A study about the role of Wip1 in renal brosis by modulating macrophage phenotype

objective: To investigate the effect of wild type p53-induced phosphatase 1 (Wip1) on regulation the phenotype of macrophages and to participate in renal brosis. Method: RAW264.7 macrophages were stimulated by lipopolysaccharide (LPS) and IFN-γ for 24h into M1 macrophages with high expression of iNOS and TNF-α. RAW264.7 macrophages were stimulated by interleukin 4(IL-4) for 24 h to induce M2 macrophages with high expression of Arg-1 and CD206. In the meantime, RAW264.7 macrophages were transduced with Wip1 lentivirus by overexpressing it, and transduced with Wip1 RNAi by reducing the Wip1 expression. Furthermore, after coculture the transformed macrophages and mice primary renal tubular epithelial cells, and the expression levels of E-Cadhrin, Vimentin and α-SMA were measured by RT-PCR. Result: it was found that macrophages with Wip1 overexpression had no statistical changes on the expression of iNOS and showed the decreased expression of TNF-α; macrophages with Wip1 siRNA showed the increased expression of iNOS and TNF-α compared to control group. In the meantime, macrophages with Wip1 overexpression had increased expression of Arg-1 and CD206; macrophages with Wip1 siRNA showed decreased expression of Arg-1 and CD206 compared to control group. Macrophage RAW264.7could be transformed into the M2 macrophage after transducing with Wip1 lentivirus by overexpressing it, and the RAW264.7 macrophages could transform into the M1 macrophages by reducing the Wip1 expression via the Wip1 RNAi. Furthermore, after coculture the M2 macrophage and mice primary renal tubular epithelial cells, there was a decreased E-Cadherin expression, increased Vimentin, and α-SMA on the mRNA levels. Conclusion: Wip1 in renal brosis by transforming them into the M2 macrophage phenotype. α-smooth actin; TGF-β: Transforming growth factor-β.


Introduction
Renal brosis is mainly characterized by glomerulosclerosis and tubular interstitial brosis, and eventually develops into chronic renal failure, which is a serious threat to human health and life. The patients with chronic kidney failure have poor quality of life and high medical costs, which impose a severe burden on society and families. Therefore, to explore the pathogenesis of chronic renal brosis and to develop effective treatment methods has become an important challenge faced by health departments all over the world 1 .
Clinical studies have found that most kidney diseases are characterized by macrophage accumulation, indicating that macrophages are involved in the occurrence and development of renal brosis 2 . Li et al. 3 modi ed macrophages to enable them to express the co-stimulating molecule VSIG4, thereby reducing the damage of renal tubulointerstitium by inhibiting T cell in ltration and secretion of in ammatory mediators. Zhao et al. [4][5][6][7] found that the mouse bone marrow and peripheral blood macrophages with Wip1 gene knockout secreted in ammatory factors, and the number of neutrophils increased signi cantly, and their functions of phagocytosis, in ltration, in ammatory response and production of reactive oxygen species were also signi cantly enhanced, mainly through the p38MAPK-STAT1 and NF-κB pathways. In vitro, macrophage cell line Raw264.7 was transfected or RNAi interfered Wip1 gene and co-cultured with broblasts to establish microenvironment of renal brosis. The effect of Wip1 on the phenotype and function of macrophages and the mechanism of regulating renal brosis were studied, by inhibiting or slowing down the progression of renal brosis, the prevention and treatment of renal brosis may bring the new perspective.

Materials And Methods
Experimental reagent RAW264.7 mouse mononuclear macrophage leukemia cells cells were purchased from the Kunming cell bank of the committee of typical culture preservation, Chinese academy of sciences. TGF-β1 IFN-γ TNFα, purchased from Peprotech company; LPS purchased from Sigma; iNOS IL-4 Arg-1 CD206, purchased from Cell signaling technology. The genetic primer for Wip1 was synthesized by Takara.
Plasmid extraction using the Omega plasmid large extraction kit. The protocol of study was approved by the ethics committee of Xi'an Jiaotong University.

Culture, Induction and identi cation of macrophages
The RAW264.7 macrophages in good condition were cultured and resuspended in 1640 culture medium of 1%FBS. The cell counting plate was used to count the cells, and the cell concentration was adjusted to 5×10^5/ml. The cells were seeded into a 6-well plate and 2ml of the above cell suspension was added into each well. Lipopolysaccharide (LPS 100 ng/ml) and IFN-γ (2.5 ng/ml) were added to each well for 12 hours to induce M1 macrophages. IL-4 (10ng/ml) was cultured in each well for 12 h to induce M2 macrophages. The cells in the 6-well plate were divided into 3 groups. After the induction period, the cells were gently washed with PBS for 3 times, replacing the liquid, and the changes in cell morphology were observed. The macrophage phenotype was detected by ELISA method: the level of iNOS, TNF-α, Arg-1 and CD206 in culture media were measured to determine the changes of macrophage phenotype. Take out the supernatant of the culture solution, adjust the sample gun to 100μl, add the standard sample and the sample to be tested, 100μl per hole. Add enzyme labeled coupling solution, 50μl per hole. After slightly shaking the pores, they were put into the water bath box and incubated for 60min. Wash plate 10 minutes before, prepare liquid A and liquid B and store them away from light. After incubation, take out 96-well plates and place them on the washing machine. Wash the plates 5 times. Add A, B liquid, after adding the sample, place the cover of the water bath box and incubate for 15 minutes in dark. Record time; At the end of incubation time, the enzyme plate was taken out and the water was sucked dry on the lter paper.
Add termination uid; OD value was measured on the enzyme marker for three times.

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Construction of WIP1 overexpression and RNAi interference by lentivirus transfection

Construction and identi cation of lentiviral vector lenti-Wip1
The gene sequence of Wip1 Ppm1d-001 was searched in Genbank, the PCR primers(The sequence is Wip1 F 5'-ggttaattaaATGGCGGGGCTGTACTCGCTG-3' Wip1 R 5'-ccgttaccTCAGCACACACACACTGTTTTCC-3')were designed with Primer-3, and PacI and PmeI cleavage sites were added at the two ends of the primers, the fragment size was 1832 bp.

Transfection of 293T cells with lentiviral vector
Prepare 293T cells (cell density ~70%-80%) for transfection, and evenly spread 0.5~1×10^6 cells in a 5ml cell culture dish one day before transfection. The prepared transfection reaction system was added to the prepared culture dish of 293T cells, and the virus supernatant was collected after 48 hours of culture, then ltered and stored at-80 °C.

Construction of lentiviral vector LV-shWip1
According to the RNA interference (RNAI) sequence of Wip1 gene selected from websites, the rst 3 pairs of the 10 pairs of sequences are usually selected in order to synthesize the target sequence (the approximate fragment size is 50 ~ 60 bp) After enzyme digestion, the vector was linked to the synthetic target sequence, and the positive vector was con rmed by sequencing. The virus was packaged in 293T, the virus was collected, mononuclear macrophages were infected, the positive transfected cells were screened for drugs, RNA of positive cells was extracted, and the silencing e ciency of Wip1 gene was detected. Protein extraction, Western blot was used to detect the expression of lentiviral vector.
The phenotypic changes of macrophages were determined by ELISA When mouse derived macrophage RAW264.7 was interfered with Wip1 overexpression and RNAi interference vector, it was found that macrophages with Wip1 overexpression had high expression of iNOS and TNF-α, while macrophages with RNAi interference had high expression of Arg-1 and CD206.
The Primary Cultured Renal Tubular epithelial cells were cocultured with the above-mentioned macrophages, and the changes of brosis indexes were detected by RT-PCR The kidney was taken from 5-6 weeks old healthy male BalB/C mice and put into a sterile petri dish; The kidney tissue was cut into pieces repeatedly with ophthalmic scissors until it was cut into 0.5 ~ 1mm 3 tissue pieces; 0.25% trypsin; Grind the kidney tissue on a sterile petri dish lter; 1ml was taken from the ltered cell suspension and the cell density after dilution was 5×105 ~ 1×106; The diluted cell suspensions were divided into culture vials, cultured in a CO2 incubator at 37 °C, and co-cultured with lentiviral over-expression vector Wip1 and RNAi vector to interfere with murine derived macrophages Raw264.7. After 72 hours, the cells were collected and the mRNA was extracted. The PCR product band was semi-quantitatively analyzed by gel image processing software and converted into numerical variables. Taking GADPH as internal reference, E-Cadhrin / GADPH, Vimentin / GADPH, α-SMA / GADPH, and computing the relative expression of E-Cadhrin, Vimentin, α-SMA.

Statistical
The data were processed by statistical analysis software SPSS13.0, and the measurement data were presented as mean ± standard deviation (). For inter-group comparison, the data were rst tested for normality and homogeneity of variance. For multi-group comparison, one-way ANOVA was used, if the variance was equal to LSD test and if the variance was unequal to Dunnett t 3 tests, the difference was statistically signi cant (p < 0.05).

Results
Induction and identi cation of macrophages RAW264.7 macrophages were cultured with lipopolysaccharide (LPS 100 ng/ml) and IFN-γ (2.5 ng/ml) for 12 h to induce M1 macrophages. The results showed that Raw264.7 macrophages were stimulated with LPS and IFN-γ for 24 hours to induce M1 macrophages with high expression of iNOS and TNF-α. RAW264.7 macrophages were stimulated with interleukin 4(IL-4) for 24 h to induce the M2 macrophages with high expression of Arg-1 and CD206 ( gure 1).
Lentivirus was used to infect 293 cells and broblast cells respectively. After 24h of infection, the cells were obviously enlarged and spherical, the nuclei became larger, the adherence ability, decreased and the cells were easy to fall off. The infected positive cells were observed to be green under uorescence microscope for 48h and 72h ( gure 2A), and the infection e ciency was 83.28% as detected by ow cytometry. Western blot analysis of TIPE2 expression showed that the recombinant lentivirus infected 293T cells with Wip1 protein at 48h, and the infectious effect was the strongest at 72h, which was statistically different from the control group (p<0.05, see gure 2B,2D Primary culture of mouse renal tubular epithelial cells, coculture with above mentioned macrophages, and determination of changes in brosis indicators by RT-PCR It was found that after co-culture with M2 and non-M1-type macrophages, the mRNA levels of E-Cadhrin in renal tubular epithelial cells decreased, and the mRNA levels of Vimentin and α-SMA increased, with statistically signi cant differences (P < 0.01, see gure 4).

Discussion
The early stage of renal brosis is mainly the in ltration and activation of T lymphocytes and macrophages to initiate renal brosis 8 9 . Macrophages can be divided into classically activated macrophages (M1) and activated macrophages (M2) types according to different characteristics such as inducible activation factors, secreted effector cytokines and phenotype. M1 macrophages release NO and promote the release of in ammatory factors through oxygen explosion, causing tissue damage and brosis. However, M2 macrophages were connected by FCγR, which combined with IgG immune complex, activates the ITAM motif and the downstream SyK, leads to the activation of PI3K, and converts the phenotype of the immune response to Th2 type, which has anti-in ammatory and tissue repair effects. Thus, the phenotypic and functional heterogeneity of macrophages, with strong plasticity, ultimately determines chronic in ammation and irreversible tissue damage 10 11 .
Wip1 is a nucleoprotein belonging to the serine/threonine phosphatase family, encoded by the PPM1D (protein phosphatase magnesium-dependent 1 delta) gene, which is expressed in various organs of adult and embryonic mice 12  It was found that m1-type macrophage in ltration was dominant in the early 48 hours of renal ischemia injury, which aggravated the kidney injury. If macrophages were eliminated before injury, renal injury could be alleviated by promoting the proliferation and repair of renal tubular epithelial cells; During the recovery period, M2 type macrophages were predominant, which promoted the regeneration and repair of Renal Tubular epithelial cells 21 . There are also studies that suggest that M2-type macrophages produce a large amount of TGF-β, which promotes renal brosis. It is not completely clear whether this transformation of macrophage phenotype is bene cial or harmful to the body, but it is still di cult to con rm it in vivo 22 .
Our study found that after co-culture with M2-type macrophages and non-M1-type macrophages, the mRNA level of E-Cadhrin in renal tubular epithelial cells decreased, and the mRNA levels of Vimentin and α-SMA increased, but the mechanism of M2-type macrophages and Wip1 in the role of renal brosis are still unclear. Speci cally, which subtype of macrophages directly promotes renal brosis and the role of Wip1 still needs to be further explored. Next, we plan to study the anti-renal brosis mechanism of Wip1 through macrophage and verify our hypothesis.

Figure 2
Construction and identi cation of lentiviral vector LV-shWip1 The 293 cells and broblast cells were infected by Lentivirus and the infected positive cells were observed to be green under uorescence microscope for 48h and 72h ( gure 2A). Wip1 expression was the strongest with the recombinant lentivirus infected 293T cells at 72h, which was statistically different from the control group (p<0.05, see gure 2B,2D). The expression of Wip1 protein in constructing lentiviral vector LV-shWip1 infected 293T cells was decreased, which was statistically different from the control group (p<0.05, gure 2C,2E).

Figure 3
The phenotypic changes of macrophages were detected by ELISA The macrophages with Wip1 overexpression had high expression of iNOS and TNF-α, while macrophages with RNAi interference had high expression of Arg-1 and CD206 after co-culture with the renal tubular epithelial cells and the macrophages transformed ( gure 3).