC treatment, and dose.

Results

The evaluations of hemodynamics variables are shown in Fig. 1, where CKD caused an increase in the SBP (37.9%, Fig. 1A), DBP (27%, Fig. 1B), MAP (28.53%, Fig. 1C), and HR (37.51%, Fig. 1D) at the end of treatment. These elevations are related to CKD-caused hypertension. Meanwhile, the CPC treatment during four weeks in rats with CKD prevented the elevation of SBP, DBP, MBP, and HR preventing hypertension development.

Fig. 1
figure 1

Hemodynamic variables after four weeks of CPC treatment: systolic blood pressure (A), diastolic blood pressure (B), mean arterial pressure (C), and heart rate (D). Values represent the mean ± SEM (n = 6). (**) P < 0.001, (***) P < 0.0005, (****) P < 0.0001; Two-way ANOVA and Tukey post hoc

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The renal function evaluation is illustrated in Fig. 2. CKD caused an increase in creatinine (18%, Fig. 2A), BUN (114%, Fig. 2B), uric acid (43%, Fig. 2C), and proteinuria (15-fold over control, Fig. 2D). Meanwhile, the CPC treatment in CKD partially mitigates the increase of serum creatinine by about 8% (Fig. 2A), BUN by about 59% (Fig. 2B), uric acid by about 21% (Fig. 2C). Also, the proteinuria was reduced by CPC treatment 11 times compared to the CKD group (Fig. 2D).

Fig. 2
figure 2

Evaluation of serum markers of renal function: seric creatinine (A), BUN (B), uric acid (C), and proteinuria (D). Values represent the mean ± SEM (n = 6). (*) P < 0.05, (**) P < 0.01, P < 0.001 (***) compared to the Sham + vehicle group. (*) P < 0.05, (**) P < 0.001, (***) P < 0.0005, (****) P < 0.0001; Two-way ANOVA and Tukey post hoc

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The pharmacology of the respective agonists was determined in isolated aortic rings to determine whether the antihypertensive effects of CPC are due to changes in Mas and AT2 receptor-mediated vasodilatory responses or AT1 receptor-mediated contractile responses. In the Sham + vehicle group, Ang 1–7 (Mas receptor agonist) produced concentration-dependent vasorelaxant responses with a maximum effect (Emax) of 58.42 ± 2.55% and potency (pEC50) of 7.39 ± 0.07 (Fig. 3A). In the sham group, CPC administration showed similar Emax (p = 0.9508) and pEC50 (p = 0.1422) compared to the sham + vehicle group (Fig. 3A). On the other hand, the vasorelaxant response was lower in the CKD + vehicle group compared to the sham + vehicle group (Fig. 3B; Emax: 23.81 ± 1.83%, p = 0.0432; pEC50: 6.92 ± 0.09, p = 0.0008). Remarkably, the administration of CPC in animals with CKD prevented the alterations in the relaxing responses to Ang 1–7 compared to the CKD + vehicle group (Fig. 3C; Emax: 47.21 ± 3.56, p = 0.0376; pEC50: 7.38 ± 0.11, p = 0.0054). Concerning concentration-response curves to Ang II as an agonist of AT1 and AT2 receptors, the sham + vehicle group showed an Emax: 0.24 ± 0.01 g and a pEC50: 7.01 ± 0.02. In sham animals, CPC administration failed to modify the contractile responses (Fig. 3D, Emax: 0.23 ± 0.03 g, p = 0.9729; pEC50: 7.03 ± 0.03, p = 0.9218). Opposite to the vasorelaxant responses, the CKD + vehicle group showed an increase in the contractile responses to Ang II compared to the sham+vehicle group (Fig. 3E, Emax: 0.32 ± 0.02, p = 0.0326; pEC50: 7.28 ± 0.02, p = 0.0199). Interestingly, the treatment in CKD + CPC group prevented CKD-caused increases in contractile responses due to Ang II (Fig. 3F, Emax: 0.31 ± 0.02 g, p = 0.0480; pEC50: 6.94 ± 0.02, p = 0.0070) (Table 1).

Fig. 3
figure 3

Concentration-response curves for cumulative concentrations of angiotensin 1–7 (A–C) and angiotensin II (D–F) in thoracic aortas. Data represent the mean ± SEM of 6 animals. (*) compared with sham + vehicle at same dose, (**) compared with CKD + vehicle; RM Three-way ANOVA and Tukey post hoc

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Table 1 Negative logarithm of the effective concentration 50 (pCE50) and maximum efficacy (Emax) of relaxant responses induced by Ang (1–7), and contractile responses induced by Ang II
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Figure 4 illustrates the expression of Ang (1–7) and Ang II receptors (AT1, AT2, and Mas receptors. CKD up-regulated the expression of AT1 (Fig. 4A, ~81%) and AT2 (Fig. 4B, ~47%), and down-regulated the Mas receptor expression (Fig. 4C, ~50%) compared to the sham + vehicle group. CPC treatment in CKD partially prevented the alteration of AT1 (Fig. 4A, 52%, p < 0.001) and the Mas receptor (Fig. 4C, 25%, p < 0.05) expressions. Also, CPC treatment normalized the AT2 receptor in CKD and AT2 receptors compared to the sham group (Fig. 4B).

Fig. 4
figure 4

Protein expression of AT1 (A), AT2 (B), Mas (C) receptors, and (D) representative Western blots of each experimental group. Values represent the mean ± SEM (n = 4). (*) P < 0.05, (***) P < 0.0005, (****) P < 0.0001; Two-way ANOVA and Tukey post hoc

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Discussion

SAH treatment plays a central role in managing CKD because patients with CKD have an excessive burden of cardiovascular disease associated with endothelial dysfunction. Consequently, SAH management in CKD is an important issue for physicians and scientists because nonpharmacologic methods alone are insufficient in controlling SAH. Also, CKD patients were treated with three or more antihypertensive medications since SAH is resistant [8, 16]. Therefore, to manage SAH and its related cardiovascular complications, nutraceuticals such as Spirulina and its CPC could be used to control SAH. Although there was a proposed clinical trial with Spirulina, the complete antihypertensive action remains unknown [17]. We have proposed that CPC is one of the responsible proteins of the nutraceutical antihypertensive action [15] because it avoids endothelial dysfunction [13]. Although the complete pharmacological CPC antihypertensive mechanism is still undescribed, our study demonstrated that CPC prevents the development of vascular dysfunction through Ang II and Ang (1–7) physiological response. The pharmacological assays where the contractile responses of the aortic rings were obtained by adding cumulative Ang II concentrations showed that CPC treatment avoids the aberrant excessive contractile responses associated with vascular dysfunction. In addition, a lower expression of AT1 and AT2 receptors was found compared to animals with CKD, which is directly related to the lower response to Ang II and the antihypertensive effect of the treatment. Activating AT1 receptors by Ang II generates a vasopressor effect, increasing systemic blood pressure related to CKD-caused SAH [3]. AT1 receptor activation triggers intracellular responses associated with endothelial dysfunction in the vascular wall, like NO production disturbance, NADPH oxidase activation, VEGF, and COX2 over-expression. AngII activates protein phosphatase 2A, which dephosphorylates eNOS reducing NO production [18]. NADPH oxidase activation causes oxidative stress through ROS over-production [19]. High VEGF levels promote angiogenesis, fibrosis, and vascular remodeling [20]. Meanwhile, the COX2 in macula densa increases the prostanoids, which is a positive feedback of the RAAS exacerbating SAH [21]. In the case of CPC and its hydrolyzed peptides on RAAS, several in silico and in vitro reports have demonstrated its potential to inhibit angiotensin-converting enzyme (ACE) [22, 23]. Thus, a low Ang II level in circulation reduces its vasoconstrictor effect on the endothelium. Nevertheless, CPC is a protein (about 124.2 kDa) not absorbed directly by the gastrointestinal tract. Thus, CPC must be metabolized into peptides and chromo-peptides containing phycocyanobilin (PCB) and PCB. CPC and PCB have the potential to avoid endothelial dysfunction due to their antioxidant, anti-inflammatory [13, 24], and signaling pathway modulators on NADPH oxidase [25], COX2 [26], VEGF [27], and eNOS expression [13].

On the other hand, it has been previously demonstrated that AT2 receptor over-expression is related to vascular injury compensation against the Ang II-activated AT1 receptor [28]. Indeed, Ang II stimulates AT2 receptors reducing endothelial remodeling and delaying the increase in vascular resistance because its activation promotes vasodilation [29] and antifibrotic and antithrombotic effects [30]. Also, AT2 receptors decrease endothelial cell migration as an antiproliferative effect [31]. In rats with CKD, the up-regulation of the AT2 receptor did not compensate for AT1 receptor activation due to the exacerbated vasoconstrictor response to Ang II observed in our results. However, CPC treatment in CKD delays the vasoconstrictor response to Ang II, notwithstanding the increase of the AT1 receptor without modification in the expression of the AT2 receptor. Therefore, CPC treatment prevents CKD-caused vascular dysfunction and SAH because CPC reduces the Ang II contractile response through AT1 receptors.

Another molecule responsible for regulating the vascular tone is Ang (1–7), a heptapeptide that opposes Ang II action. Ang (1–7) exerts vasodilatory and antiproliferative effects with vasorelaxant action on arterioles through the Mas receptor [32]. In the pharmacological evaluations of the relaxing responses, by activating the Mas receptor using cumulative concentrations of Ang (1–7), we observed that animals with CKD presented a lower relaxing response and a lower Mas receptor expression than the sham group. This evidence suggests that CKD developed vascular dysfunction. In CKD animal models, the levels of Ang (1–7) are elevated [33], but the down-regulation of Mas receptors reduces vasorelaxation. Concerning CPC treatment in CKD, we observed a mild reduction in Mas receptor expression compared with CKD, but this beneficial effect improved the vasorelaxant effect of Ang (1–7) in the aorta rings. Also, Ang (1–7) can activate another compensatory mechanism associated with the delay of endothelial dysfunction, such as maintaining the REDOX balance to avoid the increase of ROS [34]. Additionally, the vascular tone is not only the product of the vasorelaxant action of Ang (1–7) but also of other molecules, such as bradykinin and acetylcholine. In fact, CPC treatment in CKD avoids the disturbance in relaxation response due to acetylcholine [13].

Finally, CKD increases serum levels of creatinine, BUN, and uric acid, waste products eliminated through the kidney and whose accumulation reflects a decrease in GFR [35]. During CKD, there is a progressive decrease in GFR, which causes many solutes and waste products to accumulate in the blood, increasing plasma osmolarity, exacerbating SAH, and causing long-term renal deterioration [36]. As previously mentioned, SAH and CKD are closely related; one situation can exacerbate the development of the other condition. When we administrated CPC to treat CKD, the accumulation of the metabolites mentioned above was reduced, proving that CPC positively affected GFR, revealing its nephroprotective role, as previously reported [37, 38].

With the results obtained, we can propose CPC as a nutraceutical for CKD that retard the cardiovascular complication associated with endothelial dysfunction, which induces SAH and kidney failure.

In summary, C-phycocyanin prevents the development of vascular dysfunction in a model of CKD through its antihypertensive and nephroprotective properties. The mechanism that explains the antihypertensive effect is associated with preventing the down-expression of Mas receptors and reducing the vasorelaxant response induced by angiotensin (1–7). Also, the CPC prevents the exacerbated vasoconstriction caused by angiotensin II through the disturbance in AT1/AT2 expression.