MiR‐802 causes nephropathy by suppressing NF‐κB‐repressing factor in obese mice and human

Abstract Obesity is associated with significant microvascular complications including renal injuries and may induce end‐stage renal disease. Emerging studies have demonstrated microRNAs (miRNAs) are potential mediators in the pathophysiological process of nephropathy. The present study aimed to investigate the role of miR‐802 in obesity‐related nephropathy and potential molecular mechanisms. Through utilizing obese mouse model and human subjects, we explored the therapeutic benefits and clinical application of miR‐802 in protecting against nephropathy. Renal miR‐802 level was positively correlated with functional parameters, including blood urea nitrogen and creatinine in obese mice. Specific silencing of renal miR‐802 improved high fat diet (HFD)‐induced renal dysfunction, structural disorders and fibrosis. The up‐regulated inflammatory response and infiltrated macrophages were also significantly decreased in miR‐802 inhibitor‐treated obese mice. Mechanistically, miR‐802 directly bond to 3ʹ‐UTR of NF‐κB‐repressing factor (NRF) and suppressed its expression. In clinical study, the circulating miR‐802 level was significantly increased in obese subjects, and positively correlated with plasma creatinine level but negatively correlated with creatinine clearance. Taken together, our findings provided evidence that miR‐802/NRF signalling was an important pathway in mediating obesity‐related nephropathy. It is a possible useful clinical approach of treating miR‐802 inhibitor to combat nephropathy.

chronic kidney disease (CKD) and end-stage renal disease (ESRD). 4 However, the molecular changes of obesity-related nephropathy are complicated. To protect against obesity-associated nephropathy, it is urgent need to explore the underlying molecular mechanism.
Inflammatory response is the main phenotype of obesity-related nephropathy. Excess nutrition, especially saturated fatty acids and its metabolites, injures the renal structure and function. Toxic-free fatty acids can bind to toll-like receptors (TLRs) resulting in the activation of IκB kinase/NF-κB (IKK/NF-κB) signalling pathways, which up-regulate the synthesis and secretion of chemokines, leading to the infiltration of pro-inflammatory immune cells. 5,6 Meanwhile, the obesity-down-regulated anti-inflammatory signalling, such as NF-E2-related factor-2 (Nrf2) and NF-kappa-B-repressing factor (NRF), further exaggerates the inflammatory response. 7,8 Many findings also support renal inflammation mainly contributes to obesity-associated nephropathy, whereas immunosuppressive strategies attenuate the development of diabetic nephropathy. 6,9,10 Elevated circulating levels of inflammatory cytokines are positively correlated with renal injuries. 11 Therefore, better controlling of inflammatory response is critical for suppressing the pathphysiological process of obesityrelated nephropathy.
MicroRNAs (miRNAs) are small non-coding RNAs that are increasingly recognized as critical players in gene regulation and various diseases. Interestingly, a cluster of miRNAs is reported to be involved in the inflammatory process. 12 Among them, recent studies show that several inflammation-associated miRNAs are highly expressed in kidney. Renal glomerular miR-192 regulates the process of diabetic nephropathy via inhibition of E-box repressors. 13 Renal miR-146a positively correlates with the development of chronic renal inflammation and dysfunction. 14 MiR-21 promotes renal inflammation and fibrosis in diabetic mice. 15 These findings support renal miRNAs are new promising biomarkers for diagnosis and therapeutic targets in nephropathy. MiR-802, an emerging important mediator, is also increased in renal tissues of obese mice. 16 The upregulation of inflammatory response also induces miR-802 expression, and promotes cell proliferation in cholesteatoma. 17 MiR-802 participates in palmitate acid-induced damage to pancreatic β cells through repression of sirtuin 6. 18 Up-regulation of miR-802 induces apoptosis of keratinocytes in oral lichen planus. 19 However, whether miR-802 participates in the process of obesity-associated nephropathy or the underlying molecular mechanism is still unclear.
Present study aimed to explore the potential role of miR-802 in obesity-associated nephropathy through high fat diet-fed mouse model and human subjects. Here, we not only found a close correlation between miR-802 and nephropathy parameters, but also suppression of miR-802 could effectively improve cellular inflammatory response and renal function in obese mice. Mechanistically, NRF, an NF-κB suppressor, was a potential direct target gene of miR-802 in mediating renal disorders. Present study comprehensively provided the pathophysiological and clinical role of miR-802 in obese-associated nephropathy.

| Reagents
The biochemical assays for measuring blood urea nitrogen (BUN) and creatinine were purchased from Sigma chemicals (Sigma, St. Louis, USA). The lentivirus encoding miR-802 sponge or control sponge was gifted from Dr Xiang Shen (Zhengzhou University).

| Animal experiment
Six-week-old male C57BL/6J mice were randomly assigned to normal chow (NC) or high fat diet (HFD, Cat#D12492, Research diets) for 16 weeks. Another set of animal study included obese mice fed high fat diet for 12 weeks were locally treated with 1 × 10 9 lentivirus particles encoding miR-802 sponge or control sponge by ultrasound-microbubbles. 20 Briefly, the lentivirus particle was mixed with Optison (Mallinckrodt, St. Louis, MO) in 50% v/v ratios, and injected into the renal artery. Ultrasound transducer (Sonitron 2000, NEPA GENE, Co.) exposed directly onto one side of the kidney with a continuous wave output of 1 MHz ultrasound for 1 minute. The infusion cannula is then removed, and the wound closed. These mice were fed HFD for another 4 weeks. Then, the mice were sacrificed, the serum and kidneys were collected for further analysis. The experimental procedure described here was approved by the Institutional Animal Use and Care Committee at the Wenzhou University (Wenzhou, China).

| Renal histological analysis
Mouse kidneys were fixed in 4% paraformaldehyde for 24-hour and embedded in paraffin. Paraffin sections of 5 µm were prepared and stained with haematoxylin and eosin solution or Sirius red staining kit. For immune histological for renal macrophages, slides were processed with antigen retrieval and 3% bovine serum albumin (BSA) blocking. Then, the slides were stained with anti-CD68 antibody, secondary antibody and DAB HRP substrate for label-positive stained cells. To measure the histological changes, the renal images were observed under a light microscope (400× amplification, Nikon, Tokyo).

| Total RNA extraction, cDNA synthesis, reverse transcription and real-time PCR
The total RNA was homogenized in TRIzol and isolated from mouse Small nucleolar RNA 202 (Sno202) was used as an internal control for comparison of relative changes in miRNA.

| NRF gene 3ʹ UTR luciferase reporter assay
To generate NRF gene 3ʹ UTR luciferase reporter constructs, the miR-802-binding sites were synthesized by annealing the oligos: NRF 3ʹUTR forward: CTTCTTAATGCTTTCACCCCTCCGAA CACACACCG; reverse: CTAATTGTGCAGGTACAGGAATTGTTCCA CCAGCATTAATA. The products were ligated into the pMIR-REPORT vector (Ambion). To create a mutant 3ʹ UTR, mutations were introduced at two miR-802-seeding sequence regions with the following sites: CA were changed to GC, and AGG were changed to GCC. HEK-293T cells were transfected with one of the above plasmids using PEI (Polyplus) according to the manufacture's instruction. Luciferase activity was measured using the Dual-Luciferase Reporter Assay (Promega). Data are presented as ratio of renilla to firefly luciferase activity.

| Human anthropometric and biological measurement
The following data of all participants were collected from medi-

| Statistical analysis
Data were presented as mean ± SEM. The Students' t test was used for comparing two groups, and one-way ANOVA was used for comparing four groups. GraphPad Prism 7 (GraphPad, San Diego, CA) was used to analyse the statistical significance between sets of data.
Differences were considered to be significant at P < 0.05.

| Elevated expression of renal miR-802 is positively associated with obese mouse renal functional parameters
Previous study has showed miR-802 participated in the development of metabolic diseases, 16 but whether miR-802 involved in renal pathophysiology is still unknown. First, we measured the expression of miR-802 in different tissues, including heart, visceral adipose tissue, brain and kidney. As Figure 1A showed, high fat diet (HFD) obviously increased miR-802 level in kidney (P < 0.001).
Blood urea nitrogen (BUN) and creatinine are key markers for diagnosing nephropathy. 21 We then measured serum levels of

| Renal miR-802 inhibitor effectively improves high fat diet-induced nephropathy
To determine the pathophysiological role of miR-802 in dietinduced renal injuries, we utilized ultrasound-based microbubble carrying lentivirus delivery method to silence renal miR-802 and investigated the changes of renal function and structure.
The synthesized inhibitor, miR-802 sponge, did not change the body weight ( Figure 2A) and fasting blood glucose levels ( Figure 2B), as compared with control (Ctrl) sponge treatment.
However, miR-802 sponge decreased the gain of kidney weight in obese mice ( Figure 2C, P < 0.05). MiR-802 sponge also effectively inhibited serum levels of BUN ( Figure 2D, P < 0.01) and creatinine ( Figure 2E, P < 0.001), as compared with Ctrl sponge-treated obese mice. Abnormal glomerular enlargement and fibrosis are characters of nephropathy. 22 The results of renal H&E staining Severe inflammatory response initiates and further deteriorates renal injuries in obese status. 11 Real-time PCR results showed that HFD obviously increased gene levels of several inflammatory factors, including TNF-α, IL-6, iNOS and MCP-1 ( Figure 3A). Treatment of miR-802 sponge effectively suppressed the mRNA levels of these inflammatory factors. NF-κB signalling is the most important pathway to regulate inflammatory response in metabolic diseases. 5,6 As Figure 3B,C showed, miR-802 sponge could inhibit the phosphorylation and degradation of IκB in obese mice, as compared with Ctrl sponge-treated obese mice (P < 0.01). MiR-802 sponge also decreased the translocation of p65 and p50 to nuclear, as compared with Ctrl sponge-treated obese mice ( Figure S1). The local inflammatory cytokines also recruit macrophage infiltration and amplify the inflammatory response. 11 As showed Figure 3D,E, HFD stimulated CD68 + macrophage infiltration into renal tissues, whereas miR-802 sponge significantly blocked macrophage accumulation (P < 0.01).

| MiR-802 regulates renal inflammatory response through directly suppressing NF-κBrepressing factor
We next wanted to find out the direct target of miR-802 in mediating inflammatory response. Previous studies have reported the activation of IκB is mainly controlled by IκB kinase (IKK) and NF-κB-repressing factor (NRF). 23,24 To this end, we utilized dual-luciferase reporter assay to determine their interaction to miR-802. Our showed that miR-802 did not inhibit the luciferase density of IKK 3ʹ-UTR ( Figure 4A), but significantly suppressed the luciferase activity of NRF 3ʹ-UTR ( Figure 4B, P < 0.001). These findings indicated NRF was a direct target of mir-802.
Furthermore, we overexpressed miR-802 level in mouse mesangial cells (Figure 4C), and western blot analysis showed that overexpression of miR-802 almost completely suppressed protein level of NRF ( Figure 4D). In obese mice treated with miR-802 sponge, miR-802 inhibitor also significantly increased NRF gene level ( Figure 4E) and protein level ( Figure 4F,G). Above results strongly supported miR-802-induced renal inflammatory response and injuries through suppressing NRF in kidney.

| Evaluated circulating miR-802 is positively correlated with renal functional parameters in human subjects
To explore the clinical application of miR-802 in diagnosing renal dysfunction in individuals, we collected plasma samples from 25 lean (BMI ≤ 23) and 20 obese (BMI > 28) human subjects. The clinical characteristics of the human subjects according to BMI categories were summarized in Table S1. As expected, obese subjects had significantly more adverse metabolic profiles including abnormal lipid profiles, hyperglycaemia and impaired insulin sensitivity.
Then, we measured and compared the circulating levels of miR-802 in lean and obese subjects. As showed in Figure 5A, plasma from obese subjects had higher level of miR-802 than from lean subjects (P < 0.01), which was consistent with mouse renal miR-802 level. Then, we measured plasma creatinine levels and calculated the creatinine clearance (Ccr) in these subjects. As Figure 5B,C showed, plasma miR-802 was positively correlated with creatinine levels ( Figure 5B; r = 0.5781, P < 0.001), but negatively correlated with Ccr ( Figure 5C; r = −0.6863, P < 0.001). E-G. Six-week-old male C57BL/6J mice were fed normal chow (NC) or high fat diet (HFD) for 12 wk. 1.2 × 10 9 lentivirus particles encoding miR-802 sponge or control sponge were delivered into renal tissue by ultrasound-based microbubbles for 4-wk. Real-time PCR analysis of NRF mRNA level (E), western blot analysis of NRF expression in renal tissues (F) and quantitative analysis of relative protein density (G) (n = 6). Significance was assessed by ANOVA test (A-B, E, G) and Students' t test (C). Data are shown as mean ± SEM (**P < 0.01 and ***P < 0.001)

| D ISCUSS I ON
Increasing evidence shows that obesity-induced renal inflammation and its consequent structural disorders are a critical process leading to end-stage of nephropathy. Present study demonstrated that suppression of renal miR-802 protected against the progressive renal injury through down-regulation of renal inflammatory response in obese mice. Mechanistically, our findings also supported the protective role of miR-802 inhibitor on nephropathy might be associated with the suppressive effect NF-κB activity by its suppressing factor NRF. In human study, we also found the levels of miR-802 were closely correlated with clinical parameters of nephropathy.
The pathogenesis of obesity-related complications is characterized by activation of complex molecular pathways, and emerging evidence now suggests that inflammatory pathways have a central role in the development of these diseases, such as obesity-related nephropathy. One of the major signalling pathways implicated in this inflammatory reaction is nuclear factor-κB (NF-κB), a transcription factor that is activated by many stimuli relevant to obesity-related nephropathy, such as pro-inflammatory cytokines and mechanical forces. In turn, activation of NF-κB stimulates the synthesis and production of inflammatory molecules. 11 Inhibition of tumour necrosis factor (TNF)-α improved markers of glomerular and tubulointerstitial injury with diabetic nephropathy. 25 Abnormalities of interleukin (IL)-6 expression altered glomerular endothelial cell permeability, mesangial cell proliferation and expression of fibronectin. 26 Local infiltration of immune cells, especially macrophages, exacerbated inflammatory response in the process of nephropathy. 11 Mechanistically, the accumulated macrophages and inflammatory cytokines damaged endothelial cell permeability, metabolic homeostasis and structure. Consistently, present study also found the abnormal inflammatory activation in obese mice, but suppression of miR-802 effectively improved high fat diet-induced renal dysfunction in mice. ing sites. 24 Then, lots of studies continued to explore the biological roles of NRF in different molecular signalling. NRF participated in basal repression of pro-inflammatory cytokine IL-1-induced activation of IL-8. 29 NRF was a negative mediator of inducible nitric-oxide synthase (iNOS) in human A549 and HeLa cells. 8 In severe COPD patients, there existed significant reduction of NRF expression, but enhanced NF-κB activation. 30 Consistently, present study also found the renal level of NRF was obviously decreased, but the inflammatory response was increased in obese mice. Luciferase assay also determined 3ʹ-UTR of NRF was a direct binding site for miR-802, which firstly provided the links between miRNAs and NRF.
A cluster of studies have reported miR-802 was involved in the tissue dysfunction. MiR-802 was identified as an important mediator in controlling different pathological process. Intestinal miR-802 affected the biological efficacy of angiotensin II in gastrointestinal system. 31 In obesity, up-regulated hepatic miR-802 impaired glucose metabolism through silencing of Hnf1b. 16 In type 2 diabetic patients, the circulating miR-802 was also significantly increased and closely correlated with metabolic parameters. 32 These studies also addressed miR-802 and inflammatory response had a positive feedback in the development of tissue dysfunction. Furthermore, a multitude of miRNAs have also been found to be associated with the process of renal diseases. The down-regulation of circulating miR-130b might be involved in the pathological development of diabetic nephropathy through inducing lipid metabolic disorders, oxidative stress, extracellular matrix deposition and renal fibrosis. 33 Circulating miR-217 was positively associated with the levels F I G U R E 5 Circulating miR-802 level is closely correlated with renal functional parameters in human subjects. Plasma from 25 lean (BMI ≤ 23) and 20 obese (BMI > 28) individuals were collected and subjected to analysis. A, Plasma level of miR-802. Significance was assessed by Students' t test. B-C. Correlation between plasma miR-802 level and creatinine (B) and creatinine clearance (Ccr, C). Correlation was assessed by non-parametric Spearman's test. Data are shown as mean ± SEM (**P < 0.01) of proteinuria in type 2 diabetes patients. 34 Besides, one study also identified several urea miRNAs had the capability of significantly differentiating patients with acute kidney disease (AKI) from individuals without AKI. 35 All these findings supported miRNA is a useful clinical biomarker of nephropathy. More importantly, renal miR-802 could mediate potassium channel activity through suppressing caveolin-1 expression. 36 In present study, we found up-regulation of miR-802 closely associated with obesity-induced renal dysfunction, including structural disorders, fibrosis and accumulated inflammatory response. Furthermore, we firstly demonstrated that serum levels of miR-802 were significantly increased in obese subjects with renal dysfunction.
However, there were still several limitations in present study. Yan and Chi Zhang discussed the study and edited this manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest.