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access type Open Access

The role of vitamin D receptor agonist on podocyte injury induced by high glucose

Published Online: 01 Aug 2022
Volume & Issue: AHEAD OF PRINT
Page range: -
Received: 18 Oct 2020
Accepted: 11 May 2022
Journal Details
License
Format
Journal
eISSN
2719-3500
First Published
30 Jun 2021
Publication timeframe
4 times per year
Languages
English
Abstract Background

The effects of vitamin D receptor (VDR) agonist paricalcitol on the podocyte injury induced by high glucose (HG) were investigated in conditioned immortalized mouse podocytes (MPC-5).

Methods

(1) Grouped according to different glucose concentrations: normal group (NG): 5.6 mmol/L glucose; HG stimulation group: 25 mmol/L glucose (25HG); high osmotic control group (NG + M): 5.6 mmol/L glucose + 19.4 mmol/L D-mannitol. The expression levels of VDR, podocyte marker proteins podocin, nephrin and mesenchymal marker proteins α-smooth muscle actin (α-SMA), matrix metalloproteinases (MMP9) in MPC-5 were measured, respectively. (2) Effect of VDR agonist-paricalcitol on podocyte epithelial-mesenchymal transition (EMT) induced by HG: cultured podocytes are divided into NG group, NG with dimethylsulfoxide (DMSO) group (NG+D), NG with paricalcitol (0.1 μmol/L) group (NG+P), HG group, HG with DMSO group (HG+D), and HG with paricalcitol (0.1 μmol/L) group (HG+P). The expression levels of VDR, podocyte marker proteins, marker proteins of mesenchymal cells, and the albumin flow in each group were then detected.

Results

(1) Under HG conditions, the expressions of VDR, podocin, and nephrin were decreased, while the expressions of α-SMA and MMP9 were increased (all P < 0.05). After administration of paricalcitol, the levels of VDR, podocin, and nephrin were increased, while the expressions of α-SMA and MMP9 were decreased compared with the control groups (all P < 0.05). (2) The results of albumin flow showed that the albumin flow of podocytes increased under the condition of HG, while it decreased after the treatment of paricalcitol.

Conclusion

The podocyte injury induced by HG could be partly rescued by Paricalcitol.

Keywords

Introduction

Diabetic nephropathy (DN) is one of the major microvascular complications of diabetes [1]. Approximately 30% of diabetic patients will gradually progress to issues with their kidneys, eventually leading to DN [2, 3, 4]. Previous studies have suggested that the early stage of DN is concentrated in the glomerulus [5, 6]. Under the light microscope, glomerular hypertrophy, hyperplasia, basement membrane thickening, and mesangial cell proliferation were shown in DN [7]. With the progress of DN, glomerular sclerosis will gradually appear. However, these pathological changes can only represent a part of the reason for the change in the glomerular filtration rate [8, 9]. With further research on glomerular epithelial cells (podocytes), it is found that changes in the number and related functions of podocytes also play an important role in the progression of DN [5, 10].

The vitamin D receptor (VDR) belongs to the superfamily of nuclear hormone receptors, and encompasses receptors for steroid and thyroid hormones [11, 12]. VDR can be found in multiple kinds of tissues and cells, such as hepatocytes, islet cells, renal tubular epithelial cells, glomerular podocytes, and so on [13]. A recent study has shown that compared with wild-type mice, diabetic mice with VDR gene knockout are more prone to present severe proteinuria and glomerulosclerosis, along with the loss of podocytes [14, 15]. VDR activation or Vitamin D analogs can effectively reduce proteinuria in patients with DN [16, 17]. Therefore, VDR is closely related to the development of DN.

Paricalcitol (19-nor-1, 25-hydroxy-vitamin D2), a synthetic vitamin D analog, can take multiple protective effects on the kidney [18, 19]. In this experiment, VDR agonist paricalcitol was used to study the protective effect of VDR on podocyte phenotype and functional damage caused by high glucose (HG) in vitro.

Materials and Methods
Laboratory instruments and reagents

Ultra-low temperature refrigerator (SANYO Company, Osaka, Japan), liquid nitrogen container (Hengao Biotechnology Co., Ltd., Beijing, China), electronic balance (ARl530, OHAUS Company, American), dry type thermostat (GL.150B, Qilin Bell instrument company, Haimen, China), Ice machine (IEC-25, Xueni, Suzhou, China), western blot system (Tanon, Shanghai, China), infrared laser imaging system (Tanon, Shanghai, China), Paricalcitol (Abbott Company, San Francisco, USA), Dimethylsulfoxide (Dingguo Changsheng Co., Ltd., Beijing, China), cDNA synthesis kit for real-time quantitative PCR (FSQ-101, Toyobo, Japan), SYBR rapid quantitative PCR Kit (KR-0389, Kapa Company, Kapa Biosystems, Inc., Corston, UK), rabbit anti-mouse VDR polyclonal antibody, rabbit anti-mouse podocin polyclonal antibody, rabbit anti-mouse nephrin polyclonal antibody, and mouse anti-mouse α-smooth muscle actin (α-SMA) monoclonal antibody were obtained from Abcam, USA. Rabbit anti-mouse matrix metal-loproteinases (MMP9) polyclonal antibody was purchased from BBI Life Science (Shanghai, China) and mouse anti-mouse GAPDH monoclonal antibody was purchased from Zhongshanjinqiao Bio Company, Beijing. Horseradish peroxidase-labeled goat anti-mouse secondary antibody and goat anti-rabbit secondary antibody were acquired from Beijing Dingguo Changsheng Biotechnology Company.

Cell culture

First, the frozen podocytes were removed from liquid nitrogen, and thawed in 37°C to 42°C waters. Then, 3 mL culture medium was added, and centrifuged at 1000 rpm for 10 min. The supernatant was discarded, then resuspended by 3–4 mL of culture medium. The cell suspension was transferred to a culture flask and cultured in a 33°C incubator. After 16 h, adherence of the cells was observed and the medium was exchanged. For the podocytes in 33°C, the medium was exchanged every 2–3 d. The podocytes were able to undergo passage at approximately 80%–90% confluence. After trypsinization, pipette 1–2 mL of the cell suspension into a new culture flask, and then 2–3 mL of culture medium was added. The new culture flask was then placed in a 37°C incubator and the medium was exchanged every 2–3 d. The podocytes were cultured for 10–14 d for the subsequent experiments.

Western blotting analysis

The total protein of each group was extracted from the podocytes and the concentration of proteins was measured by BCA assay (Thermo Fisher, Waltham, USA). The total proteins were heated and denatured at 99°C for 5 min. Then the loading samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked in 5% BSA at room temperature for 2 h. The primary antibody, VDR (1:200), nephrin (1:1000), podocin (1:1000), α-SMA (1:200), and MMP-9 (1:500), was incubated overnight at 4°C. The goat anti-mouse IgG secondary antibody (1:2000) and goat anti-rabbit secondary antibody (1:2000) were reacted at room temperature for 2 h. The image was performed by using the infrared laser imaging system, and the gray value of strips were measured by Image J and analyzed with GADPH as internal reference.

Real-time PCR

Total RNA was extracted using Trizol reagent (Invitrogen Inc.), according to the manufacturer’s instructions. The RNA purity was determined using absorbance at 260 nm and 280 nm (A260/280) and the cDNA was synthesized using the cDNA synthesis kit. The sequences of specific primers used in the present study are shown in Table 1. In KAPA SYBR rapid quantitative PCR Master Mix, the cDNA (2 μL) was mixed with 10 nmol/L forward and reverse primers (0.8 μL each), 2 × PCR Master Mix (10 μL) and RNase free water (in total volume of 20 μL); the reaction was performed in a thermocycler (BD Diagnostics, Sparks, MD, USA) under the conditions of cycle as follows: (1) pre-denaturation at 95°C for 3 min; (2) denaturation at 95°C for 5 s; (3) annealing at 60°C for 30 s; and (4) extension at 72°C for 35 s; a total of 40 cycles.

The sequences of primers used in the present study.

Name Sequences
VDR Upstream: 5′TCCTTCCTCTGCTGGTAT3′
Downstream: 5′CTCCTTGGTTAGTGTGGTAG3′
Nephrin Upstream: 5′ACTGGAGGAATGTAGGTAATG3′
Downstream: 5′TGTGTTCTTGCTTCTGTGA3′
Podocin Upstream: 5′AGAAGAGGAGAAGGAGTT3′
Downstream: 5′TTGGAGTTGAATGGTGTT3′
α-SMA Upstream: 5′AACTGTGAATGTCCTGTG3′
Downstream: 5′CATAGGTAACGAGTCAGAG3′
MMP9 Upstream: 5′ACACGACATCTTCCAGTA3′
Downstream: 5′CACCTTGTTCACCTCATT3′
GAPDH Upstream: 5′AGTGGCAAAGTGGAGATT3′
Downstream: 5′GTGGAGTCATACTGGAACA3′

VDR, vitamin D receptor; α-SMA, α-smooth muscle actin; MMP9, matrix metalloproteinases; GADPH, Glyceraldehyde-3-phosphate dehydrogenase.

Detection of podocyte monolayer barrier function

When the podocytes grew to a certain density and matured, the cells were uniformly inoculated onto the fibrous membrane of the Transwell chamber. After 24–36 h, when the cells grew to 80%–90% confluence, they were starved by replacing with fetal bovine serum-free medium for 6–8 h. Different treatment conditions were added to the upper layer of the Transwell chamber, and after 36 h, the cells were washed with sterilized PBS thrice. Then, the medium in the upper part of the chamber was discarded and replaced with 0.3 mL roswell park memorial institute (RPMI) 1640 medium containing 40 mg/mL albumin, while the medium in the lower part was replaced with 1 mL RPMI 1640 medium. The cells were cultured under 37°C for 1 h, then the sub-compartment medium was collected and the protein concentration was detected using the bicinchoninic acid (BCA) assay kit.

Statistical analysis

The data are expressed as means ± SD. SPSS 17.0 software (Chicago, IL, USA) was used for the statistical analysis and the test of intergroup significance of the measurement data was performed by one-way analysis of variance.

Results
Changes in podocytes phenotype and function under high-glucose treatments
Changes in podocyte phenotypes under different concentrations of glucose

To determine the effect of HG on the changes in podocyte phenotypes, we exerted different glucose concentrations on the podocytes. The results of western blot showed that the expression levels of nephrin, podocin, α-SMA, and MMP9 in the normal group (NG) were not significantly different compared with that of the hypertonic control group (NG+M, P > 0.05); Under the stimulation of HG (12.5G and 25G), the expressions of VDR, nephrin, and podocin proteins were reduced compared with that of the NG and the NG+M. The protein levels were decreased followed with the increase of glucose concentration (P < 0.05). Moreover, compared with the NG, the protein expressions of α-SMA and MMP9 were increased under the condition of HG stimulation, and the protein expressions were also upregulated with the increase in glucose concentration (P < 0.05) (Figure 1A,B). The results of qPCR were consistent with its protein levels (Figure 1C,D). Compared with the normal glucose concentration, the expression levels of VDR and podocyte marker proteins, nephrin and podocin, were decreased in the HG environment, and its mRNA levels decreased with the increase in glucose concentration (P < 0.05). Additionally, the mRNA levels of α-SMA and MMP9 were elevated with the increase in glucose concentration (P < 0.05).

Figure 1

The protein and mRNA levels of nephrin, podocin, α-SMA, and MMP9 under different concentrations of glucose. The lysate of cultured podocytes was used to do protein blotting and RNA extraction. (A) Western blotting analysis showed podocin, nephrin, VDR, and GAPDH expressions. (B) Protein expressions of MMP9, α-SMA, and GAPDH. (C) and (D) qRT-PCR showed mRNA expression of podocyte marker proteins, VDR and podocyte injury proteins. Compared with the NG group, Pabc < 0.05. α-SMA, α-smooth muscle actin; VDR, vitamin D receptor; 12.5G, 12.5 mmol/L glucose; 25G, 25 mmol/L glucose; NG, normal group; M, Mannitol; MMP9, matrix metalloproteinases; GADPH, Glyceraldehyde-3-phosphate dehydrogenase.

Changes in podocyte monolayer barrier function under HG conditions

To detect whether the podocytes with altered phenotypes under HG conditions exist functional impairment, albumin influx measured by the Transwell chamber were utilized to assess the monolayer barrier function of podocytes. The results showed that albumin inflow was increased in the 12.5 G group and the 25 G group compared with the NG (Figure 2 and Table 2), and the difference was statistically significant (P < 0.05). Our results indicated that the podocytes with altered phenotypes under HG also presented impaired monolayer barrier functions.

Figure 2

Function of podocytes under hyperglycemic treatment. Transwell analysis was used to analyze the albumin influx. Companied with HG treatment, the albumin influx was increased, indicating podocyte injury. Note: Compared with NG group, Pa < 0.05. 12.5G, 12.5 mmol/L glucose; 25G, 25 mmol/L glucose; HG, high glucose; NG, normal group.

Albumin flow at different glucose concentrations (mean ± SD).

Group Albumin flow (mg/mL)
NG 1.20 ± 0.16
12.5 G 1.29 ± 0.16a
25 G 2.06 ± 0.17a

NG, normal group; 12.5G, 12.5 mmol/L glucose; 25G, 25 mmol/L glucose. Compared with the NG group, Pa < 0.05.

Protective effect of paricalcitol on the changes in podocyte phenotype and the impairment of barrier function induced by HG
Protective effects of paricalcitol on the phenotypic damage of podocytes induced by HG

To examine the effect of paricalcitol on the changes in podocyte phenotype, western blot and qPCR were utilized to assess its protective role on HG induced podocyte injury. The results of qRT-PCR (Figure 3) showed mRNA expressions of these factors. The mRNA levels of VDR, nephrin, and podocin were also decreased in HG concentration conditions compared with that of the normal control group (P < 0.05). However, the mRNA expression levels of VDR, nephrin, and podocin were increased in the HG group after adding paricalcitol (P < 0.05). Moreover, compared with that of the normal control group, α-SMA, MMP9 mRNA levels were elevated in HG conditions (P < 0.05), and the expressions of α-SMA and MMP9 conversely decreased when paricalcitol was added to podocytes under HG (P < 0.05).

Figure 3

The protective role of VDR agonist (paricalcitol) on podocytes injury induced by HG. qRT-PCR showed mRNA expression of VDR, nephrin, podocin, α-SMA, and MMP9 under different treatments. VDR, nephrin, and podocin expressions were increased by paricalcitol, but α-SMA and MMP9 expressions were decreased. Compared with NG the group, Pa < 0.05 compared with 25G group, Pb < 0.05. α-SMA, α-smooth muscle actin; HG, high glucose; 25G, 25 mmol/L glucose; NG, normal group; VDR, vitamin D receptor; P, Paricalcitol; D, dimethylsulfoxide.

Protective effects of paricalcitol on damage of podocyte barrier function caused by HG

In order to find out whether paricalcitol can alleviate the damage of podocyte barrier function caused by HG, we measured the albumin flow using the Transwell to evaluate the podocyte monolayer barrier function. The study found that the albumin influx increased in the 25HG group compared with the normal control group, and the albumin flow decreased in the paricalcitol group compared with the 25HG group (Figure 4 and Table 3), and the difference was statistically significant (P < 0.05). The results indicated that paricalcitol can relieve the damage of podocyte barrier function caused by HG.

Figure 4

Effect of paricalcitol on barrier function of mature podocytes under the HG treatment. Transwell analysis was used to analyze the albumin influx. Compared with the NG group, Pa < 0.05, compared with 25G group, Pb < 0.05. 25G, 25 mmol/L glucose; NG, normal group; PAR, Paricalcitol.

Albumin flow under different treatment (mean with SD).

Groups Albumin flow (mg/mL)
NG 1.14 ± 0.10
25G 2.13 ± 0.06a
PAR 1.53 ± 0.02b

Compared with the NG group, Pa < 0.05, compared with 25G group, Pb < 0.05. NG, normal group; 25G, 25 mmol/L glucose; PAR, Paricalcitol.

Discussion

The capillary loops of glomerulus comprise three structures: capillary endothelial cells, glomerular basement membrane (GBM), and visceral epithelial cells (i.e. podocytes). Among them, podocytes are terminally differentiated cells coated on the basement membrane of the glomerulus. From the cell body, podocytes can give off larger primary processes, and then give off secondary processes to the GBM, which is the foot process [10, 20]. A 20–30 nm wide crack is formed between adjacent foot processes and they are connected by a permeable slit-diaphragm (SD). A variety of podocyte marker proteins and backbone proteins are distributed on the cracked diaphragm, and their integrity is key to determining the permeability of the glomerular filtration barrier [21]. Therefore, they constitute the last barrier of plasma albumin filtration together [22, 23, 24]. With the continuous in-depth research on glomerular visceral epithelial cells (podocytes), it has also been found that in the early stage of DN, pathological changes such as podocyte hypertrophy, foot process enfacement, reduced number of foot processes, and the increased gap between them accompanied by structural dysfunction were observed under electron microscope. As such, the impairment and loss of podocytes are involved in the development of proteinuria in DN. Podocytes are considered to be key to the development and progression of DN [25, 26]. In addition, recent studies have shown that the injury of podocytes is an important link in the development and progression of multiple kidney diseases [27].

VDR is a member of the superfamily of steroid and parathyroid hormone nuclear receptors, and it can be found in a variety of tissues and cells, such as hepatocyte, pancreatic islet cell, renal tubular epithelial cells, and glomerular podocytes [12, 28]. Vitamin D (VD)—VDR signaling system not only plays a role in maintaining the balance of calcium and phosphorus metabolism in the body, but also affects the synthesis of some proteins by regulating the transcription and expression of related genes, which plays an imperative role in the physiological and biochemical regulation of the body [29]. Compared with diabetic wild-type mice, VDR knockout mice have increased expression of fibronectin and reduced nephrin levels [30]. The results of this study also showed that the expression of VDR in podocytes decreased and podocyte injury occurred under the HG treatment.

Nephrin and podocin are distributed on the SD of podocyte, which are encoded by the NPHS1 and NPHS2 genes, respectively [31]. They are the structural proteins on the SD, which can be interconnected with other molecules in the SD, such as backbone proteins, to maintain the integrity and normal permeability of the SD [32, 33]. The results of this experiment showed that the protein expression of nephrin and podocin decreased under HG stimulation, and conversely, the expression of nephrin and podocin increased relatively after the administration of paricalcitol, the VDR agonist [34].

α-SMA and MMP9 are specific marker proteins of mesenchymal cells [35]. Overexpression of α-SMA in non-smooth muscle cells usually represents the occurrence of mesenchymal trans-differentiation [36]. In this study, the protein levels of α-SMA and MMP9 are increased under the stimulation of HG, and its expression will be relatively reduced after adding the VDR agonist paricalcitol.

The detection of podocyte monolayers barrier function treated with different treatments by Transwell suggest that HG induces epithelial-mesenchymal transition (EMT) in podocytes, resulting in the loss of structural proteins on the SD, which in turn damages the integrity of the SD and leads to impaired glomerular filtration barrier function. However, the podocyte albumin flow was decreased after the treatment of VDR agonist paricalcitol, indicating that paricalcitol can alleviate the impairment of podocyte barrier function caused by HG.

In short, podocyte expression of VDR is decreased and podocyte injury occurs in the HG environment, and the VDR agonist paricalcitol can alleviate the damage of podocyte phenotype and barrier function caused by HG. Therefore, VDR activation is one of the pathways to block podocyte EMT. However, the inner regulatory mechanism of VDR’s protective effect on the kidney has not been clarified yet, and further research is required.

Figure 1

The protein and mRNA levels of nephrin, podocin, α-SMA, and MMP9 under different concentrations of glucose. The lysate of cultured podocytes was used to do protein blotting and RNA extraction. (A) Western blotting analysis showed podocin, nephrin, VDR, and GAPDH expressions. (B) Protein expressions of MMP9, α-SMA, and GAPDH. (C) and (D) qRT-PCR showed mRNA expression of podocyte marker proteins, VDR and podocyte injury proteins. Compared with the NG group, Pabc < 0.05. α-SMA, α-smooth muscle actin; VDR, vitamin D receptor; 12.5G, 12.5 mmol/L glucose; 25G, 25 mmol/L glucose; NG, normal group; M, Mannitol; MMP9, matrix metalloproteinases; GADPH, Glyceraldehyde-3-phosphate dehydrogenase.
The protein and mRNA levels of nephrin, podocin, α-SMA, and MMP9 under different concentrations of glucose. The lysate of cultured podocytes was used to do protein blotting and RNA extraction. (A) Western blotting analysis showed podocin, nephrin, VDR, and GAPDH expressions. (B) Protein expressions of MMP9, α-SMA, and GAPDH. (C) and (D) qRT-PCR showed mRNA expression of podocyte marker proteins, VDR and podocyte injury proteins. Compared with the NG group, Pabc < 0.05. α-SMA, α-smooth muscle actin; VDR, vitamin D receptor; 12.5G, 12.5 mmol/L glucose; 25G, 25 mmol/L glucose; NG, normal group; M, Mannitol; MMP9, matrix metalloproteinases; GADPH, Glyceraldehyde-3-phosphate dehydrogenase.

Figure 2

Function of podocytes under hyperglycemic treatment. Transwell analysis was used to analyze the albumin influx. Companied with HG treatment, the albumin influx was increased, indicating podocyte injury. Note: Compared with NG group, Pa < 0.05. 12.5G, 12.5 mmol/L glucose; 25G, 25 mmol/L glucose; HG, high glucose; NG, normal group.
Function of podocytes under hyperglycemic treatment. Transwell analysis was used to analyze the albumin influx. Companied with HG treatment, the albumin influx was increased, indicating podocyte injury. Note: Compared with NG group, Pa < 0.05. 12.5G, 12.5 mmol/L glucose; 25G, 25 mmol/L glucose; HG, high glucose; NG, normal group.

Figure 3

The protective role of VDR agonist (paricalcitol) on podocytes injury induced by HG. qRT-PCR showed mRNA expression of VDR, nephrin, podocin, α-SMA, and MMP9 under different treatments. VDR, nephrin, and podocin expressions were increased by paricalcitol, but α-SMA and MMP9 expressions were decreased. Compared with NG the group, Pa < 0.05 compared with 25G group, Pb < 0.05. α-SMA, α-smooth muscle actin; HG, high glucose; 25G, 25 mmol/L glucose; NG, normal group; VDR, vitamin D receptor; P, Paricalcitol; D, dimethylsulfoxide.
The protective role of VDR agonist (paricalcitol) on podocytes injury induced by HG. qRT-PCR showed mRNA expression of VDR, nephrin, podocin, α-SMA, and MMP9 under different treatments. VDR, nephrin, and podocin expressions were increased by paricalcitol, but α-SMA and MMP9 expressions were decreased. Compared with NG the group, Pa < 0.05 compared with 25G group, Pb < 0.05. α-SMA, α-smooth muscle actin; HG, high glucose; 25G, 25 mmol/L glucose; NG, normal group; VDR, vitamin D receptor; P, Paricalcitol; D, dimethylsulfoxide.

Figure 4

Effect of paricalcitol on barrier function of mature podocytes under the HG treatment. Transwell analysis was used to analyze the albumin influx. Compared with the NG group, Pa < 0.05, compared with 25G group, Pb < 0.05. 25G, 25 mmol/L glucose; NG, normal group; PAR, Paricalcitol.
Effect of paricalcitol on barrier function of mature podocytes under the HG treatment. Transwell analysis was used to analyze the albumin influx. Compared with the NG group, Pa < 0.05, compared with 25G group, Pb < 0.05. 25G, 25 mmol/L glucose; NG, normal group; PAR, Paricalcitol.

Albumin flow at different glucose concentrations (mean ± SD).

Group Albumin flow (mg/mL)
NG 1.20 ± 0.16
12.5 G 1.29 ± 0.16a
25 G 2.06 ± 0.17a

Albumin flow under different treatment (mean with SD).

Groups Albumin flow (mg/mL)
NG 1.14 ± 0.10
25G 2.13 ± 0.06a
PAR 1.53 ± 0.02b

The sequences of primers used in the present study.

Name Sequences
VDR Upstream: 5′TCCTTCCTCTGCTGGTAT3′
Downstream: 5′CTCCTTGGTTAGTGTGGTAG3′
Nephrin Upstream: 5′ACTGGAGGAATGTAGGTAATG3′
Downstream: 5′TGTGTTCTTGCTTCTGTGA3′
Podocin Upstream: 5′AGAAGAGGAGAAGGAGTT3′
Downstream: 5′TTGGAGTTGAATGGTGTT3′
α-SMA Upstream: 5′AACTGTGAATGTCCTGTG3′
Downstream: 5′CATAGGTAACGAGTCAGAG3′
MMP9 Upstream: 5′ACACGACATCTTCCAGTA3′
Downstream: 5′CACCTTGTTCACCTCATT3′
GAPDH Upstream: 5′AGTGGCAAAGTGGAGATT3′
Downstream: 5′GTGGAGTCATACTGGAACA3′

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