Cell culture media, Dulbecco's modified Eagle medium (DMEM) and F12, were obtained from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Recombinant human TGF-β1 was obtained from PeproTech, Inc. (Rocky Hill, NJ, USA). KMUP-1 was synthesized in the laboratory of the authors, the stock solution (10 mM) was prepared by dissolving KMUP-1 in the solvent (10% absolute alcohol, 10% propylene glycol and 2% 1 N HCl), according to previous studies (8–10).

Mouse kidney mesangial cells (MES13) were purchased from the American Type Culture Collection (cat. no. CRL-1927; Manassas, VA, USA). Cells were grown in DMEM/F12 (3:1) medium (with 6.67 mM glucose) supplemented with 5% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified 5% CO₂ incubator at 37°C. Cells were starved in serum-free (0.5% FBS) media for 24 h prior to experiments in 5% FBS medium. All cell culture materials were obtained from Gibco (Thermo Fisher Scientific, Inc.).

MES13 cells were cultured in 24-well plates (5×10³/well). After 24 h, cells were treated with the control (KMUP-1 solvent at a final concentration of 1%) or KMUP-1 for 72 h. MTT (0.5 mg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was incubated at 37°C for 4 h prior to harvesting. Following removal of the culture medium, cells were dissolved in dimethylsulfoxide (DMSO) and shaken for 10 min. Formazan was dissolved by DMSO and the assay was quantified by determining the absorbance at 540 nm using an ELISA reader (Dynex Technologies GmbH, Denkendorf, Germany).

Human TGF-β1 promoter activity was detected using the phTG5 plasmid, which was donated by Dr Jean-Louis Virelizier (Unité d'Immunologie Virale, Institut Pasteur, Paris, France) (11). The TGF-β1 bioactivity reporter p3TP-lux, which contains the plasminogen activator inhibitor-1 promoter, was donated by Dr. Joan Massagué (Memorial Sloan Kettering Cancer Center, New York, USA) (12). Cells were cultured in 6-well plates at a density of 1.5×10⁴ cells/well and transfected with 1 µg of the phTG5 or p3TP-lux plasmid using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. Following 6 h of transfection, cells were treated with HG (30 mM) or combined with KMUP-1 (10 µM) for 72 h. Luciferase activity was measured using the Dynatech ML1,000 luminometer (Dynatech Laboratories, Inc., Chantilly, VA, USA).

Briefly (13), MES13 cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 1 mM Na₃VO₄, 50 mM NaF and 10 mM β-glycerol phosphate) containing 0.1% protease inhibitor cocktail (Merck KGaA). Cell proteins were extracted and quantified with a DC™ protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA). Proteins (50 µg/well) were separated by 10% SDS-PAGE and transferred to PolyScreen polyvinylidene difluoride membranes (PerkinElmer, Inc., Waltham, MA, USA). Following blocking for 2 h at room temperature with 5% non-fat milk and rinsing with PBS, the membranes were probed with the primary antibodies overnight at 4°C and washed with 0.1% PBS-Tween-20 (PBST) 3 times (5 min each). The primary antibodies used were phosphorylated-Smad2/3 (p-Smad2/3; 1:1,000; cat. no. 8828), Smad2/3 (1:2,000; cat. no. 3102), Suv39h1 (1:1,500; cat. no. 8729) and H3K9me3 (1:2,000; cat. no. 9754), antibodies were obtained from Cell Signaling Technology, Inc., Danvers, MA, USA). Fibronectin (1:10,000; cat. no. AJ1297b) and collagen IV (1:2,000; cat. no. AP7369a) antibodies were obtained from Abgent, Inc. (San Diego, CA, USA), GAPDH (1:5,000; cat. no. sc-25778) and α-tubulin (1:5,000; cat. no. MS-581-P) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and Labvision/NeoMarkers (Thermo Fisher Scientific, Inc.), respectively. The membranes were then incubated in horseradish peroxidase (HRP)-conjugated anti-rabbit (1:5,000; cat. no. AP132P) or anti-mouse (1:5,000; cat. no. AP124P) secondary antibodies (Merck KGaA) for 1 h at room temperature and washed with 0.1% PBST 5 times (5 min each). The protein bands were detected by using the enhanced chemiluminescence ECL system (PerkinElmer, Inc.) and the bands were quantified by densitometric analysis (Image Studio Lite 5.25; LI-COR Biosciences, Lincoln, NE, USA).

Cell hypertrophy was determined by the ratio of whole cell protein lysates to total cell numbers (14). Briefly, MES13 cells were cultured in 6-well plates to 50% confluence and then treated with HG (30 mM) or HG + KMUP-1 (10 µM) for 3 days. Following trypsinization, cells were washed twice with PBS and cell numbers were counted using a hemocytometer. Equal numbers of cells were lysed in RIPA buffer containing the protease inhibitor cocktail. Total protein content was measured by using the DC^™ protein assay kit (Bio-Rad Laboratories, Inc.).

Male Sprague-Dawley rats (n=23) weighing 200–250 g (aged 7 weeks) were purchased from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan) and were housed (3 per cage) in a temperature-(22±2°C) and humidity (50±10%)-controlled room with a 12 h light/dark cycle in the Animal Center of Kaohsiung Medical University (Kaohsiung, Taiwan). Rats were divided into three groups: Control, streptozotocin (STZ)-diabetic and diabetic + KMUP-1. Briefly (15), after overnight fasting, rats received a single intraperitoneal injection of 55 mg/kg STZ (Sigma-Aldrich; Merck KGaA) in 0.1 M citrate buffer (diabetic, n=9; diabetic + KMUP-1, n=9) or citrate buffer (control, n=5). Thereafter, rats were given free access to food and water. Diabetic rats received Lantus insulin (Sanofi S.A., Paris, France) to maintain non-fasting blood glucose levels between 19.4 and 27.8 mmol/l. The diabetic + KMUP-1 treatment group was intraperitoneally injected with KMUP-1 (dissolved in distilled water, 5 mg/kg/day) daily (16). Rats were perfused with normal saline and anesthetized intraperitoneally with sodium pentobarbital (50 mg/kg; Abbott Pharmaceutical Co., Ltd., Lake Bluff, IL, USA) at week 8. Kidneys were removed and kidney slices were immersed in 4% paraformaldehyde at room temperature for 24 h. Kidney slices were embedded in the paraffin block and cut into sections (thickness, 4 µm) for immunohistochemical studies. All animal procedures were conducted in accordance with the national guidelines (17) and were approved by the Kaohsiung Medical University Animal Experiment Committee.

Briefly (15), tissue sections were rehydrated and deparaffinized in xylene and ethanol. The sections then underwent antigen retrieval in 10 mM sodium citrate buffer by microwaving for 30 min. Following this, preincubation of tissue sections with the blocking buffer was conducted for 30 min prior to incubation with primary antibodies overnight at 4°C. Primary antibodies used were fibronectin (1:400, cat. no. AJ1297b) obtained from Abgent, Inc. and collagen IV antibodies (1:500, cat. no. ab6586) obtained from Abcam (Cambridge, UK). Following washing with PBST, sections were stained and incubated with DAB+ and the ready-to-use (undiluted) HRP-conjugated anti-rabbit secondary antibodies contained in the Dako REAL™ EnVision™ Detection system (cat. no. K5007; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) for 30 min at room temperature, according to the manufacturer's instructions, and counterstained with hematoxylin.

Statistical analyses were performed by using Stata 13.1 software (StataCorp LP, College Station, TX, USA). Data were expressed as the mean ± standard error. Unpaired Student's t-tests were used for the comparison between two groups. P<0.05 was considered to be statistically significant.

The present study investigated the effects of KMUP-1 on cell viability of MES13 cells to determine the optimum concentration of KMUP-1. MES13 cells were treated with KMUP-1 (1–100 µM) for 72 h and cell viabilities were measured using an MTT assay. The optimum concentration of KMUP-1 was determined to be 10 µM (Fig. 1).

TGF-β1 gene transcriptional activity or TGF-β1 bioactivity was measured by transient transfection of phTG5 or p3TP-lux, respectively. It was identified that KMUP-1 (10 µM) attenuated HG (30 mM)-induced TGF-β1 gene transcriptional activity or TGF-β1 bioactivity at 72 h (Fig. 2).

Since TGF-β1 gene transcriptional activity and TGF-β1 bioactivity were attenuated by KMUP-1, the present study examined whether KMUP-1 also attenuated TGF-β1-Smad signaling pathway. TGF-β1 (5 ng/ml) increased phosphorylation of Smad2/3 in a time-dependent manner (5–30 min; Fig. 3). Furthermore, KMUP-1 (10 µM) attenuated TGF-β1-induced p-Smad2/3 expression at 5–10 min (5 ng/ml; Fig. 3).

Both HG and TGF-β1 have been previously reported to increase fibronectin and collagen IV protein expression in mesangial cells (18,19). As a result, the present study investigated the effects of KMUP-1 on HG or TGF-β1-induced fibronectin and collagen IV protein expression. KMUP-1 (10 µM) was demonstrated to attenuate HG (Fig. 4A) or TGF-β1 (5 ng/ml)-induced fibronectin and collagen IV protein expression at 72 h (Fig. 5).

A previous study identified that KMUP-1 decreased cardiac hypertrophy via the NO/cGMP pathway (20). In addition to extracellular matrix accumulation, DN is characterized by renal (including mesangial cell) hypertrophy (21). In addition, DN is associated with NO deficiency (22). For example, eNOS knockout mice develop diabetic renal hypertrophy while soluble guanylate cyclase enhancers attenuate DN (23,24). Therefore, the authors studied the effects of KMUP-1 on HG-induced cell hypertrophy. KMUP-1 (10 µM) attenuated HG-induced cell hypertrophy at 72 h (Fig. 4B).

Suv39h1 is a histone lysine methyltransferase, which induces the gene-silencing H3K9me3, while HG increases pro-inflammatory genes concomitantly with decreased levels of H3K9me3 and Suv39h1 at their promoters in vascular smooth muscle cells (25). However, the roles of Suv39h1 in diabetic nephropathy remain unclear. As a result, the study investigated whether HG-induced pro-fibrotic collagen IV and fibronectin is associated with reduced Suv39h1 levels and the effects of KMUP-1 on HG-decreased Suv39h1 levels. KMUP-1 (10 µM) was demonstrated to attenuate HG-decreased Suv39h1 (Fig. 6A) and H3K9me3 (Fig. 6B) levels at 72 h.

To corroborate the in vitro findings, the effects of intraperitoneal KMUP-1 (5 mg/kg/day) treatment on STZ-diabetic rats were investigated at 8 weeks. Increased glomerular and tubular expression of collagen IV were both attenuated by KMUP-1 in diabetic rats (Fig. 7). Furthermore, increased peri-glomerular and tubulointerstitial expression of fibronectin was attenuated by KMUP-1 in diabetic rats (Fig. 8).