C57BL/KsJ-db/db mice and their littermate db/m mice were purchased from the Model Animal Research Center at Nanjing University (Nanjing, China) and housed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The experimental protocols of the present study were approved by the Animal Care Committee at Hubei University of Medicine (Shiyan, China). The db/db mice were determined to be diabetic on the basis of obesity and hyperglycemia at ~4 weeks of age. The mice were subject to right uninephrectomy under anesthesia at various time-points, depending on the experiment design. Anesthesia was performed using intraperitoneal injection of pentobarbital (90 mg/kg; Shanghai Chemical Reagent Co., Ltd., Shanghai, China), and the mice were sacrificed by cervical dislocation following anesthesia.

Mouse kidney tissue (~100 mg), taken from the renal cortices of DN or control mice, was added into 1 ml TRIzol® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) in a 1.5-ml Eppendorf tube. The tissue was homogenized by RNase-free pestles followed by the addition of chloroform (400 µl). Total RNA was extracted from the TRIzol-chloroform mixture using the miRNeasy Mini kit (Qiagen, Valencia, CA, USA). Total RNA was eluted in 100 µl nuclease-free water, and the concentration was measured using the NanoDrop™ 8000 spectrophotometer (Thermo Fisher Scientific, Inc., Wilmington, DE, USA). Samples with an A₂₆₀/A₂₈₀ ratio >1.8 were considered pure. The RNA integrity was verified by rRNA visualization following electrophoresis in an agarose gel.

The differential expression profile of the miRNAs was assayed using the miRMouse miRNA array (Exiqon miRCURY LNA™ miRNA; Exiqon A/S, Vedbaek, Denmark), which contained 1,181 capture probes, in db/db DN mice and db/m controls. Following RNA isolation from the samples, the microarray labeling and array hybridization procedure were performed according to the manufacturer's instructions. Upon the completion of the hybridization procedure, the slides were produced and dried by centrifugation. The slides were then scanned using a SureScan Microarray Scanner (Agilent Technologies, Santa Clara, CA, USA).

qPCR was performed to validate the miRNA expression profiles identified with the miRNA array. The first-strand cDNA was synthesized using the PrimeScript Reverse Transcription reagent kit (Clontech Laboratories, Inc., Mountain View, CA, USA) with oligonucleotide dTs. The cDNA levels for each tested miRNA and GAPDH gene transcripts were measured using a 7900HT Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Each amplification reaction was performed in triplicate in separate tubes. The average calculated from the three threshold cycles was used to determine the number of transcripts in the sample. miRNA expression was quantitatively expressed in arbitrary units as the ratio of the target sample quantity to the calibrator or to the mean values of the control samples. All values were normalized to the endogenous control GAPDH. qPCR was also used to detect SMAD3/4 expression, with primers provided by Sangon Biotech Co., Ltd. (Shanghai, China).

The miR-346 expression vector was constructed according to the miR-346 sequence (accession no. MI0000634; http://www.mirbase.org/) using the SacI cloning site of pGenesil-1. Recombinant pGenesil-miR-346 and control pGenesil-1 were provided by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Starting at week 8, three groups of db/db DN mice (6 mice/group) were intravenously injected an miR-346-bearing plasmid (pGenesil-miR-346, 30 mg/kg), a plasmid control (pGenesil-1, 30 mg/kg) and an untreated control (same volume of saline), respectively. The injection was administered through the tail vein once a week until week 16. The urinary protein content, body weight and blood glucose were dynamically monitored. At week 16, all animals were sacrificed, and their renal cortex tissues were sampled for molecular analysis and morphological examinations.

Immunoblot analysis was used to detect SMAD3/4 expression in the renal tissues. The renal tissue samples were harvested from miR-346-treated (pGenesil-miR-346), plasmid-treated (pGenesil-1) and untreated control mice at week 16. The tissue samples were homogenized with Kontes pellet pestle cordless motor (Thermo Fisher Scientific, Inc.) and lysed with cell lysis solution (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and the tissue lysates were subjected to an immunoblotting analysis as previously described (11). The total protein was quantified using Pierce BCA protein assay kit (Life Technologies). Briefly, tissue lysates (50 µg) were separated through 12% denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was incubated overnight with rabbit anti-SMAD3/4 polyclonal antibodies (1:1,000; #9523; Cell Signaling Technology, Inc., Boston, MA, USA). After three washes with Tris-buffered saline/0.05% Tween-20, the membrane was incubated for 1 h with goat anti-rabbit immunoglobulin G conjugated to horseradish peroxidase (1:2,500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Then, the membrane was washed three times with Tris-buffered saline/0.05% Tween-20, followed by a final wash with Tris-buffered saline without Tween-20. The blot was developed using enhanced chemiluminescence detection (Beyotime Institute of Biotechnology, Shanghai, China). β-actin was used as a loading control.

The Periodic acid Schiff (PAS) protocol was used for renal tissue staining. Tissue sections (~5 µm) were mounted on slides and stained with a PAS staining kit (Sigma-Aldrich, St. Louis, MO, USA), as per the manufacturer's instructions.

Scanned images from the microarray were imported into miRCURY LNA software (Exiqon A/S) for grid alignment and data extraction. Expressed data were normalized using median normalization. The numerical results are expressed as the mean ± standard error of the mean, with ‘n’ representing the number of animals. Groups were compared using one-way analysis of variance and subsequently using the Student-Newman-Keuls and Dunnett tests for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

The blood glucose, body weight and urinary proteins of the DN mice were dynamically observed for 16 weeks (Table I). Starting from week 4, the blood glucose level and body weight of the db/db mice were significantly higher than those of the db/m control mice (P<0.05), thereby confirming the development of DN in the db/db mice. Fig. 1 shows the glomerular pathologies of the db/db and db/m mice at the end of the observation period (week 18). Typical DN changes were observed in the db/db mouse kidney, including glomerular hypertrophy, mesangial cell proliferation and matrix accumulation. The results indicated the development of DN in the db/db mice.

An miRNA microarray was used to identify the miRNA profiles of the mouse DN model. To ensure the reliable performance of the microarray and qPCR, RNA samples isolated from the renal cortices of DN and control animals were verified prior to the miRNA array being performed. The RNA purity was confirmed by a 260/280 optical density value of 1.8–2.0 in both DN (n=6) and control groups (n=6). The RNA samples were also assessed for degradation status by agarose gel electrophoresis. Three RNA samples were randomly selected from the DN and control groups and were subjected to subsequent Cy3 and Cy5 labeling and hybridization with the Exiqon miRNA array. The microarray data demonstrated that the db/db DN mice had a significantly altered miRNA profile compared with the db/m control mice. An overview of the miRNA expression is shown in Fig. 2. In total, 772 and 433 miRNAs were detected for expression in the DN and control mice, respectively. Compared with the control, 219 miRNAs were differentially expressed by >2-fold, and 71 miRNAs had a difference of >4-fold in the DN mice. Among these 71 miRNAs, 39 miRNAs were upregulated (Fig. 3A) and 31 miRNAs were downregulated (Fig. 3B).

To validate the expression profile obtained from the miRNA microarray analysis, 10 downregulated miRNAs (miR-215, miR-451, miR-200a, miR-200b, miR-200c, miR-141, miR-21, miR-346, miR-709 and miR-187) and 8 upregulated miRNAs (miR-29c, miR-192, miR-216a, miR-17, miR-377, miR-196a, miR-166 and miR-98) were quantitatively examined using qPCR analysis. As shown in Table II, consistent fold changes were observed between the qPCR and microarray for the selected miRNAs, indicating the reliability of the miRNA microarray analysis under the present experimental conditions.

Target predictions were based on the detected expression profiles of the miRNAs. Considering glomerular fibrosis as a major process during the pathogenesis of DN, the target searching was focused on fibrosis-related mRNAs. As guided by Krek et al (12), the PicTar algorithm and the microRNA.org website (http://www.microrna.org/) were used to identify potential mRNA targets of the miRNAs found to be differentially regulated in the DN mice. Among the 71 most differentially expressed miRNAs, miR-346 was identified a SMAD family-related miRNA that has not been well characterized in this regard. Of the 95 potential targets for miR-346, SMAD3 and SMAD4 were closely associated with fibrogenesis. As shown in Fig. 4, miR-346 has 6–11 complementary binding sites in the 3′-untranslated region (3′-UTR) of SMAD3/4.

As shown in Fig. 5A and B, the expression of SMAD3/4 was significantly attenuated by miR-346 administration, which was demonstrated in the analysis of both mRNA and protein levels.

As shown in Fig. 6A, a significant improvement in the proteinuria was observed with pGenesil-miR-346 administration, although the changes in the body weight and blood glucose level remained consistent with the controls. As can be noted in Fig. 6B, typical DN glomerular alterations were present in both the plasmid-treated and untreated controls, exhibiting matrix accumulation, glomerular hypertrophy and mesangial cell proliferation; however, the db/db mice treated with pGenesil-miR-346 exhibited a normal glomerular histology.