Exosomes derived from SIRT6 transfected urine-derived stem cells improves renal fibrosis

Purpose: The treatment of renal fibrosis caused by long-term obstructive nephropathy is limited. The purpose of this study was to establish a mouse model of renal fibrosis with unilateral ureteral obstruction (UUO) and to treat it with exosomes derived from SIRT6 transfected urine-derived stem cells (USCs), which will determine whether exosomes have the effect of anti-fibrosis Methods: The renal fibrosis model of UUO mice was established by ligating unilateral ureter. USCs were extracted from human urine and transfected with SIRT6 lentivirus plasmid. The SIRT6-USCs-exosomes(SIRT6-USCs-exos) were collected and identified by transmission electron microscope, nanoparticle tracking analysis and western blot. SIRT6-USCs-exos were injected into the tail vein of mice and the renal tissue of mice was stained with HE. The relative gene expressions of α-SMA, E-cadherin and TGF-β1 were analyzed by RT-qPCR. Results: In this study, we successfully constructed the renal fibrosis model of UUO mice and hUSCs with overexpression of SIRT6. The SIRT6-USCs-exos significantly improved the renal fibrosis in UUO mice. The relative mRNA Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 18 March 2021 doi:10.20944/preprints202103.0472.v1 © 2021 by the author(s). Distributed under a Creative Commons CC BY license. expressions of fibrosis-related genes α-SMA, E-cadherin and TGF-β1 in renal tissue were significantly down-regulated after SIRT6-USCs-exos treatment. Conclusion: Our results indicate that SIRT6-USCs-exos can effectively alleviate renal fibrosis caused by long-term obstructive nephropathy. The findings may be promising for dealing with renal fibrosis.


Introduction
Obstructive nephropathy refers to the obstruction of renal pelvis, ureter, bladder and urethra caused by a variety of reasons, which is common in urinary tract obstruction caused by urinary calculi, benign prostatic hyperplasia, neurogenic bladder, abnormal urinary tract anatomy in children and so on. In recent years, the diseases of urinary calculi and benign prostatic hyperplasia show a rising and complicated trend in clinic [1,2]. Long-term urinary tract obstruction caused by these diseases reduces the blood supply of renal tissue, resulting in renal tubular cell atrophy and interstitial fibrosis. Chronic progressive renal fibrosis can lead to irreversible renal failure, gradually decline the quality of life of patients, and cause a huge medical burden to the society. At present, for the renal fibrosis caused by long-term obstructive nephropathy, surgery can relieve the obstruction, but can not further improve the renal fibrosis. Therefore, to find out an effective treatment of renal fibrosis is an urgent clinical problem to be solved. Sirtuins are an evolutionarily conserved family of deacetylases that depend on nicotinamide adenine dinucleotide (NAD+), which are associated with proliferation, DNA repair, mitochondrial energy homeostasis, and antioxidant activity. Studies have shown that SIRT6 attenuates or inhibits the fibrosis process of heart [11,12], lung [13,14], liver [15,16] and renal [17,18] by affecting cell differentiation, TGF-β1/Smads signal pathway and NF-κB signal pathway.
Therefore, as an excellent anti-fibrosis factor, we chose SIRT6 to transfect USCs and obtain SIRT6-USCs-exos.
In this study, we found exosomes derived from SIRT6 transfected urine-derived stem cells (SIRT6-USCs-exos) inhibited renal fibrosis of UUO mice. RT-qPCR showed the relative mRNA expressions of fibrosis-related genes α-SMA, E-cadherin and TGF-β1 in renal tissue were significantly down-regulated after SIRT6-USCs-exos treatment.

Animal model
One week after adaptation, the mice were randomly divided into four groups: sham operation group, unilateral ureteral obstruction (UUO) group and GFP-USCs-exos group and SIRT6-USCs-exos group. The right ureter was ligated and cut through the right back incision in mice, but only free ureter was not ligated in Sham group. The experimental animals were given drugs three days before the establishment of the model. 10ugI SIRT6-USCs-exos was injected into the tail vein of rats in the SIRT6-USCs-exos group for one week.
10ugI GFP-USCs-exos was injected into the tail vein of rats in the GFP-USCs-exos group for one week. The mice were killed on the 7th day after the establishment of the model, and the serum and renal tissue samples were taken.

Cell procedure
Urine samples of healthy adult volunteers (22-35 years old) were collected, and all volunteers signed informed consent forms. Under aseptic condition, 200 mL fresh midstream urine of healthy adult volunteers was collected in a disposable aseptic container, divided into 4 tubes with a 50mL centrifuge tube and centrifuged for 10 minutes. Then the supernatant was absorbed and discarded. PBS containing 1% penicillin streptomycin was added to the supernatant and washed evenly. After repeated centrifugation, the supernatant was discarded. Finally, the remaining sediment was re-suspended with a special culture medium for 5mL urine-derived stem cells and inoculated on a 24-well plate. After 3 days of static culture in a 5% CO2 incubator at 37℃, the cell growth was observed, the medium was changed, and the non-adherent cells were discarded, and then the liquid was changed every 3 days. When the primary cultured urine-derived stem cells reached 80% of 90% fusion, they were digested with 0.25% trypsin and inoculated at 1:2 ratio. The morphological changes of the cells were observed under inverted microscope.

Isolation and identification of exosomes
At 80-90% confluence, USCs were cultured in vesicle-depleted medium for 48-72 hours. The medium was gathered and subjected to gradient centrifugation. Briefly, the medium was first centrifuged at 1000g for 10 min, at 3000g for 30 min, at 10,000g for 60 min, at 100,000g for 4 hours. All centrifugal steps were performed at 4 ℃ . Exosomes were stored at −80°C or used for downstream experiments. Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blotting were used to identify the collected exosomes.

USCs transfection, selection, and SIRT6 expression
A green fluorescent protein (GFP) label for a lentiviral vector plasmid system carrying the SIRT6 gene was constructed by Shanghai Jikai Gene Technology Co., Ltd. USCs were transfected with lentiviral vectors at an appropriate multiplicity of infection (MOI=20) according to the manufacturers' instructions.
GFP expression was observed via fluorescence microscopy at 1, 3, and 5 days after lentiviral vector transfection. The protein expression of SIRT6 in USCs after SIRT6 transfer was verified by western blot. The protein expression of SIRT6 in both SIRT6-USCs-exos and GFP-USCs-exos was measured by western blot.

Western blot
Cells were lysed in RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS) containing a Complete Protease Inhibitor Cocktail. Protein concentration was determined by Bio-Rad DC protein assay. Total protein (30 ug) from the cell lysate was separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked in 5% non-fat milk in PBS overnight and then incubated with primary antibody at 37℃ for 2 h. After washing for 30 min, the membrane was incubated with 1:3000 secondary goat anti-mouse IgG in TBS-Tween for 1 h at 37 ℃ . After further washes, the proteins of interest were detected using a Chemiluminescent HRP Antibody Detection Kit and the signals were captured using an electro chemiluminescent system. The anti-CD63, anti-TSG1001, anti-ALIX and anti-SIRT6 antibody was used at a 1:1000 dilution, the anti-GAPDH was used at a 1:1500 dilution.

RT-qPCR
Total RNA was extracted from renal tissues and cells using TRIzol reagent according to the manufacturer's instructions (Invitrogen, USA), and one microgram of RNA was reverse transcribed to first-strand cDNA using the

Characterization of exosomes
We successfully constructed USCs that stably overexpressed SIRT6 and collected exosomes derived from USCs. The exosomes purified from USCs medium were identified by transmission electron microscope (TEM). The diameter of exosomes was about 100nm (Fig. 1A). Then the expression of exosome markers CD63, TSG101 and ALIX was confirmed by Western blot (Fig. 1B). Next, the concentration and size distribution of exocrine bodies were analyzed by NanoSightLM10 Nanoparticle Tracking Analysis (NTA). In USCs medium, the concentration of exosome in control group was 8.42 × 10 8 /ml, and that in overexpression SIRT6 group was 4.39 × 10 8 /mL. The number of exocrine bodies in USCs medium overexpressing SIRT6 was lower than that in the control group, and NTA estimated that the size of vesicles was between 60 and 140nm (Fig. 1C). Finally, the expression of SIRT6 in the exosome secreted by USCs was identified by Western blot. The results showed that compared with the exosome of the control USCs, the USCs overexpressing SIRT6 and the SIRT6 contained in the exosome were significantly increased (Fig. 1D).

Discussion
Cell-based regenerative therapy has been widely used in patients who have no other treatment options as an alternative treatment. More and more evidences show that stem cells have a better effect on improving damage and fibrosis. In this study, we demonstrated that administration of USCs-exos on UUO model ameliorated tubular interstitial fibrosis. We further used SIRT6-USCs-exos to show better repair ability of renal fibrosis in UUO mice than GFP-USCs-exos. The TGF-β signaling pathway is a key mediator involved in the process of organ fibrosis, and TGF-β1 is considered to be an important cytokine for the transition from inflammatory diseases to fibrosis [19,20]. Studies have found that bone marrow-derived exosomes can regulate fibrosis repair of damaged endometrium by down-regulating TGF-β1 [21]. In this study, mouse ureteral obstruction caused high expression of TGF-β1 and α-SMA in renal tissue. After treatment with SIRT6-USCs-exo, the expression of fibrosis-related proteins TGF-β1 and α-SMA could be down-regulated.
In summary, this study shows that SIRT6-USCs-exos can effectively alleviate renal fibrosis induced by UUO in mouse, but whether it can inhibit human renal fibrosis remains to be confirmed. In addition, this experiment is aimed at the interventional treatment in the early stage of renal fibrosis, and whether there is a relieving effect on the renals in the middle and late stages of failure requires further experiments.

Conflicts of interest
All authors declare that there is no conflict of interest associated with this manuscript.