Melatonin ameliorates renal fibroblast‐myofibroblast transdifferentiation and renal fibrosis through miR‐21‐5p regulation

Abstract Fibroblast‐myofibroblast transdifferentiation (FMT) is widely recognized as the major pathological feature of renal fibrosis. Although melatonin has exerted antifibrogenic activity in many diseases, its role in renal FMT remains unclear. In the present study, the aim was to explore the effect of melatonin on renal FMT and the underlying mechanisms. We established the transforming growth factor (TGF)‐β1 stimulated rat renal fibroblast cells (NRK‐49F) model in vitro and unilateral ureteral obstruction (UUO) mice model in vivo. We assessed levels of α‐smooth muscle actin (α‐SMA), col1a1 and fibronectin, STAT3 and AP‐1, as well as miR‐21‐5p and its target genes (Spry1, PTEN, Smurf2 and PDCD4). We found that melatonin reduced the expression of α‐SMA, col1a1 and fibronectin, as well as the formation of α‐SMA filament in TGF‐β1‐treated NRK‐49F cells. Meanwhile, melatonin inhibited STAT3 phosphorylation, down‐regulated miR‐21‐5p expression, and up‐regulated Spry1 and PTEN expression. Moreover, miR‐21‐5p mimics partially antagonized the anti‐fibrotic effect of melatonin. For animal experiments, the results revealed that melatonin remarkably ameliorated UUO‐induced renal fibrosis, attenuated the expression of miR‐21‐5p and pro‐fibrotic proteins and elevated Spry1 and PTEN expression. Nevertheless, agomir of miR‐21‐5p blocked the renoprotective effect of melatonin in UUO mice. These results indicated that melatonin could alleviate TGF‐β1‐induced renal FMT and UUO‐induced renal fibrosis through down‐regulation of miR‐21‐5p. Regulation of miR‐21‐5p/PTEN and/or miR‐21‐5p/Spry1 signal might be involved in the anti‐fibrotic effect of melatonin in the kidneys of UUO mice.


| INTRODUC TI ON
Globally, chronic kidney disease (CKD) has gradually grown a primary economic burden in many countries and seriously threatens life quality of patients. The prevalence of CKD is predicted to be 8%-16% all over the world, even higher afterwards. 1 The present counter measure for remedying CKD mainly includes the control of systemic blood pressure, dyslipidaemia and hyperglycaemia. 2 Effective therapeutic strategies for CKD are still lacking in the clinical treatment. As a consequence, efforts to find more new therapeutic strategies that treat CKD or retard progression of CKD are urgently needed.
Renal fibrosis is a common pathological process from the progression of all CKD to end-stage renal disease. 3 As the main effector cells, myofibroblasts play an important role in the process of renal fibrosis, and its large accumulation in the renal interstitial is the main pathological feature of fibrosis. 4 During the development of progressive fibrosis, myofibroblasts appear to acquire smooth muscle cell characteristics, mainly expressing alpha-smooth muscle actin (α-SMA). Accordingly, α-SMA-positive myofibroblasts are closely related to interstitial extracellular matrix aggregation and fibrosis-induced tissue disruption. Extensive investigations suggest that myofibroblasts may generate from activated resident fibroblasts or pericytes 5 ; or perivascular fibroblasts, 6 or circulating bone marrow-derived fibrocytes 7 ; or epithelial and endothelial cells, which go through epithelial-to-mesenchymal transition (EMT) 8 and endothelial-to-mesenchymal transition (EndoMT), 9 respectively. Recent evidence indicated that resident renal fibroblasts tend to directly differentiate to form myofibroblasts under the pro-fibrotic stiff microenvironment, for example the stimulation of transforming growth factor (TGF)-β1. 10 Regardless of the origin of myofibroblasts, blocking the pathways that promote myofibroblast expansion should be an option worth considering in treating renal fibrosis.
It has been demonstrated that expression of miR-21 greatly increased during renal fibrogenesis. 11,12 Overexpression of miR-21 has been discovered in the injured kidney and closely related to the stage of renal fibrosis. [11][12][13] Additionally, knock-down of miR-21 in renal cells acquires good effects against renal fibrosis. 14 Even more intriguingly, there is some evidence that a prominent elevation of miR-21 primarily located in myofibroblasts in the kidneys of unilateral ureteral obstruction (UUO) mice and patients' kidneys with severe renal fibrosis. 15 Collectively, these findings imply that miR-21 plays an important role in renal fibrosis.
Melatonin is an indolamine product produced by the pineal gland and is believed to have a significant effect on regulating a series of biological functions, including anti-inflammation, anti-oxidation, inhibition of intrarenal renin-angiotensin system (RAS) activation and preservation of renal function. 16,17 Our recent findings have also shown that melatonin administration can potently protect against bilateral ureteral obstruction-induced renal injury. 18 However, as far as we know, there is currently no information about melatonin in the context of fibroblast-myofibroblast transdifferentiation (FMT) during renal fibrosis. In the present study, the TGF-β1-treated rat renal fibroblast cells (NRK-49F) and UUO mice were used to investigate whether melatonin could attenuate renal FMT and whether the miR-21-5p signal might serve a role in melatonin-induced renal protective effect.

| Cell culture and treatment
Rat renal fibroblast cell line NRK-49F were purchased from the American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 5% foetal bovine serum (FBS) (Gibco), 100 IU/mL penicillin and 100 IU/mL streptomycin at 37°C in a 5% CO 2 atmosphere. Cells were seeded on 6-well plate at a density of 2 × 10 5 cell/well and allowed to adhere 24 hours.
Afterwards, the medium was changed to DMEM containing 0.1% FBS. Cells were incubated with TGF-β1 (2 ng/mL) alone, or TGF-β1 (2 ng/mL) with different concentrations of melatonin for the indicated periods before harvesting. For the specific experiment, the medium was supplemented with 10 µmol/L Stattic dissolved in dimethyl sulphoxide (DMSO) (SIGMA-ALDRICH) for the indicated period before collection.
The control mimics, miR-21 mimics, negative control siRNA for Spry1, Spry1 siRNA, negative control siRNA for PTEN or PTEN siRNA were transfected with Lipofectamine RNAiMAX Reagent (CAT No: 13778150, Thermo Fisher Scientific) based on the manufacturer's protocols before TGF-β1 administration or co-treatment with melatonin. Then, cells were incubated with different doses of melatonin. After 3 days, 10 µL of CCK-8 solution was supplemented to each well, and the absorbance was measured at 450 nm in the microplate reader after 4-hours incubation. This experiment was repeated independently three times.

| Luciferase reporter assay
The pGL3-AP-1 firefly luciferase reporter plasmid and renilla luciferase control reporter vector were transfected into NRK-49F cells with Lipofectamine 2000 reagent following the manufacturer's protocols. Twenty-four hours after transfection, the cells were serum free in DMEM with 0.1% FBS overnight, then treated with TGF-β1 (2 ng/mL) alone or TGF-β1 combined with melatonin (10 µmol/L) for 48 hours and lysed in passive lysis buffer.
Bioluminescence signals were determined using dual-luciferase reporter assay system (Promega). The signal intensity from the firefly luciferase reporter gene was normalized to that of the renilla luciferase reporter vector.
The cells were fixed with ice-cold 1:1 methanol/acetone solution for immunofluorescence analysis. Afterwards, these sections were blocked with 10% normal goat serum, incubated with 100 nmol/L working stock of rhodamine phalloid at 4°C in the dark overnight.
After washing with PBS three times, the slides were counterstained with 200 µL of 100 nmol/L 4,6-diamidino-2-phenylindole (DAPI) in PBS. In the end, images were collected on a Zeiss laser-scanning confocal microscope (Olympus). F-actin level in every single experimental group was quantified using Image Pro Plus 6.0 analysis software by taking measurements of the average pixel intensities in 5-10 random fields.

| Establishment of an UUO animal model
Seven-week-old male C57BL/6 mice weighing 23-28 g were purchased from Zhengzhou University Laboratory Animal Center and then were kept in separate cages in a light-and temperature-con- Unilateral ureteral obstruction (UUO) was conducted using an established procedure as described in previous published papers. 18 In brief, mice were anaesthetized with isoflurane inhalation, and the left proximal ureter was exposed and ligated with 6-0 silk sutures.
Mice in sham group were subjected to operations that were identical to the ones done for the mice with UUO except that the ureters were not ligated. Forty-eight weight-matched C57BL/6 mice were randomly allocated to six groups (n = 8).

| Histopathological evaluation
Paraffin-embedded kidney specimens were cut into 4-µm sections and stained with haematoxylin and eosin (HE) and Masson's trichrome staining. Slides were examined and pictures taken using a Leica DM4B microscope equipped with Leica X software. The degree of collagen accumulation and renal interstitial impairment was assessed as described previously. 19

| Real-time reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from cultured NRK-49F cells and renal tis- Sangon Biotech. β-actin was used as an endogenous reference control gene for mRNA and U6 RNA for miRNA. The comparative threshold cycle (CT) method was used to evaluate gene relative expression.

| Western blot
Western blot analysis was performed according to the previous published paper. 20

| Statistical analysis
The experimental results were expressed as means ± standard derivation (SD) of at least three independent experiments. Statistical analysis was performed using a Statistical Package for Social Sciences (version 19.0 SPSS Inc). Data were analysed with a two-sided Student's t test, and the one-way ANOVA followed by a post hoc test.
Differences in P-values < .05 were considered statistically significant.

| Effect of melatonin on cell viability in NRK-49F cells
In order to find the proper dosage of melatonin for exploring its role on renal fibroblast transdifferentiation, we used the CCK-8 assay to analyse the cell survival after treated with various concentrations of melatonin (1, 5, 10, 20, 40, 80 µmol/L) for 72 hours. 21,22 The results revealed that cell viability in the melatonin treatment groups (1, 5, 10 µmol/L) was not markedly different compared with that of the vehicle group, indicating that these three different doses of melatonin exerted no cytotoxicity in the NRK-49F cells ( Figure 1A). Thus, two dosages (1 and 10 µmol/L) were confirmed for the further experiment of melatonin's effect on renal fibroblast transdifferentiation.

| Effect of melatonin on α-SMA expression in TGF-β1-stimulated NRK-49F cells
Acquiring α-SMA phenotype is supposed to be a helpful marker for FMT during renal fibrosis. 23 Figure 1E, melatonin reduced the level of α-SMA protein expression in a time-dependent manner. Therefore, all these data suggested that melatonin was capable of suppressing renal FMT.

| Effect of melatonin on the production of extracellular matrix (ECM) components in TGF-β1stimulated NRK-49F cells
A notably up-regulated production of fibronectin and col1a1 (type I collagen) is one important feature of fibroblast differentiation. As the main ingredients of ECM, the expression of col1a1 and fibronectin was evaluated in the present study. Our findings showed that melatonin blocked the improved expression of col1a1 and fibronectin in the TGF-β1-treated NRK-49F cells in a dose-dependent manner ( Figure 1C and D). Furthermore, 10 µmol/L of melatonin reduced the level expression of both proteins with time ( Figure 1E).
Although 1 µmol/L of melatonin (low dose) had no effect on the expression of α-SMA mRNA and protein in the TGF-β1-treated NRK-49F cells, it inhibited the production of ECM components ( Figure 1C and D). The results suggested even low dose of melatonin can exert an inhibitory effect on fibroblast activation. In a word, our findings F I G U R E 1 Melatonin alleviated renal fibroblast transdifferentiation and ECM production with TGF-β1 incubation. A, The effect of melatonin on cell viability was evaluated by CCK-8 assay (n = 6). NRK-49F cells were treated with melatonin (1, 5, 10, 20, 40 and 80 µmol/L) for 72 h. The results showed that three different doses of melatonin (1, 5, 10 µmol/L) had no cytotoxicity in the NRK-49F cells. *P < .05, #P < .01 compared with Vehicle group. (B) The level of α-SMA mRNA in TGF-β1-stimulated NRK-49F cells treated with melatonin (1 and 10 µmol/L) for 48 h was determined by RT-PCR; n = 6, *P < .01 compared with control group, #P < .01 compared with TGF-β1 + Vehicle group. (C, D) The levels of α-SMA, col1a1 and fibronectin protein expression in TGF-β1-stimulated (2 ng/mL) NRK-49F cells treated with 1 and 10 µmol/L of melatonin for 48 h were examined by Western blot; n = 6, *P < .01 compared with control group, #P < .01 compared with TGF-β1 + Vehicle group. (E) The levels of α-SMA, col1a1 and fibronectin protein expression in TGF-β1-stimulated NRK-49F cells treated with 10 µmol/L of melatonin at different time points (24,48 and 72 h) were assayed by Western blot; n = 6, *P < .01 compared with control group, #P < .01, &P < .05 compared with TGF-β1 + Vehicle group indicated that melatonin could attenuate the differentiation of renal fibroblast and prevent ECM production after TGF-β1 treatment.

| Effect of melatonin on α-SMA filament formation in NRK-49F cells treated with TGF-β1
FMT is characterized by the occurrence of incrassate α-SMAinclusive cytoplasmic filaments. 25 The results from filament staining displayed that melatonin (10 µmol/L dose) obviously repressed stress fibres formation and F-actin level (Figure 2 and Figure S1).
These observations indicated that melatonin could arrest a transition from fibroblast phenotype to myofibroblast phenotype.

| Effect of melatonin on the miR-21-5p expression in NRK-49F cells treated with TGF-β1
There is some evidence that elevation of renal miR-21 plays an important role in regulating expression of ECM and fibroblast transdifferentiation during renal fibrosis. 26 However, whether the effect of melatonin against fibrosis was associated with miR-21-5p has not been reported. To determine the role of miR-21-5pin melatonin-blocked renal FMT, we evaluated the miR-21-5p expression in NRK-49F cells co-treated with TGF-β1 and melatonin (1 or 10 µmol/L dose) for 48 hours. As shown in Figure 3A, miR-21-5p expression significantly decreased in TGF-β1-stimulated NRK-49F cells after melatonin administration.
These findings indicated that melatonin could inhibit the miR-21-5p expression, which might be associated with the inhibitory effect of melatonin on renal FMT.

| The inhibitory effect of melatonin on α-SMA expression induced by TGF-β1 was reversed by miR-21-5p mimics
In order to verify melatonin works by inhibiting the miR-21-5p signal, we transfected a miR-21-5p mimics into NRK-49F cells. Then, NRK-49F cells were co-treated with TGF-β1 and melatonin (10 µmol/L dose) for another 48 hours. The results found that the level of α-SMA protein was remarkably up-regulated in TGF-β1-treated NRK-49F cells co-incubation with melatonin and miR-21-5p mimics compared to those of co-incubation with melatonin and control mimics ( Figure 3B-D). Our data revealed that up-regulation of miR-21-5p expression could partially block the inhibitory effect of melatonin on α-SMA, indicating that melatonin could alleviate renal FMT through down-regulating miR-21-5p expression.

| Effect of melatonin on TGF-β1-induced STAT3 activation and AP-1 activity
It has been demonstrated that several transcription factors can significantly up-regulate miR-21-5p expression, for example activation protein 1 (AP-1 family) and STAT3. 27 To investigate whether melatonin-mediated change of transcription factors activities participated in the biological consequences of melatonin in TGF-β1-induced renal fibroblast differentiation, we detected STAT3 phosphorylation level and AP-1 activity. As shown in Figure 3E, AP-1 activity was markedly increased after TGF-β1 stimulation, which was not changed after melatonin treatment. Next, the detection results of STAT3 phosphorylation showed that melatonin administration apparently inhibited the activation of STAT3 F I G U R E 2 Melatonin decreased the formation of stress fibre in TGF-β1stimulated NRK-49F cells. The formation of stress fibre in TGF-β1-stimulated NRK-49F cells treated with or without 10 µmol/L of melatonin for 48 h was evaluated by cell immunofluorescence staining. Magnification ×400, Scale bar = 50 µm ( Figure 3F and G). Altogether, our findings indicated that melatonin might down-regulate miR-21-5p expression through blocking STAT3 activation induced by TGF-β1, but not AP-1 activity in NRK-49F cells. Moreover, in order to determine whether STAT3 activation was important for miR-21-5p expression, we evaluated miR-21-5p expression in NRK-49F cells relative to STAT3 activation status. Activation by tyrosine phosphorylation at residue 705 (Y705) is required for STAT3 to induce downstream changes in gene expression in response to cytokine stimulation. 28 We therefore confirmed the effect of a specific small molecular inhibitor of STAT3 phosphorylation: Stattic. 29 The results showed that Stattic did not affect the basal expression level of miR-21-5p in untreated NKK-49F cells ( Figure S2), suggesting that Stattic itself was unable to modulate miR-21-5p expression.

| Effect of melatonin on miR-21-5p targets expression
Depending on the availability of a certain number of mRNA targets, a single miRNA may regulate multiple pathways and lead to multiple phenotypes in different cellular environments. 30 Target validation experiments demonstrated that phosphatase and tensin homolog (PTEN) and Sprouty1 (Spry1) are two of potential targets of miR-21. 31,32 Sprouty1 (Spry1), a negative regulator of ERK/MAPK signalling, has been revealed to be associated with increased fibrogenic epithelial-to-mesenchymal transition of TGF-β1. 33 PTEN negatively regulates Akt/PKB activation, which has been implicated in renal fibrosis. 34 To explore whether melatonin treatment works through modulating miR-21-5p targets, firstly we examined the protein expression of Spry1 and PTEN. The results showed the level of Spry1 and PTEN protein was evidently improved after melatonin administration ( Figure 4A and B). Additionally, another two miR-21-5p targets, Smurf2 and PDCD4, were important factors for fibroblast differentiation and fibrogenic process. 35,36 However, after detecting the protein level of Smurf2 and PDCD4, we found melatonin did not significantly affect their expression ( Figure 4C and D). These data indicated that melatonin treatment could regulate miR-21-5p targets Spry1 and PTEN expression, which might lead to the inhibition of fibroblast differentiation.

| The renoprotective effect of melatonin was reversed by miR-21-5p agomir in UUO mice
Compared with sham group, the mice after 7 days of UUO showed significantly elevated BUN and SCr values, as well as 24 hours urinary protein levels ( Table 1), suggesting that UUO caused renal dysfunction. Unsurprisingly, melatonin treatment markedly reduced the upregulation of BUN, SCr and 24-hour urinary protein caused by UUO.
However, the protective effect of melatonin on renal function was reversed after the additional miR-21-5p agomir injection in group 5.
Despite this, compared with the UUO + agomir group (group 6), melatonin could still promote the recovery of renal function to a certain extent in agomir-injected UUO mice (group 5). These data suggested that miR-21-5p might be involved in the pathophysiological process of renoprotection by melatonin.
We further investigated the protective effect of melatonin against obstructive nephropathy. Normal kidney tissue structure is necessary to maintain normal kidney function. Histological examination showed that there were obvious tubular dilatation and atrophy, renal interstitial widening, interstitial inflammatory cells infiltration and a large amount of collagen deposition in the kidneys of UUO mice ( Figure 5A and B). However, in the melatonin alone treated group, these above histopathological changes, including interstitial fibrosis and collagen deposition, were markedly improved compared with those in the UUO group ( Figure 5C and

| D ISCUSS I ON
In the current study, we investigated the inhibitory action of melatonin on renal FMT in TGF-β1-exposed NRK-49F cells and the renoprotective effect of melatonin on renal fibrosis in UUO mice.
According to the analysis of the current experimental results, we firstly revealed that melatonin significantly prevented the expression In fibrogenesis, fibroblasts are thought to transform to the socalled myofibroblasts, during which accompanied with the accumulation of numerous ECM components including fibronectin and col1a1. 10 The excessive ECM production in the interstitial area is the important pathological character of renal fibrosis and results in progressive renal function decline. 38  animal experiments indicate that miRNAs play vital roles in the pathophysiology of kidney-related diseases. 26 It has been confirmed in many experimental models of renal fibrosis that the increased expression of miR-21 is closely related to the progression of fibrosis. 11 STAT pathway. 43 In mammals, STAT3 has been widely investigated due to its numerous functions, including cell growth regulation and inflammation. 44 STAT3 is activated by tyrosine phosphorylation, predominantly JAK, leading to its phosphorylation on activating tyrosine residues, dimerization and nuclear translocation. 43 The pharmacological effects of melatonin have been reported to be closely related to JAK2/STAT3 signalling. 22 22 In the present study, we observed that melatonin can significantly inhibit the activation of STAT3 phosphorylation in the TGF-β1-treated NRK-49F cells, but its underlying mechanism still needs further investigation.
Moreover, target validation experiments have revealed that PTEN and Spry1 are target genes of miR-21-5p. 31,32 MiR-21-5p has been found to inhibit PTEN to increase PI3K and Akt phosphorylation, and then promotes MMP-2 overexpression. 32 As Spry1 is an inhibitor of Ras/MEK/ERK, inhibition of Spry1 by miR-21-5p will activate ERK to promote TGF-β1 induced fibrosis. 46 Our findings showed that melatonin treatment significantly up-regulated the level of PTEN and Spry1 protein expression in renal fibroblasts response to TGF-β1 induction and kidneys of UUO mice, indicating that PTEN and Spry1 protein may participate in the effects of melatonin on renal fibroblast transdifferentiation and renal fibrosis. As a consequence, our present exploration revealed that melatonin could ameliorate renal FMT and renal fibrosis via miR-21-5p/PTEN and/ or miR-21-5p/Spry1 signalling pathway, which may be a vital anti-fibrotic mechanism of melatonin.
In conclusion, the present study confirmed that melatonin could alleviate renal FMT and renal fibrosis. Further analysis of the underlying mechanisms implied that melatonin protected renal against fibrosis through reducing expression of miR-21-5p, which might be related to decreased phosphorylation of STAT3. Regulation of miR-21-5p/PTEN and/or miR-21-5p/Spry1 signalling pathway is also participated in the anti-fibrotic action of melatonin in kidney. These signals could restore PTEN and/or Spry1 expression and prevent the expression of pro-fibrotic genes. The present study puts forward a novel mechanism for melatonin action and further provides new evidence on the anti-fibrotic effects of melatonin. However, more detailed and in-depth studies on melatonin, miR-21-5p and signal pathways need further exploration in the future.

ACK N OWLED G EM ENTS
This study was supported by 'The National Natural Science

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

AUTH O R CO NTR I B UTI O N S
ZL conceived and designed the study. NL FG and YL performed the experiments. NL FG and JZ analysed the specimens. NL and ZL wrote and prepared the original draft.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.