C57BL/6 mice were caged individually under controlled temperature (23±2°C) and humidity (55±5%) with an artificial light cycle. Six-week-old male C57BL/6 mice were divided into three groups: mice fed with a normal chow diet (ND, N=4); mice fed with a high-fat chow diet (HFD, 60% of total calories from fat, 5.5% from soybean oil, and 54.5% from lard; N=4); and GIT27-treated mice fed with a HFD (N=7). GIT27 was purchased from Tocris Bioscience (Tocris Cookson, Ellisville, MI, USA) and administered via an intraperitoneal route at a dose of 20 mg/kg daily for 12 weeks. We measured the food and water intake, urine volume, body weight, fasting blood glucose concentration, and HbA1c level every month. Plasma glucose level was measured using the glucose oxidase method, and HbA1c level was calculated using the IN2IT system (Bio-Rad Laboratories, Hercules, CA, USA). Plasma creatinine level was determined using high-performance liquid chromatography. Plasma insulin and adiponectin levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Linco Research, St Charles, MO, USA). The homeostasis model assessment index (HOMA-IR) was calculated using the following formula: [fasting glucose (mmol/L)×fasting insulin (mU/L)/22.5]. Plasma triglyceride and cholesterol analyses were performed using a GPO-Trinder kit (Sigma, St Louis, MO, USA). For the glucose tolerance test, we performed oral gavage of 3 g dextrose/kg in mice after a 5-hour fast and collected blood samples through the tail vein. Individual mice were separated in a metabolic cage where urine was collected and measured for 24 hours every month to determine the amount of urinary albumin excretion by competitive ELISA (Shibayagi, Shibukawa, Japan), which was corrected by the urinary creatinine concentration. The urinary level of 8-isoprostane was measured using an ELISA kit (Cayman Chemical, Ann Arbor, MI, USA). The levels of proinflammatory cytokines (IFN-γ and IL-2) in urine were measured using the Millipore Milliplex mouse cytokine/chemokine kit. At the end of the study period, systolic blood pressure was measured using tail-cuff plethysmography (LE 5001-Pressure Meter; Letica SA, Barcelona, Spain). Mice were sacrificed under anesthesia with intraperitoneal injections of sodium pentobarbital (50 mg/kg). Their heart, epididymal fat, liver, and kidney tissues were extracted and subsequently snap-frozen in liquid nitrogen. Lipids from the hepatic, adipose, and renal cortical tissues were extracted using the Bligh and Dyer method [22]. Total cholesterol and triglyceride contents were measured using a commercial kit (Wako Chemicals, Richmond, VA, USA). Plasma and tissue FFA levels were also measured using a commercial kit (Abcam Plc, Cambridge, MA, USA). The extent of peroxidative reaction in the hepatic tissue, adipose tissue, and kidneys was determined by direct measurement of lipid hydroperoxides (LPO) using an LPO assay kit (Cayman Chemical), as described previously [23]. We performed all the experiments in accordance with the guidelines of the National Institutes of Health and with the approval of the Korea University Institutional Animal Care and Use Committee.

We fixed kidney tissues in 4% paraformaldehyde and embedded them in paraffin. They were then cut into 4-μm-thick slices and stained with periodic acid–Schiff. Glomerular mesangial expansion was scored semiquantitatively, and the percentage of mesangial matrix occupying each glomerulus was rated from 0 to 4 as follows: 0, 0%; 1, <25%; 2, 25–50%; 3, 50–75%; and 4, >75%. After the removal of paraffin, the slices were dehydrated in xylene followed by in graded alcohol and immersed in distilled water. The free kidney sections were transferred to 10 mmol/L citrate buffer adjusted to a pH of 6.0 for various staining methods. Sections were microwaved for 10–20 minutes to retrieve the antigens for transforming growth factor β 1 (TGFβ1) and F4/80, and transferred to Biogenex Retrievit (pH 8.0) (InnoGenex, San Ramon, CA, USA) for PAI-1 staining or trypsin treatment (Sigma) for 30 minutes at 37°C, to detect type IV collagen. For blocking the endogenous peroxidase activity, 3.0% H₂O₂ in methanol was applied to the tissues for 20 minutes, followed by incubation at room temperature for 60 minutes with 3% bovine serum albumin (BSA)/3% normal goat serum (type IV collagen), 90 minutes with 5% normal goat serum, and either 15 minutes with 10% powerblock (PAI-1) or 30 minutes with 20% normal sheep serum (TGFβ1). Slides were incubated overnight at 4°C with rabbit polyclonal anti-TGFβ1 antibody (1:200; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), mouse monoclonal anti-F4/80 antibody (1:100; Serotec, Oxford, UK), rabbit polyclonal anti-type IV collagen antibody (1:200; Santa Cruz Biotechnology Inc.), or rabbit polyclonal anti-PAI-1 antibody (1:50; American Diagnostica, Stanford, CT, USA). After overnight incubation, the slides were incubated again with a secondary antibody for 30 minutes and detected by incubation at room temperature with a mixture of 0.05% 3,3′-diaminobenzidine containing 0.01% H₂O₂ and counterstaining with Mayer’s hematoxylin. Negative control sections were stained under identical conditions, but with a buffer solution instead of the primary antibody. For the evaluation of immunohistochemical staining for type IV collagen, TGFβ1, PAI-1, and F4/80, the glomerular fields were graded semiquantitatively under a high-power field containing 50–60 glomeruli, and the average score was calculated as described previously [23]. A pathologist examined and scored the histological changes in a blinded manner.

We extracted total RNA from the experimental cells using the Trizol reagent. Primers were designed for their respective gene sequences using the Primer 3 software, and the secondary structures of the templates were examined and excluded using the mfold program. Quantitative gene expression was performed using a LightCycler 1.5 system (Roche Diagnostics Corporation, Indianapolis, IN, USA) using SYBR Green technology. In 96-well real-time polymerase chain reaction (PCR) plates, 10 μL SYBR Green master mix was added to 1 μL of RNA (corresponding to 50 ng of total RNA) and 900nM of both forward and reverse primers for a total reaction volume of 20 μL. Real-time reverse transcriptase PCR was performed for 10 minutes at 50°C and for 5 minutes at 95°C. Thirty PCR cycles of denaturation for 10 seconds at 95°C and annealing with extension for 30 seconds at 60°C were then conducted. Gene-level ratios relative to β-actin (relative gene expression number) were calculated by subtracting the threshold cycle number of the target gene from that of β-actin and squaring this difference. We evaluated the specificity of each PCR product using melting curve analysis, followed by agarose gel electrophoresis.

We extracted nuclear and cytoplasmic proteins from renal cortical tissues and cells using a commercial nuclear extraction kit according to the manufacturer’s instructions (Active Motif, Carlsbad, CA, USA). Protein concentration was determined using the bicinchoninic acid method (Pierce Pharmaceuticals, Rockford, IL, USA). For Western blotting, 40 μg of protein was electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) mini-gel. Proteins were transferred onto a polyvinylidene difluoride membrane and the membrane was hybridized in blocking buffer overnight at 4°C with goat polyclonal anti-TLR4 antibody (1:500, Santa Cruz Biotechnology Inc.), goat polyclonal anti-TRAF6 antibody (1:500, Santa Cruz Biotechnology Inc.), rabbit polyclonal anti-NOX4 antibody (1:500, Novus Biologicals), rabbit polyclonal anti-NFκB (p65) antibody (1:1,000, Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal anti-inhibitory kappa B (IκB)β antibody (1:1,000, Cell Signaling Technology), anti-p-AMP-dependent kinase (AMPK) antibody, anti-AMPK antibody, anti-sterol regulatory element binding transcription protein1 (SREBP1) antibody, and mouse monoclonal anti-β-actin antibody (1:5,000, Sigma). The membrane was subsequently incubated with horseradish peroxidase-conjugated secondary antibody (diluted 1:1,000) for 60 minutes at room temperature. We detected the specific signals using an enhanced chemiluminescence method (Amersham, Buckinghamshire, UK).

We cultured podocytes to elucidate the molecular mechanism of TLR4 because TLR4 expression was detected preferentially in glomerular podocytes. Conditionally immortalized mouse podocytes were cultured as described previously [24]. Briefly, frozen podocytes were first grown under permissive conditions at 33°C in RPMI 1640 media containing 10% fetal bovine serum (FBS), 50 U/mL IFN-γ, and 100 μg/mL antibiotics in collagen-coated flasks. The IFN-γ was gradually reduced to 10 U/mL in successive passages. Cells were then trypsinized and subcultured without IFN-γ (nonpermissive conditions) and allowed to differentiate at 37°C, with changing the medium on alternate days. Differentiation of the podocytes grown for at least 8 days at 37°C was confirmed by identification of the podocyte differentiation marker synaptopodin using Western blotting (data not shown). The 3T3-L1 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and were maintained in Dulbecco’s modified Eagle’s medium containing 10% FBS and 100 μg/mL antibiotics at 37°C under a humidified 5% CO₂ atmosphere. Differentiation of the preadipocytes into adipocytes was induced by the addition of a hormone cocktail, as described previously [25]. Briefly, 2 days after confluence, the medium was replaced with a standard adipocyte differentiation induction medium containing 0.5 mmol/L isobutylmethylxanthine, 1 μmol/L dexamethasone, and 10 μg/mL insulin. Identification of oil-red O-stained lipid droplets in the cytoplasm, quantification of the intracytoplasmic lipids, and the expression of peroxisome proliferator activator receptorγ (PPARγ) (data not shown) were used to confirm the differentiation of adipocytes. For palmitate preparation, palmitate-free acids and low-endotoxin BSA (FFA free) were purchased from Sigma-Aldrich. A 20mM solution of palmitate was dissolved in 0.01M NaOH, incubated at 70°C for 30 minutes, and complexed to 10% BSA at a molar ratio of 6.6:1 in podocytes and 3:1 in adipocytes. The solution was shaken overnight at 37°C under N₂, sonicated for 10 minutes, filter sterilized, and stored at 20 °C prior to use [26], [27]. Cells were cultivated on 100-mm dishes and serum restricted for 24 hours. These were subsequently exposed to normal glucose (NG; 5.5mM) or high glucose (HG; 30mM) with or without palmitate (100μM) for 24 hours. To evaluate the effects of TLR antagonism, GIT27 was added to the cells 2 hours prior to exposure at a concentration of 10 μg/mL. After the experiment, the cell lysates and conditioned media were centrifuged at 15,000 rpm for 10 minutes at 4°C, and the supernatants were collected. All experimental groups were cultured in triplicate and harvested at 24 hours.

We used nonparametric analysis for the relatively small number of samples. Results are expressed as mean±standard error of the mean. Comparisons were made using the Wilcoxon rank sum test and Bonferroni correction. A Kruskal–Wallis test was used for comparison of more than two groups, followed by a Mann–Whitney U test. A P value of <0.05 was considered statistically significant. The statistical analyses were performed using SPSS for Windows, version 13.0 (SPSS Inc., Chicago, IL, USA).

The physical and biochemical parameters for each group of the experimental animals are shown in Table 1. As expected, body weights of HFD mice were much higher than those of ND mice, despite the former group taking smaller amounts of food and water. However, interestingly, GIT27 treatment reduced body weight in HFD mice without any changes in food and water intake. We observed significant changes in fat content of each organ and in kidney weight. Fat weight increased significantly in HFD mice and decreased with GIT27 treatment. However, kidney weight decreased in HFD mice and was restored with GIT27 treatment. No changes were observed in the weight of the heart and liver. In HFD mice, larger urinary volumes were recorded, which were not affected by GIT27. HFD mice did not show any significant changes in systolic blood pressure and serum creatinine level as compared to ND mice. The plasma glucose and HbA1c levels were not different among the three groups. The plasma insulin level was higher in HFD mice than in ND mice, but this difference was not significant. Among the three groups, no changes were observed in plasma adiponectin. HOMA-IR was significantly lower in HFD mice treated with GIT27 for 8 weeks compared to those without GIT27 treatment.

To evaluate glucose tolerance, we measured blood glucose levels after oral dextrose gavage every 4 weeks. Interestingly, GIT27 treatment improved blood glucose levels in HFD mice significantly at 4, 8, and 12 weeks, although variable levels were observed in ND mice after 12 weeks (Fig. 1A–D). In accordance with glucose tolerance, lipid abnormalities also improved significantly in HFD mice by GIT27 treatment (Fig. 2). In addition, lipid contents in the kidneys, fat, and liver also showed a significant decrease by GIT27 treatment (Fig. 3A and B). We also compared the oxidative stress in each organ among the three groups. An assessment of the LPO contents and urinary 8-isoprostane levels of the kidneys, fat, and liver showed that the LPO contents were markedly higher in HFD mice, which were decreased significantly by GIT27 treatment (Fig. 3C). The level of urinary 8-isoprostane was increased markedly in HFD mice and decreased in GIT27-treated HDF mice, but the differences were not significant (Fig. 3D).

To examine the effect of GIT27 on renal injury, we first measured urinary excretion of protein and albumin. Urinary protein excretion was increased markedly in HFD mice compared with that in ND mice, as shown in Fig. 4A. However, urinary protein excretion was improved significantly after treatment with GIT27. Fig. 4B shows the urine albumin excretion in 24 hours. After 12 weeks, urinary albumin excretion was also improved significantly in GIT27-treated HFD mice compared with HFD mice without treatment. Next, we evaluated the anti-inflammatory effect of GIT27 on obesity-induced renal injury after confirming metabolic improvement and an antiproteinuric effect. We also compared the urinary levels of proinflammatory cytokines (TNFα and IL-2) among the three groups based on an analysis of the 24-hour urine samples using an ELISA kit. The levels of TNFα and IL-2 in the urine were increased in HFD mice and decreased in GIT27-treated HFD mice. In particular, the IL-2 level was improved significantly in GIT27-treated HFD mice compared with HFD mice without treatment (Fig. 5A). Moreover, we evaluated the gene expression of the adipose and kidney tissues to confirm the anti-inflammatory and antifibrotic effects of GIT27 treatment. The gene expression of proinflammatory cytokines such as MCP-1, PAI-1, and TNFα in the adipose tissue was increased in HFD mice and decreased by GIT27 treatment (Fig. 5B). No significant change was recorded in PPARγ and adiponectin gene expression. AMPK gene expression was increased significantly in HFD mice, which decreased by GIT27 treatment. Gene expressions of proinflammatory and profibrotic cytokines in the kidney tissue tended to increase in HFD mice and decrease in those treated with GIT27. In particular, gene expressions of type IV collagen, PAI-1, MCP-1, and IL-1b were decreased significantly in GIT27-treated HFD mice compared with HDF mice without treatment (Fig. 5B and C). With regard to lipid metabolism in the kidney tissue, SREBP1c gene expression was reduced by GIT27 treatment.

Fig. 6 shows the representative immunohistochemical findings for each group of experimental animals. We observed higher extracellular matrix expansion and tubulointerstitial fibrosis, and increased expression of type IV collagen, TGFβ1, and PAI-1 in HFD mice. Consistent with the results of the urinary albumin excretion, the expression of these genes was decreased by GIT27 treatment compared to that in HFD mice without treatment. The expression of F4/80 as a macrophage marker was increased in HFD mice and reduced in GIT27-treated HFD mice. This result suggests that GIT27 restored the renal inflammatory changes by suppressing macrophage accumulation in the kidneys.

To understand the mechanism of the beneficial effects of GIT27 relating to the TLR4 pathway, we investigated TLR4, NFκB, IκBβ, and NOX4 protein synthesis in the kidneys of ND mice and HFD mice with and without GIT27 treatment. The expression of TLR4 showed a tendency to increase in HFD mice, which decreased with GIT27 treatment; however, these changes were not significant. The expression of NFκB, IκBβ, and NOX4 also showed no change in HFD mice with and without GIT27 treatment (Fig. 7).

We confirmed previously that TLR4 mRNA expression was increased markedly under FFA and HG conditions in cultured podocytes [18]. This effect was notable upon exposure to both HG and FFAs. Consistent with these changes, proinflammatory cytokine secretion into the culture supernatant was also increased tremendously in FFA conditions. Moreover, HG and FFA also increased NFκB activation. However, GIT27 interestingly inhibited TLR4, TNFα, NOX4 expression, NFκB activation, and proinflammatory cytokine synthesis. The protein synthesis results of this study agreed with the findings of previous studies. In particular, FFA induced NFκB activation and IκBβ inhibition in podocytes (Fig. 8). Additionally, GIT27 suppressed NFκB activation and reactivated IκBβ inhibition significantly. The protein expression of TLR4, TRAF6, NOX4, p-AMPK, and SREBP1 tended to increase under FFA conditions, but the trend was reversed by GIT27 treatment; however these changes were not significant in Western blot analysis. In addition, with regard to adipocytes, no significant changes were observed in the protein expression of SREBP1, TLR4, NFκB, IκBβ, NOX4, and p-AMPK (Fig. 9).