miR‐218 regulates diabetic nephropathy via targeting IKK‐β and modulating NK‐κB‐mediated inflammation

2.1. Animal models

The Experimental Animal Ethical Committee of Second Hospital of Jilin University approved all study protocols, which were conducted in a manner consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague Dawley rats (250 ± 20 g; 12 weeks of age) came from the Shanghai Laboratory Animal Center (Shanghai, China) and were housed in a specific pathogen‐free facility at 21 ± 2°C with 55 ± 2% humidity. All animals were given 1 week to acclimate, after which they were randomized into one of four groups (n = 12/group): a control group, a DN group, a DN+lentivirus (LV) negative control (NC) group, and a DN+LV‐miR‐218 group. We established a DN model system in these rats received a single intraperitoneal injection of 50 mg/kg STZ which was dissolved in 0.1 M citrate acid buffer (Sigma‐Aldrich), and then assessing levels of glucose in the blood of these animals 3 days later. If animals had a blood glucose level >16.7 mmol/L, the DN model system was deemed successful. Animals in the two lentiviral groups were injected with an appropriate adenovirus expressing miR‐218 or a control sequence (100 µl volume, 1 × 10⁵ IU/µl). Twelve weeks later, rats were killed, and kidney tissue was isolated for analysis.

2.2. Podocyte treatment

We purchased an immortalized murine podocyte cell line from The Cell Culture Center (PUMC, CAMS, Beijing, China), growing these cells in Roswell Park Memorial Institute‐1640 containing 10% fetal bovine serum (Gibco, Grand Island, NY), 5.6 mM glucose, 10 U/ml murine interferon‐γ (Sigma‐Aldrich), and penicillin/streptomycin according to the previously experimental protocols (Mundel et al., 1997). Lipofectamine 2000 (Invitrogen, Carlsbad, CA) was used to transfect cells with miR‐218 mimics or appropriate controls. To establish an HG stimulation model in these podocytes, cells were incubated without serum for 12 hr, after which they were then treated with either normal glucose (con, 5.6 mM) or HG (30 mM) levels as appropriate.

2.3. Biochemical analyses

Following sacrifice of study animals, we collected blood from the abdominal aorta. This blood was allowed to clot with anticoagulant for 2 hr, after which it was spun at 2,000g for 10 min and the supernatant plasma was collected for measurement of blood glucose (F006‐1‐1; Jiancheng Biotech, Nanjing, China), serum creatinine (C011‐2‐1; Jiancheng Biotech), and blood urea nitrogen (C013‐2‐1; Jiancheng Biotech) levels based on commercially available kits. We also measured protein levels in the blood via a Bradford Protein Assay Kit (Bio‐Rad).

2.4. Cell Counting Kit‐8 assay

We incubated podocytes overnight in 96‐well plates. We then added 100 μl of cell counting kit (CCK‐8; 96692; Sigma) reagent to each well after 24, 48, and 72 hr, and analyzed samples via microplate reader (Bio Tek Instruments) after 4 hr.

2.5. 5‐Ethynyl‐2′‐deoxyuridine proliferation assay

To assess cell proliferation, we treated transfected cells (48 hr posttransfection) using 50 μM 5‐Ethynyl‐2′‐deoxyuridine (EdU). We then utilized Apollo staining as well as 4′,6‐diamidino‐2‐phenylindole to identify EdU‐positive cells via fluorescence microscopy.

2.6. Apoptosis analysis

To assess apoptotic cell death, we dual‐stained podocytes using a FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA) and analyzed the stained cells via a FACScan flow cytometer (BD Biosciences).

2.7. Real‐time reverse transcription‐polymerase chain reaction

TRIzol (Invitrogen) was used for the extraction of podocyte RNA, which then underwent quantification using a NanoDrop 2000/2000c machine (Thermo Fisher Scientific). Next, 1 µg of extracted RNA from each sample was used in a reverse transcription assay to yield complementary DNA (cDNA) via a high capacity cDNA Reverse Transcription Kit (TaKaRa, Tokyo, Japan). We then used the SYBR Premix ExTaq II Kit (TaKaRa) and appropriate gene‐specific primers to conduct quantitative PCR assays with Applied Biosystems 7900 Real‐Time PCR instrument (Applied Biosystems). The $2^{-\mathrm{\Delta }\mathrm{\Delta }{C}_{\mathrm{t}}}$ method was used to determine relative gene expression, normalizing target genes to β‐actin.

2.8. Cytokine enzyme‐linked immunosorbent assays

We homogenized kidney samples and podocytes, spun homogenates at 9000g for 30 min at 4°C, and then measured homogenate or supernatant levels of IL‐1β (ab100704), IL‐6 (ab100712), TNF‐α (ab208348), and MCP‐1 (ab100721) with appropriate ELISA Kits (Abcam).

2.9. Western blotting

Protein samples were isolated and then separated them via electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels followed by transfer onto polyvinylidene fluoride membranes (Millipore). Membranes underwent blocking with 5% nonfat dry milk and were then probed overnight using appropriate primary antibodies followed by the use of a secondary HRP‐conjugated secondary antibody. Western blotting detection reagents were then used to identify immunoreactive protein bands on these blots. The primary antibodies are list as following: β‐actin (#3700), IKK‐β (#8943; Cell Signaling Technology), cleaved caspase‐3 (ab13847), Bcl‐2 (ab32124), p‐p65 (ab86299; Abcam), and p65 (sc71675; Santa Cruz Biotechnology).

2.10. miRNA target prediction

We used miRDB (http://www.mirdb.org/), miRanda (http://www.microrna.org/), TargetScan (http://www.targetscan.org/), and Starbase (http://starbase.sysu.edu.cn/) for predicting miR‐218 target mRNAs.

2.11. Luciferase reporter assay

We synthesized wild‐type (WT) or mutant (MUT) IKK‐β sequences with normal or disrupted miR‐218 binding, and then cloned these constructs into the pGL3 basic vector (Promega, Madison, WI). We then plated cells in 12‐well plates overnight, before transfecting them with miR‐218 mimics as well as 10 μg pLUC‐WT‐IKK‐β or pLUC‐MUT‐IKK‐β and then using a dual‐luciferase reporter assay system (Promega) to measure subsequent luciferase activity.

2.12. Statistical analysis

GraphPad Prism 6.0 (Graphpad Software Inc.) was used for all statistical analyses. Data are means ± standard deviation from triplicate experiments. Student's t test and one‐way analysis of variance were used to compare results, with p < .05 as the threshold of significance.

3.1. miR‐218 downregulation and IKK‐β upregulation are evident in DN models

We began by measuring the levels of miR‐218 and IKK‐β in DN model rats to explore the relationship between these two molecules and disease progression. Our finding showed that miR‐218 expression was decreased in DN model rats (Figure 1a), whereas there was a corresponding increase in the mRNA levels of IKK‐β (Figure 1b,c). To extend these observations in vitro, we treated murine podocytes with a 30 mM HG stimulation and assessed miR‐218 dynamics overtime. We found that there was both a time‐dependent decrease in miR‐218 in these cells and a corresponding time‐dependent increase in IKK‐β mRNA levels (Figure 1d–f). Together these results suggest a potential link between miR‐218, IKK‐β, and DN progression.

3.2. Overexpression of miR‐218 was associated with reduced kidney damage in response to STZ

To formally confirm the relevance of miR‐218 to the progression of DN, we next injected rats intravenously with a LV that encoded miR‐218 or a control virus, leading to a significant increase in miR‐218 expression in rats injected with the LV‐miR‐218 construct (Figure 2a). We found that DN model rats had significantly reduced body weights relative to control animals, whereas miR‐218 overexpression significantly increased the body weight of these animals after 12 weeks (Figure 2b). We further found that DN model rats exhibited elevated levels of blood glucose, blood urea nitrogen, serum creatinine, and total protein in the blood, while miR‐218 overexpression reduced all these parameters (Figure 2c–f). Together, these results thus confirm that miR‐218 could ameliorate renal injury in STZ‐induced DN rats.

3.3. miR‐218 overexpression impaired HG‐induced apoptosis and growth inhibition of mouse podocytes in vitro

To explore how miR‐218 influences podocyte growth in vitro, we next transfected these cells with miR‐218 mimics or control constructs, leading to significantly elevated miR‐218 levels in appropriately transfected cells (Figure 3a). We next used CCK‐8 assays to assess the survival of these cells, revealing that HG impaired cell survival, whereas miR‐218 transfection was able to boost survival (Figure 3b). In addition, in an EdU‐based proliferation assay, we found that HG levels impaired podocyte proliferation, while miR‐218 transfection was able to overcome this impairment (Figure 3c). These results thus suggest that miR‐218 overexpression is linked to enhanced podocyte proliferation.

We further performed analyses of the apoptotic death of podocytes under HG treatment conditions, revealing that HG treatment induced substantial podocyte apoptosis, while miR‐218 transfection substantially attenuated this phenotype (Figure 3d). This was consistent with the results of western blotting experiments, revealing elevated cleaved caspase‐3 levels in HG‐treated cells, as well as decreased Bcl‐2 levels in these same cells, whereas miR‐218 transfection reversed both of these results (Figure 3e). Together, these findings suggest that miR‐218 was able to suppress the apoptotic death of podocytes induced in response to HG levels.

3.4. miR‐218 reduced the inflammation triggered by STZ in vivo and by glucose in vitro in podocytes

To explore how miR‐218 influences the expression of inflammatory cytokines in our DN model rats, we measured renal cortical levels of IL‐6, IL‐1β, MCP‐1, and TNF‐α by ELISA, revealing these proteins to be highly expressed in DN model animals, whereas this expression was reduced when rats were injected with the miR‐218 expressing LV (Figure 4a–d). This result suggests that overexpressing miR‐218 can reduce inflammatory cytokine production in DN models of rats.

We further sought to assess how miR‐218 influences inflammatory cytokine production in our in vitro podocyte model system. Consistent with our in vivo findings, levels IL‐6, IL‐1β, MCP‐1, and TNF‐α were elevated in cells treated with high levels of glucose, and transfection of these cells with miR‐218 mimics significantly reduced the expression of these cytokines (Figure 4e–h). This thus confirms that overexpressing miR‐218 can inhibits the inflammatory response induced by HG levels in podocyte cell line.

3.5. miR‐218 inhibited NF‐κB pathway activation both in vitro and in vivo

Given that the IKK‐β/NF‐κB pathway is known to play a key role in activating and regulating inflammatory responses, we next wanted to assess whether the protective effects of miR‐218 that we observed in our model systems were linked to regulation of this pathway. We therefore measured the IKK‐β and NF‐κB p65 levels in our DN rat model before or after miR‐218 administration, revealing that both total IKK‐β (Figure 5a,b) and the p‐p65/p65 ratio (Figure 5a,c) were significantly increased in DN rat model relative to control animals, whereas miR‐218 overexpression significantly reduced these inflammatory parameters, leading to reduced p65 phosphorylation and IKK‐β expression (Figure 5a–c). Together, these results suggest that miR‐218 is thus able to interfere with the IKK‐β/NF‐κB pathway activation in a rat model of STZ‐induced DN.

We further assessed how miR‐218 influences the IKK‐β/NF‐κB pathway in our podocyte HG treatment model system. We found that IKK‐β levels (Figure 5d,e), as well as the p‐p65/p65 ratio (Figure 5d,f) were significantly increased in podocytes following HG treatment, and transfection with the miR‐218 mimic significantly reduced these levels suggesting that miR‐218 is able to disrupt the activation of this IKK‐β/NF‐κB pathway in podocytes treated with HG concentrations.

3.6. miR‐218 directly targets the IKK‐β mRNA

We next used available pathway databases to predict potential miR‐218 targets, which led to the identification of IKK‐β as one such predicted target. We, therefore, constructed WT IKK‐β and MUT IKK‐β (without the requisite miR‐218 binding sequence) plasmids (Figure 6a), and cotransfected these into cells along with miR‐218 mimics. We observed decreased reporter activity when cells were cotransfected with the WT construct and miR‐218 (Figure 6b), and found IKK‐β to also be inhibited by miR‐218 in these same cells (Figure 6c,d). These results thus confirmed that IKK‐β was a direct miR‐218 target.