Male C57BL/6J mice (n=18; age, 6 weeks; weight, 20±2 g; Sibeifu Biology Technology Co., Ltd., Beijing, China) were housed in a 50~60% humidity and temperature (20–26°C)- and light (light/dark cycle: 12 h light, 12 h dark)-controlled animal room of specific-pathogen-free cleanliness grade. Mice were randomly divided into the following 3 groups: i) Control group (n=6), which were fed a common diet ad libitum that contained fat accounting for 10% kcal (Beijing Huafukang Biological Technology Co., Ltd., Beijing, China); ii) ORG model group (n=6), which were fed a high-fat diet that contained fat accounting for 60% kcal (Research Diet, Inc., New Brunswick, NJ, USA) as described by us previously (26); and iii) Spironolactone intervention group (n=6), which also were the high-fat diet, and at week 9 received daily subcutaneous injections of spironolactone (20 mg/kg/d; catalog no. Y0001295; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), a non-selective MR antagonist, that was dissolved in 60% propylene glycol; mice in the Control group and in the ORG model group received daily subcutaneous injections of 60% propylene glycol only. All mice were sacrificed at the end of 12th week and the kidneys collected. Mice were scarified by 100% carbon dioxide. The filling rate was 20% of the volume of the chamber per minute to achieve animal unconsciousness rapidly and reduce animal pain. The sign of death is the lack of respiration and fading of the eye-color. Once these two phenomena were observed, mice were removed from the box. A portion of the kidney tissue was fixed in 4% neutral formaldehyde solution for 48 h in room temperature, for light microscopy; the remaining of the kidney tissue was rapidly frozen in liquid nitrogen for use in reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting assays.

All animal care and experimental protocols complied with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals (publication no. 85–23, 1996) and were approved by the Institutional Animal Care and Use Committee of Capital Medical University (Beijing, China).

A conditionally immortalized mouse podocyte cell line was kindly provided by Professor Maria Pia Rastaldi (San Carlo Hospital, University of Milan, Italy) and cultured as described previously (25). Briefly, podocytes were grown in RPMI-1640 with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Waltham, MA, USA) with mouse recombinant interferon-γ (20 U/ml (IFN-γ; Sigma-Aldrich; Merck KGaA) at 33°C, and subsequently differentiated in RPMI-1640 without interferon-γ at 37°C. Results from preliminary experiments were similar to previously described results of podocytes cultured in serum-containing medium and serum-free medium with 2% FBS (data not shown) following aldosterone and/or eplerenone stimulation (27–29). Based on these date, serum-free medium was used for podocyte culture for subsequent experiments post-stimulation.

In the first set of experiments, podocytes were incubated in RPMI-1640 medium only, in medium containing ALDO (cat. no. A9477, Sigma-Aldrich; Merck KGaA), in medium containing eplerenone (cat. no. E6657, Sigma-Aldrich; Merck KGaA) or in medium containing both eplerenone and ALDO (cells in this group were pre-incubated with eplerenone for 30 min at 37°C, and subsequently incubated with both eplerenone and ALDO). The concentration of ALDO and eplerenone in the media was 10⁻⁷ and 10⁻⁵ mol/l, respectively. Cells were incubated for 12 or 24 h at 37°C and harvested for RT-qPCR or western blotting assays, respectively.

In the second set of experiments, podocytes were incubated in RPMI-1640 medium only, in medium containing ALDO, in medium containing Dickkopf-related protein 1 (DKK1, a well-known inhibitor of Wnt/β-catenin signaling; cat. no. 5897-DK, R&D Systems, Inc., Minneapolis, MN, USA) or in medium containing DKK1 and ALDO (cells in this group were pre-incubated with DKK1 for 1 h at 37°C, followed by incubation with DKK1 and ALDO). Cells were incubated and harvested for RT-qPCR and western blotting as aforementioned.

Mouse body weight was measured every 4 weeks and body length was measured at week 12. Nocturnal 12 h urine samples were collected at weeks 0 and 12 to measure urinary protein using a Bradford Protein Assay kit (Beyotime Institute of Biotechnology, Shanghai, China), according to the manufacturer's protocol. Blood and urine samples were collected at week 12 to measure serum triglycerides, serum cholesterol and blood glucose levels, as well as serum and urine creatinine levels using an Olympus AU5400 Chemistry Analyzer (Olympus Corporation, Tokyo, Japan). Following sacrifice, the weight of visceral fat mass was measured as previously described (30) and the weight of kidney was also measured.

The follows formulas were used: Lee's obesity index=[(Body weight (g) ×1,000)1/3]/Body length (cm) (31,32); abdominal visceral fat index=[abdominal visceral fat mass (g)/body weight (g)] × 100% (30); and creatinine clearance rate (ml/min)=[(urine creatinine (µmol/l) × urine volume (ml/min)]/serum creatinine (µmol/l) (33).

Fixed kidney cortical tissues were dehydrated, embedded, sectioned (3 µm) and stained with periodic acid-Schiff reagent. Paraffin production: A portion of kidney tissues were fixed in 4% neutral formaldehyde solution for 48 h. Subsequently, they were dehydrated with 70, 80, 90, 95, and 100% ethanol gradient, xylene transparent and 60°C dipping wax. Paraffin embedding machine made wax, then making paraffin wax slicing machine. PAS slice thickness was 3 µm, and 58°C for 1 h. PAS staining: 3 µm thick paraffin sections were dewaxed, washed with 1% periodic acid for 10 min, then were rinsed using tap water for 5 times and washed once with distilled water. Subsequently, Schiff reagent (cat. no. TR1337, ZSGB-BIO; OriGene Technologies, Inc., Beijing; China) was added for 30 min in the dark, rinsed with tap water for 5 times and washed 3 times with distilled water. Subsequently, hematoxylin Mayer dye was added for 5 min, rinsing with tap water for 5 times and washed 3 times with distilled water, then using hydrochloric acid alcohol differentiation for 10 sec, and rinsing with ethanol 3 times. Finally, xylene was transparent and neutral resin sheet was produces. A total of 20 images of glomerular maximal profiles with vascular pole and/or urinary pole were captured using a confocal microscope (×400 magnification). The two longest perpendicular diameters of the glomerular capillary tufts were measured using the Nikon NIS-Elements Basic Research Image Analysis software (Nikon NIS-Elements Basic Research Image Analysis software 3.0; Nikon Corporation, Tokyo, Japan), and the mean value was calculated (25,26,34). In addition, images of 20 random visual fields containing only tubules and interstitium were captured (×200 magnification), and the relative area of tubules displaying vacuolar degeneration of cytoplasm in each visual field was assessed using a semi-quantitative method, and then their mean value was calculated. A semi-quantitative method described as: ‘Each slice randomly selected with 20 visual fields, the area of vacuolated tubules that accounted for <25, 26–50, 51–75 or >75% of the visual field were given lesion scores of 1, 2, 3 and 4, respectively’ (35). Calculating mean value in every slice, then calculating the mean value of every group.

Total RNA (50 µg) was extracted from the renal cortex of mouse kidney or 30–40,000 cultured podocytes/dish using the TRIzol RNA isolation system (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. RNA (2 µg) was reverse transcribed to cDNA with Moloney Murine Leukemia Virus reverse transcriptase (Beijing TransGen Biotech Co., Ltd., Beijing, China) as follows: Incubation for 30 min at 42°C, inactivation in 85°C for 5 min, followed by incubation at 12°C. The synthetic cDNA is preserved at −20°C. qPCR was performed using SYBR-Green labelled kit (TransGen Biotech, Beijing, China) under a Rotor-Gene 6000 thermocycler (Qiagen GmbH, Hilden, Germany). The thermocycling profile was as denaturation follows: Initial denaturation at 95°C for 60 sec, followed by 40 cycles of at 95°C (15 sec), annealing at 60°C (15 sec) and extension at 72°C (45 sec). Gene-specific primers were designed using GenBank (www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) and synthesized by Beijing SBS Genetech Co., Ltd. (Beijing, China) (Table I). A no template control was used as a negative control. mRNA expression levels of various genes were calculated following normalizing with GAPDH expression. Reactions were performed in triplicate and quantification cycle (Cq) numbers were averaged. Relative mRNA expression levels were calculated according to the formula: 2^{−(target gene Cq - control gene Cq)} ×10³ (36).

The tissue was removed from the liquid nitrogen and placed on ice and into the homogenizer, where Ripa lysis buffer (R0010-100 ml; Beyotime Institute of Biotechnology) with protease inhibitors was added following homogenization. Subsequently, the suspension was transferred to the sterilization of the EP tube (Eppendorf AG, Hamburg, Germany), and centrifuged at a low temperature high speed centrifuge at 10,000 × g for 10 min, the supernatant was transferred to the EP tube. All incubations were performed on ice. Total protein was extracted from mouse renal cortex or cultured podocytes (200 µg) using Cell Lysis Buffer (Cell Signaling Technology, Inc., Danvers, MA, USA). Lysate samples were boiled for 5 min, and proteins (40 µg) were separated by 10% SDS-PAGE followed by transferred to nitrocellulose membranes (GE Healthcare Life Sciences, Little Chalfont, UK). Following blocking with 5% skim milk in PBS containing 0.1% Tween-20 for 1 h at room temperature, the membranes were incubated with various primary antibodies at 4°C overnight, followed by incubation with the corresponding fluorophore-labeled secondary antibodies at room temperature for 1 h. The primary and secondary antibodies are listed in Table II. The blotted proteins were quantified using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). β-actin was used as internal loading control. The relative expression level of each target protein was normalized to β-actin. All assays were performed at least in triplicate. The relative level of phosphorylated (p)-β-catenin was expressed as the ratio of p-β-catenin/total β-catenin using image J 1.51j8 (Image J, National Institutes of Health, Bethesda, MD, USA).

All data of continuous variables were expressed as the mean ± standard deviation and analyzed using SPSS 16.0 statistical software (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance followed by the Dunnett's test was performed to evaluate the significant differences among groups. P<0.05 was considered to indicate a statistically significant difference.

No significant differences in body weight were identified among the three groups week 0 (P>0.05; Fig. 1A). At week 4, there were no difference in any group (P>0.05). At week 8, body weights in the Model group and the Spironolactone group were significantly heavier compared with the Control group (P<0.05), whereas no significant difference was identified between the Model group and the Spironolactone group w (P>0.05). However, at week 12, body weight in the Spironolactone group was significantly lower compared with the Model group (P<0.05), and the difference between Spironolactone group and Control group was not significant (P>0.05).

At week 12, Lee's index, abdominal visceral fat index and kidney weight were significantly increased in the Model group compared with the measurements of these parameters in the Control group (P<0.01 or P<0.05; Table III), whereas these parameter measurements were significantly decreased in the Spironolactone group compared with the Model group (P<0.01 or P<0.05).

Also at week 12, the levels of serum triglyceride and cholesterol in the Model group were significantly increased compared with the Control group (P<0.05 and P<0.01, respectively; Table III), whereas these parameters were significantly decreased in the Spironolactone group compared with the Model group (P<0.05). No significant difference was identified for blood glucose levels among the three groups (P>0.05; Table III).

There was no significant difference identified for the nocturnal 12 h urinary protein excretion among the three groups at week 0 (P>0.05; Fig. 1B); however, at week 12, urinary protein excretion in the Model group was significantly higher compared with the Control group (P<0.05), and mice treated with spironolactone exhibited a significantly lower level of urine protein compared with the Model group (P<0.05). In addition, creatinine clearance rate in the model group was significantly higher compared with the Control group (P<0.05; Table III), whereas this level was significantly reduced in the Spironolactone group compared with mice in the Model group (P<0.05).

The average glomerular diameter in kidneys from mice in the Model group was significantly larger compared with the Control group (P<0.01; Fig. 2A and B), and the glomerular diameter in the Spironolactone group was significantly smaller compared with the Model group (P<0.05). The average tubular lesion score in the kidneys from Model group mice was significantly higher compared with mice in the Control group (P<0.01; Fig. 2C and D), whereas the lesion score in the Spironolactone group was significantly lower compared with the Model group (P<0.01).

The mRNA and protein expression levels of podocyte-associated molecules, including nephrin, podocin, podoplanin and podocalyxin, in the kidney cortical tissue was significantly downregulated in Model group mice compared with mice in the Control group (Fig. 3A and B, respectively). Treatment with spironolactone significantly upregulated the mRNA and protein expression of these molecules, compared with the model group.

The mRNA expression levels of Wnt1, Wnt2b, Wnt6 and β-catenin in the kidney cortical tissue were significantly upregulated in ORG model mice compared with the respective expression levels in the Control group (P<0.05; Fig. 4A). ORG model mice treated with spironolactone exhibited significantly downregulated expression levels of Wnt1, Wnt2b, Wnt6 and β-catenin compared untreated mice in the Model group (P<0.05).

The expression level of p-β-catenin protein was significantly decreased in Model group mice compared with expression in the Control group (P<0.01; Fig. 4B); p-β-catenin protein expression level was significantly increased in Spironolactone group mice compared mice in the Model group (P<0.05).

The mRNA expression levels of Wnt1, Wnt2b, Wnt6 and β-catenin in cultured podocytes were significantly upregulated in the ALDO-treated group compared with the respective expression levels in the untreated Control group (P<0.05; Fig. 5A). ALDO-treated cells that were co-treated with eplerenone exhibited significantly downregulated expression levels of these podocyte-associated molecules compared with the ALDO-only group (P<0.05). The level of p-β-catenin protein expression in cultured podocytes was significantly decreased in the ALDO group compared with the control group (P<0.05; Fig. 5B) and significantly increased in the eplerenone + ALDO group compared with the ALDO-only group (P<0.05).

The mRNA and protein expression levels of additional podocyte-associated molecules, including nephrin, podocin, podoplanin and podocalyxin in cultured podocytes were significantly reduced in the ALDO group compared with expression levels in the Control group (P<0.05, P<0.01; Fig. 6A and B, respectively); the mRNA and protein expression levels of these molecules were significantly upregulated in cells co-treated with eplerenone and ALDO, compared with the respective expression levels in the ALDO-only treatment group (P<0.05, P<0.01).

The expression level of p-β-catenin protein in cultured podocytes was significantly decreased in the ALDO group compared with the Control group (P<0.01; Fig. 7), and p-β-catenin protein expression was significantly increased in cells co-treated with ALDO and DKK1 compared with the expression level of those in the ALDO-only treatment group (P<0.05).

The mRNA and protein expression levels of the podocyte-associated molecules nephrin, podocin, podoplanin and podocalyxin in cultured podocytes were significantly reduced in cells treated with ALDO compared with untreated cells in the Control group (P<0.05, <0.01; Fig. 8A and B, respectively); the expression levels of these molecules was significantly upregulated in cells co-treated with ALDO and DKK1 compared with the expression levels in those cells in the ALDO-only treatment group (P<0.05).