SRT1720

MiR-155-5p promotes renal interstitial fibrosis in obstructive nephropathy via inhibiting SIRT1 signaling pathway

Zhan Wang, Rui Chen, Zheming Xu, Wei Ru, Hongjuan Tian, Fan Yang and Chang Tao
a Department of Urology, The Children’s Hospital Zhejiang University School of Medicine, Hangzhou, China;
b Department of Neonatal surgery, The Children’s Hospital Zhejiang University School of Medicine, Hangzhou, China

Introduction
Obstructive nephropathy refers to a group of diseases includ- ing kidney stones, polycystic kidney disease, and renal artery stenosis [1]. In obstructive nephropathy, the discharge of urine is blocked due to abnormal structure and/or function of the urinary tract [2]. The main pathophysiological changes are hydronephrosis, increased pressure in the renal pelvis and tubules, renal interstitial edema, inflammatory cell infil- tration, matrix protein aggregation and proliferation of renal interstitial fibroblasts, which ultimately lead to renal intersti- tial fibrosis and kidney atrophy [3]. Tubulointerstitial fibrosis is a common pathway for the progression of obstructive kid- ney disease into end-stage renal failure induced by various causes [4]. Hence, protecting kidney cells or tissues from fibrosis plays a crucial role in the management of obstructive nephropathy, and a comprehensive understanding of renal interstitial fibrosis and its underlying mechanism may pro- vide a new treatment method or management strategy for obstructive nephropathy.
The main pathological changes of obstructive nephrop-athy are renal tubular epithelial cell reduction, renal tubular atrophy, local deposition of extracellular matrix (ECM), andmassive myofibroblast accumulation in the plasma [5]. Epithelial-mesenchymal transition (EMT) performs a crucial role in renal interstitial fibrosis [6,7]. It induces cell cycle arrest and parenchymal damage in renal fibrosis [8]. MicroRNAs (miRNAs) are a class of endogenously non-coding RNAs with 19–25 nucleotides in length [9]. Widely present in various species, miRNAs mainly silence specific genes via binding to the complementary base in the 30-untranslated regions (30-UTR) of mRNAs of the target gene [10]. Besides, they also regulate cell differentiation, cell cycle, cell metabol- ism and other cell life activities [11]. In recent years, miRNAs have been reported to affect EMT or interstitial fibrosis. MiR- 573 is reported to function as a tumor suppressor by inhibit- ing EMT [12]. MiR-487a-3p is also thought to be related to EMT [13]. The knowledge about the roles of miRNAs in renal fibrosis in obstructive nephropathy may provide an insight into their potential application in the diagnosis, treatment, and prognosis of obstructive nephropathy. During EMT, both the mesenchymal biomarker alpha-smooth muscle actin (a-SMA) and the crucial mediator transforming growth factor beta (TGF-b) are increased [14,15]. TGF-b1 can improve the levels of collagens and fibronectin in myofibroblasts [16,17].
According to literature, all these proteins (a-SMA, TGF-b, col- lagens and fibronectin) are related with EMT or renal intersti- tial fibrosis [18]; and their expressions can be used to indicate the level of renal interstitial fibrosis and test the function of miRNAs in renal interstitial fibrosis.
MiR-155-5p is reported to exert multiple biological cellular functions in diverse pathology, including hepatocellular car- cinoma [19], cardiovascular disease [20], and acute pancrea- titis [21]. MiR-155-5p has also been proven to improve cancer-associated fibroblasts by inducing the proangiogenic switch [22]. In addition, it is reported that miR-155-5p plays a role in the process of diabetic kidney disease which is char- acterized by tubulointerstitial fibrosis [23]. However, the molecular mechanisms of miR-155-5p in renal fibrosis in obstructive nephropathy are still not clear. Also, whether miR-155-5p has a similar function to that described in previ- ous reports is worthy of further study. Therefore, we studied the roles and possible mechanism of miR-155-5p in renal interstitial fibrosis with rat renal stromal fibroblast NRK-49F cells and mice unilateral ureteral obstruction (UUO) model, which may provide a potential endogenous molecule against renal interstitial fibrosis in obstructive nephropathy. A better understanding of the role and underlying mechanism of miR-155-5p in renal fibrosis in obstructive nephropathy may provide a new route for developing a potential therapy and management strategies for obstructive nephropathy.

Methods
Culture of NRK-49F cells
Rat renal stromal fibroblast NRK-49F cells (CRL-1570, ATCC, https://www.atcc.org) were cultivated with DMEM high glu- cose medium (12800017, Solarbio, China, http://www.solar- bio.com) containing 10% fetal bovine serum (FBS, YZ- 1270548, Solarbio, China) in a CO2 incubator (37 ◦C, 5% CO2). The culture medium was refreshed every 2 days. When a pav- ing stone-like monolayer was formed during cell fusion, the NRK-49F cells were digested with 0.25% trypsin (T1360, Solarbio, China) and passaged. Well-grown 2nd–5th gener- ation NRK-49F cells from were selected for experiments.

Cell transfection
SIRT1 plasmid (SIRT1) was constructed by cloning the chem- ically synthesized complementary DNA of SIRT1 into the KpnI and BamHI sites of pcDNA expression vector (V36620, Thermo Fisher Scientific, America). The sequence of SIRT1 siRNA was designed with siDirect version 2.0 (http://sidirect2. rnai.jp) and synthesized by Thermo Fisher Scientific. NRK-49F cells were transfected with miR-155-5p mimic (miR10003301- 1-5, Guangzhou RiboBio Co., Ltd., China, https://www.ribobio. com/en/), miR-155-5p inhibitor (miR20003301-1-5, Guangzhou RiboBio Co., Ltd., China, https://www.ribobio. com/en/), SIRT1 plasmid and/or SIRT1 siRNA by using Lipofectamine 2000 transfection reagent (11668019, Thermo Fisher Scientific, America) according to the instructions. The sequences of miR-155-5p mimic, miR-155-5p inhibitor, andSIRT1 siRNA were listed in Table 1. In brief, 50 lL of DMEM medium (12491015, Thermo Fisher Scientific, America) was used to dilute plasmid or RNA (1.0 lg). DMEM medium (50 lL) was employed to dilute Lipofectamine 2000 reagent (1 lL), and the dilution was then placed at 25 ◦C for 5 min. Then DNA or RNA was mixed with the diluted Lipofectamine 2000 reagent and placed at 25 ◦C for 20 min, followed by 24 h of incubation (37 ◦C, 5% CO2). After transfection for 24 h, gene level was assessed for further experiment.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Levels of miR-155-5p and SIRT1 in NRK-49F cells or mice kid- ney tissue were measured with qRT-PCR. Total RNA was extracted from NRK-49F cells using TRIzol reagent (15596018, Thermo Fisher Scientific, America). Primers (Table 2) used in this study were designed with PrimerBank (https://pga.mgh. harvard.edu/primerbank/) or Primer3 Plus (http://www.pri- mer3plus.com/cgi-bin/dev/primer3plus.cgi) and synthesized by Thermo Fisher Scientific. RNA reverse transcription was performed using a PrimeScript RT Reagent Kit with gDNA Eraser (RR047B, Takara, America, https://www.takarabio.com). PCR was performed using a Thermo Fisher Scientific 7500 Real-Time PCR System (4351105, Thermo Fisher Scientific, America) under the following condition: at 95 ◦C for 5 s, at 60 ◦C for 30 s, for 40 cycles. The data were analyzed by the 2–DD Ct method. The housekeeping gene U6 and GAPDH (F: 5’-TGTGGGCATCAATGGATTTGG-3’; R: 5’-ACACCATGTATTCCGGGTCAAT-3’) served as the control.

Western blot
Levels of SIRT1 and related proteins (TGF-b1, a-SMA, Collage I and fibronectin) in NRK-49F cells or mice kidney tissue were assessed by Western blot. Total protein was extracted by lysis with Western and IP cell lysates (P0013J, Beyotime, China, https://www.beyotime.com) and centrifugation(12,000 rpm, 10 min, 4 ◦C). The protein concentration was measured with a BCA Protein Assay Kit (PC0020, Solarbio, China) following the protocol. Then protein lysate (30 lg) was separated by 12% sodium dodecyl sulfate polyacryl- amide gel electrophoresis (SDS–PAGE; P0012A, Beyotime, China) and transferred to polyvinylidene fluoride (PVDF) mem- branes (FFP28, Beyotime, China). Then the PVDF membranes were blocked with skimmed milk for 2 h and then incubated overnight at 4 ◦C with Mouse anti-SIRT1 antibody (ab110304, Abcam, England, https://www.abcam.cn/), Rabbit anti-TGF-b1 antibody (ab92486, Abcam, England), Rabbit anti-a-smooth muscle actin antibody (ab5694, Abcam, England), Mouse anti- Collagen I antibody (ab6308, Abcam, England), Rabbit anti- fibronectin antibody (ab2413, Abcam, England), and Mouse anti-GAPDH antibody (ab8245, Abcam, England). After that, the PVDF membranes were incubated with Goat anti-Rabbit IgG (ab205718, Abcam, England) or Goat anti-Mouse IgG (ab205719, Abcam, England) at 25 ◦C for 2 h. Then immunode- tection of the PVDF membranes was conducted with enhanced chemiluminesence (ECL) reagent (PE0020, Solarbio, China). Image J software (Wayne Rasband, America) was used to normalize the relative expression to GADPH.

Bioinformatics analysis and dual-luciferase reporter assay
The target gene of miR-155-5p was analyzed through TargetScan. Dual-luciferase reporter assay was conducted to verify the interaction between miR-155-5p and the SIRT1 gene. In NRK-49F cells, miR-155-5p binding sites for SIRT1, including mutant type (MUT) and wild type (WT), were ana- lyzed using dual-luciferase reporter assay based on the protocol of Dual-Luciferase Reporter Assay System (11402ES60/80, Yeasen Biotech Co., Ltd., China, https://www. yeasen.com). Briefly, pmirGLO luciferase Vector (E1330; Promega, CA, USA), which contained the 30 untranslated region of SIRT1 mutant type (SIRT11-30-UTR-mut) or wild type (SIRT11-30-UTR), was co-transfected with miR-155-5p mimic (M) or miR-155-5p inhibitor (I) into NRK-49F cells by using Lipofectamine 2000 transfection reagent in light of the protocol. The expression of luciferase in the NRK-49F cell extract was measured after 48 h following the instructions.

Establishment of UUO model and experimental design
UUO model was built with 40 male C57 black mice (6 weeks,20 25 g; Hunan SJA Laboratory animal CO., LTD., China, http://www.hnsja.com/index.html) as described in Pang’s report [24]. Briefly, before the experiment, the 40 mice were acclimatized for 7 days on a 12 h light/dark cycle, with the relative humidity and temperature maintained at 50 ± 10% and 22 ± 1 ◦C respectively. All mice were given free access to rodent laboratory chow and tap water and fed under the same condition. For the operation, the abdominal cavity of mice was opened and the left ureter was separated under anesthesia. Then the left ureter of 20 mice in the UUO group was ligated near the renal pelvis, while the other 20 mice in the Sham group underwent no further operation. The SIRT1activator SRT1720 (100 mg/kg, Selleck Chemicals, America, https://www.selleckchem.com) was administered to 10 UUO group mice and 10 Sham group mice by tail vein injection for 3 days prior to the operation and the administration was continued for 7 days after UUO. The remaining 10 mice in each group were injected with an equal volume of saline. The dosage of SRT1720 was in accordance with Ren’s report [25]. All mice were sacrificed 7 days after operation, and then the kidneys were harvested immediately for assessment of relative protein and RNA levels, as well as tissue staining.

Masson trichrome staining
In order to assess renal fibrosis, mouse kidney tissue was stained with Masson trichrome staining kits (G1340, Solarbio, China) according to the protocol of the manufacturer. Briefly, the kidney tissue section was deparaffinized in xylene (X112051, Aladdin). Then the section was sequentially placed into prepared Weigert ferroxylin staining solution for 10 min, acid ethanol differentiation solution for 10 s, Masson bluing liquid for 3–5 min, Lichun magenta staining solution for 10 min, and aniline blue staining solution for 1–2 min. At last, the section was dehydrated and sealed with neutral gum. The Masson Trichrome positive staining area (blue, collagen tissue area) from each microscopic field (200 ×) was quanti- tatively assessed using Image J software (the National Institutes of Health, America) and photographed.

Statistical analysis
All the experiments were repeated at least 3 times. Data were expressed as mean ± standard deviation. Group differ- ences were assessed by one way analysis of variance (ANOVA) or Student’s t test when necessary with SPSS 18.0 (SPSS Inc., America). p < 0.05 was considered to indicate a statistically significant difference. Results MiR-155-5p overexpression promoted the expressions of fibroblast related proteins and inhibited the SIRT1 expression in NRK-49F cells, while miR-155-5p silencing had the opposite effects To assess the effect of miR-155-5p on the fibrosis of NRK-49F cells, qRT-PCR or Western blot was performed to assess the levels of miR-155-5p, fibroblast related proteins and SIRT1. The details in Figure 1(A) showed that miR-155-5p level was mark- edly up-regulated by miR-155-5p mimic transfection (p < 0.001). Figure 1(B) showed the expression levels of fibro- blast related proteins, and it was found that the level of SIRT1 was significantly down-regulated by miR-155-5p mimic (p < 0.001) and up-regulated by miR-155-5p inhibitor (p < 0.05); while the levels of TGF-b1, a-SMA, Collage I and fibronectin were markedly up-regulated by miR-155-5p mimic (p < 0.05) and down-regulated by miR-155-5p inhibitor (p < 0.001). These results implied that miR-155-5p promotedthe expressions of fibroblast related proteins and inhibited the SIRT1 expression. SIRT1 could bind to miR-155-5p in NRK-49F cells The target gene of miR-155-5p was analyzed by the online tool TargetScan, and dual-luciferase reporter assay was conducted to verify the binding relation of SIRT1 and miR-155-5p. Figure 2(A) exhibited the predicted binding site on SIRT1 for miR-155-5p. According to Figure 2(B), the luciferase activity in the M þ SIRT1- 30-UTR group was markedly lower than that in both the Control þ SIRT1-30-UTR and M þ SIRT1-30-UTR mut groups (p < 0.05). On the contrary, the luciferase activity of I SIRT1-30- UTR group was markedly higher than that in both the Control SIRT1-30-UTR and I SIRT1-30-UTR mut groups (p < 0.05). All these results suggested that SIRT1 could bind to miR-155-5p. MiR-155-5p inhibited the level of SIRT1 in NRK-49F cells To reveal the relation of miR-155-5p and SIRT1, the levels of miR-155-5p and SIRT1 in NRK-49F cells were assessed with qRT-PCR and Western blot, after transfection of miR-155-5p mimic, miR-155-5p inhibitor, SIRT1 plasmid or SIRT1 siRNA or co-transfection of the plasmids. As we can see in Figure 3(A), the level of miR-155-5p was significantly increased by co- transfection of the miR-155-5p mimic and SIRT1 plasmids(p < 0.001), while the SIRT1 plasmid alone had no significanteffect on the level of miR-155-5p. Interestingly, Figure 3(B) illustrated that miR-155-5p inhibitor and si-SIRT1 alone or in combination had no significant effect on the miR-155-5p level. As shown in Figure 3(C, E), the mRNA and protein lev- els of SIRT1 in NRK-49F cells were significantly down-regu-lated by miR-155-5p mimic and significantly up-regulated by the SIRT1 plasmid (p < 0.01); and the mRNA and proteinlevels of SIRT1 in NRK-49F cells co-transfected with the SIRT1 plasmid and miR-155-5p mimic were markedly higher than those transfected with miR-155-5p mimic and markedly lower than those transfected with the SIRT1 plasmid(p < 0.01). On the contrary, the data in Figure 3(D, F) showedthat the mRNA and protein levels of SIRT1 in NRK-49F cells were significantly up-regulated by miR-155-5p inhibitor and significantly down-regulated by SIRT1 siRNA (p < 0.01); andthe mRNA and protein levels of SIRT1 in NRK-49F cells co-transfected with SIRT1 siRNA and miR-155-5p inhibitor were markedly lower than those transfected with miR-155-5p inhibitor and markedly higher than those transfected with SIRT1 siRNA (p < 0.01). The results suggested that miR-155-5poverexpression promoted the level of miR-155-5p, and thatmiR-155-5p overexpression and SIRT1 silencing inhibited the level of SIRT1, while miR-155-5p silencing and SIRT1 overex- pression promoted the level of SIRT1. Fibroblast related proteins were up-regulated by miR- 155-5p and down-regulated by SIRT1 in NRK-49F cells, while the up-regulatory effect of miR-155-5p was reversed by SIRT1 To determine the roles of miR-155-5p and SIRT1 in NRK-49F cells, the levels of fibroblast related proteins (TGF-b1, a-SMA, Collage I and fibronectin) in NRK-49F cells were measured with Western blot. The detail in Figure 4(A) showed that the levels of these fibroblast related proteins in NRK-49F cellswere markedly up-regulated by miR-155-5p overexpression and down-regulated by SIRT1 expression (p < 0.05); and the levels of these fibroblast related proteins in NRK-49F cells co- transfected with miR-155-5p mimic and the SIRT1 plasmid were markedly lower than those transfected with miR-155-5p mimic and markedly higher than those transfected with the SIRT1 plasmid (p < 0.05). Oppositely, the date in Figure 4(B)exhibited that the levels of the fibroblast related proteins inNRK-49F cells were significantly down-regulated by miR-155- 5p inhibitor and up-regulated by SIRT1 silencing (p < 0.05); and the levels of the fibroblast related proteins in NRK-49Fcells co-transfected with miR-155-5p inhibitor and SIRT1 siRNA were markedly lower than those transfected with SIRT1 siRNA and markedly higher than those transfected with miR-155-5p inhibitor (p < 0.05). These data suggestedthat the levels of fibroblast related proteins in NRK-49F cellswere up-regulated by miR-155-5p overexpression and SIRT1 silencing and down-regulated by SIRT1 overexpression and miR-155-5p silencing, while the regulatory effect of miR-155- 5p on these proteins was reversed by SIRT1. MiR-155-5p expression was up-regulated and SIRT1 expression was down-regulated in the kidney tissue of UUO model mice, while the fiber deposition induced by UUO was attenuated by SRT1720 Expression levels of miR-155-5p and SIRT1 in the kidney tis- sue of mice were measured with qRT-PCR or Western blot. The mice kidney tissue was stained with Masson trichrome staining kits. As shown in Figure 5(A), miR-155-5p level in the kidney tissue of model mice was markedly higher than that in the control group (p < 0.001). In comparison, the datain Figure 5(B) showed that SIRT1 level in the kidney tissue of model mice was significantly lower than that in the control group (p < 0.001). As can be seen in Figure 5(C), positiveareas of ECM within the interstitial space stained withMasson trichrome were markedly increased after UUO injury, while they were dramatically reduced by SRT1720 administra- tion. These results implied that the level of miR-155-5p was up-regulated and SIRT1 was down-regulated in the UUO mouse model, and the collagen deposition of ECM induced by obstructive kidney was inhibited by SRT1720. SIRT1 was down-regulated and fibroblast related proteins were up-regulated in the UUO model group, while this effect was inversed by SRT1720 To determine the roles of miR-155-5p and SIRT1 in the kid- ney tissue of mice, the fibroblast related proteins (TGF-b1, a-SMA, Collage I and fibronectin) in mice kidney tissue were measured with Western blot. Figure 6 showed that SIRT1 level was markedly down-regulated in the model group(p < 0.001), while this down-regulation was markedly inversed by SRT1720 injection (p < 0.001); meantime, thelevels of the fibroblast related proteins were markedly up- regulated in the model group (p < 0.05), and this up-regula- tion was also inversed by SRT1720 (p < 0.05). These data sug- gested that SIRT1 was down-regulated and fibroblast relatedproteins were up-regulated in the UUO model group, while this effect was inversed by SRT1720. Discussion Obstructive nephropathy is characterized by blocking of urine discharge [2] and one of the main pathophysiological changes of obstructive nephropathy is renal interstitial fibro- sis [3,4]. Therefore, protecting the kidney from fibrosis is important for the management of obstructive nephropathy. In this study, we studied the effects of miR-155-5p on renal interstitial fibrosis as well as its possible mechanism using NRK-49F cells and mice UUO model. Our data may contributeto the development of the potential therapeutic methods in the treatment of obstructive nephropathy. MiRNAs have various functions, such as regulating cell dif- ferentiation and metabolism [10,11]. Recently, miRNAs are believed to participate in fibrosis regulation [23]. For example, both miR-573 and miR-487a-3p are reported to play an important role in EMT, a process related to the pro- cess of renal fibrosis [12,13]. In our research, we demon- strated that miR-155-5p promoted renal fibrosis via up- regulating the expressions of fibroblast related proteins (TGF- b1, a-SMA, Collage I and fibronectin) in NRK-49F cells. a-SMA, TGF-b, collagen and fibronectin are all important bio- markers of EMT, which is also one of the main pathological changes of renal interstitial fibrosis [14–18]. In Chen’s study, inhibited miR-155-5p was found to exert anti-fibrotic effects in silicotic mice [26]. Wang’s study pointed out that miR-155- 5p may be a crucial factor for fibrosis in diabetic kidney injury [23]. Our results indicate that miR-155-5p plays a cru- cial role in the process of renal fibrosis in obstructive nephr- opathy, and may be a promising target for the treatment of obstructive nephropathy. Then we further explored the mechanism of miR-155-5p in inhibiting fibrosis in obstructive nephropathy. Here, miR- 155-5p was proved to interact with SIRT1 throughbioinformatics analysis on TargetScan and dual-luciferase reporter assay, which was similar to Wang’s finding that miR- 155 could target the 3’UTR of SIRT1 [23]. SIRT1 is a kind of NAD -dependent histone deacetylase and exerts a strong effect on regulating gene expression, maintaining genome stability, and regulating other cells functions; what’s more, SIRT1 also has an important function in the epigenetic regu- lation of multiple diseases [27]. Additionally, it was reported that SIRT1 is related with liver fibrosis [28] and involved in the function of mitochondria [29]. Interestingly, in our research, miR-155-5p overexpression promoted the level of miR-155-5p and inhibited the level of SIRT1, while miR-155- 5p silencing caused the opposite effects. Besides, SIRT1 over- expression promotes the level of SIRT1, and SIRT1 silencing inhibits the level of SIRT1, while both SIRT1 overexpression and SIRT1 silencing had no effect on the level of miR-155-5p. Likewise, in Wang’s research, miR-155 was also discovered to reduce SIRT1 expression through binding to SIRT1 [23]. It is therefore possible that miR-155-5p promotes renal fibrosis in obstructive nephropathy via inhibiting the expression of SIRT1. We next verified the assumption that SIRT1 has a crucial role in the anti-fibrosis function of miR-155-5p using UUO mice referring to Ponnusamy’s research [30]. The resultsshowed that miR-155-5p expression is up-regulated and SIRT1 expression is down-regulated in the mouse kidney tis- sue, which are consistent with our previous results in NRK- 49F cells. At the same time, the fiber deposition in mouse kidney induced by UUO treatment was attenuated by admin- istration of the SIRT1 activator SRT1720. This result is also consistent with the previous finding that SRT1720 attenu- ates renal fibrosis in diabetic nephropathy and UUO- induced tubulointerstitial fibrosis [25,31]. Through further study, we found that SRT1720 inhibited kidney tissues fibro- sis and the expressions of fibroblast related proteins in the UUO mice model. Similarly, Ren’s study also reported that SRT1720 attenuates UUO-induced tubulointerstitial fibrosis, as manifested by reduced a-SMA, TGF-b and other fibrosis related proteins [25]. In addition, Han’s study showed that a-SMA, Collage I and fibronectin were reduced by SRT1720 administration as well [31]. Combining our results, we con- clude that SIRT1 is important for anti-fibrosis activity and it inhibits tubulointerstitial fibrosis. In conclusion, in this study, through experiments in vivoand in vitro, we found that miR-155-5p targets STIR1 andpromotes tubulointerstitial fibrosis in obstructive nephrop- athy by inhibiting STIR1 levels. 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