Mice were maintained under pathogen-free conditions at 22–26°C (humidity, 40–60%) with a 12-h light/dark cycle and free access to water and food. Male C57BL/6 mice (8–10 weeks old; weighing 20–25 g; n=18) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., (Beijing, China) To assess the effects of TLR2 activation on STZ (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany)-induced renal damage, mice were randomly divided into three groups (n=6/group): (i) Control mice (Con); (ii) mice receiving STZ (STZ); (iii) STZ-induced mice treated with TLR2 agonist Pam₃CysSK₄ (TLR2A+STZ). Mice in the STZ and TLR2A+STZ groups were administered STZ in citrate buffer by intraperitoneal injection (50 mg/kg per day for 5 days). Control mice received citrate buffer alone. After 2 weeks, fasting blood glucose was monitored using an Auto-Check meter (Roche Diagnostics, Basel, Switzerland) (21). Following confirmation of hyperglycemia (blood glucose levels >15 mmol/l), TLR2A+STZ mice received intraperitoneal injection of 100 µg Pam₃CysSK₄ (EMC Microcollections GmbH, Tübingen, Germany) in 10 µl dimethyl sulfoxide (DMSO) four times (once a week); whereas Con and STZ mice received 10 µl DMSO alone weekly for four weeks (13). To measure 24-h proteinuria, mice were placed in individual mouse metabolic cages (Tecniplast S.P.A., Buguggiate, Italy) with ad libitum access to water and food. After 4 weeks, mice were sacrificed by intraperitoneal injection of sodium pentobarbital (30 mg/kg; Abbott Laboratories, Chicago, IL, USA) and their kidneys were removed prior to examination by light and electron microscopy, immunohistochemistry, western blot analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The present study was approved by the Animal Experimentation Committee of Shenzhen University Health Science Center (Guangdong, China).

At the end of the experiment, blood samples were collected from the left ventricle and centrifuged at 5,000 × g for 10 min at 4°C. Plasma concentrations of total cholesterol, albumin, creatinine and urea nitrogen were measured using an autoanalyzer (Hitachi 917; Hitachi, Ltd., Tokyo, Japan). Urinary albumin excretion was assessed using a Mouse Albumin ELISA Quantitation Set (Bethyl Laboratories, Inc., Montgomery, TX, USA).

Prior to electron microscopic examination, renal cortex samples were cut into 1-mm³ slices on ice, immediately fixed in 2.5% buffered glutaraldehyde for 12–16 h at 4°C and embedded in epoxy resin. Ultrathin sections (0.1-µm thick) were examined by electron microscopy. To study slit diaphragm morphology, images (20 fields per sample) were captured at random fields of view at a final magnification of ×10,000. From each mouse, three glomeruli were evaluated on five 10 k images/glomerulus. Electron microscopy images were analyzed using ImageJ (v2.1.4.7; imagej.nih.gov/ij/) and the method of measuring podocyte foot process production was adapted from a previous report (22). From each image, the mean of the foot process width (FPW) was calculated as follows: FPW=π/4x(∑glomerular basement membrane length/∑foot process).

Kidney samples were fixed in 4% paraformaldehyde overnight at 4°C, then dehydrated in an ascending ethanol series, embedded in paraffin and cut into sections 4-µm thick. Sections were stained with hematoxylin/eosin and examined with a light microscope to evaluate glomerular mesangial expansion.

Immunohistochemistry was performed to detect collagen IV and F4/80. Briefly, the kidney tissues were fixed in 4% paraformaldehyde overnight at 4°C and blocked for 20 min at 37°C with 1% bovine serum albumin (cat. no. V900933; Sigma-Aldrich; Merck Millipore). The sections were subsequently incubated with primary antibodies against collagen IV (1:500; cat. no. sc-9301; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and F4/80, which is a marker of macrophage infiltration, (1:500; cat. no. MCA497; Bio-Rad Laboratories, Inc., Hercules, CA, USA) overnight at 4°C. Sections were examined using a light microscope (Olympus BX50; Olympus Corporation, Tokyo, Japan) by experimental condition-blinded researchers. To quantify the proportional area of staining, 20 randomly-selected views (magnification, ×200) were examined in the renal cortex of each slide.

Renal tissue was removed for extraction of total or nuclear protein in order to perform a western blot analysis to measure TLR2 or nuclear factor (NF) -κB p65 expression. Actin or fibrillarin were used as controls. Nuclear proteins were isolated using an NE-PER Nuclear and Cytoplasmic Extraction kit (cat. no. 78833; Pierce Biotechnology; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. Protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce Biotechnology; Thermo Fisher Scientific, Inc.). Equal amounts of proteins (40 µg) from three groups (n=6) were extracted and separated by 10% SDS-PAGE, and the proteins were transferred to a nitrocellulose membrane. The membrane was washed and blocked in 1X PBS with 0.02% Tween-20 (PBST) supplemented with 5% milk for 1 h at room temperature with gentle shaking, then incubated overnight at 4°C with the primary antibody: Anti-NF-κB p65 antibody (1:500; cat. no. sc-8008) or anti-TLR2 antibody (1:500; cat. no. sc-12504; both Santa Cruz Biotechnology, Inc.). The membrane was washed three times for 30 min in PBST and incubated with horseradish peroxidase-conjugated anti-mouse (1:5,000; cat. no. sc-358917) or anti-goat (1:5,000; cat. no. sc-2768; both Santa Cruz Biotechnology, Inc.) secondary antibody for 1 h at room temperature. Following three washes, the membrane was transferred to the Enhanced Chemiluminescence Reagent (Applygen Technologies, Inc., Beijing, China) and exposed to XBT-1 X-ray film (Kodak, Rochester, NY, USA).

Total RNA (100 µg) was extracted from the kidneys of three groups (n=6) with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Subsequently, RNA from each sample (2 µg) were reverse-transcribed in accordance with the manufacturer's protocol for the Reverse Transcription kit (Invitrogen; Thermo Fisher Scientific, Inc.). The reverse transcription mixture contained 2.0 µl template RNA, 4 µl reaction buffer (5X), 2 µl dNTPs mix (10 mmol), 1 µl Moloney murine leukemia virus reverse transcriptase (200 U/µl), 1 µl RNase inhibitor (200 U/µl) and 1 µl oligo (dT)₁₈ primer. cDNA samples were then used as templates for qPCR. A SYBR Premix ExTaq fluorescent quantitative PCR kit (Invitrogen; Thermo Fisher Scientific Inc.) and LightCycler 96 Real-time PCR System (Roche Diagnostics) were used to conduct the reaction and analysis, respectively, according to a protocol from a previous report (22). The qPCR mixture included 1 µl cDNA, 12.5 µl PCR Master mix (2X; including 0.5 µl SYBR), 1 µl forward primer, 1 µl reverse primer and 9.5 µl nuclease-free water. GAPDH was used as an internal control. The primers used were as follows: TLR2, forward 5′-CTCTTCAGCAAACGCTGTTCT-3′ and reverse 5′-GGCGTCTCCCTCTATTGTATTG-3′; myeloid differentiation primary response gene 88 (MyD88), forward 5′-ATCGCTGTTCTTGAACCCTCG-3′ and reverse 5′-CTCACGGTCTAACAAGGCCAG-3′; MCP-1, forward 5′-AATGAGTAGCAGCAGGTGAGTG-3′ and reverse 5′-GAAGCCAGCTCTCTCTTCCTC-3′; and GAPDH, forward 5′-GGTGAAGGTCGGTGTGAACG-3′ and reverse 5′-CTCGCTCCTGGAAGATGGTG-3′. DNA amplification was performed as follows: Pre-denaturation at 94°C for 2 min, denaturation at 94°C for 15 sec, annealing at 58°C for 15 sec, extension at 72°C for 15 min for total of 40 cycles, and final extension at 72°C for 10 min. The Cq value of the sample evaluated by qPCR was quantified and the GAPDH value was subtracted in the corresponding sample, thereby obtaining the ΔCq value. The mRNA expression quantity of gene was calculated according to the method (23).

Data are presented as mean ± standard error of the mean, using GraphPad Prism v5.01 software (GraphPad Software Inc., La Jolla, CA, USA). Analysis was completed with an analysis of variance and Student's t-test. P<0.05 was considered to represent a statistically significant difference.

Mice in the STZ group exhibited the typical manifestation of diabetes, including significant hyperglycemia, hyperlipidemia, weight loss and an increased kidney to body weight ratio compared with mice in the control group (Table I). Furthermore, mice in the TLR2A+STZ group exhibited a higher blood glucose level and lower body weight and serum albumin levels than mice in the STZ group (Table I).

As determined by measuring urine albumin levels over 24 h, compared with control mice, STZ-induced mice exhibited significant albuminuria (P<0.01), which was consistent with a previous report (15), and Pam₃CysSK₄-treated diabetic mice had more severe proteinuria compared with untreated STZ-treated mice (P<0.05; Fig. 1A). Electron microscopy revealed podocyte foot process production in STZ-treated mice, with or without the TLR2 agonist Pam₃CysSK₄ (Fig. 1B). From each image, the mean of the foot process width was calculated as described in the methods. It was demonstrated that Pam₃CysSK₄ treatment exacerbated STZ-induced podocyte foot process production (P<0.05; Fig. 1C). Additionally, the renal function of Pam₃CysSK₄-treated diabetic mice declined progressively, as reflected by the progressive increase in blood urea nitrogen and serum creatinine levels (Table I).

Histological sections of kidneys from the diabetic mice revealed glomeruli with signs of hypertrophy of the tuft and a mild expansion of the mesangial matrix (Fig. 2A). Glomerular fractional mesangial areas were significantly increased in Pam₃CysSK₄-treated diabetic mice compared with untreated STZ-diabetic mice (P<0.05; Fig. 2B), and in STZ mice compared with controls. No histological changes in kidney were detected in the normal controls.

Diabetes is associated with an increase in collagen IV protein expression in the kidneys (5,21). Collagen IV expression was elevated in Pam₃CysSK₄-treated diabetic mice compared with the untreated STZ-diabetic mice (P<0.05; Fig. 3A and B). F4/80-positive immunostaining was observed in the STZ-induced mouse kidney, predominantly in the renal interstitium (Fig. 3A). Additionally, there was a significant increase in F4/80 immunostaining in the kidneys of mice in the Pam₃CysSK₄ treatment group compared with the increase observed in the STZ-induced group (P<0.05; Fig. 3A and C).

To investigate the effect of Pam₃CysSK₄ on the expression of TLR2 in the kidneys of diabetic mice, renal TLR2 protein and mRNA expression were examined by western blot analysis and RT-qPCR, respectively. Renal TLR2 protein expression was significantly upregulated in STZ-treated mice compared with control mice (P<0.05), and elevated further following Pam₃CysSK₄ treatment (P<0.05; Fig. 4A). Similarly, STZ-induced mice exhibited a significant increase in renal TLR2 mRNA expression compared with normal controls (P<0.05; Fig. 4B). Furthermore, the expression of TLR2 mRNA in the kidneys of Pam₃CysSK₄-treated mice was significantly elevated compared with the untreated STZ-induced mice (P<0.05; Fig. 4B).

Western blot analysis results indicate there was significant upregulation of NF-κB p65 expression in STZ-treated mice compared with controls (P<0.05; Fig. 4C). The activation of NF-κB p65 was in accordance with the upregulation of MyD88 mRNA (Fig. 4B). In addition, the mRNA expression of MCP-1 was also markedly elevated in the diabetic kidney (Fig. 4B) and Pam₃CysSK₄-treated mice exhibited a significantly increased expression of MyD88 and MCP-1 mRNA (P<0.05; Fig. 4B), and activation of NF-κB p65, compared with the increase observed in STZ-induced mice (P<0.05; Fig. 4C).