Invest Clin 66(3): 301 - 312, 2025 https://doi.org/10.54817/IC.v66n3a06

Correspondence author: Qingpeng Hu, Department of Pediatrics, the Second Affiliated Hospital, Hengyang Medi-

cal School, University of South China, Hengyang, 421001, Hunan, China.

Email: huqingpeng163@126.com

Vitamin D attenuates epithelial-

mesenchymal transition of renal tubular

epithelial cells in infant rats with

hypothyroidism via the Traf6/TAK1

signalling pathway.

Aiyuan Cai1, Qingpeng Hu2, Haixia Wu1, Zilong Li1, Yuanhong Lin3, Jiaohua Yu1,

Hailong Huang1, Ruizhong Zhang1, Jing Xiao1 and Ping Liu1

1Department of Pediatrics, Longhua District People’s Hospital, Shenzhen, Guangdong,

China.

2Department of Pediatrics, the Second Affiliated Hospital, Hengyang Medical School,

University of South China, Hengyang, Hunan, China.

3Second Clinical Medical College, Guangzhou University of Chinese Medicine,

Guangzhou, China.

Keywords: vitamin D; hypothyroidism; kidney injuries; epithelial-mesenchymal

transition; TRAF6 protein; MAP3K7 protein (TAK1); signal transduction.

Abstract. In the hypothyroidism(HT) state, renal hemodynamics is disor-

dered, oxidative stress is intensified, and inflammatory factors are activated. Vi-

tamin D (VD) not only regulates calcium and phosphorus metabolism, but its

active form (1,25-dihydroxyvitamin D) could also exert anti-inflammatory and an-

ti-fibrotic effects by binding with the vitamin D receptor (VDR) widely distributed

in the kidney. This study aimed to elucidate the impact of VD on the epithelial-

mesenchymal transition (EMT) of renal tubular epithelial cells in HT-induced re-

nal injury in young rats, as well as its regulatory mechanism involving the tumor

necrosis factor receptor-associated factor 6 (Traf6)/transforming growth factor-β

activated kinase 1 (TAK1) pathway. An HT model in young rats was established

via propylthiouracil (PTU) gavage, and a functional rescue experiment was con-

ducted by overexpressing TAK1 (pcDNA3.1-TAK1). The animals were divided into

five groups: normal, HT, low-dose VD (HT+VD-L), high-dose VD (HT+VD-H), and

HT+VD-H+pcDNA3.1-TAK1 (HT+VD-H+pc). Each group consisted of 10 rats.

Serum creatinine (Scr) and blood urea nitrogen (BUN) levels were measured

using an automatic biochemical analyzer. Renal apoptosis (TUNEL), TGF-β1/α-

SMA/E-cadherin (immunohistochemistry), and Bcl-2/Bax/Traf6/TAK1/p-TAK1

(Western blot) expressions were assessed in renal tissue. VD significantly reduced

Scr and BUN levels in the serum of HT young rats, downregulated renal tissue

apoptosis, decreased TGF-β1 and α-SMA expressions, and upregulated E-cadherin

expression. Additionally, VD inhibited Traf6, p-TAK1, and Bax expressions while

increasing Bcl-2 expression. All differences were statistically significant.

302 Cai et al.

Investigación Clínica 66(3): 2025

Vitamina D atenúa la transformación mesenquímica-epitelial

de células epiteliales tubulares renales a través de la vía

de señalización Traf6/TAK1 en ratas bebé con hipotiroidismo.

Invest Clin 2025; 66 (3): 301 – 312

Palabras clave: vitamina D; hipotiroidismo; daño renal; transición epitelio-mesénquima;

proteína TRAF6; proteína MAP3K7 (TAK1); transducción de señales.

Resumen:El hipotiroidismo (HT) altera la hemodinámica renal, el estrés

oxidativo y la inflamación. La vitamina D (VD) regula calcio/fósforo; su forma

activa ejerce efectos antiinflamatorios/antifibróticos vía el receptor VD renal.

Este estudio se centra en observar y aclarar el efecto de la VD sobre la transi-

ción epitelial-mesenquimal (EMT) de las células epiteliales tubulares renales

en la lesión renal inducida por el HT en ratas jóvenes, así como su mecanismo

regulador que involucra la vía del receptor del factor de necrosis del tumor 6

(traf 6) / y del factor de crecimiento transformante β activado por la quinasa

1(TAK1). El modelo de HT se estableció en ratas jóvenes mediante el método

de alimentación por sonda de propil-tiouracilo (PTU) y se realizó un experi-

mento de rescate funcional mediante la sobreexpresión de TAK1 (pcDNA3.1-

TAK1). Los animales fueron divididos en cinco grupos: normal, HT, VD de baja

dosis (HT+VD-L), VD de alta dosis (HT+VD-H), HT+VD-H + PC DNA 3.1-tak

1 (HT+VD-H + PC). Cada grupo estuvo compuesto por 10 ratas. Se deter-

minaron los niveles de creatinina sérica (Scr) y nitrógeno de urea en sangre

(BUN) con un analizador bioquímico automático. Se evaluó la expresión de la

apoptosis renal (TUNEL), TGF-β1/α-SMA/E-Calcinina (immunohistoquímica)

y Bcl-2/Bax/traf 6/tak 1/p-tak 1 (Western blot) en el tejido renal. VD redujo

significativamente los niveles de SCR y BUN en suero de ratones de HT, redujo

la apoptosis en tejido renal, redujo la expresión de TGF-β1 y α-SMA y aumentó la

expresión de E-calciferina. Además, la VD inhibe la expresión de Traf6, p-TAK1

y Bax, mientras que aumenta la expresión de Bcl-2.

Received: 25-01-2025 Accepted: 26-06-2025

INTRODUCTION

Hypothyroidism (HT) is a common dis-

ease of the endocrine system, which can oc-

cur at all ages. It occurs in fetuses, newborns

and infants, which could directly affect the

development of children’s nervous system

and skeletal system, directly lead to short

stature or permanent mental retardation,

and bring unpredictable harm to patients’

families 1. Studies have shown that HT di-

rectly causes changes in renal hemodynam-

ics and glomerular filtration performance2,3,

which directly leads to certain renal inter-

stitial fibrosis. Consequently HT patients

are often accompanied by certain renal dys-

function, but the pathological mechanism of

HT renal injury has not been fully clarified,

and there is still a lack of specific drugs in

clinic, especially for infants with HT-induced

renal injury. Studies have shown that abnor-

mal apoptosis in renal tissue induced by in-

flammatory stress plays an important role in

the progression of the disease. Adan et al. 4

Vitamin D effect on transition of renal tubular epithelial cells in infant rats 303

Vol. 66(3): 301 - 312, 2025

showed that the expression of tumor necro-

sis factor-α (TNF-α) and interleukin-6 (IL-6)

is increased in the serum of patients with in-

sufficient thyroxine synthesis, and it was cor-

related with the expression of apoptosis-in-

hibiting protein B cell lymphoma-2 (Bcl-2).

It was positively correlated with the expres-

sion of Bcl-2-related X protein (Bax). Narilla

et al. 5, showed that the levels of translation

and transcription of transforming growth

factor-β 1 (TGF-β1) and α-smooth muscle

actin (α-SMA) in the serum of patients with

hypothyroidism were significantly increased.

However, the transcription and translation

levels of E-cadherin decreased significantly,

and the patients exhibited specific patho-

logical changes indicative of epithelial-mes-

enchymal transition (EMT) in renal tubules.

Tumor necrosis factor receptor-associated

factor 6 (Traf6)/ transforming growth factor

β-activated kinase 1 (TAK1) signal is an en-

dogenous pathway closely related to inflam-

matory stress and cell fibrosis. Wang et al.

6 demonstrated that inhibiting the TRAF6/

TAK1 pathway and reducing the phosphory-

lated TAK1 (p-TAK1) expression in the HK-2

renal tubular fibrosis model induced by high

glucose significantly suppressed myofibro-

blast marker α-SMA expression in HK-2 cells,

thereby blocking cell fibrosis progression.

Many clinical data and basic experimental

data at home and abroad shows that vita-

min D (VD) is not only a simple fat-soluble

vitamin, but also a steroid hormone closely

related to physiological processes such as

immune regulation, cell stress and apopto-

sis. Therefore, in this study, the HT-model of

young rats was established by intragastric

administration of propylthiouracil (PTU).

Based on the TRAF6/TAK1 signal, a function

rescue experiment was conducted by overex-

pressing TAK1 (pc DNA3.1-TAK1). The effect

of VD on the EMT of renal tubular epithe-

lial cells in young rats with HT is discussed,

aiming to provide a reliable direction for the

clinical treatment of the disease.

MATERIALS AND METHODS

Test animals, main reagents

and instruments

Fifty 3-week-old male SD rats with SPF

cleanliness, weighing 45g~55g, were used in

the experiment after being fed adaptively by

the specialized personnel in our hospital at

a temperature of 20℃~25℃ and a humidity

of 55~60%)% for 12h. Experimental reagent

vitamin D drops (Star Shark) (VD, 400U*36

pills, Star shark pharmaceutical (Xiamen)

Co., Ltd., Sinopharm zhunzi H35021450);

PTU (purity ≥99.9%) was purchased from

sigma company in USA, and overexpression

TAK1(pc DNA3.1-TAK1) and negative con-

trol pc DNA3.1-TAK1-NC were purchased

from anbote genetic engineering technology

Co., Ltd, Beijing. Other reagents and equip-

ment were: Phosphate buffer solution (PBS),

formaldehyde, xylene, neutral resin (Beijing

Suolaibao Biotechnology Co., Ltd.), rabbit

anti-rat Traf6, TAK1 and p-TAK1, Bcl-2, Bax,

glyceraldehyde triphosphate dehydrogenase

(GAPDH) monoclonal antibody (Shanghai

Biyuntian Biotechnology Co., Ltd.), Immu-

nofluorescence, immunohistochemistry and

hematoxylin and eosin (HE) staining kit

(Shanghai Wohong Biotechnology Co., Ltd.),

Protein western blot kit, deoxynucleotide ter-

minal-mediated nick end labeling (TUNEL)

kit (Nanjing Senbeijia Biotechnology Co.,

Ltd.), enzyme-linked immunosorbent assay

kit (Nanjing Jiancheng Biotechnology Co.,

Ltd.). Tissue scissors, hemostatic forceps and

other surgical instruments (Jinhua Yidi Medi-

cal Equipment Co., Ltd., Jinhua City, Zheji-

ang Province), Image Quant LAS4010 gel

imaging system (GE Company, USA), CFX96

real-time fluorescent quantitative polymerase

chain reaction (quantitative real-time poly-

merase chain reaction, QRT-PCR) amplifier

(Bio-Rad Company, USA), Leica DMI6000B

microscope (Leica Company, Germany),

Z-5000 spectrophotometer (Hitachi Compa-

ny, Japan), CK-150 high-speed freezing cen-

trifuge (Sigma Company, USA).

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Investigación Clínica 66(3): 2025

Replication of the HT rat model

According to the method introduced by

Santos et al.7, a propylthiouracil (PTU) so-

lution was prepared with normal saline at a

concentration of 0.5% (5 mg/mL). Rats were

treated by gavage with 10 mL/kg PTU solu-

tion every day, and at the same time, their

weigh was recorded, and the dosage of PTU

was adjusted every week according to their

weight. After four weeks of continuous ga-

vage, 2mL of tail vein blood of rats in each

group was taken to detect thyroid stimulat-

ing hormone (thyroid-stimulating hormone,

TSH) and total thyroxine (TT4), compared

with the normal group, the serum TSH level

of HT rats increased significantly, while TT4

level decreased significantly, which indicat-

ed that the model was successful.

Packet processing

After adaptive feeding, rats were ran-

domly divided into a normal group, HT

group, VD low-dose (HT+VD-L) group, VD

high-dose (HT+VD-H) group and HT+VD-

H+PCDNA3.1-Tak1 (HT+VD-H+PC) group,

with 10 rats in each group. Rats in the

HT+VD-L group were injected with 5×109

pfu/mL of pc DNA3.1-TAK1-NC via tail vein

every day, and treated with 0.25 mg/kg of

VD by gavage. Rats in the HT+VD-H group

were injected with 5×109 pfu/mL of pc

DNA3.1-TAK1-NC via the tail vein every day,

and perfused with 1 mg/kg of VD. Rats in

the HT+VD-H+pc group were injected with

5×109 pfu/mL of pc DNA3.1-TAK1 by the

tail vein every day, and treated with 1 mg/kg

of VD by gavage. Rats in the normal group

were injected with 5×109 pfu/mL of pc

DNA3.1-TAK1-NC by the tail vein every day,

and treated with the same dose of normal

saline by gavage for four weeks. Rats in the

HT, HT+VD-L, HT+VD-H and HT+VD-H+pc

groups still need to be treated with PTU to

maintain the decline of thyroid function in

experimental animals. During the experi-

ment, rats were raised in a single cage, with

access to drinking water and food.

Sample collection

After the last drug administration, 24-

hour urine samples were collected from rats

in each group to detect the levels of 24-hour

urinary albumin (UAlb) in the offspring

rats. Subsequently, the rats were anesthe-

tized with an intraperitoneal injection of 30

mg/kg pentobarbital sodium. Then, 5 mL

of blood was collected from the heart and

placed in a benchtop refrigerated centrifuge

for 15 minutes to obtain serum for testing.

The rats were euthanized by cervical dislo-

cation. Under a microscope and on a sterile

operating table, the kidney tissues of the off-

spring rats were dissected.

Thyroid and renal function of rats in each

group

According to the requirements of the

ELISA kit, corresponding solutions were

added to the standard wells and sample

wells, and blank wells were set up. After an-

tibody coating, plate washing, and reaction

termination, the OD450 values of each well

were measured using a multifunctional mi-

croplate reader. A standard curve was plot-

ted, and the concentrations of TSH and TT4

in animal serum were calculated. At the

same time, the levels of UAlb, serum creati-

nine (Scr), and blood urea nitrogen (BUN)

in rats from each group were detected using

an automatic biochemical analyzer.

Apoptosis in renal tissue of rats in each

group

Partial renal tissue from rats in each

group was taken and fixed, sliced, and washed

according to the preliminary requirements

of the TUNEL kit. TUNEL working solution

was added, and the samples were incubated

at 37°C in the dark. After dehydration and

mounting, microscopic examination was

performed. The apoptosis rate in renal tissue

was statistically analyzed using the Image-

Pro 6.2 software.

Vitamin D effect on transition of renal tubular epithelial cells in infant rats 305

Vol. 66(3): 301 - 312, 2025

PCR detection of gene expression in renal

tissue of rats in each group

Partial renal tissue samples from rats

were collected, and an appropriate amount

of Trizol lysis solution was added. Total RNA

was extracted according to the requirements

of the Trizol kit. After determining its con-

centration using a Z-5000 spectrophotom-

eter, cDNA was synthesized using the Prime

Script™ kit. qRT-PCR amplification was per-

formed under the following conditions: ini-

tial denaturation at 95°C for 5 seconds, de-

naturation at 95°C for 5 seconds, annealing

at 60°C for 60 seconds, for 40 cycles. The

primer sequences are shown in Table 1. The

relative expression levels of each target gene

were represented by 2-△△CT.

Immunofluorescence detection of TNF-α

and IL-6 expression in renal tissue of rats

in each group

Partial renal tissue samples from rats

were collected and subjected to fixation, em-

bedding, slicing, microwave repair, and incu-

bation in 3% hydrogen peroxide. After wash-

ing with PBS, the samples were blocked with

goat serum and then incubated with primary

antibodies against TNF-α and IL-6 (diluted

1:500) at 4°C overnight in the dark. The

next day, fluorescent secondary antibodies

were added, and the samples were incubated

in the dark. Observation was performed un-

der a microscope.

Immunohistochemical experiment to

detect the expression of E-cadherin,

α-SMA, and TGF-β1 in renal tissue

Partial renal tissue samples from rats

were fixed, embedded, and sliced, followed

by immunohistochemical staining according

to the kit instructions. Primary antibodies

against E-cadherin, α-SMA, and TGF-β1 (all

diluted 1:500) and secondary antibodies (di-

luted 1:1500) were added. Observation was

performed under a microscope, and statisti-

cal analysis was conducted using the Image

J image processing software.

Expression levels of Bcl-2, Bax, Traf6,

TAK1, and p-TAK1 in renal tissue of rats

in each group

renal tissue from rats in each group

was placed in lysis buffer at 4°C for 30 min-

utes, followed by centrifugation and dilu-

tion of the supernatant. Total protein was

extracted from the samples using RIPA ly-

sate. Fifty micrograms of protein samples

were loaded for electrophoresis, followed by

membrane transfer and blocking. Primary

antibodies against Bcl-2, Bax, Traf6, TAK1,

and p-TAK1 (all diluted 1:1000) and second-

ary antibodies (diluted 1:5000) were added,

and the samples were incubated at room

temperature. After color development for

30 minutes, GAPDH was used as a reference

to analyze the grayscale values of the target

proteins.

Statistical analysis

The SPSS19.0 software (International

Business Machines Corporation, New York,

USA) and Graphpad5.01 software (Graph-

Pad Software Inc., San Diego, CA, USA) were

used for statistical analysis of data, and the

data were expressed as mean±SD. The t-test

was conducted to compare the two groups,

and the comparison between multiple

groups was conducted by one-way analysis of

variance, with p<0.05 indicating statistical

significance.

RESULTS

Comparison of thyroid function of rats

in each group

The results of the enzyme-linked ad-

sorption kit showed that compared with

normal rats, the serum TT4 levels in the HT,

HT+VD-L, HT+VD-H and HT+VD-H+pc

groups were reduced significantly, while the

TSH level increased significantly. Compared

with the HT group, the serum TT4 levels in

the HT+VD-L group, HT+VD-H group, and

HT+VD-H+pc group increased significantly,

while the TSH level decreased significantly.

306 Cai et al.

Investigación Clínica 66(3): 2025

Compared with the HT+VD-L group, the

level of TT4 in the serum of the HT+VD-H

group was significantly higher, while the lev-

el of TSH was significantly lower. Compared

with the HT+VD-H group, the level of TT4

in the serum of the HT+VD-H+pc group de-

creased significantly, and the level of TSH

increased significantly, with statistical sig-

nificance (all p<0.05) (Fig. 1).

Changes in renal function in each group

of rats

The results showed that, compared

with the normal group, the contents of

UAlb, Scr, and BUN in serum of rats in the

HT, HT+VD-L, HT+VD-H, and HT+VD-

H+pc groups were significantly higher. In

the HT+VD-L group, HT+VD-H group and

HT+VD-H+pc group, the contents of UAlb,

Scr and BUN in serum decreased signifi-

cantly. Compared with the HT+VD-L group,

the contents of UAlb, Scr and BUN in the

serum of the HT+VD-H group decreased

significantly (Fig. 2).

Apoptosis in the renal tissue of rats

in each group

TUNEL staining results showed that com-

pared with the normal group, the apoptosis rate

in the kidney tissue of rats in the HT, HT+VD-

L, HT+VD-H and HT+VD-H+pc groups was

significantly higher. Compared with the HT

group, the cells in the kidney tissue of rats in

HT+VD-L, HT+VD-H and the HT+VD-H+pc

groups were significantly higher. The apopto-

sis rate in the kidney tissue of the HT+VD-H

group decreased significantly, and compared

with the HT+VD-H group, the apoptosis rate in

the kidney tissue of the HT+VD-H+pc group

increased significantly, with statistical signifi-

cance (all p<0.05) (Fig. 3).

Expression of related genes in the renal

tissue of rats in each group

The results of PCR showed that com-

pared with the normal group, the mRNA

expressions of TNF-α, IL-6, α-SMA, TGF-β1,

Traf6 and TAK1 in the renal tissues of the

HT, HT+VD-L, HT+VD-H and HT+VD-H+pc

groups were significantly increased, while

the mRNA expression of E-cadherin was sig-

nificantly decreased.

Fig. 1. Changes of Thyroid stimulating hormone

(TSH) and Total thyroxine (TT4) in rats in

each group. Note: One-way ANOVA. Com-

pared with the Normal group, *: p<0.05

compared with HT group; #:p<0.05 com-

pared with HT+VD-L group; △:p<0.05 com-

pared with HT+VD-H group; ☆: p<0.05).

Fig. 2. Changes of 24-hour urinary albumin (UAlb),

Serum creatinine (Scr) and Blood urea ni-

trogen (BUN) in rats of each group (Note:

One-way ANOVA. Compared with the Nor-

mal group, *:p<0.05 compared with the HT

group; #:p<0.05 compared with HT+VD-L

group; △:p<0.05 compared with HT+VD-H

group; ☆:p<0.05)

Hormone concentration

Compound concentration

Vitamin D effect on transition of renal tubular epithelial cells in infant rats 307

Vol. 66(3): 301 - 312, 2025

Compared with the HT group, the

mRNA expressions of TNF-α, IL-6, α-SMA,

TGF-β1, Traf6, and TAK1 in renal tissue of

the HT+VD-L, HT+VD-H, and HT+VD-

H+pc groups decreased significantly, while

the mRNA expression of E-cadherin in-

creased significantly.

Compared with the HT+VD-L group,

the mRNA expression of TNF-α, IL-6, α-SMA,

TGF-β1, Traf6, and TAK1 in the HT+VD-H

group decreased significantly, while the mRNA

expression of E-cadherin increased signifi-

cantly. Compared with the HT+VD-H group,

the mRNA expressions of TNF-α, IL-6, α-SMA,

TGF-β1, Traf6 and TAK1 in the kidney tissue

of the HT+VD-H+pc group were significantly

increased, while the mRNA expression of E-cad-

herin was significantly decreased, with statisti-

cal significance (all p<0.05), (Fig. 4).

Expression of TNF-α and IL-6 in the renal

tissue of rats in each group

The results of immunofluorescence stain-

ing showed that compared with the normal

group, the fluorescence intensity and expres-

sion of TNF-α and IL-6 in the renal tissue of the

HT, HT+VD-L, HT+VD-H and HT+VD-H+pc

groups increased significantly. Compared with

HT+VD-L group, the fluorescence intensity

of TNF-α and IL-6 in the HT+VD-H group de-

creased obviously. Compared with the HT+VD-

H group, the fluorescence intensity of TNF-α

and IL-6 in the HT+VD-H+pc group increased

significantly (Fig. 5).

Fig. 3. The cell apoptosis of renal tissue and chan-

ges of renal function in rats of each group.

Note: One-way ANOVA.Compared with the

Normal group, *:p<0.05 compared with

the HT group; #:p<0.05 compared with

HT+VD-L group; △:p<0.05 compared with

HT+VD-H group; ☆:p<0.05).

Fig. 4. The expression of related genes in renal tissue in rats of each group (Note: One-way ANOVA. Com-

pared with the Normal group, *:p<0.05 compared with the HT group; #:p<0.05 compared with

HT+VD-L group; △:p<0.05 compared with HT+VD-H group; ☆:p<0.05).

Relative mRNAs expression of the gene

Apoptosis rate (%)

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Investigación Clínica 66(3): 2025

Expression of E-cadherin, α-SMA and

TGF-β1 in the renal tissue of rats in each

group

Immunohistochemical results showed

that, compared with the normal group, the

percentage of α-SMA and TGF-β1 positive

cells in the renal tissue of rats in the HT,

HT+VD-L, HT+VD-H and HT+VD-H+pc

groups increased significantly. In contrast,

the percentage of E-cadherin-positive cells

decreased significantly, and the expres-

sions of α-SMA, TGF-β1, and E-cadherin in-

creased significantly. Compared with the

HT group, the percentage of α-SMA and

TGF-β1 positive cells in kidney tissue of

the HT+VD-L, HT+VD-H and the HT+VD-

H+pc groups decreased significantly, and

the percentage of E-cadherin positive cells

increased significantly. The expressions of

α-SMA and TGF-β1 decreased significantly,

and the expression of E-cadherin increased

significantly, which was similar to that of

HT. In the HT+VD-H group, the percent-

age of α-SMA and TGF-β1 positive cells in

renal tissue decreased significantly, and

the percentage of E-cadherin positive cells

increased significantly. The expressions of

α-SMA and TGF-β1 decreased significantly,

and the expression of E-cadherin increased

significantly. Compared with the HT+VD-

H group, in the HT+VD-H+pc group, the

percentage of α-SMA and TGF-β1 positive

cells in rat kidney tissue increased signifi-

cantly. In contrast, the percentage of E-

cadherin-positive cells decreased signifi-

cantly, and the expressions of α-SMA and

TGF-β1 increased significantly, with statis-

tical significance (all p<0.05)(Fig. 6).

Expression of Bcl-2, Bax, Traf6 and

p-TAK1 in renal tissue of rats in each

group

Western blot results showed that com-

pared with the normal group, the expres-

sions of Bax, Traf6, and p-TAK1 in renal tis-

sue of rats in HT, HT+VD-L, HT+VD-Hand,

and HT+VD-H+pc groups were significantly

increased, while the expression of Bcl-2

was significantly decreased. The expres-

sions of Bax, Traf6 and p-TAK1 in the renal

tissue of rats in the HT+VD-L, HT+VD-H

and HT+VD-H+pc groups decreased sig-

nificantly, while the expression of Bcl-2 in-

creased significantly. Compared with the

HT+VD-L group, the expressions of Bax,

Traf6 and p-TAK1 in renal tissue of rats in

the HT+VD-H group decreased significantly.

In the HT+VD-H+pc group, the expressions

of Bax, Traf6 and p-TAK1 in the renal tissue

of rats were significantly increased, while

the expression of Bcl-2 was significantly de-

creased, with statistical significance (all p <

0.05), while the expression of TAK1 was not

statistically significantly different (p>0.05),

(Fig. 7).

Mechanism of VD alleviating renal injury

in HT rats

When HT occurs in rats, Traf6/TAK1

signal in renal tissue is activated, the level

of p-TAK1 is increased, the level of transcrip-

tion and translation of related inflammatory

and interstitial fibrosis genes is increased,

the level of inflammatory stress in animal

renal tissue is increased, and the apoptosis

rate of cells is increased, which promote

the EMT progress of renal tubular epithelial

cells and further affects the renal function

Fig. 5. The expression of TNF-α and IL-6 in renal tissue

in rats of each group (Note: One-way ANOVA.

Compared with the Normal group, *:p<0.05

compared with the HT group; #:p<0.05 com-

pared with HT+VD-L group; △:p<0.05 com-

pared with HT+VD-H group; ☆:p<0.05).

Relative uorescence intensity

Vitamin D effect on transition of renal tubular epithelial cells in infant rats 309

Vol. 66(3): 301 - 312, 2025

of animals. Following intervention with VD,

the Traf6/TAK1 signal activation was signifi-

cantly inhibited, leading to decreased levels

of p-TAK1, reduced transcription and trans-

lation of related genes, lower apoptosis rates

in cells within the animal kidney, and limited

EMT progression. The rescue experiment was

carried out using pCDNA3.1-TAK1, which

overexpresses TAK1. The results showed that

pcDNA3.1-TAK1 could partially reverse the

protective effect of VD on renal tissue, and it

was inferred that VD might play a protective

role in renal tissue by inhibiting the Traf6/

TAK1 signal (Fig. 8).

Fig. 6. Percentage of E-cadherin, α-SMA and TGF-β1 positive cells in renal tissue of rats in each group

(Note: One-way ANOVA. Compared with the Normal group, *:p<0.05 compared with the HT group;

#:p<0.05 compared with HT+VD-L group; △:p<0.05 compared with HT+VD-H group; ☆:p<0.05).

Fig. 7. The expression of Bcl-2, Bax,Traf6, TAK1 and p-TAK1 in renal tissue in rats of each group (Note: One-

way ANOVA. Compared with the Normal group, *:p<0.05 compared with the HT group; #:p<0.05

compared with HT+VD-L group; △:p<0.05 compared with HT+VD-H group; ☆:p<0.05).

Proportion of positive cells (%)

The relative expression of protein

310 Cai et al.

Investigación Clínica 66(3): 2025

DISCUSSION

HT is an endocrine system disorder

characterized by a series of metabolic im-

pairments due to insufficient synthesis and

secretion of thyroid hormones by the thy-

roid gland. The pathological mechanisms

of this disease are highly complex, affect-

ing multiple organs and tissues such as the

liver, kidneys, and brain. Currently, patients

could only manage the condition through

lifelong oral administration of thyroid hor-

mone supplements, and there is no defini-

tive cure 8. Renal injury induced by HT is one

of the most prominent complications in the

progression of the disease. Lathiya et al.9 in-

dicated that renal injury due to thyroid hor-

mone deficiency was closely related to the

progression of renal interstitial fibrosis in

patients. Therefore, an in-depth exploration

of the molecular mechanisms underlying

epithelial-mesenchymal transition (EMT) in

renal tubular epithelial cells in HT and the

identification of reliable therapeutic tar-

gets are of great significance for the clinical

treatment of the disease.

Vitamin D (VD), one of the essential vi-

tamins for growth, development, and stress

response, primarily participates in bone ho-

meostasis by regulating calcium and phos-

phorus metabolism in cells. However, re-

cent studies have shown 9 that VD exhibits

multiple physiological effects, playing a role

in pathophysiological processes such as in-

flammatory stress, immune regulation, neu-

rotransmitter development, and antitumor

activity. A clinical study by Durmuş et al.10

revealed that supplementing COVID-19 pa-

tients with a certain amount of VD signifi-

cantly reduced their serum levels of α-SMA

and TGF-β1, decreased their urine albumin

(UAlb) content and serum levels of creati-

nine (Scr) and blood urea nitrogen (BUN),

thereby improving renal dysfunction. Wu et

al. demonstrated that exogenous VD supple-

mentation in a model of pituitary secretion

deficiency significantly inhibited the expres-

sion of TNF-α and IL-6 in renal tissue, down-

regulated the apoptosis rate in animal renal

tissue, and alleviated pathological damage

to renal tissue. Li et al. 11 showed that in a

model of HK-2 cell injury induced by high

glucose, VD intervention significantly inhib-

ited the expression of Bax in cells, increased

the expression of E-cadherin, and inhibited

the EMT process in cells. In this study, af-

ter the construction of an HT model in rats

using propylthiouracil (PTU), results from

Fig. 8. The schematic diagram of the mechanism of VD attenuating renal injury in HT rats.

The inflammatory stress in renal tissue

The renal tissue pathological damage

The mesenchymal transdifferentiation

The apoptosis rate

of renal tubular epithelial cells

The inflammatory stress in renal tissue

The renal tissue pathological damage

The mesenchymal transdifferentiation

The apoptosis rate

of renal tubular epithelial cells

Vitamin D effect on transition of renal tubular epithelial cells in infant rats 311

Vol. 66(3): 301 - 312, 2025

enzyme-linked immunosorbent assay kits

and hematoxylin and eosin (HE) staining of

thyroid tissue showed that compared with

the normal group, HT rats had significantly

elevated serum TSH levels, significantly de-

creased TT4 levels, and apparent thyroid

pathological damage, indicating successful

modeling. After VD intervention, renal func-

tion parameters (UAlb, Scr, BUN) in HT rats

improved significantly, pathological damage

to renal tissue was markedly reduced, the

apoptosis rate of renal tissue cells decreased

significantly, the expression of inflammatory

mediators TNF-α and IL-6 in renal tissue was

significantly lowered, and EMT in renal tu-

bules was significantly restricted, confirm-

ing the protective effect of VD on the kid-

neys of HT rats.

To better evaluate the interventional

effects of drugs and seek reliable therapeu-

tic targets, it was necessary to delve deeper

into the drug targets to promote their wide-

spread use in clinical treatment. The acti-

vation of Traf6/TAK1 signalling was closely

related to the progression of renal diseases

such as acute kidney injury, chronic kidney

disease, renal aging, and renal cell carcino-

ma, primarily exerting pro-inflammatory ef-

fects, promoting fibrosis, and regulating cell

apoptosis and pyroptosis, thereby participat-

ing in the occurrence and development of re-

lated renal diseases. Wei et al.12 showed that

inhibiting the phosphorylation of TAK1 in a

diabetic mouse model significantly reduced

the expression of mesenchymal cell marker

α-SMA in renal tissue and decreased the per-

centage of collagen deposition in renal tis-

sue. The results of this study showed that the

expression of Traf6 and p-TAK1 was signifi-

cantly reduced in the renal tissue of rats in

both high and low-dose VD groups. Function-

al rescue experiments using pcDNA3.1-TAK1

overexpressing TAK1 showed that pcDNA3.1-

TAK1 could partially reverse the protective

effect of VD on renal tissue, suggesting that

VD may exert its protective effect on renal

tissue by inhibiting Traf6/TAK1 signalling.

In summary, vitamin D can inhibit EMT

in renal tubular epithelial cells of HT rats,

reduce the apoptosis rate in renal tissue, al-

leviate pathological damage to renal tissue,

and improve renal function. These effects are

related to the inhibition of Traf6/TAK1 sig-

nalling activation. However, more detailed

and systematic research is needed before vi-

tamin D can be comprehensively used in the

clinical treatment of the disease.

Acknowledgement

The article was supported by the Nat-

ural Science Foundation of Hunan Prov-

ince (2023JJ30532), Key Project of Hu-

nan Provincial Department of Education

(21A0265), Key Project of Hunan Provincial

Health Planning Commission-Guiding Proj-

ect (20200549) and the District-level scien-

tific research project of Medical and Health

Institutions in Longhua District (2022093).

Conflicts of interest

The authors declare that they have no

conflicts of interest to report regarding the

present study.

ORCID number of authors

• Aiyuan Cai(AC):

0000-0002-2978-5476

• Qingpeng Hu(QH):

0009-0002-0586-5364

• Haixia Wu(HW):

0009-0009-1503-8187

• Zilong Li(ZL):

0009-0002-9174-9246

• Yuanhong Lin(YL):

0009-0009-5397-2587

• Jiaohua Yu(JY):

0009-0002-1098-7913

• Hailong Huang(HH):

0009-0001-7321-5710

312 Cai et al.

Investigación Clínica 66(3): 2025

• Ruizhong Zhang(RZ):

0009-0005-9678-3226

• Jing Xiao(JX):

0009-0001-4989-3909

• Ping Liu(PL):

0009-0007-9178-5278

Contribution the authors

AC: conceived the study and drafted the

manuscript. QH, HW: supervised the project

and acquired funding, and critically revised the

intellectual content. ZL, YL,JY, HH, RZ, JX, PL:

performed the experiments and collected data.

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