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ABSTRACT
Background: Renal diseases represent a major public health problem. The
demonstration that maladaptive repair of acute kidney injury (AKI) can lead to the
development of chronic kidney disease (CKD) and end-stage renal disease has
generated interest in studying the pathophysiological pathways involved. Animal
models of AKI–CKD transition represent important tools to study this pathology.
Submitted 30 January 2019 We hypothesized that the administration of multiple doses of folic acid (FA) would
Accepted 10 May 2019 lead to a progressive loss of renal function that could be characterized through
Published 21 June 2019 biochemical parameters, histological classification and nuclear magnetic resonance
Corresponding author (NMR) profiling.
Paula Cordero-Pérez, Methods: Wistar rats were divided into groups: the control group received a daily
paucordero@yahoo.com.mx
intraperitoneal (I.P.) injection of double-distilled water, the experimental group
Academic editor received a daily I.P. injection of FA (250 mg kg body weight-1). Disease was
David Kennedy
classified according to blood urea nitrogen level: mild (40–80 mg dL-1), moderate
Additional Information and
(100–200 mg dL-1) and severe (>200 mg dL-1). We analyzed through biochemical
Declarations can be found on
page 20 parameters, histological classification and NMR profiling.
DOI 10.7717/peerj.7113
Results: Biochemical markers, pro-inflammatory cytokines and kidney injury
biomarkers differed significantly (P < 0.05) between control and experimental
Copyright
2019 Perales-Quintana et al. groups. Histology revealed that as damage progressed, the degree of tubular injury
increased, and the inflammatory infiltrate was more evident. NMR metabolomics
Distributed under
Creative Commons CC-BY 4.0 and chemometrics revealed differences in urinary metabolites associated with CKD
progression. The main physiological pathways affected were those involved in energy
How to cite this article Perales-Quintana MM, Saucedo AL, Lucio-Gutiérrez JR, Waksman N, Alarcon-Galvan G, Govea-Torres G,
Sanchez-Martinez C, Pérez-Rodríguez E, Guzman-de la Garza FJ, Cordero-Pérez P. 2019. Metabolomic and biochemical characterization of a
new model of the transition of acute kidney injury to chronic kidney disease induced by folic acid. PeerJ 7:e7113 DOI 10.7717/peerj.7113
production and amino-acid metabolism, together with organic osmolytes. These data
suggest that multiple administrations of FA induce a reproducible model of the
induction of CKD. This model could help to evaluate new strategies for
nephroprotection that could be applied in the clinic.
INTRODUCTION
Interest in kidney health has recently increased mainly because of the alarming statistics.
It has been estimated that 5–10 million people die annually from either chronic kidney
disease (CKD) or acute kidney injury (AKI) (Luyckx, Tonelli & Stanifer, 2018). Previous
studies have estimated a worldwide prevalence of 8–16% for CKD (Jha et al., 2013) and
1–25% (Lameire et al., 2013) for AKI. Although these two diseases were originally considered
to be totally unrelated syndromes, the evidence suggests that maladaptive repair from
AKI can lead to the development of CKD, and that either of these two syndromes can lead to
the development of end-stage renal disease (Chawla et al., 2014). At present, there is no
specific treatment to reverse or stop kidney disease. The currently used treatments attempt to
identify the syndromes at an early stage and arrest or delay their natural history of
progression before the need for dialysis or transplantation (Eknoyan et al., 2004).
However, we cannot ignore that the cost of treating kidney diseases and their associated
complications is a challenge for health services around the world; for example, the
annual costs of CKD treatment range between US$35,000 and $100,000 per patient
(Levin et al., 2017).
In clinical practice, urea and creatinine levels have been traditionally used as markers of
renal function for early identification of kidney disease. However, it has been reported that
these tests have limitations and may under-estimate the disease stage. To overcome this
restriction, exploration using “omics” sciences (such as proteomics and metabolomics)
has allowed the identification of new non-invasive biomarkers of renal function
(Saucedo-Yanez et al., 2018).
Studies have suggested that a persistent inflammatory response mediated by
pro-inflammatory cytokines such as interleukin 1 beta (IL-1β), interleukin 6 (IL-6) and
tumor necrosis factor alpha (TNF-a) contributes to the perpetuation of kidney damage
(Furuichi, Kaneko & Wada, 2009). Other proteins including kidney injury molecule-1
(KIM-1), neutrophil gelatinase associated with lipocalin (NGAL) and cystatin C (Cys-C)
also have a major impact on the development of kidney disease. KIM-1, NGAL and Cys-C
were originally proposed as markers of AKI, but recently have been used to describe
the evolution of CKD (Gil et al., 2016; Lobato et al., 2017). In addition, the omics sciences
allow us to evaluate globally the different molecules of a family, rather than a single
protein or metabolite, in such a way that the physiological state of an organism can be
described by the presence or absence of these molecules (Amaro et al., 2016). In particular,
developments in metabolomics allow the identification of metabolites in a living organism,
Renal histopathology
Kidneys from all rats of each group were fixed in 10% buffered formaldehyde solution
(pH 7.4). After fixation, the tissue was embedded in paraffin and cut into 4-mm sections,
which were deparaffinized and stained with haematoxylin and eosin (H&E). These sections
were subsequently evaluated under an optical microscope for indicators of cell damage,
such as: the increase in Bowman’s space area, degenerative tubular changes (edema
and cytoplasmic vacuolization), tubular dilation, tubular necrosis, vascular congestion,
intratubular proteins and neutrophil casts, leukocyte interstitial infiltration, leukocyte
intratubular infiltration, interstitial fibrosis and tubular atrophy. Damage was evaluated
using a scale ranging from zero to five: not present (grade 0), 1–20% injuries (grade 1),
21–40% injuries (grade 2), 41–60% (grade 3), 61–80% (grade 4) and 81–100% (grade 5).
Lastly, the total histopathologic score was calculated, as the sum of all grades of the
different injuries.
Statistical analysis
Data for body weight, urine output, creatinine and BUN levels are expressed as the
mean ± SD and were analyzed by one-way analysis of variance followed by Tukey’s test for
multiple comparisons; significance was set at P < 0.05. Analysis was performed using
GraphPad Prism software (v. 6.0; GraphPad, San Diego, CA, USA).
RESULTS
Biochemical markers, pro-inflammatory cytokines and kidney injury
biomarkers
The serum BUN and creatinine of the control group were significantly lower than those of
the FAIGs (P < 0.0001 each). Furthermore, the BUN and creatinine levels gradually
increased as kidney injury worsened.
The serum levels of IL-1β and IL-6 showed similar trends: the control group differed
significantly only from that in the FAIG-Mo and FAIG-S subgroups; moreover, these
damage groups differed significantly from the FAIG-Mi group. In contrast, in TNF-a the
levels differed significantly between the control group and the FAIGs, and there were
significant differences between each stage of damage (Fig. 1).
For the serum NGAL levels, two of the damage groups differed significantly from the
control group: the levels were higher in the FAIG-Mi group but lower in the FAIG-S group.
The serum NGAL levels decreased markedly with increasing damage. The serum
KIM-1 level was significantly higher in all FAIGs than in the control group. Among the
FAIG subgroups, the KIM-1 levels differed significantly between the FAIG-Mo and FAIG-S
groups and between the FAIG-Mi and FAIG-S groups. Serum concentrations of Cys-C
differed significantly between the control group and the FAIGs, but there was no significant
difference between the FAIG subgroups (Fig. 1).
Renal histopathology
Representative renal histology of the different experimental groups is shown in Fig. 2.
Histological score of kidney damage is shown in Table 1. Examination of H&E-stained
tissue sections showed normal renal parenchyma, tubules and glomeruli in the control
group (Fig. 2A). By contrast, the architecture of the kidneys from the FAIGs was affected,
as it is summarized in Table 1. It is shown that total histological score increased in
FAIGs, when compared to the control group. In the FAIG-Mi group, the interstitium was
Creatinine (mg.dL-1)
****
BUN (mg.dL-1)
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IL-1β (ng.mL-1)
IL-6 (ng.mL-1)
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Figure 1 Serum levels of biochemical markers, pro-inflammatory cytokines and kidney injury
biomarkers. (A) Serum level of BUN; (B) Serum level of creatinine; (C) Serum level of interleukin 1β;
(D) Serum level of interleukin 6; (E) Serum level of tumor necrosis factor alpha; (F) Serum level of neu-
trophil gelatinase associated with lipocalin; (G) Serum level of kidney injury molecule-1; (H) Serum level of
cystatin C. Values are expressed as means ± SD. N = 6 in each group. P < 0.05 vs. C. †P < 0.05 vs. FAIG-Mi.
#
P < 0.05 vs. FAIG-Mo. FAIG-Mi: Mild damage, FAIG-Mo: Moderate damage, FAIG-S: Severe damage.
Full-size DOI: 10.7717/peerj.7113/fig-1
C D
Figure 2 Representative light microphotographs of sections of renal tissue from each experimental group,
stained with haematoxylin and eosin. (A) Control group, (B) FAIG-Mi: Mild damage, (C) FAIG-Mo:
Moderate damage, (D) FAIG-S: Severe damage. Full-size DOI: 10.7717/peerj.7113/fig-2
Table 1 1H-NMR chemical shift assignment of differential metabolites observed in urine from
control and FA-treated rats and their change trends (increased/decreased) compared with the
control group.
Parameters Groups
apparent because of the presence of an inflammatory mixed infiltrate, and the tubules were
affected with degenerative changes and tubular dilatation (Fig. 2B). The FAIG-Mo group
was characterized by decreased levels of preserved tissue, which was surrounded by
an abundant inflammatory infiltrate. Degenerative tubular changes, tubular dilatation and
tubular necrosis were present. In addition, intratubular casts become more apparent with
Figure 3 1H-NMR spectra of rat urine from the different experimental groups. (A) 1H-NMR spectra
of urine samples of each experimental group. (B) High field of the 1H-NMR spectra of urine samples of
each experimental group. (C) Low field of the 1H-NMR spectra of urine samples of each experimental
group. Identified metabolites: (1) valine, (2) lactate, (3) alanine, (4) acetate, (5) succinate, (6) oxoglu-
tarate, (7) citrate, (8) dimethylamine, (9) trimethylamine, (10) creatinine, (11) creatine, (12) malonate,
(13) taurine, (14) TMAO, (15) glycine, (16) phenylacetylglycine, (17) hippurate, (18) allantoin, (19) urea,
(20) aconitate, (21) kynurenate, (22) n-1-methylnicotinamide (23) trigonelline. ( ) Removed peaks
correspond to the folic acid signals. Full-size DOI: 10.7717/peerj.7113/fig-3
Phenylacetylglycine (PAG) and TMAO. The increased signals of acetate and creatine,
altogether with a decrease in the signals of allantoin, creatinine and taurine, contribute for
the cluster in FAIG-S (Fig. 7).
Metabolic pathway analysis using Metaboanalyst (https://www.metaboanalyst.ca/), led
to the identification of several pathways significantly affected by the AKI-CKD transition
induced by FA. The most relevant pathways in FAIG-Mi group were citrate cycle,
pyruvate metabolism, glycolysis or gluconeogenesis and glycine serine and threonine
metabolism. An overall evaluation showed that in the FAIG-Mo group the disturbs
A
0.4
0.3
PC 1 (37.53%)
0.2
0.1
-0.1
9 8 7 6 5 4 3 2 1
0.4
0.3
B
0.2
0.1
PC 2 (23.22%)
-0.1
-0.2
-0.3
-0.4 5 4 3 1
9 8 7 6 2
0.4
c
0.3
0.2
PC 3 (14.16%)
0.1
-0.1
-0.2
-0.3
9 8 7 6 5 4 3 2 1
Figure 6 Loading plots from PCA applied to 1H-NMR spectra of rat urine samples from control rats
and rats with AKI–CKD transition induced by folic acid. Loading plots from (A) PC1, first principal
component; (B) PC2, second principal component and (C) PC3, third principal component.
Full-size DOI: 10.7717/peerj.7113/fig-6
pathway correspond to both the citrate cycle, and the arginine and proline metabolism,
while in the FAIG-S group taurine and arginine metabolism were the most relevant
pathways (Fig. 7).
D E F
Increased Decreased Increased Decreased Increased Decreased
metabolites metabolites metabolites metabolites metabolites metabolites
Figure 7 Pathway analysis of identified metabolites in the different groups. The upper panels (A–C)
represent the relevant metabolic pathways on the basis of the urine metabolites of each group using the
MetaboAnalyst. The lower tables (D–F) correspond to the metabolites whose NMR signal increased or
decreased in comparison to control group. (A) and (D) FAIG-Mi: Mild damage; (B) and (E) FAIG-Mo:
Moderate damage; (C) and (F) FAIG-S: Severe damage. Full-size DOI: 10.7717/peerj.7113/fig-7
DISCUSSION
In recent years, the study of the development of CKD from severe or repeated episodes of
AKI has attracted increasing attention (Belayev & Palevsky, 2014; Goldstein et al., 2013).
The development of experimental models that show pathophysiological characteristics
similar to those seen during the transition from AKI to CKD in humans is vital to
understand the progression of the disease and to generate strategies that allow an early
diagnosis. Several models of the AKI–CKD transition have been described, including
those using IRI or nephrotoxic substances such as cisplatin or aristolochic acid.
In the present study, we focused on evaluating FA as an inducer of kidney damage. High
doses of FA have been used to induce an acute renal reaction to the generation of FA crystals
that precipitate in the renal tubules (Klingler, Evan & Anderson, 1980) and produce acute
tubular necrosis followed by epithelial regeneration and cortical healing (Fink, Henry &
Tange, 1987). In addition, it has been shown that FA has nephrotoxic activity at various
levels of the nephron because it induces a pro-oxidant state by increasing lipid binding and
reducing protective anti-oxidant enzymes (Gupta et al., 2012). It has been suggested
recently that the residual structural damage that is produced by FA could lead to CKD
pathology (Fu et al., 2018).
Creatinine and BUN levels have been used traditionally in clinical practice as markers of
renal function. Using a surgical model, Ormrod & Miller (1980) established three levels
of renal damage based on BUN levels: mild (40–80 mg dL-1), moderate (100–200 mg dL-1)
and severe (>200 mg dL-1). These levels were used in the present study for the first time as
CONCLUSIONS
The characterization of this model of kidney disease induced by repeated doses of FA in
rats by analysis of biochemical markers, histopathology, pro-inflammatory cytokines and
kidney injury biomarkers revealed that this model can be used to study the transition
between AKI and CKD, and that the transition can be divided into three well-differentiated
phases. The metabolomic analysis of rat urine made it possible to identify potential
Funding
This work was supported by National Council on Science and Technology–Conacyt
(project 2017-01-5652). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Grant Disclosures
The following grant information was disclosed by the authors:
National Council on Science and Technology—Conacyt (project 2017-01-5652).
Competing Interests
The authors declare that they have no competing interests.
Author Contributions
Marlene Marisol Perales-Quintana conceived and designed the experiments, performed
the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed
drafts of the paper, approved the final draft.
Alma L. Saucedo conceived and designed the experiments, performed the experiments,
analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or
tables, authored or reviewed drafts of the paper, approved the final draft.
Juan Ricardo Lucio-Gutiérrez conceived and designed the experiments, performed
the experiments, analyzed the data, contributed reagents/materials/analysis tools,
prepared figures and/or tables, authored or reviewed drafts of the paper, approved the
final draft.
Noemí Waksman contributed reagents/materials/analysis tools, authored or reviewed
drafts of the paper, approved the final draft.
Gabriela Alarcon-Galvan analyzed the data, authored or reviewed drafts of the paper,
approved the final draft.
Gustavo Govea-Torres performed the experiments, prepared figures and/or tables,
approved the final draft.
Concepcion Sanchez-Martinez conceived and designed the experiments, performed the
experiments, analyzed the data, prepared figures and/or tables, approved the final draft.
Edelmiro Pérez-Rodríguez conceived and designed the experiments, contributed
reagents/materials/analysis tools, approved the final draft.
Francisco J. Guzman-de la Garza conceived and designed the experiments, contributed
reagents/materials/analysis tools, approved the final draft.
Animal Ethics
The following information was supplied relating to ethical approvals (i.e., approving body
and any reference numbers):
All animal experiments were performed in accordance with the principles and regulations
of the Mexican Official Norm NOM-062-ZOO-1999 and were approved by the Animal Care
and Use Committee of Universidad Autónoma de Nuevo León (HI17-00004).
Data Availability
The following information was supplied regarding data availability:
The raw data is available in Dataset S1.
Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
peerj.7113#supplemental-information.
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