MicroRNA‐26a alleviates tubulointerstitial fibrosis in diabetic kidney disease by targeting PAR4

Abstract Our previous study found that miR‐26a alleviates aldosterone‐induced tubulointerstitial fibrosis (TIF). However, the effect of miR‐26a on TIF in diabetic kidney disease (DKD) remains unclear. This study clarifies the role and possible mechanism of exogenous miR‐26a in controlling the progression of TIF in DKD models. Firstly, we showed that miR‐26a was markedly decreased in type 2 diabetic db/db mice and mouse tubular epithelial cells (mTECs) treated with high glucose (HG, 30 mM) using RT‐qPCR. We then used adeno‐associated virus carrying miR‐26a and adenovirus miR‐26a to enhance the expression of miR‐26a in vivo and in vitro. Overexpressing miR‐26a alleviated the TIF in db/db mice and the extracellular matrix (ECM) deposition in HG‐stimulated mTECs. These protective effects were caused by reducing expression of protease‐activated receptor 4 (PAR4), which involved in multiple pro‐fibrotic pathways. The rescue of PAR4 expression reversed the anti‐fibrosis activity of miR‐26a. We conclude that miR‐26a alleviates TIF in DKD models by directly targeting PAR4, which may provide a novel molecular strategy for DKD therapy.

and knockdown of miR-21 mitigates fibrosis in DKD. 8 Another research illustrates that miR-30 and miR-146a serve as antifibrotic roles in DKD. 6These data suggest that targeting specific miRNAs could be a promising therapeutic strategy for renal fibrosis in DKD.
Our previous study found that miRNA-26a expression levels are diminished in both heart and kidney tissues of CKD mice, and exogenous application of miR-26a depressed renal fibrosis in CKD mice by targeting connective tissue growth factor (CTGF). 9,10Of note, we also revealed that exogenous miR-26a can ameliorate ALD-induced TIF. 11Despite this, the role miR-26a on TIF of DKD remains unclear.
We used bioinformatics analysis to screen the target mRNAs of miR-26a and found that miR-26a has potential binding sites in the 3′-UTR of protease-activated receptor 4 (PAR4).As a subfamily of Gprotein-coupled receptors, PAR4 has been reported to be involved in the regulation of various diseases, [12][13][14][15] but its contribution to kidney diseases remaining unknown.In the current study, we analysed the expression of miR-26a in DKD models and further investigated that miR-26a improves TIF in DKD via silencing PAR4.Our results will provide new insights into the pathogenesis and treatment of DKD.

| Animal and treatment
Eight-week-old male C57BLKS/J Lepr background db/db mice (40-43 g) were used as a type 2 diabetes model, and their heterozygous 8-week-old male db/m mice (19-21 g) served as controls.Mice were purchased from GemPharmatech LLC (Nanjing, China) at the same time and were fed adaptively at 23 ± 2°C, 55% ± 5% relative humidity, with 12-h of light and 12-h of darkness under specific pathogenfree conditions.The db/db mice developed hyperglycemia from about 8 weeks of age, and the blood glucose was increased to more than 28.6 ± 13.2 mM at 20 weeks of age, 16 accompanied by deterioration of renal function，renal hypertrophy, diffuse expansion of mesangial matrix and TIF. 17 Therefore, db/db mice are a classic tool for studying human DKD.At 12 weeks of age, adeno-associated virus miR-26a (AAV-miR-26a, GenePharma, Shanghai, China) was used to overexpress miR-26a, and adeno-associated virus GFP (AAV-GFP, GenePharma, Shanghai, China) was used as a negative control (NC) in mice.The db/m mice were randomly divided into the following groups (n =

| Cell culture and transfection
Mouse tubular epithelial cells (mTECs) were gifted by Zhongda Hospital Southeast University.Human embryonic kidney (HEK) 293T cells were purchased from Procell Life Science &Technology Co., Ltd (Wuhan, China) for luciferase binding experiments.Both cell lines were cultured in Dulbecco's modified eagle medium (DMEM) containing 10% foetal bovine serum (FBS) at 37°C in 5% CO 2 .The mTECs in high glucose (HG) group were treated with 30 mM glucose for 24 h, 36 h or 48 h, while the cells in normal glucose (NG) group were treated with 5 mM glucose.

| Real-time quantitative PCR (RT-qPCR)
Total RNAs (including mRNA and microRNA) was purified from kidney and mTECs using TRIzol® reagent (Life Technologies; Agilent, Inc., USA).RT-qPCR was performed and analysed as described previously. 19The primers sequences are shown in Table 1.The thermocycling conditions were presented in Tables 2 and 3.

| Statistical analysis
All data analyses were assessed with the GraphPad Prism 9.3.1 software (GraphPad Software, Inc., California, USA).The data are presented as mean ± SD of at least three independent experiments.The data are analysed using unpaired Student's t-test (two groups) and one-way ANOVA (three or more groups), followed by a Tukey's post hoc test.
p-value <0.05 were considered to indicate a significant difference.

| Expression of miR-26a decreased and TIF aggravated in diabetic mice
To explore the role of miR-26a in TIF of DKD, a type 2 diabetic model with db/db mice was first established to simulate human diabetic kidney disease.As shown in Figure 1A, 24 h-UTP and serum creatinine (Scr) levels were markedly increased in db/db mice at 20 weeks of age.
Histologically, masson staining showed an increase in tubulointerstitial collagen expression in db/db group (Figure 1B).Additionally, the expression of the fibrotic markers COL I, α-SMA and FN were upregulated in the kidneys of db/db group on immunofluorescence (Figure 1C) and immunohistochemistry (Figure 1D).Consistently, Western blot (Figure 1E) and RT-qPCR (Figure 1F,G) assays depicted that the tubular injury marker KIM-1 and fibrotic markers (COL I, α-SMA and FN) were higher in the kidneys of db/db group.Furthermore, we observed that the miR-26a level in the kidney tissue of db/db mice considerably decreased contrast with db/m group (Figure 1H).These results suggest that the reduced miR-26a expression may contribute to TIF in DKD.

| Expression of miR-26a decreased and ECM deposition increased in mTECs under HG condition
Given that tubular epithelial cells (TECs) play a key role in the process of TIF in DKD, 5  Therefore, 48 h of HG treatment was conducted thereafter.On Western blot, the expression of KIM-1, FN, COL I and a-SMA were increased in the HG group compared with the NG group (Figure 2C).
These data suggest that HG-induced ECM deposition in mTECs may be related to downregulated miR-26a expression levels.

| Overexpression of miR-26a alleviated TIF in diabetic mice
To further investigate the effect of ectogenous miR-26a on TIF in DKD, db/db mice were injected with AAV-miR-26a (le + 12 vg) at 12 weeks of age and were sacrificed at 20 weeks of age.The exogenously added AAV-miR-26a replenished miR-26a in the kidney of the db/db mice (Figure 3A).Elevated Scr and 24h-UTP levels in db/db mice were significantly ameliorated by AAV-miR-26a treatment (Figure 3B).Further, masson staining showed a reduction in tubulointerstitial collagen expression in db/db + miR-26a group (Figure 3C).In addition, immunofluorescence revealed that COL I and a-SMA was remarkably attenuated by miR-26a overexpression in dbdb mice (Figure 3D).Moreover, the expressions of KIM-1, FN, COL I and α-SMA were markedly reversed by AAV-miR-26a treatment in db/db mice as shown by RT-qPCR (Figure 3E), consistent with the Western blot results (Figure 3F).
The above results demonstrated that exogenous miR-26a inhibited TIF in DKD.

| Overexpression of miR-26a reduced HG-induced ECM deposition in mTECs
To characterize the biological functions of exogenous miR-26a in ECM accumulation, mTECs were treated with Ad-miR-26a to enhance the expression of miR-26a (Figure 4A,B).Furthermore, as shown by the results of RT-qPCR (Figure 4C) and Western blot (Figure 4D), Ad-miR-26a could dramatically inhibit the expression of KIM-

| Elevated PAR4 in mice and mTECs under diabetic condition
With the attempt to explore the molecular mechanism of the antifibrotic activity of miR-26a, we used bioinformatics databases (TargetScan and miRDB) to predict the target genes of miR-26a and found that there existed the presence of binding sites between miR-26a and the 3′UTR of PAR4 (Figure 5A).Considering that PAR4 was reported to be involve in the fibrosis process in various organs, 20 we hypothesized that PAR4 might be a potential target of miR-26a.
As expected, RT-qPCR (Figure 5B) and Western blot (Figure 5D) results revealed higher level of PAR4 in db/db group compared with db/m group.When miR-26a was overexpressed in db/db mice, PAR4 level was downregulated (Figure 5B,D).Consistently, PAR4 levels were increased in HG-induced mTECs (Figure 5C,E) and were decreased following the ectopic expression of miR-26a by Ad-miR-26a (Figure 5C,E).These data indicated that the increased PAR4 expression may be responsible for in DKD and be negatively regulated by miR-26a.

| DISCUSS ION
DKD is a serious complication of diabetes that results in kidney function loss.TIF correlates the best with DKD progression. 21The involvement of miRNAs in the progression of DKD has been proposed in previous studies. 22,23The present study demonstrates that ectogenic miR-26a effectively alleviated renal TIF in diabetic mice, and overexpression of miR-26a significantly ameliorated ECM deposition in mTECs via suppressing PAR4 expression (Figure 7).These findings may provide a promising approach for the treatment of TIF in DKD using exogenous miR-26a.
5][26][27][28] A previous study demonstrated that miR-26a-5p protects cardiomyocytes from damage caused by hypoxia/reoxygenation through the negative regulation of WNT5A via the Wnt/β-catenin signalling pathway. 24It was reported that miR-26a-5p directly targets ADAM17 to inhibit cardiac hypertrophy and dysfunction in mice with cardiac hypertrophy. 25R-26a enhances the tumorigenesis of colorectal cancer by decreasing the acetylation of RREB1 to activate the AKT signalling pathway. 26However, studies regarding the function of miR-26a in kidney disease are limited.Chen et al. discovered that miR-26a-5p play a protective role in septic acute kidney injury. 29In addition, our previous research discovered that miR-26a improved TGFβtriggered ECM accumulation by silencing CTGF and that miR-26a level was diminished in the glomeruli of diabetic mice. 10In this study, miR-26a expression showed a remarkable attenuation in the renal tissue of db/db mice and HG-treated mTECs.In addition, the overexpression of miR-26a alleviated renal tubular injury and renal dysfunction in db/db mice, highly suggesting a role of miR-26a in mediating the tubular function in DKD.
Growing evidence suggests that TIF is critic in the pathological progress of DKD.Recently, the emerging effect of miRNAs in the development of TIF has been highlighted.Specifically, miR-30e reduces the progression of epithelial-mesenchymal transdifferentiation in proximal tubular cell during TIF through blocking Snail, Slug and Zeb2 expression and rescuing E-cadherin expression. 30R-192 function as a TIF repressor in DKD by targeting Egr1 to promote TGF-β1 and FN degradation. 31miR-22 is upregulated in the kidney tissues of rats with DKD and exerts autophagyinhibiting and TIF-promoting effects by targeting phosphatase and tensin homologues. 32However, no biological effects of miR-26a on TIF in DKD have been reported previously.In this study, we observed that miR-26a overexpression protected diabetic mice from the development of TIF, with a remarkable drop in the expression of FN, COL I and α-SMA in vivo and in vitro.These results revealed that increased miR-26a levels may play a crucial role in ameliorating TIF in DKD.
Another interesting finding of our study was that miR-26a targets PAR4.PAR4 is expressed in several types of cells and is an important mediator that facilitates direct communication between proteases and intracellular signalling mechanisms.shown that appropriate doses of PAR4 antagonists decrease progressive pulmonary fibrosis. 38However, whether PAR4 mediate the progression of renal fibrosis remained to be explored.Here, we showed that miR-26a overexpression dramatically restrained PAR4 expression and then revealed that miR-26a downregulated PAR4 by directly binding to the 3′ UTR of PAR4 mRNA.
Furthermore, we found that PAR4 overexpression reversed the protective effect of exogenous miR-26a on ECM deposition in HG condition.Collectively, the above results demonstrated that upregulating miR-26a may inhibit the progression of TIF in DKD by targeting PAR4.
Nevertheless, there are still some limitations in this study.It remains to be established whether the similar results could be also observed in other cell lines or renal biopsies from patients with DKD.
Additionally, further investigation is required to illustrate the downstream molecular mechanism of miR-26a alleviating TIF in DKD.
In conclusion, the findings of our study is proposed for the first time that miR-26a alleviates TIF progression in DKD by targeting
we next established an in vitro model by stimulating mTECs with high glucose (HG, 30 mM glucose).RT-qPCR data depicted a descended miR-26a expression and ascended expressions of tubular injury marker KIM-1 and fibrosis-related proteins (FN, COL I and a-SMA) in mTECs treated with HG for 24 h, 36 h and 48 h (Figure 2A,B).The above-mentioned expression changed more significantly when the intervention time was 48 h.

F I G U R E 1
mir-26a expression was reduced and TIF was aggravated in DKD mice.(A) Scr and 24-h urine total protein in the mice at 20 weeks of age.n = 6 per group.(B) Representative images of Masson staining in the kidney tissue of mice.Scale bar, 50 μm.(C) Representative images of COL I (green) and α-SMA (red) expression assessed by immunofluorescence analyses in the kidney tissue of mice.DAPI (blue) was used to stain the nuclei.Scale bar, 50 μm.(D) Representative images of FN expression assessed by immunohistochemistry analyses in the kidney tissue of mice.(E) Western blot analyses of KIM-1, FN, COL I and α-SMA protein expressions in the kidney tissue of mice.(F) RT-qPCR analysis of KIM-1 in the kidney tissue of mice.(G) RT-qPCR analyses of FN, COL I and α-SMA in the kidney tissue of mice.(H) RT-qPCR analysis of miR-26a in the kidney tissue of mice.All data are expressed as mean ± SD; n = 6 in each group.*p < 0.05 versus db/m group.miR, microRNA; TIF, tubulointerstitial fibrosis; DKD, diabetic kidney disease; Scr, serum creatinine, COL I, collagen I; α-SMA, α-smooth muscle Actin; KIM-1, kidney injury molecule 1; FN, fibronectin; RT-qPCR, reverse transcription-quantitative PCR.

| 9 of 13 QU
et al. significantly counteracted the protective effect of miR-26a on HGinduced ECM deposition in mTECs, as evidenced by the increased levels of KIM-1, FN, COL I and α-SMA.The obtained data indicate that miR-26a may inhibit the progression of TIF in DKD by directly downregulating PAR4.

PAR4.
The current findings may not only provided new insights into the molecular mechanisms of TIF progression in DKD, but also opened new avenues for the treatment of renal fibrosis.AUTH O R CO NTR I B UTI O N S Gaoting Qu: Data curation (equal); investigation (equal); methodology (equal); validation (equal); writing -original draft (equal).Xingyue Li: Data curation (equal); methodology (equal).Ran Jin: Software (equal).Dian Guan: Methodology (equal); software (equal).Jialing Ji: Data curation (equal).Shanwen Li: Writing -review and editing (equal).Huimin Shi: Writing -review and editing (equal).Pingfan Tong: Formal analysis (equal).Weihua Gan: Conceptualization (equal); writing -review and editing (equal).Aiqing Zhang: Conceptualization (equal); project administration (equal); writing -review and editing (equal).This work was fully supported by National Natural Science Foundation of China (81970664), Natural Science Foundation of Jiangsu Province (BK20191082 and BK20211385), 789 Outstanding Talent Program of SAHNMU (789ZYRC202080119 and 789ZYRC202090251), Science and Technology Development Foundation of Nanjing Medical University (NMUB2020052).

F I G U R E 7
Schematic illustration of ectogenous miR-26a for the treatment of DKD.Exogenous miR-26a through the tail vein in mice ameliorate tubular injury and TIF by a mechanism that directly targets PAR4.