Bortezomib attenuates renal interstitial fibrosis in kidney transplantation via regulating the EMT induced by TNF‐α‐Smurf1‐Akt‐mTOR‐P70S6K pathway

Abstract Allograft interstitial fibrosis was characterized by massive extracellular matrix deposition caused by activated fibroblasts and myofibroblasts. Epithelial‐mesenchymal transition (EMT) is recognized as an important source of myofibroblasts contributing to the pathogenesis of allograft interstitial fibrosis. Smad ubiquitination regulatory factor 1 (Smurf1) has been recently reported to be involved in the progression of EMT. Our study was to detect the effect of Bortezomib and Smurf1 in the EMT and allograft interstitial fibrosis. Biomarkers of EMT, as well as Smurf1, were examined in human proximal tubular epithelial cells (HK‐2) treated with tumour necrosis factor‐alpha (TNF‐α) in various doses or at various time points by Western Blotting or qRT‐PCR. We knockdown or overexpressed Smurf1 in HK‐2 cells. Furthermore, rat renal transplant model was established and intervened by Bortezomib. Allograft tissues from human and rats were also collected and prepared for HE, Masson's trichrome, immunohistochemical staining and western blotting assays. As a result, we found that TNF‐α significantly promoted the development of EMT in a time‐dependent and dose‐dependent manner through Smurf1/Akt/mTOR/P70S6K signalling pathway. More importantly, Bortezomib alleviated the progression of EMT and allograft interstitial fibrosis in vivo and in vitro by inhibiting the production of TNF‐α and expression of Smurf1. In conclusion, Smurf1 plays a critical role in the development of EMT induced by TNF‐α. Bortezomib can attenuate the Sumrf1‐mediated progression of EMT and renal allograft interstitial fibrosis, which could be suggested as a novel choice for the prevention and treatment of renal allograft interstitial fibrosis.


| INTRODUC TI ON
Renal transplantation (RT) is the most effective therapeutic treatment for end-stage renal disease. 1 T-cell mediated acute rejection was effectively reduced by immunosuppressants. 2 However, longterm survival rate of allografts and recipients is still limited because of the progressive deterioration of allograft function and (CAD). 3,4 Kidney interstitial fibrosis, the most important pathological lesion of CAD, was derived from the extracellular matrix deposition caused by activated fibroblasts. 5 Epithelial-mesenchymal transition (EMT), as a source of myofibroblasts, is characterized that epithelial cells are transformed to α-smooth muscle actin (α-SMA) positive myofibroblasts and lose their epithelial markers such as E-cadherin. 6,7 EMT is regulated by various bio-mediators such as transforming growth factor-beta (TGF-β), Ang II and microRNAs. [8][9][10] Our previous research has shown that tumor necrosis factor-alpha (TNF-α) could induce the progression of EMT. HK-2 cells lose the expression of E-cadherin and overexpress α-SMA and fibronectin after the treatment of TNF-α. 11 Smad ubiquitination regulatory factor 1 (Smurf1), an E3 ubiquitin ligase, was reported that it was involved in the EMT in lens epithelial explants, breast cancer and chronic kidney diseases (CKD) induced by TGF-β. [12][13][14] In recent studies, Smurf1 was reported that it was involved in the fibrosis of diabetic kidneys and obstructive nephropathy. 15,16 But the role of Smurf1 in the interstitial fibrosis of transplant kidney is still unknown. Depending on these reported findings, we hypothesized that Smurf1 might play a critical role in the transplant kidney interstitial fibrosis caused by EMT.
Bortezomib, a proteasome inhibitor, is used to prevent the production of alloantibody in the treatment of antibody-mediated rejection (ABMR) through the induction of plasma cells apoptosis. 17 It was also reported that could alleviate the fibrosis of skin, lung and CKD stimulated by TGF-β. 18,19 However, the effect of Bortezomib in the treatment of allograft kidney interstitial fibrosis caused by EMT still remain to be determined. The aim of this study was to investigate the role and the potential molecular mechanisms of Smurf1 in the EMT induced by TNF-α and to detect the anti-fibrogenic effect of Bortezomib. Our results show that Smurf1 plays a critical role in regulating the EMT induced by TNF-α. Bortezomib can attenuate the Sumrf1-mediated progression of EMT and renal allograft interstitial fibrosis.

| Ethics statement
The study protocol was in accordance with the ethical standards of the Declaration of Helsinki and Istanbul. The study protocol involving human kidney tissues was approved by the local ethics committee of the First Affiliated Hospital of Nanjing Medical University.
Written informed consent was obtained from all transplant recipients and nephrectomy patients.

| Sample collection
Allograft kidney sections were obtained from 30 patients, which were collected from transplanted kidney nephrectomy or kidney biopsy of recipients. All patients, who had undergone kidney transplantation at our centre from January 2001 to October 2017 was diagnosed with CAD according to their allograft biopsy results.
Patients diagnosed with CAD was classified as CAD group. In addition, 25 normal kidney samples obtained from more than 5 cm away from the tumour tissues were collected from radical nephrectomy and considered as control group. The baseline characteristics of patients in the CAD group and control group are given in Table 1.

| Animals
Adult male F344 and Lewis rats (200-250 g) were procured from Charles River Laboratories (Beijing, China). The animal centre provided the rats with the clean tap water and standard rat chow.

| Kidney transplantation
Orthotopic left kidney transplantation was performed between F344 and Lewis rats as previously described. 20 Nephrectomy of the right kidney was performed 10 days after the surgery of the trans-

| Pharmaceutical treatment and tissue harvest
From the first day after the kidney transplantation, Bortezomib (0.2 mg/kg; Selleck Chemicals, USA) was iv injected to isogeneic or allogeneic recipient rats twice a week.
At 4 weeks, 8 weeks, 12 weeks and 16 weeks, organs were harvested and divided into two parts, which was fixed in paraffin or snap-frozen in N2 and stored at −80°C.

| Enzyme-linked immunosorbent assay
The levels of rat serum TNF-α were quantified by the rat TNF-α ELISA kit (MUTISCIENCES; China). Rat serum and cell culture medium supernatant level of TGF-β1 were quantified by the TGF-β1 ELISA kit (MUTISCIENCES; China). The assays were performed as described in the manufacturer's instruction.

| Histology and morphometry
Histological analysis was performed by using haematoxylin and eosin (H&E) and Masson trichome staining. H&E and Masson trichome staining were performed as detailed elsewhere, which was used to evaluate the severity of chronic kidney rejection and the area of renal interstitial fibrosis separately. 21 Image-Pro Plus (Media Cybernetics, Rockville, MD) was used by two pathologists blinded to the experimental design independently to quantify the morphometric change of the kidney sections.

| Renal function assessment
Concentrations of rat blood creatinine and urea nitrogen were tested by instructions of manufacturer of the kits (Jiancheng, Beijing, China).

| Quantitative real-time PCR analysis
Total RNA was extracted from cells with the RNA extraction kits (TIANGEN, Beijing, China). cDNA was synthesized with a PrimeScriptTMRT reagent kit (TaKaRa Biotechnology, Japan). mRNA expression was normalized to b-actin expression. Every experiment described previously was repeated at least three times.

| Western blot analysis
Western blotting was performed according to the protocol described CST, USA). GAPDH was used to measure the relative abundance of proteins as an internal reference. The intensity of the protein signals was detected by the NIH image analysis software.

| Statistical analysis
All data were presented as the mean ± SD values determined from three independent experiments. After demonstration of homogeneity of variance with Bartlett test, inter-group comparisons were made using oneway analysis of variance (ANOVA). Multiple means were compared using Tukey's test. The differences between two groups were determined by Student t test. Values of P < 0.05 were considered statistically significant. All the assays were repeated at least three times independently.

| Smurf1 exhibits remarkably high expression in the kidney tissues of renal interstitial fibrosis from CAD patients
In this study, we used human specimens to confirm the high expression of Smurf1 in the CAD patients with allograft renal interstitial

| Progression of EMT in HK-2 cells could be stimulated by TNF-α
To investigate the pathogenesis of EMT stimulated by TNF-α, The relative abundance of proteins and relative intensity of E-cadherin and α-SMA was presented as the mean ± SD values of three independent experiments. a P < 0.05 vs sh-con cells without TNF-α treatment, b P < 0.05 vs shcon cells stimulated by TNF-α, c P < 0.01 vs sh-con cells without TNF-α treatment, d P < 0.01 vs sh-con cells stimulated by TNF-α, e P < 0.05 vs sh-con cells without TNF-α treatment, f P < 0.05 vs sh-con cells stimulated by TNF-α, g P < 0.05 vs sh-con cells without TNF-α treatment, h P < 0.05 vs sh-con cells stimulated by TNF-α, *P < 0.05 vs sh-con cells or ov-con cells stimulated by TNF-α to a dose-dependent decline in the expression of E-cadherin at the same time (

| Smurf1 plays a critical role in the EMT induced by TNF-α
To confirm the role of Smurf1 in the EMT induced by TNF-α, we examined the effect of Smurf1 silencing on these events by constructing Smurf1 stable knockdown HK-2 cells. As shown in the Figure 3A, relative intensity of α-SMA in sh-Smurf1 group stimulated by TNF-α was significantly less than sh-con group according to the outcome of immunofluorescence. However, the relative intensity of E-cadherin was remarkably higher than sh-con group.
Knockdown of Smurf1 blocked the expression of α-SMA, up-regulated the expression of E-cadherin and alleviated the progression of EMT ( Figure 3B-D). When the Smurf1 was overexpressed, the progression of EMT was significantly promoted ( Figure 3E-H).
These data supported our conclusion that Smurf1 plays a critical role in the EMT of HK-2 cells.

| Bortezomib alleviates the Smurf1-meditated EMT of HK-2 cells by inhibiting TNF-α-Akt-mTOR-P70S6K pathway
To evaluate the signalling pathways involved in TNF-α-induced EMT in HK-2 cells, we examined phosphorylation of factors involved in the canonical signalling pathways by western blotting. We observed the activation of the Akt-mTOR-P70S6K signalling pathways when HK-2 cells were treated with TNF-α ( Figure 4A). The Akt inhibitor MK-2206 could reduce the expression of α-SMA and restore the expression of E-cadherin ( Figure 4B-C). Immunofluorescence staining of α-SMA and E-cadherin confirmed our conclusions ( Figure S2A-C). On the other hand, MK-2206 could not reduce the high expression of Smurf1 stimulated by TNF-α ( Figure 4B,D). The Smurf1 stable knockdown HK-2 cells treated by TNF-α showed remarkably less phosphorylation of Akt, mTOR and P70S6K than sh-con cells ( Figure 4F). This effect was reversed between the Smurf1 stable overexpression HK2 cells and OV-con cells ( Figure 4G). These results revealed

| Bortezomib ameliorates the inflammatory response and renal interstitial fibrosis in rat allograft kidney after transplantation
In this study, we applied Bortezomib to explore the role of the pro-

| Bortezomib alleviated the Smurf1-meditated EMT and renal interstitial fibrosis by reducing the secretion of TNF-α in kidney transplantation
The outcome of immunohistochemistry assay revealed the remarkably high expression of TNF-α and Smurf1 in allo-recipient group ( Figure 6A). After the treatment of 0.2 mg/kg Bortezomib for 12 weeks, the positive area of TNF-α and Smurf1 in allograft kidney was dramatically decreased ( Figure 6A). The results of immunohistochemistry assay also indicated that Bortezomib could down-regulate the expression of α-SMA, fibronectin and restore the expression of E-cadherin in the rat allograft kidney ( Figure 6A).
Bortezomib could significantly decreased the serum TNF-α level determined by the ELISA assay ( Figure 6B). Western blot confirmed the outcome of immunohistochemistry assay ( Figure 6C-G).
The anti-fibrosis effect of Bortezomib was not only to block the TNF-α-Smurf1-Akt-mTOR-P70S6K pathway by reducing the expression of Smurf1, but also to decrease the secretion of inflammatory cytokines TNF-α ( Figure 6H).

| Bortezomib prevents allograft renal function impairment and prolongs survival of allogeneic recipients
In order to test the impact of Bortezomib on allograft renal function and long-term survival of allogeneic recipients, the native right kidneys of recipients were removed 2 weeks after left kidney transplantation. One week after the resection of right kidney, the renal function including the blood concentration of creatinine and urea nitrogen, started to be continuously deteriorated until their death.
However, the increase of blood creatinine and urea nitrogen was inhibited and they remained at a relatively low level by the treatment of Bortezomib ( Figure 7A-B). The survival rate of allogeneic recipients was enhanced by the treatment of Bortezomib ( Figure 7C).
Allogeneic recipients treated with Bortezomib enjoyed a higher survival rate compared with allo-recipients treated by vehicle at both 12 and 16 weeks.

| D ISCUSS I ON
In present study, we reported that Bortezomib could attenuate the fibrosis of renal interstitial in kidney transplantation via reg- Data are expressed as the mean ± SD of each group (n = 3) from 3 separate experiments (H) A model is proposed to illustrate the fibrotic mechanism involved in EMT induced by TNF-α in the pathogenesis of allogeneic kidney interstitial fibrosis and the therapeutic mechanism of Bortezomib. ## P < 0.01 vs syngeneic recipients treated with vehicle. ### P < 0.001 vs syngeneic recipients treated with vehicle. *P < 0.05 vs allogeneic recipients treated with Bortezomib. **P < 0.01 vs allogeneic recipients treated with Bortezomib. ***P < 0.001 vs allogeneic recipients treated with Bortezomib. + P < 0.05 vs syngeneic recipients treated with Bortezomib. ++ P < 0.01 vs syngeneic recipients treated with Bortezomib. +++ P < 0.001 vs syngeneic recipients treated with Bortezomib  31 The mechanism that Smurf1 down-regulated the Akt-mTOR-P70S6K pathway could be the degradation of PTEN.
To confirm the anti-fibrogenic effect of Bortezomib, our vivo findings revealed that Bortezomib could reduce the inflammatory response and renal interstitial fibrosis in allograft kidney. Bortezomib

D I S C LO S U R E S TAT E M E N T
The authors confirm that there are no conflicts of interest. F I G U R E 7 Bortezomib prevents allograft renal function impairment and prolongs survival of allogeneic recipients. The native right kidneys of recipient rats were resected 14 days after left kidney transplantation. (A-B) The blood creatinine and urea nitrogen were continuously increased in allogeneic recipients treated with vehicle. Treatment of Bortezomib remarkably prohibited the increases of blood creatinine and urea nitrogen. (C) The survival rate curve showed that Bortezomib significantly increased the long-term survival of allogeneic recipients. Data are expressed as the mean ± SD of each group from three separate experiments. *P < 0.05 vs allogeneic recipients treated with vehicle. **P < 0.01 vs allogeneic recipients treated with vehicle. Log-rank test showed the Bortezomib significantly increased the survival rate of allogenic recipients. P < 0.05 vs allogeneic recipients treated with vehicle