The present study aimed to investigate the antifibrogenic effects of genistein (GEN) on the kidney in streptozotocin (STZ)-induced diabetic rats and to determine the associated mechanisms. Rats were randomized into four groups: Normal control (N), STZ (S), L (STZ + low-dose GEN) and H (STZ + high-dose GEN). After 8 weeks, the fasting blood glucose (FBG) level, the ratio of kidney weight to body weight (renal index), 24-h urine protein, blood urea nitrogen (BUN), serum creatinine (SCr), renal total antioxidant capacity (T-AOC), superoxide dismutase (SOD), lipid peroxidation (LPO), malondialdehyde (MDA) and hydroxyproline (Hyp) contents were measured. The histomorphology and ultrastructure of the kidney were also assessed. In addition, mRNA expression levels of transforming growth factor-β1 (TGF-β1) and protein expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO1), TGF-β1, mothers against decapentaplegic homolog 3 (Smad3), phosphorylated (p)-Smad3 and collagen IV were estimated. Compared with group N, the levels of FBG, renal index, 24-h urine protein, BUN, SCr, LPO, MDA and Hyp were increased, whereas the levels of T-AOC and SOD were decreased in group S. The structure of renal tissue was damaged, and the expression of Nrf2, HO-1 and NQO1 were reduced, whereas the expression of TGF-β1, Smad3, p-Smad3 and collagen IV were increased in group S. Compared with group S, the aforementioned indices were improved in groups L and H. In conclusion, GEN exhibited reno-protective effects in diabetic rats and its mechanisms may be associated with the inhibition of oxidative stress by activating the Nrf2-HO-1/NQO1 pathway, and the alleviation of renal fibrosis by suppressing the TGF-β1/Smad3 pathway.
Chronic kidney disease is increasing worldwide at an annual rate of 8%, with the prevalence higher in developing countries (
The nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway is a crucial cytoprotective regulator in mammalian cells in response to endogenous and exogenous stress (
Oxidative stress is defined as a disturbance in the balance between the production of reactive oxygen species (ROS) and antioxidant defenses (
Genistein (GEN) is a major isoflavone in soybeans that interacts with estrogen receptors
Although the positive effects of GEN on renal function are well known, the effects of GEN on renal fibrosis in T1DM remain unclear. Therefore the present study aimed to investigate whether GEN exerts an antifibrogenic effect on T1DM kidney and to determine its associated mechanism.
A total of 24 male Sprague-Dawley rats (age, 6–7 weeks; weight, 160–200 g) were obtained from the Animal Administration Center of Bengbu Medical College (Bengbu, China). The rats were housed in conventional animal facility with a 12-h light/dark cycle at a constant temperature of 21–23°C and 50–60% relative humidity. All the rats were fed with normal laboratory rodent diet and water
Rats were randomized into four groups (n=6 rats/group): i) Normal control group (N); ii) STZ group (S); iii) STZ + low-dose GEN group (L); and iv) STZ + high-dose GEN group (H). T1DM was induced as previously described (
At 8 weeks post-induction, the rats were placed in metabolic cages for 24-h urine collection and consequent albuminuria measurement prior to being anesthetized with chloral hydrate (400 mg/kg; i.p.). Blood was obtained from the tail vein to measure FBG using a portable glucometer (Accu-Chek, Roche, Mannheim, Germany). Then, 4 ml blood from abdominal aorta was collected. The fresh blood was placed in serum tubes and centrifuged at 765 × g for 20 min at 4°C. The serum was collected and the levels of 24-h urine protein, BUN and SCr were measured using a Urine Protein Test Kit (cat. no. C035-2), Urea Assay Kit (cat. no. C013-2) and Creatinine Assay Kit (cat. no. C011-2; Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) according to the manufacturer's protocols.
Rats were sacrificed and their bilateral kidneys were excised and placed in ice-cold normal saline, and the ratio of kidney weight to body weight (renal index) was calculated.
Renal tissue (0.1 g) was homogenized in 0.9 ml normal saline. The supernatant fluid was collected following centrifugation for 20 min (825 × g at 4°C). Following measurement of the protein concentration using a Bicinchoninic Acid (BCA) Protein Assay kit (cat. no. P0010; Beyotime Institute of Biotechnology, Shanghai, China), the renal T-AOC, SOD, LPO, MDA and Hyp levels were measured according to the protocols of Total Antioxidant Capacity Assay kit (cat. no. A015-2), Total Superoxide Dismutase Assay kit (cat. no. A001-1-1), Lipid Peroxidation Assay kit (cat. no. A106), Malondialdehyde Assay kit (cat. no. A003-1) and Hydroxyproline Assay kit (cat. no. A030-3; Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
For histological analysis, fresh left renal tissue collected from each group was fixed using 4% paraformaldehyde for 12 h at 4°C. Tissues were embedded in paraffin, cut into 5-µm thick section and stained using hematoxylin for 5 min and 0.5% eosin for 2 min (H&E) at room temperature. For Masson's trichrome staining, paraffin sections were dewaxed with xylene, rehydrated with graded ethanol at room temperature and stained with Regaud dye hematoxylin for 10 min at room temperature. Following rinsing with water, sections were stained with Ponceau Fuchsin acid solution for 10 min at room temperature, immersed in 2% acetic acid aqueous solution and treated with a 1% aqueous solution of phosphomolybdic acid for 5 min at room temperature. Without rinsing with water, the sections were stained with 2% aniline blue for 5 min at room temperature, immersed in 0.2% acetic acid aqueous solution, in 95% alcohol, anhydrous alcohol, permeabilized with xylene and mounted with neutral resin. The sections were observed and images were captured using a NanoZoomer 2.0 RS Digital Pathology slide scanner (Hamamatsu Photonics K.K., Hamamatsu, Japan). Renal collagen volume fraction (CVF) was analyzed using Image-Pro Plus 6.0 analysis software (Media Cybernetics, Inc., Rockville, MD, USA). A total of five fields of view per sample were randomly chosen and the average was determined for analysis.
Fresh left kidneys were cut into 1×1×1 mm cubes and fixed using 2.5% glutaraldehyde for 4–6 h at 4°C. Tissues were washed with 0.1 mol/l phosphate buffer and post-fixed in 1% osmium tetroxide for 1 h at room temperature. Tissues were embedded in Epon812 for 2 h at room temperature, put into an oven at 45°C for 12 h and at 65°C for 48 h then cut into 70-nm thick ultrathin sections and stained with uranyl acetate for 30 min and lead citrate for 15 min at room temperature. These sections were examined using a JEM-1230 JEOL transmission electron microscope (TEM; Tokyo, Japan).
The mRNA expression levels of TGF-β1 in renal tissue were determined by RT-PCR. Briefly, total RNA was extracted from renal tissue (0.1 g) using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. cDNA was synthesized according to the protocol of RevertAid First Strand cDNA Synthesis kit (cat. no. K1622; Invitrogen; Thermo Fisher Scientific, Inc.). TGF-β1 and β-actin genes were amplified using PCR Master Mix (cat. no. K0171; Thermo Fisher Scientific, Inc.). All PCR reactions were performed with a T-Gradient thermocycler (Biometra GmbH, Göttingen, Germany). The primer sequences were as follows: TGF-β1, forward 5′-CCAAGGAGACGGAATACAGG-3′, reverse 5′-ATGAGGAGCAGGAAGGGTC-3′ (expected size, 156 bp); β-actin, forward 5′-GATGGTGGGTATGGGTCAGAAGGAGG-3′, reverse 5′-GCTCATTGCCGATAGTGATGACC-3′ (expected size, 632 bp). The thermocycling conditions were as follows: Initial denaturation at 95°C for 3 min; followed by 32 cycles of denaturation at 95°C for 50 sec; annealing at 54.5°C (TGF-β1) or 59.4°C (β-actin) for 50 sec; and extension at 72°C for 60 sec; followed by final extension at 72°C for 10 min. The PCR products were electrophoresed in 1.5% agarose gel and stained with ethidium bromide. Densitometric analysis of TGF-β1 gene expression was normalized to the corresponding β-actin gene using Tanon 3.1.2 software (Tanon Science and Technology, Co., Ltd., Shanghai, China).
Rat renal tissue (0.1 g) was lysed using Cell Lysis Buffer for western blotting and IP (cat. no. P0013; Beyotime Institute of Biotechnology) with phenylmethanesulfonyl fluoride (cat. no. ST506; Beyotime Institute of Biotechnology). The protein concentration was measured using the BCA Protein Assay kit. A total of 50 µg protein was separated by 10% SDS-PAGE and subsequently electroblotted onto polyvinylidene difluoride membranes. Membranes used for Nrf2, HO-1, NQO1, TGF-β1, Smad3, collagen-IV and GAPDH detection were blocked in TBS + 0.1% Tween 20 (TBST) containing 5% nonfat dry milk at 37°C for 2 h, and membranes used for phosphorylated (p)-Smad3 were blocked in TBST containing 5% bovine serum albumin (cat. no. B2064; Sigma-Aldrich; Merck KGaA) at 37°C for 2 h. The membranes were incubated with primary antibodies against the following proteins: Nrf2 (1:1,000; cat. no. ab137550; Abcam, Cambridge, MA, USA), HO-1 (1:1,000; cat. no. ab52947; Abcam), NQO1 (1:300; cat. no. PB0526; Wuhan Boster Biological Technology, Ltd., Wuhan, China), TGF-β1 (1:1,000; cat. no. ab92486; Abcam), Smad3 (1:1,000; cat. no. ab40854; Abcam), p-Smad3 (1:1,000; cat. no. ab52903; Abcam), collagen IV (1:300; cat. no. A01411; Wuhan Boster Biological Technology, Ltd.) and GAPDH (1:2,000; cat. no. ab181602; Abcam) at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (1:2,000; cat. no. BL003A; Biosharp Biotechnology, Hefei, China) for 1 h at room temperature. The blots were detected using an enhanced chemiluminescent reagent (cat. no. WBKLS0100; Millipore Corporation, Billerica, MA, USA) and scanned with ChemiDoc XRS system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The intensities of the protein bands were quantified and normalized to GAPDH using Quantity One Software Version 4.6.6 (Bio-Rad Laboratories, Inc.).
All statistical analysis was conducted using SPSS Software, version 17.0 (SPSS, Inc., Chicago, IL, USA). All tests were repeated three times. Data are expressed as the mean ± standard deviation. Statistical comparisons were analyzed using one-way analysis of variance followed by the Newman-Keuls test. P<0.05 was considered to indicate a statistically significant difference.
The levels of FBG, 24-h urine protein, BUN and SCr were significantly increased in the S group compared with the respective levels in the N group (P<0.01;
Compared with group N, BW was significantly decreased (P<0.01;
The levels of T-AOC and SOD in renal tissue were significantly decreased (P<0.01;
H&E staining indicated that the glomerular structure was normal and the thickness of glomerular basement membrane was uniform in the kidneys of rats in group N (
Masson's trichrome staining demonstrated normal glomerular and renal tubules with less collagen deposition in group N kidneys (
In group N kidneys, TEM demonstrated that the normal glomerular basement membrane and mesangial matrix, as well the podocyte foot processes were neatly arranged (
The mRNA expression levels of TGF-β1 in renal tissue were significantly upregulated in group S compared with group N (P<0.01;
The protein expression levels of renal Nrf2, HO-1 and NQO1 were significantly reduced compared with the respective expression levels in the N group (P<0.01;
DN is a major diabetic microvascular complication and is the leading cause of chronic renal failure and end-stage renal disease (
Oxidative stress serves a pivotal role in the occurrence and development of DN (
GEN is a plant phytoestrogen that has an anti-oxidative effect. For example, a previous study reported that GEN prevents cisplatin-induced renal injury by reducing ROS levels and inhibiting nuclear factor-κB activation (
Previous studies have revealed that GEN treatment inhibits tyrosine kinase and has an anti-proliferative effect on many types of cells (
Renal fibrosis, characterized by the continuous accumulation and excessive deposition of ECM, is a major pathological feature of chronic kidney diseases (
In fibrotic diseases, TGF-β1 is synthesized by all cell types in the kidney and is a crucial pro-fibrotic mediator; the expression of TGF-β1 is significantly upregulated in damaged kidneys in both animal models and patients (
In summary, results from the present study demonstrated that GEN may attenuate renal fibrosis and exhibit reno-protective effects in T1DM model rats. The beneficial effects of GEN treatment on kidney injury may be attributed to its ability to suppress oxidative stress by activating the Nrf2/HO-1/NQO1 pathway and to downregulate the TGF-β1/Smad3 pathway to regulate collagen IV protein expression.
Not applicable.
The present study was supported by The Natural Science Research Project of The Education Commission of Anhui Province (grant nos. KJ2017A216 and KJ2018A0994) and The Natural Science Research Project of Bengbu Medical College (grant nos. BYKY1621ZD and BYKF1706).
The analyzed data sets generated during the study are available from the corresponding author on reasonable request.
QJ, RY and LW produced substantial contributions to the conception and design of the present study. QJ, XFL and SFM performed the experiments. QJ and RY analyzed the data and wrote the manuscript. All authors read and approved the manuscript.
The present study was approved by the Animal Ethics Committee of Bengbu Medical College (Bengbu, China).
Not applicable.
The authors declare they have no competing interests.
Levels of kidney function markers in rats. (A) FBG, (B) 24-h urine protein, (C) BUN and (D) SCr level in rats. Data are presented as the mean ± standard deviation; n=6; **P<0.01 vs. N; #P<0.05 and ##P<0.01 vs. S. BUN, blood urea nitrogen; FBG, fasting blood glucose; H, streptozotocin + high-dose genistein group; L, streptozotocin + low-dose genistein group; N, normal control group; S, streptozotocin group; SCr, serum creatinine.
GEN improved the pathological state of renal tissue from diabetic rats. Morphology of renal tissues by hematoxylin and eosin staining; magnification, ×400. N group: The glomerular structure was normal. S group: The glomerular structure was damaged. L group: Damage to the glomerular structure was ameliorated. H group: Damage to the glomerular structure was notably ameliorated. Arrows indicate the glomerular structure. H, streptozotocin + high-dose genistein group; L, streptozotocin + low-dose genistein group; N, normal control group; S, streptozotocin group.
Effects of GEN on renal fibrosis. (A) Representative Masson's trichrome stained tissues from each group; magnification, ×200. Arrows indicate collagen. (B) Semi-quantitative analysis of CVF, based on (A). Data are presented as the mean ± standard deviation; n=6; **P<0.01 vs. N; #P<0.05 and ##P<0.01 vs. S. CVF, collagen volume fraction; GEN, genistein; H, streptozotocin + high-dose GEN group; L, streptozotocin + low-dose GEN group; N, normal control group; S, streptozotocin group.
GEN improved the ultrastructure of renal tissue from diabetic rats. Alterations of renal ultrastructure under transmission electron microscopy in rats; magnification, ×15,000. Red arrows indicate the basement membrane and black arrows indicate the podocyte foot processes. N group: The normal glomerular basement membrane and the podocyte foot processes were neatly arranged. S group: The basement membrane was thickened and foot processes were fused. L group: The fusion of foot processes were ameliorated. H group: The fusion of foot processes were ameliorated notably. H, streptozotocin + high-dose genistein group; L, streptozotocin + low-dose genistein group; N, control group; S, streptozotocin group.
Expression levels of renal TGF-β1 mRNA in the different groups. Representative images and semi-quantitative analysis of TGF-β1 mRNA expression levels, as determined by reverse transcription-polymerase chain reaction, in renal tissues of rats. Data are presented as the mean ± standard deviation; n=6; **P<0.01 vs. N; #P<0.05 and ##P<0.01 vs. S. H, streptozotocin + high-dose genistein group; L, streptozotocin + low-dose genistein group; N, normal control group; S, streptozotocin group; TGF-β1, transforming growth factor-β1.
Protein expression levels of Nrf2, HO-1 and NQO1 in the different groups. Representative western blotting images and semi-quantitative analysis of Nrf2, HO-1 and NQO1 proteins; GAPDH was used as a loading control. Data are presented as the mean ± standard deviation; n=6; **P<0.01 vs. N; #P<0.05 and ##P<0.01 vs. S. H, STZ + high-dose GEN group; L, STZ + low-dose GEN group; HO-1, heme oxygenase-1; N, normal control group; Nrf2, nuclear factor erythroid 2-related factor 2; NQO1, NAD(P)H:quinone oxidoreductase 1; S, STZ group.
Protein expression of TGF-β1, Smad3, p-Smad3 and collagen IV in the different groups. Representative western blotting images and semi-quantitative analysis of TGF-β1, Smad3, p-Smad3, p-Smad3/Smad3 and collagen IV proteins; GAPDH was used as a loading control. Data are presented as the mean ± standard deviation; n=6; **P<0.01 vs. N; #P<0.05 and ##P<0.01 vs. S. H, streptozotocin + high-dose genistein group; L, streptozotocin + low-dose genistein group; N, normal control group; p, phosphorylated; S, streptozotocin group; Smad3, mothers against decapentaplegic homolog 3; TGF-β1, transforming growth factor-β1.
Alterations of BW, KW and renal index in diabetic model rats.
Group | BW (g) | KW (mg) | Renal index |
---|---|---|---|
N | 421.54±33.62 | 2.92±0.22 | 7.02±0.56 |
S | 232.15±23.07 |
3.52±0.15 |
13.70±0.64 |
L | 257.46±27.94 | 3.42±0.12 | 12.78±0.62 |
H | 290.30±31.28 |
3.28±0.14 |
11.88±0.46 |
Renal index, kidney weight to body weight ratio.
P<0.01 vs. N.
P<0.05 vs. S.
P<0.01 vs. S. Data are presented as the mean ± standard deviation; n=6. BW, body weight; H, streptozotocin + high-dose genistein group; KW, kidney weight; L, streptozotocin + low-dose genistein group; N, normal control group; S, streptozotocin group.
Levels of T-AOC, SOD, LPO, MDA and Hyp in renal tissues of diabetic model rats.
Group | T-AOC (U/mg) | SOD (U/mg) | LPO (µmol/g) | MDA (nmol/mg) | Hyp (µg/mg) |
---|---|---|---|---|---|
N | 87.55±6.97 | 64.10±7.95 | 15.24±2.62 | 3.78±0.49 | 2.95±0.28 |
D | 51.65±5.52 |
31.22±4.70 |
31.46±3.41 |
6.92±0.72 |
5.67±0.55 |
L | 60.12±5.68 |
39.56±5.24 |
27.19±3.23 |
6.18±0.54 |
5.13±0.43 |
H | 67.49±6.11 |
47.83±5.58 |
22.65±2.87 |
5.72±0.53 |
4.46±0.41 |
P<0.01 vs. N.
P<0.05 vs. S.
P<0.01 vs. S. Data are presented as the mean ± standard deviation; n=6. H, streptozotocin + high-dose genistein group; Hyp, hydroxyproline; L, streptozotocin + low-dose genistein group; LPO, lipid peroxidation; MDA, malondialdehyde; N, normal control group; S, streptozotocin group; SOD, superoxide dismutase; T-AOC, total antioxidant capacity.