Original Research Paper

Natural Product Communications

Volume 18(3): 1–12
Antioxidant Effect of Jatropha dioica Extract on © The Author(s) 2023
Article reuse guidelines:
Immunoreactivity of Claudin 2 in the Kidney of sagepub.com/journals-permissions
DOI: 10.1177/1934578X231157183
Rats with Induced Diabetes journals.sagepub.com/home/npx

Agustina Ramírez-Moreno1, Rubén García Garza2, David Pedroza Escobar3,

Adolfo Soto Dominguez4, Erika Flores-Loyola1, Irais Castillo Maldonado3,
Hady Keita5, Ibrahim Sharara Núñez2 and Dealmy Delgadillo Guzmán2

Abstract
Introduction: Diabetes is related to different complications, such as nephropathy. In kidneys, the tight junctions (TJ) regulate the
paracellular transport of solutes and water. The claudins are the most significant component in the TJ and are expressed throughout
the epithelium and modulated permeability. Objective: To evaluate the antioxidant effect of Jatropha dioica (JD) extract and claudin 2
(CLDN 2) in the kidney of rats with induced diabetes. Methods: It is an experimental, explanatory, prospective, longitudinal, and
analytical study. The effect of hydro-alcoholic extract from JD root on the presence of CLDN 2 in rats with induced diabetes was
evaluated. The experiment lasting 21 days, treated 40 rats with and without induced diabetes by an intraperitoneal (IP) injection of
alloxan. The study groups were exposed to JD extract at 25, 50, and 100 mg/kg. There was no significant morphological difference
in the renal tissue morphology of the treated and control groups. Conclusion: These results demonstrate that JD extract at 50 mg/
kg ameliorated the tubular damage induced by diabetes, and the mechanism might be related to the CLDN2 expression. Finally, JD
could also increase total antioxidant capacity to protect the kidney from oxidative stress.

Keywords
Polyphenols, claudin 2, diabetic nephropathy, kidney histology, plants extract

Received: July 8th, 2022; Accepted: January 27th, 2023.

Introduction Various reports indicate that G officinalis is useful as an adjuvant

Diabetes is a chronic disease characterized by the fact that the
pancreas is insufficient to produce insulin due to an autoimmune
process, type 1 diabetes (T1D), or the body does not effectively
use the insulin secreted by the pancreas, type 2 diabetes (T2D).
The elevation of blood glucose manifests in both T1D and
T2D. T1D is also referred to as insulin-dependent or juvenile
and requires the daily administration of this hormone. It has
been postulated that T1D has a genetic origin and cannot be pre-
vented. On the other hand, T2D, also called non-insulin-depen-
dent, occurs in adulthood, represents 90% of diabetes cases,
and can be prevented or, in some cases, reversed via treatment.1
High glucose levels in diabetes cause severe damage and micro-
vascular complications such as retinopathy, heart disease, and neu-
ropathy.2,3 Medicinal plants may be essential in managing blood
glucose through different mechanisms. For example, Galega offici-
nalis could be beneficial for treating diabetes and reducing fast
blood sugar by improving the serum level of insulin and prevent-
ing kidney tissue damage by antioxidant features.4 G officinalis is
known as Galega belonging to the Fabaceae family. It is known
to grow in the Middle East and southeastern Europe.4–6
treatment for diabetes mellitus, with a known origin from the
Middle Ages.4,6 It has been identified that G officinalis contains fla-
vonoids, tannins, saponins, glycosides, resins, and steroids with
antioxidant properties.6
1
Facultad de Ciencias Biológicas, Universidad Autónoma de Coahuila, Torreón,
Mexico
2
Departamento de Farmacologíaa, Departamento de Histologíab, Facultad de
Medicina, Universidad Autónoma de Coahuila, Torreón, Mexico
3
Departamento de Bioquímica, Facultad de Medicina, Centro de Investigación
Biomédica, Universidad Autónoma de Coahuila, Torreón, México
4
Departamento de Histología, Facultad de Medicina, Universidad Autónoma de
Nuevo León, Monterrey, México
5
División de estudios de posgrado, Universidad de la Sierra Sur, Miahuatlán de
Porfirio Díaz, Oaxaca, México
Corresponding Author:
Dealmy Delgadillo Guzmán, Departamento de Farmacología, Facultad de
Medicina, Universidad Autónoma de Coahuila. Morelos #900. Col.
Centro. C. P. 27000, Torreón, Coahuila, Mexico.
Email: dealmydelgdilloguz@uadec.edu.mx

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2 Natural Product Communications
At the renal level, the functions of blood filtration are per-
formed in the glomerular capillaries. There, the reduction of
liquid volume and modification of the composition by the phe-
nomenon of tubular resorption and tubular secretion forms the
urine that enters the renal pelvis.7 The glomerular and tubular
epithelial cells have as their primary function the handling of
organic and inorganic solutes. Therefore, the homeostasis of
extracellular fluids depends on their efficient functioning.
Renal tight junction proteins (TJ proteins) are involved in para-
cellular solute and water transport.
These proteins are adhesion complexes that form a contin-
uous belt around the circumference of the cells8 allowing a good
diffusion through the paracellular route that includes TJ pro-
teins located in the apical portion in the epithelial cells.9
There are different integral proteins, such as claudins and occlu-
dins. The claudins are the most abundant proteins, constituting
large chains of TJ proteins, and expressed throughout the mod-
ulation of epithelial permeability.10
The TJ proteins are responsible for the size and charge selec-
tive conductance properties of paracellular pathways.11 The
ocludins are expressed along the segments of the epithelial
tubular nephrons.12
Diabetic nephropathy (DN) manifests by reduction of the
glomerular filtration13 where creatinine clearance values
decrease to values below 60 mL min/1.73 m2. The primary
marker of renal damage is a urinary albumin excretion.14 In
addition, the pathogenesis involves various elements, such as
decreased nitric oxide production, increased protein kinase
activity, overproduction of reactive oxygen species (ROS).
Although the generation of free radicals (FRs) is a normal char-
acteristic of cell function, excessive generation or insufficient
removal of FRs can cause oxidative stress and lead to injury
or destruction of the tissue. There are innate defense mecha-
nisms to counteract the high production of FR or ROS, pre-
dominantly the action of enzymes such as catalase,
superoxide dismutase, and glutathione peroxidase.15 On the
other hand, Syzygium (S.) aromaticum is a dried flower bud
belonging to the Myrtaceae family native to the Moluccas
islands in Indonesia, also cultivated on different continents
for its gastronomic utility.16,17 Recently, it has been shown
that medicinal plant extracts such as S aromaticum exert a protec-
tive action on kidney tissue against oxidative damage and
decrease urea and creatinine levels, kidney weight, glomerular
diameter, and thickness of the glomerular basement mem-
brane.18 As external help, ascorbic acid, tocopherol,
β-carotenes, and other carotenoids, as well as selenium, have
been helpful for their complementary antioxidant effect to
combat ROS. Likewise, the secondary metabolites present in
roots and plants such as curcumin, flavonoids, terpenoids, phe-
nylpropanoids, tannins, coumarins, and lignins capture FRs due
to their high metal-reducing and chelating capacity. In addition,
these mechanisms neutralize ROS.19,20 It has been proposed
that the antioxidants present in medicinal roots such as curcu-
min could help in the preventive treatment of pathologies
such as serum lipoperoxidation and liver and liver diseases.21
Jatropha dioica (JD) is endemic plant of South United States
(Texas) and most of Mexico. It grows in dry and semi-dry
climate. This plant belongs to the Kingdom Plantae, the
spurge family (Euphorbiaceae) and is commonly known as
leatherstem, leatherwood or sangre de drago (“dragon blood”
within Spanish speaking area). It owes its common name to
the presence of a colorless liquid that changes to red in
contact with oxygen. The bush typically grows to the height
of 50 to 150 cm.22,23 The presence of terpenoids compounds,
flavonoids, reducing sugars and alkaloids are possibly responsi-
ble for the antioxidant effect observed using model radical scav-
enger (1, 1-diphenyl-2-picrylhydrazyl, DPPH) for the root
extracts of JD The presence of terpenoid compounds, flavo-
noids, reducing sugars, and alkaloids has been identified by
observing their antioxidant activity that counteracts FRs in JD
root extract.24
In our previous work it was determined that the ethanolic
extracts (70%) obtained from JD were not toxic.25 Similarly,
aqueous extracts do not have genotoxic or cytotoxic effect as
shown in analysis of micronucleus in mouse peripheral
blood.26 Also, it has been reported that this plant extract exhibits
a protective behavior on mouse hepatocytes, renal and medulla
bone cells.27 The JD extracts contain phenolics which are bioac-
tive compounds displaying antioxidant function.28 In addition,
other properties of this plant extracts have also been reported
such as an antibiotic,22 anti-hyperglycemic,29 and antiviral.30
Previous research studied the antioxidant properties of the
hydroalcoholic extract of JD from the plant, stem, and root
aerial parts. We resulted in the latter with the highest quantifica-
tion of polyphenols.31 Therefore, this study aimed to evaluate
the effect of the hydroalcoholic extract of the JD root on the
morphology of renal tissue in diabetic rats. In addition, the
immunoreactivity of claudin 2 (CLDN 2) within the same
tissue was evaluated since it is believed that the extracts of JD
have a protective behavior on the cells.
Materials and Methods
Extract Preparation
The plant material was collected in May 2015 locally in Torreon,
Coahuila, Mexico (103°26 33” W and 25°32 40” N). It was
cleaned, cut into small pieces, and dried. In an amber jar, 2 g
of the dry powdered root of JD was placed in 50 mL of a hydro-
alcoholic solution containing 75 wt% of water and was stirred
for 48 h at room temperature (RT). Then, it was filtered and
condensed on a rotary evaporator at low temperatures, and
the concentrate was placed in an oven at 60°C until full
dryness was achieved. The collected powder was resuspended
in water at the required concentrations.
Study Groups
To determine the dose to be administered to the animals, a toxic
activity lethality test of the extracts in Artemia salina larvae was
Ramírez-Moreno et al
carried out in a lethal dose of 2183.84 ppm. At 2183.84 ppm of
extract, half of the brine shrimp were exposed to a vegetable
test with an LC50 value greater than 1000 ppm. Therefore, this
extract is not considered toxic.32 The concentrations examined
were 25, 50, and 100 mg/kg, according to Araujo Espino 2017. 26
In this study, 40 young female Wistar rats, 7 to 8 weeks old,
and weighing 180 to 260 g were used. They were maintained at
RT (25°C ± 2°), and with 12 h light/darkness cycles. The com-
mercial food and water were ad libitum access. The animals
underwent a 7-day adaptation period before starting the treat-
ment. Also, the animals were cared according to the Mexican
standard NOM-062-ZOO-1999, after getting the approval
from the Bioethics Committee with reference number
01-04-16 of the Faculty of Medicine of the Autonomous
University of Coahuila.
Diabetes Induction
The diabetes was induced by an intraperitoneal (IP) injection of
alloxan (Sigma)24–26 at 125 mg/kg of weight (0.5 mL/100 g of
body weight) by IP. The rats included showed values higher than
200 mgdL−1 of glucose in blood for 14 days. After 2 weeks of
being induced to diabetes, without reversing hyperglycemic state,
the rats received a dose administration daily of 25, 50, 100 mg/
kg−1 extract diluted in purified water, without feeding between
7:00 and 10:00 AM The treatment of all groups lasted for a period
of 21 days. Weight and glucose levels were measured at the beginning
and at the end of the experiment to observe animals’ behavioral ten-
dencies. Blood samples were taken by cardiac puncture with a previ-
ous use of 30 mg/kg−1 anesthesia, then the samples were
centrifuged and conserved at −70°C (until needed for the antioxi-
dant capacity and lipid peroxidation quantifications).
Glucose was measured with a glucometer (brand:
Accu-Chek®). The treated animals that showed fasting glucose
levels larger than 250 mg/dL after a period of 14 days were used
for the following experiment. Glucose measurements were per-
formed with a glucometer (Accu-Chek Nano Performa®, Roche).
Administration of JD Extract
Two weeks after diabetes induction, and without presenting
hypoglycemic shock, the animals were administered daily
doses of 25, 50, or 100 mg of extract in purified water/kg
weight, in fasting conditions between 7:00 and 10:00 AM The
animals (n = 35) were divided into 7 groups (n = 5): CN: neg-
ative control; CD: diabetic control treated with only water ad
libitum; D + E 25 mg/kg: diabetic rats treated with 25 mg/kg
of JD extract; D + E 50 mg/kg: diabetic treated with 50 mg/
kg of JD extract; D + E 100 mg/kg: diabetic rats treated
with 100 mg/kg of JD extract; E: rats without diabetes
treated with 50 mg/kg of JD extract; O: rats without diabetes
treated with olive oil; and D + VE: diabetic treated with
100 mg/kg of vitamin E. All the groups were treated for a
period of 21 days. Weight and glucose levels were measured
at the beginning and end of the study to observe the behavioral
3
trend of the animals. The animals were sacrificed by cardiac
puncture following anesthesia with 30 mg/kg of pentobarbital
IP kidney samples were then collected for histopathological
and immunohistochemical analysis.
Histopathological Analysis
The kidney tissue was fixed in 10% v/V formalin solution
(100 mL of 37%-40% formaldehyde with 900 mL of 0.9%
saline solution) to preserve the tissue structure during 48 h.
Then, kidney samples were cut crosswise, and processed by
the conventional histologic technique: washed in PBS 1X, dehy-
drated in gradual ethanol solutions, xylene treated to remove the
alcohol, and embedded in paraffin blocks. Next, histological
slices of 5 to 7 µm thick were obtained with a microtome
Leitz 1512®, placed directly in a water bath at 40°C with
0.01% of egg albumin, and dewaxed in an oven at 56°C for
1 h. Most reagents used were purchased from Merck®.
Staining with Hematoxylin and Eosin
Dewaxed sections were placed in xylene, xylene: ethanol solution
(1:1), and absolute ethanol; then were rinsed in distilled water and
immersed in the hematoxylin according to Harris formula
(Golden Bell). Next, the sections were washed (water, acid
alcohol, water) and immersed 6 times in eosin solution
(Harleco Dade de México S.A.; 0.25% w/V dissolved in
ethanol 80% with 0.5% of glacial acetic acid). Following, they
were washed according with the next order: ethanol 96°, absolute
ethanol, ethanol: xylene solution (1:1), xylene, and finally the sec-
tions were mounted in synthetic resin. They were allowed to dry
at RT for 48 h. The samples were observed with an Olympus®
optical microscope under 40x magnification.
Preparation of Samples by the Technique of Tissue Array
Once the areas of interest were identified in the H&E
kidney-stained slides, they were collected from the donor
blocks to prepare the tissue arrays according with techniques
already described by our research team.33 Briefly, the tissue
cores from the donor blocks were inserted into the perforations
made in the acceptor blocks. Later these blocks were placed in
the stove to melt the paraffin and to cover the wells. Once the
tissue arrays were ready, they were cut by microtomy to 5 µm
slices thickness.
Immunohistochemical Method
First, the slides were covered with a 10% poly-L-Lysine adhe-
sive solution (Sigma-Aldrich®) in distilled water for 15 min,
then were dried at 56°C, and stored until their use.
For the immunohistochemistry analysis for the detection of
CLDN 2, tissue slices of 5 μm were deparaffinized and rehy-
drated, and immunolabeling was performed according to the
manufacturer’s protocol. Briefly, slices were incubated for 1 h
4 Natural Product Communications
in a solution of Target Retrieval Solution (TRS) high pH, 1x at
100°C for the antigen recovery, and then slices were washed 3
times in tris-buffered saline + Triton X-100 0.1% (TBST) (TRS
and TBST solutions were purchased in Dako Cytomation Inc.).
After that, slices were incubated in 3% H2O2 in methanol at RT
for 10 min, followed by incubation with 10% horse serum in
PBS 1x at 37°C for 30 min. Later, the sections were incubated
with monoclonal specific antibody mouse anti-CLDN 2
(MA5-11711, 1:50 dilution, Thermo Scientific Pierce
Antibodies) at 4°C overnight. Then, slices were washed 3
times with TBST at RT, and were incubated 1 h with the
Mouse and Rabbit specific HRP/DAB/ABC detection
system (ab64264 Abcam®) and 3,3′ diaminobenzidine were
used to visualize antibody staining. Finally, sections were coun-
terstained with Harris’ hematoxylin (Sigma Chemical Co). For
negative controls, primary antibody was omitted, and sections
were incubated with TBST alone.
High-resolution digital images were obtained using an
Eclipse 50i microscope (Nikon Tokyo Japan) with an
NIS-Elements software system. All samples were evaluated by
3 independent specialists in Morphology using previously pub-
lished criteria.34
Morphometric Analysis
In this study, a morphometric analysis was performed in the
immunolabeling method by light microscopy, at a 40x magnifi-
cation in 8 representative fields scattered in the preparations as
follows.34
The color parameters, hue distribution, saturation, and lumi-
nance were established in the capture software and were the
same for all the images obtained. From the images obtained,
the brown hue of the positivity to CLDN 2 was manually
selected, and the program (with the detection threshold of the
control sample pre-calibrated) converted the interval color to
grayscale and the other tissue components to white. Then, the
processed images were automatically analyzed to determine
the percentage of the area and intensity of the density (Int
Dent) of elastic fibers in each sample. Quantitative analysis of
positive area was performed in triplicate sections using
ImageJ v1.51 (NIH).35
Statistical Analysis
For the analysis of the comparison of weight results, glucose
levels, histopathological, and morphometric analysis, a
one-way variance analysis (ANOVA) was performed consider-
ing a statistical significance value of 95% and to compare the
significant differences P < .05. The Student’s t-test for paired
samples was used.
Results
A qualitative analysis of flavonoids and triterpenes was per-
formed after pulverization and preparation of the extracts
both by the hydroalcoholic route and by infusion, to determine
the anatomical area with the highest polyphenol content, whole
plant matter registered the presence of flavonoids. In contrast,
the triterpenes were not identified in the hydroalcoholic extract
of the stem. When quantifying the concentrations of polyphe-
nols in the extracts, statistically significant differences were
found (ANOVA: P< .001). The concentration range found in
the 4 extracts was from 23.0 to 44.03 µg/mL of ac. gal., with
a mean of 35.32 ± 7.85 (95% CI 30.32 to 40.31). The values
for the mean and standard deviation (SD) of polyphenol con-
centration in descending order were EHR > IR > IT > EHT
(43.44 ± 0.723; 40.56 ± 0.446; 33.24 ± 1.02; 24.05 ±
0.927 µg/mL ac. gal., respectively). The EHR extract showed
the highest concentration of 43.67 ± 0.72 µg/mL ac. gal.
(95% CI 41.65 to 45.24) while the EHT extract presented
the lowest concentration of 24.05 ± 0.92 µg/mL ac. gal.
(95% CI 21.75 to 26.35) (ANOVA P < .0001); therefore, the
concentration of polyphenols in EHR is 44.64% higher than
that of EHT.
Induction of Diabetes
The diabetes was induced by an IP injection of alloxan
(Sigma)24–26 at 125 mg/kg of weight. After 2 weeks, the
animals without hypoglycemic shock and fasting glucose
levels larger than 250 mg/dL were considered as with diabetes
and chosen for the subsequent experiments. In Table 1, the
results of rat weight and blood glucose levels are shown.
These parameters were determined at the beginning and the
end of the experimental treatment period of 21 days. These
results show homogeneous glucose values from the beginning
to the end of the treatment in all the groups. On the other
hand, the weight data in the diabetic group show a significant
decrease, with an extract dose of 25 mg/kg (P<.05) is compared
with the initial weight. However, with doses of 50 and 100 mg/
kg of the extract, weight tended to increase, without and statis-
tical significant difference.
Determination of Histological Parameters
In the kidney sections of the negative control group, we
observed the specific features of the tissue: glomerulus,
urinary space, proximal and distal tubules. The structural
dimensions of the urinary space, glomerular capillaries, and
renal capillaries are shown in Figure 1. No statistically signifi-
cant differences were observed in the structural dimensions
between the study groups. All of these with normal morpholog-
ical characteristics (Figure 2A). In contrast, in diabetic groups
(Figure 2B), diabetic + 25 mg/kg of extract (Figure 2C), dia-
betic + 50 mg/kg of extract (Figure 2D), diabetic + 100 mg/
kg of extract (Figure 2E), single extract (Figure 2F), only olive
oil (Figure 2G), and diabetic plus 100 mg/kg of vitamin E
(Figure 2H), also no morphological alterations were observed.
Only some areas with mild to moderate vascular congestion
were observed in a few cases.
Ramírez-Moreno et al 5

Table 1. Blood Glucose Levels and Body Weight at the Start and end of Treatment in the Different Groups.
Blood glucose (mg/dL) Body weight (g)
Groups
Initial Final P Initial Final P
CN 111.75 ± 4.5 104.25 ± 5.05 .15 223.3 ± 4.86 224.8 ± 6.39 .456
CD 507.25 ± 96.64 450 ± 22.53 .28 204.50 ± 9.67 214 ± 7.8 .360
D+ E 25 mg/kg 591.5 ± 9.84 581.25 ± 37.5 .67 195.48 ± 9.85 167.4 ± 15.26* .022
D+ E 50 mg/kg 436 ± 69.75 398.8 ± 40.88 .17 196.20 ± 12.91 216 ± 38.89 .327
D+ E 100 mg/kg 443.25 ± 74.47 367 ± 60.55 .47 197.25 ± 24.67 206.5 ± 22.45* .022
E 98.75 ± 9.56 98.25 ± 2.75 .91 210.75 ± 0.95 216 ± 7.62 .247
O 112 ± 10.42 98.75 ± 14.66 .11 209.5 ± 10.9 211.5 ± 15.8 .566
D+ VE 449 ± 48.15 416.75 ± 145.63 .64 175.5 ± 19.87 182.34 ± 24.55 .177
The data are presented as average with standard deviation.
D + E 25mg/kg: diabetes treated with 25 mg/kg of JD extract.
D + E 50 mg/kg: diabetes treated with 50 mg/kg of JD extract.
D + E 100 mg/kg: diabetes treated with 100 mg/kg of JD extract.
*Significant difference P < .05. Student’s t-test paired samples.
Abbreviations: CN, negative control; CD, diabetes control without treatment, only water ad libitum; D VE, diabetic treated with 100 mg/kg of vitamin E; E, extract; O,
olive oil.

Figure 1. Structural dimensions of the urinary space, glomerular capillary, and renal corpuscle. NC: negative control. Kruskal–Wallis test and
Dunn’s multiple comparisons test and DC group. *P < 0.05.
Abbreviations: DC, diabetes control without treatment, only water ad libitum; D + E 25 mg/kg: diabetes treated with 25 mg/kg of JD extract. D + E
50 mg/kg, diabetes treated with 50 mg/kg of JD extract. D + E 100 mg/kg, diabetes treated with 100 mg/kg of JD extract; D VE, diabetic treated
with 100 mg/kg of vitamin E; E, extract; NC, negative control; O, olive oil.
6 Natural Product Communications

Figure 2. Histological micrographs of the kidney. Treatment time: 21 days. (A) CN: negative control; (B) CD: diabetes control; (C) D + E 25 mg/
kg: (D) D + E 50 mg/kg; (E) D + E 100 mg/kg; (F) Extract; (G) olive oil, and (H) D + VE. The cuts were paraffin-soaked and stained with
hematoxylin and eosin. Magnification: 40x.
Ramírez-Moreno et al 7

Immunoreactivity of CLDN 2 in Renal Tissue In addition, a study by Storz in 2020 showed that beta cells are
susceptible to damage from exposure to diets rich in saturated
In the negative control group, the presence of immunoreactivity
fat while the intake of polyunsaturated fatty acids exerts a protec-
to CLDN 2 was observed in distal and proximal convoluted
tive effect on beta cells.40 Not only animal models have the effect
tubules, the glomeruli did not exhibit reactivity (Figure 3A).
of polyphenols on the recovery of pancreatic cells. For example, a
In contrast, in the diabetic group, an apparent decrease in
study in patients with T2D given infusions of Somarinus officinalis
immunoreactivity of CLDN 2 was observed in distal and prox-
for 90 days found decreased percentages of glycated hemoglobin,
imal convoluted tubules (Figure 3B).
insulin resistance, and improved pancreatic β-cells function.
In the diabetic group with 25 mg/kg of extract, also an
Currently, the use of drugs such as sulfonylureas, whose site of
apparent decrease in CLDN 2 immunoreactivity in distal and
action depends on the ability of the pancreas to secrete insulin
proximal convoluted tubules was visible (Figure 3C). Here,
and, therefore, requires functional beta cells to exert a therapeutic
the presence of cytoplasmatic inclusions was observed.
effect, is still being considered. However, in the long term, it gen-
In the diabetic group with 50 mg/kg of extract, cytoplasmic
erates pancreatic damage due to overexploitation of the insulin-
inclusions were observed, but also a greater reactivity of the
secreting function by the pancreatic beta cells.41 The active ingre-
CLDN 2 in distal and proximal convoluted tubules compared
dients present in vegetables, such as JD, can be an alternative
to the diabetic group with 25 mg/kg of the extract was detected
treatment for hyperglycemic states resulting from damage to
(Figure 3D). The diabetic group treated with 100 mg/kg of
the pancreatic beta cell, which allows restoring the physiology
extract (Figure 3E) showed less reactivity to CLDN 2 compared
of glucose transport.
to the other diabetic group with extract treatment which deter-
The results of rat weight and blood glucose levels shown that
mines this dose as overdose. The nondiabetic group that admin-
diabetic animals with the treatment of 50 and 100 mg/kg
istered only extract (Figure 3F), only olive oil (Figure 3G), and
showed a decrease in glucose levels and a weight gain without
diabetic plus 100 mg/kg of vitamin E (Figure 3H) presented
a significant difference versus diabetic control. On the other
immunoreactivity of CLDN 2 in distal convoluted tubules and
hand, the weight data in the diabetic group treated with an
proximal convoluted tubules like the negative control group.
extract dose of 25 mg/kg showed a decrease in the significance
We performed an analysis of the densitometry resulting from
(P < .05) compared to the initial weight. The treatment group of
the immunohistochemical assay, which showed a statistically
25 mg/kg showed no change in glucose levels. Weight decrease
significant difference between the groups (P = .02). In the mul-
could suggest a positive influence of the treatment with 25 mg/
tiple comparisons, the group ND has differences with D +
kg of the JD extract. However, due to lack in visible trend in the
E50 (P = .003) and D + E100 (P = .0003), decreasing the
other groups studied that conclusion cannot be proved. In
densitometry of the anti-CLDN2 antibody significantly in the
similar research21 lack of trend was also observed and it was
groups that were administered the JD extract. No differences
suggested that it might be due to the length and levels of treat-
were observed between the ND group and the rest of the exper-
ment. Here, we add that absence of a trend might be as well
imental groups (Figure 3).
attributed to the low number of subjects in each group.
Compared to the DC group, the densitometric analysis of
Various mechanisms are associated with impaired pancreatic
claudin reactivity in renal tissue shows a statistically significant
secretion and insulin resistance in T2D, for example, vitamin D
difference between the groups treated at different doses with
deficiency,42 or oxidative phenomenon in the cell membrane. In
JD (25 mg, 50 mg, and 100 mg). On the contrary, the groups
the case of polyphenols, they improve insulin resistance by
treated with vitamin E and the vehicle (olive oil) did not show dif-
inhibiting the phosphorylation of IRS serine proteins, which
ferences in the densitometry concerning the DC group (Figure 4).
increases the phosphorylation of protein kinase B (Akt), a
protein that promotes the translocation of GLUT4 to the cell
membrane. Likewise, due to its high reducing capacity, it
Discussion sequesters ROS superoxide. In summary, some polyphenols
In this study, supplementation with an extract from JD was have been shown to enhance gene expression of proteins
administered to healthy nondiabetic rats and rats with involved in insulin signaling.43
induced diabetes. The diabetes was induced by an IP injection In other hand, no change in the renal structure was
of alloxan (Sigma) at 125 mg/kg of weight.36–38 After 2 observed, which indicates that the extract does not cause
weeks, the animals without hypoglycemic shock and fasting damage to the studied organ. Chandran et al. reported44 that
glucose levels larger than 250 mg/dL were considered diabetic. the polyphenolic compounds from plant extracts have protec-
The treatments included a daily dosage of 25, 50, and 100 mg/ tive ability in organs such as kidney, liver, pancreas, and heart,
kg of JD extract, 50 mg/kg of vitamin E. Control groups were due to their antioxidant capacity. In this work, although no
negative group, diabetes group, nondiabetic rats treated with morphological changes were observed in tissues of extract
50 mg/kg JD extract, and nondiabetic rats given olive oil. exposed animals, there were areas of mild to moderate vascular
Previously, we have shown that the damage caused to the islets congestion visible on diabetic groups with the treatment of 25
of Langerhans induced by alloxan is due to damage to the beta cell, and 100 mg/kg of extract. On the other hand, the diabetic
causing dysfunction that manifests with hyperglycemia and diabetes.39 group that was treated with dose of 50 mg/kg of JD extract
8 Natural Product Communications

Figure 3. Immunoreactivity of claudin 2 (CLD2) in renal tissue. Treatment time: 21 days. (A) CN: negative control; (B) CD: diabetes control; (C) D
+ E 25 mg/kg: (D) D + E 50 mg/kg; (E) D + E 100 mg/kg; (F) Extract; (G) olive oil, and (H) D + VE. The cuts were paraffin-soaked and stained
with hematoxylin and eosin. Magnification: 40x.

did not show any damage or vascular congestion. This leads to Hyperglycemia causes a high production of ROS. In
suggest that this is a suitable concentration to induce cell response to oxidative stress, numerous modifications are man-
protection. ifested at the cellular and molecular level. Kidney damage
Ramírez-Moreno et al
Figure 4. Densitometric analysis of claudin. D + E 25 mg/kg: diabetes treated with 25 mg/kg of JD extract. D + E 50 mg/kg. diabetes treated
with 50 mg/kg of JD extract. D + E 100 mg/kg, diabetes treated with 100 mg/kg of JD extract. Kruskal–Wallis test and Dunn’s multiple
comparisons test and DC group. **P < .001, ***P < .0001.
Abbreviations: DC, diabetes control without treatment, only water ad libitum; oilD VE: diabetic treated with 100 mg/kg of vitamin E; D + E 25 mg/
kg, diabetes treated with 25 mg/kg of JD extract. D + E 50 mg/kg, diabetes treated with 50 mg/kg of JD extract. D + E 100 mg/kg, diabetes
treated with 100 mg/kg of JD extract; E, extract; O, olive.
resulting from hyperglycemia and oxidative stress is the result of
the activation of immunological pathways, including the inflam-
matory process, fibrosis and apoptosis.45 The polyphenols or
bioactive compounds present in plants considered medicinal
improve the pathological conditions of chronic diseases, such
as DM.45 These data suggest that polyphenols can modify the
progression of DN by modulating premature cell aging, reduc-
ing oxidative stress, the inflammatory state, apoptosis, and
therefore the production of collagen in fibrosis. Resveratrol, a
polyphenol, has been shown to prevent kidney damage
caused by oxidative stress, enhance antioxidant capacity, and
attenuate inflammatory and fibrotic responses.46 The main
function of the kidney is the elimination of xenobiotics, includ-
ing drugs and environmental toxic agents. Due to the mecha-
nism of secretion, the cells of the proximal tubules are the
main site of accumulation of toxic materials and location for
primary damage of the nephron. Subsequently, damage pre-
sented in glomerulus, distal tubules, and loop of Henle
produce alterations in renal function. It is important to empha-
size that the oxidative stress that occurs in the kidney can start
as an alteration of the thigh junction function. Thus, identifying
the role of antioxidants in preventing oxidative stress could be
useful in preventing damage to the JT structure.47–49
In diabetes, the increase in glucose decreases the expression
of gap junctions, a phenomenon that explains the presence of
microvascular complications of diabetes.50,51 A study with
Zucker rats demonstrated that hyperglycemia enhanced oxida-
tive stress. This result in the malfunction of gap junctions in the
juxtaglomerular apparatus. Finally, the observations from
molecular biology experiments suggest that the maintenance
of the gap junction is fundamental in the autoregulation of
the filtration processes and exchange of glomerular-tubular
solutes, which is altered in diabetes.50,52 Here a greater
9
immune reactivity of the CLDN 2 was observed in the negative
control group, in the diabetic group exposed to 50 mg/kg of
extract and in the diabetic group treated with vitamin E. On
the contrary, the diabetic group showed the lowest immune
reactivity of CLDN 2, followed by the diabetic group exposed
to 25 mg/kg and the group subjected to 100 mg/kg. In the neg-
ative control group, the presence of immunoreactivity to CLDN
2 was observed in distal and proximal convoluted tubules. In a
similar study were vitamin A (retinoic acid) was administered to
diabetic rats decreases of CLDN 2 and occludins in the proxi-
mal tubules and lowering of CLDN 5 in the glomerulus were
showed.53 The nondiabetic group that was administered
extract, olive oil, diabetic rats exposed to 100 mg of vitamin
E, presented immunoreactivity of CLDN 2 in distal and prox-
imal convoluted tubules like the negative control group.
Comparable levels of CLDN 2 in the nondiabetic group
exposed to JD extract and negative control show that this
extract is not hindering the correct kidneýs functions.
The diabetic group treated with 50 mg/kg of extract JD dis-
played typical renal tissue morphology and increased CLDN 2
immunoreactivity suggesting that this dosage is the most bene-
ficial in the treatment of diabetes.
Conclusions
The histological parameters analysis of kidney tissue shows that
the extract of JD is not toxic. There was no morphological
damage observed in the kidney structure in any of the
groups, with only mild to moderate vascular congestion
observed in the diabetic groups. The diabetic group treated
with 50 mg/kg of extract JD displayed typical renal tissue mor-
phology and increased CLDN 2 immunoreactivity suggesting
this dosage is the most beneficial in the treatment of diabetes.
10
Communication mediated by TJ plays a role in the pathogen-
esis of T2D. An alteration in TJ due to genetic or environmental
disorders affects the endocrine dysfunctions of the pancreas
and kidneys that contribute to the appearance and evolution
of this metabolic disorder.
Acknowledgments:
The authors would like to thank Vivarium of Universidad Juárez del
Estado de Durango for their contribution on animal donation.
Author’s Contribution
Conceptualization: Dealmy Delgadillo Guzmán and Agustina Ramírez
Moreno; Formal analysis: Agustina Ramírez Moreno; Funding acquisi-
tion: Rubén García Garza and Adolfo Soto; Project administration:
Dealmy Delgadillo Guzmán; Resources: Erika Flores Loyola and
Irais Castillo Maldonado; Supervision: Dealmy Delgadillo Guzmán;
Validation: Ibrahim Sharara Núñez, Hady Keita, and David Pedroza
Escobar; Writing—original draft, Agustina Ramírez Moreno, Adolfo
Soto Domínguez Writing—review and editing, Rubén García Garza.
All authors have read and agreed to the published version of the
manuscript.”
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
The authors disclosed receipt of the following financial support for the
research, authorship, and/or publication of this article: This research
was funded by Consejo Nacional de Ciencia y Tecnología for the doc-
toral scholarship (grant number 574703).
ORCID iD
Dealmy Delgadillo Guzmán https://orcid.org/0000-0001-6165-7282
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