PART Ⅰ: Role Of SIK1 in The Transition Of Acute Kidney Injury Into Chronic Kidney Disease

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

Jinxiu Hul, JiaoQiaot, Qun Yul, Bing Liu1 & et al.

Background

Acute kidney injury(AKI), a common disease characterized by a decrease in glomerular filtration rate(GFR)and an increase in serum creatinine [1], is regarded as a risk factor for the development and progression of CKD[2,3]. Recently, the AKI-CKD(Acute kidney injury into chronic kidney disease) transition has become one of the hotspots in the study of kidney diseases. Although it is reported that inflammation, EMT, and fibrosis play a vital role in the progress of AKI-CKD(Acute kidney injury into chronic kidney disease) transition [4,5], the exact molecular mechanism of AKI-CKD(Acute kidney injury into chronic kidney disease) transition is still unclear.

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Salt Inducible Kinase 1(SIK1) is a member of the AMP-activated protein kinases(AMPKs) family, which play pivotal roles in regulating metabolism, cell survival, and growth [6, 7]. Studies have demonstrated that inhibition of SIK1(Salt Inducible Kinase 1) promotes EMT, leading to migration and metastasis of tumors[8-10]. Besides, it was reported that SIK1(Salt Inducible Kinase 1) negatively regulates the TLR4-induced activation of NF-kB and attenuated expressions of proinflammatory cytokines [11]. In addition, emerging evidence showed a link between SIK1(Salt Inducible Kinase 1) and kidney diseases [6,12]. For instance, upregulation of SIK1(Salt Inducible Kinase 1) reversed the high glucose-induced mesangial cells proliferation, and extracellular matrix(ECM) accumulation by inhibiting the expression of FN and PAI-1, both of which are involved in fibrotic disorders, such as glomerulosclerosis [6,13]. Thus, we speculate that SIK1(Salt Inducible Kinase 1) might be involved in the progression of AKI-CKD(Acute kidney injury into chronic kidney disease) transition, which is characterized by EMT, inflammation, and renal fibrosis.

The WNT/β-catenin pathway is a complex, highly conserved signaling pathway that regulates various biologic processes, such as organ development, tissue homeostasis, and carcinogenesis [14, 15]. Although silent in the normal adult kidney, it is found to be reactivated in a variety of kidney diseases, including acute kidney injury, diabetic nephropathy, interstitial fibrosis, and cystic kidney diseases [16]. Moreover, it is reported that silencing of the WNT/β-catenin pathway ameliorates renal fibrosis, delaying the progression of AKI-CKD(Acute kidney injury into chronic kidney disease) in I/R-induced injury [17]. But whether SIK1(Salt Inducible Kinase 1) could participate in the AKI-CKD(Acute kidney injury into chronic kidney disease) transition by modulating the WNT/β-catenin pathway remains to be further clarified.

Emerging evidence has shown that Snail and Twistl, the EMT transcription factors, play important roles in the pathogenesis of renal fibrosis[18-20]. It was reported that conditional deletion of Twistl or Snail in proximal tubular epithelial cells inhibited EMT, attenuating interstitial fibrosis in experimentally induced renal fibrosis in mice [21]. Considering Snail and Twist1 are significant molecules located downstream of the WNT/β-catenin pathway, we speculated that they may also play a vital role in SIK1(Salt Inducible Kinase 1) mediated AKI-CKD(Acute kidney injury into chronic kidney disease) transition.

Thus, in this study, we aimed to elucidate the role and mechanism of SIK1(Salt Inducible Kinase 1) in AKI-CKD(Acute kidney injury into chronic kidney disease) transition, which will provide a new therapeutic target for clinical prevention and treatment of renal fibrosis and will provide a new way to delay the progress of AKI-CKD(Acute kidney injury into chronic kidney disease) transition.

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Materials and methods

Patients and tissue samples

The study was approved by the Clinical Research Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong University. Human kidney samples were collected from patients with AKI(Acute kidney injury) diagnosed based on renal biopsy, and the kidney samples in the control groups were obtained from paracancerous tissue of patients with kidney tumors who underwent surgical resection. The tissue samples were preserved in liquid nitrogen and the informed consent was signed by all patients in accordance with the Declaration of Helsinki.

Construction of AAV9-Sik1(Salt Inducible Kinase 1)

Recombinant adeno-associated virus(AAV9-Sik1)and adeno-associated virus-negative control (AAV9-NC) were constructed by GENECHEM(Shanghai, China). Recombinant AAV9-Sik1 adeno-associated virus vector GV467 includes a promoter, antibody coding region, EGFP green fluorescent protein-coding region, and 3FLAG tag protein-coding region. The ability of AAV9 vector to trans-duce kidneys has been verified(Additional file 1).

Animals and modeling methods

Modeling and follow-up experimental programs have been approved by the Animal Care and Use Committee of Shandong University. C57BL/6 mice aged 4-6 weeks and weighing 15-20 g were supplied by the Experimental Animal Center of Shandong University and were randomly allocated into 4 groups: control group(Control), normal mice treated with AA group(AA), AA mice treated with AAV9-Sik1 group(AAV9-Sik1+AA), AA mice treated with AAV9-NC group (AAV9-NC+AA). The mice in AA group were injected with AA (10 mg/kg)intraperitoneally, while the mice in the control group were injected with PBS. On the 3rd, 7th,14th, and 28th after AA injection, the 24 h urine of mice was collected in a metabolic cage and the supernatant was stored at-20℃C after centrifugation. Blood was centrifuged at 3000r for 10 min, and the supernatant was stored at-20°C.24 h urinary protein, serum creatinine(Scr), and blood urea nitrogen (BUN) were measured by the automatic biochemical analyzer. Serum IL-1β and TNF-α were detected by ELSA. Then, the mice were sacrificed and weighed. After that kidneys were excised and weighed. All kidney samples were divided into two parts, one of which was fixed in 4% paraformaldehyde for histological staining, while the other was stored under-80°C for real-time PCR and Western blot detection.

Histology and immunohistochemistry

After fixing in 4% paraformaldehyde, the kidney samples were dehydrated, transparent, embedded in paraffin, and sectioned into 4 μm-thick slices. Subsequently， the slides were dewaxed, hydrated, and stained. For renal histological analysis, HE staining, PAS staining, and Masson's trichrome staining were used according to the manufacturer's instructions. For immunohistochemical analysis, sections were incubated with primary antibody against SIK1(Salt Inducible Kinase 1)(51045-1-AP, Proteintech, USA), E-cadherin (20874-1-AP, Proteintech, USA),α-SMA (ab32575, Abcam, USA), COL1(bs-10423R, Bioss, Beijing, China)at 4 ℃ overnight. After incubation with the secondary antibody, the DAB was added, followed by nuclear counterstaining with hematoxylin. Finally, the images were observed under a microscope(Leica, Germany). The percentage of positive staining for SIK1(Salt Inducible Kinase 1), E-cadherin, α-SMA, and COLl was measured by using a quantitative Image-Pro plus 6.0 software (Media Cybernetics, Silver Spring, MD) [22].

Cell culture and treatment

Human proximal tubular epithelial cells(HK2) were cultured in DMEM medium (Gibco, USA)supplemented with 10% fetal bovine serum(FBS)(Gibco,USA) and 1% penicillin-streptomycin(Sigma, USA) at 37℃C in a humidified atmosphere containing 5% CO,. Aristolochic acid(AA)(Sigma, USA) was dissolved in DMSO to 1 mg/mL and cells were treated with the final concentration at 10 μmol/L， 20 μmol/L， 40 μmol/L, and 60 μmol/L， and the control group was treated with the same amount of DMSO.

Lentiviral vector transduction and siRNA transfection The lentiviral shRNA constructs target SIK1(Salt Inducible Kinase 1), β-catenin and a scrambled shRNA, as well as lentiviral overexpression vector for SIK1(Salt Inducible Kinase 1) and empty control vector were constructed by Cyagen(Guangzhou, China). The target sequences are listed as follows: SIKI shRNA:5'-GCGCGTGCATTGATTACTATC-3';

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Results

SIK1(Salt Inducible Kinase 1) was down-regulated in AKI(Acute kidney injury)

Considering that SIK1(Salt Inducible Kinase 1) plays a significant role in kidney injury, we firstly detected the expression of SIK1(Salt Inducible Kinase 1) in kidney tissues of AKI(Acute kidney injury) patients and AKI(Acute kidney injury) mice. Immunohistochemistry staining revealed that, not only in patients but in mice, the SIK1(Salt Inducible Kinase 1) expression of renal tubules in control is higher than that in AKI(Acute kidney injury) group, which indicated that SIK1(Salt Inducible Kinase 1) was down-regulated during AKI(Acute kidney injury) (Fig.la,b). Besides, we conducted HE and Masson's trichrome staining to analyze the histopathological changes in kidney samples of AKI(Acute kidney injury) patients (Fig. 1c). Compared with control, renal tubular epithelial cells swelling, vacuolar degeneration, and interstitial edema were observed in the AKI group by HE staining. Moreover, compared with control, notably flattened tubular cells, detached brush border, and enlarged tubular linens were observed in the AKI group by Masson's trichrome staining. Taken together, all these findings indicated that SIK1(Salt Inducible Kinase 1) might play a role in the tubular injury.

Fig.1 SIK1 was down-regulated in AKI(Acute kidney injury). a Representative immunohistochemical staining images of SIK1 in Control and AKI(Acute kidney injury) patients. Scale bar=50 μm. The graph showed the quantitative analysis of SIK1 in immunohistochemically stained sections.b Representative immunohistochemical staining images of SIK1 in Control and AKI(Acute kidney injury) mice. Scale bar=50 μm. The graph showed the quantitative analysis of SIK1 in immunohistochemically stained sections. c Representative images of HE and Masson’s trichrome staining in kidney tissues of AKI(Acute kidney injury) patients. Scale bar=10 μm. Data are shown as mean±s.d. *P<0.05 vs Control. All experiments were performed in triplicate

SIK1(Salt Inducible Kinase 1) was down-regulated in AA-induced AKI-CKD(Acute kidney injury into chronic kidney disease) transition mice

To obtain a mice model of AKI-CKD(Acute kidney injury into chronic kidney disease) transition, we injected AA intraperitoneally into mice. Compared with control, the mice treated with AA exhibited increased production of inflammatory factors, EMT, and renal fibrosis (Fig. 2a-d). In addition, the kidney index, serum Scr, BUN, and 24 h urinary protein-enhanced upon AA treatment (Fig. 2e). Furthermore, AA impaired renal structure, leading to renal tubular epithelial cells atrophy, limen enlargement, and different extent of collagen fiber deposition in tubulointerstitium(Fig.2f). All above indicated that the AA-induced AKI-CKD(Acute kidney injury into chronic kidney disease) transition model was successfully established. To explore the role of SIK1(Salt Inducible Kinase 1) in AA-induced AKI-CKD(Acute kidney injury into chronic kidney disease) transition, we assessed its expression in the samples of mice kidneys injected with AA. We found SIK1(Salt Inducible Kinase 1) and p-SIK1(Salt Inducible Kinase 1)(Thr182)were decreased in a time-dependent manner after AA injection(Fig.2g), which indicates a potential role for SIK1(Salt Inducible Kinase 1) in regulating AA-induced AKI-CKD(Acute kidney injury into chronic kidney disease) transition.

Fig.2 SIK1 was down-regulated in AA-induced AKI-CKD(Acute kidney injury into chronic kidney disease) transition mice. an ELISA analysis of IL-1β, and TNF-α expression in different groups of mice. b Western blot and real-time PCR analysis of inflammation and fibrosis markers (Caspase1/p20/p10, FN and COL1) expression; c Western blot and real-time PCR analysis of EMT markers (E-cadherin, Vimentin, and α-SMA) expression. d Representative immunohistochemical staining images of E-cadherin in different groups of mice. Scale bar=50 μm. The graph showed the quantitative analysis of E-cadherin in immunohistochemically stained sections. e The kidney index, Scr, BUN, and 24 h urinary protein levels in different groups of mice. f Representative histological staining (HE, PAS, and Masson’s trichrome staining) images of kidney tissues in different groups of mice. Scale bar=50 μm. g Western blot analysis of SIK1 and p-SIK (Thr182) protein levels in C57BL/6 mice. Data are shown as mean±s.d. *P<0.05 vs Control. All experiments were performed in triplicate

Overexpression of SIK1(Salt Inducible Kinase 1) alleviated AA-induced AKI-CKD(Acute kidney injury into chronic kidney disease) transition

To specify the function of SIK1(Salt Inducible Kinase 1) in AA-induced AKI-CKD(Acute kidney injury into chronic kidney disease) transition, we injected AAV9-SikI into the tail vein of mice to overexpress SIK1(Salt Inducible Kinase 1). We found after being injected with AAV9-Sik1, AA-induced renal dysfunction was significantly ameliorated, as evidenced by reduced levels of the kid-ney index, Scr, BUN, and 24 h urinary protein (Fig.3a). Real-time PCR revealed that AAV9-Sik1(Salt Inducible Kinase 1) relieved AA-induced inflammatory response (Fig. 3b). In addition, AAV9-Sik1(Salt Inducible Kinase 1) significantly improved the histopathological damage induced by AA(Fig.3c). Furthermore, the results of immunohistochemical staining suggested that AAV9-Sik1(Salt Inducible Kinase 1) alleviated interstitial fibrosis and EMT in the process of AA-induced AKI-CKD(Acute kidney injury into chronic kidney disease) transition (Fig.3d). Overall, these results indicated that SIK1(Salt Inducible Kinase 1) played a protective role in AA-induced AKI-CKD(Acute kidney injury into chronic kidney disease) transition.

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SIK1(Salt Inducible Kinase 1) was down‑regulated in AA‑treated HK2 cells A large number of studies have shown that the proximal tubule of the kidney is one of the main targets of injury in AKI(Acute kidney injury), and the injury of proximal tubule may play an important pathophysiologic role in the development of AKI-CKD(Acute kidney injury into chronic kidney disease) transition. To further assess whether SIK1(Salt Inducible Kinase 1) was down-regulated in vitro, we focused on HK2 cells in this study. By performing CCK8 assays， we choose 10 μmol/L AA as the optimal concentration for subsequent experiments(Additional file 2). Consistently, AA treatment exhibited increased inflammation, EMT, and fibrosis in HK2 cells(Fig.4a-c), suggesting AA-induced HK2 cells injury in vitro. Subsequently, we examined the protein levels of SIK1(Salt Inducible Kinase 1) and observed a notably decreased SIK1(Salt Inducible Kinase 1) and p-SIK1(Thr182) in HK2 cells in the presence of AA (Fig. 4d). Furthermore, we detected the location of SIK1(Salt Inducible Kinase 1) protein in HK2 cells before and after exposure to AA by Immunofluorescence staining. In the unstimulated cells, SIK1(Salt Inducible Kinase 1) was observed in both the nucleus and cytoplasm. When treated with AA, the expression of SIK1(Salt Inducible Kinase 1) was reduced and the SIK1(Salt Inducible Kinase 1) was gradually detected in the nucleus (Additional file 3). Collectively, these results elucidated that SIK1 was involved in AA-induced HK2 cells injury.

Fig. 3 Overexpression of SIK1 alleviated AA-induced AKI-CKD(Acute kidney injury into chronic kidney disease) transition. a. The kidney index, Scr, BUN, and 24 h urinary protein levels in different groups of mice. b Real-time PCR analysis of Caspase 1, and IL-1β mRNA levels in different groups of mice. c HE, PAS, and Masson’s trichrome staining were used to assess the histological changes and the extent of tubulointerstitial fibrosis. Scale bar=50 µm. d Representative immunohistochemical staining images of E-cadherin, α-SMA, and COLI protein in different groups of mice. Scale bar=50 µm. The graphs showed the quantitative analysis of E-cadherin, α-SMA, and COL1 in immunohistochemically stained sections. Data are shown as mean ± s.d. *P< 0.05 vs Control, #P< 0.05 vs AA. All experiments were performed in triplicate

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Overexpression of SIK1(Salt Inducible Kinase 1) improved AA-induced HK2 cells injury

To further elucidate the role of SIK1(Salt Inducible Kinase 1) in AA-induced HK2 cells injury, we constructed cell lines that stably up-regulated SIK1(Salt Inducible Kinase 1) by lentivirus infection of HK2 cells(Additional file 4). Overexpression of SIK1 led to a significantly increased E-cadherin and repressed Caspasel/p20/p10, Vimentin, COLI, and Fspl when compared with the control cells stimulated with AA alone (Fig. 4e, f). Besides, overexpression of SIK1(Salt Inducible Kinase 1) inhibited the migration ability of HK2 cells induced by AA (Fig.4g). Together, these findings demonstrated that SIK1(Salt Inducible Kinase 1) was protective against AA-induced injury in HK2 cells.

Fig.4 SIK1 was down-regulated in AA-treated HK2 cells and overexpression of SIK1 improved AA-induced HK2 cells injury. a. ELISA detection of IL-1β and TNF-α levels in the supernatant of HK2 cells treated with 10 μmol/L AA for 0 h, 24 h, 48 h, and 72 h. b Western blot analysis of Caspase 1/p20/p10, E-cadherin, ZO-1, Vimentin, and α-SMA protein levels in HK2 cells treated with 10 μmol/L AA for 0 h, 24 h, 48 h, and 72 h, with the greatest effect after 72 h of treatment. β-actin was used as a control. c Real-time PCR analysis of early fibrosis indicators (COLI, PAI-1, and MMP9) mRNA levels in HK2 cells treated with 10 μmol/L AA for 72 h. d Western blot analysis of p-SIK1(Thr182) and SIK1 levels in HK2 cells treated with 10 μmol/L AA for 0 h, 24 h, 48 h, and 72 h. e Western blot analysis of Caspase1/p20/p10, E-cadherin, Vimentin, Fsp1, and COL1 in HK2 cells that are treated with SIK1 vector (SIK1 lentiviral overexpression vector) in the presence of 10 µmol/L AA or treated with 10 µmol/L AA alone for 72 h. f Representative immunofluorescence images of E-cadherin and Vimentin in HK2 cells. Scale bar=50 μm. g Representative migration results of HK2 cells. Scale bar=50 μm. Data are shown as mean±s.d. *P<0.05 vs Control, #P<0.05 vs AA. All experiments were performed in triplicate

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SIK1(Salt Inducible Kinase 1) regulated WNT/β-catenin signaling pathway in vivo and in vitro

Considering the critical role of the WNT/β-catenin pathway in AKI-CKD(Acute kidney injury into chronic kidney disease) transition, we explored whether SIK1(Salt Inducible Kinase 1) regulated the WNT/β-catenin pathway. In vivo experiments, we observed that overexpression of SIK1(Salt Inducible Kinase 1) inhibited the protein levels of WNT1, p-β-catenin (Y654), and nuclear β-catenin in AA-induced AKI-CKD(Acute kidney injury into chronic kidney disease) mice (Fig.5a). To further explore whether SIK1(Salt Inducible Kinase 1) regulated the WNT/β-catenin pathway in vitro, we silenced the expression of SIK1(Salt Inducible Kinase 1) by shRNA in HK2 cells (Additional file 5). Consistent with the in vivo results, silencing of SIK1 resulted in a notably increased β-catenin, TCF4, and LEF1 mRNA levels(Fig.5b). Besides, the knockdown of SIK1(Salt Inducible Kinase 1) increased the protein lev-els of β-catenin and p-β-catenin(Y654)(Fig. 5c). In addition, the knockdown of SIK1(Salt Inducible Kinase 1) promoted the nuclear translocation of β-catenin (Fig.5d). Collectively, these data suggested that SIK1(Salt Inducible Kinase 1) regulated the WNT/β-catenin pathway in vivo and in vitro.

Fig.5 SIK1 regulates the WNT/β-catenin pathway in vivo and in vitro. a. Western blot analysis of SIK1, WNT1,p-β-catenin (Y654), and nuclear β-catenin protein levels in different groups of mice. b Real-time PCR analysis of β-catenin, TCF4 andLEF1 in HK2cells treated with SIK1 shRNA or Scramble shRNA. c Western blot analysis β-catenin, and p-β-catenin(Y654) in HK2 cells treated with SIK1 shRNA or Scramble shRNA.d Representative immunofluorescence images of β-catenin in HK2 cells. Scale bar=50 um. Data are shown asmean±s.d.*P<0.05 ys Control. *P<0.05 ys AA. All experiments were performed in triplicate

A WNT/β-catenin signaling pathway is involved in AA-induced HK2 cells injury

To explore whether the WNT/β-catenin pathway played a role in AA-induced HK2 cells injury, we tested the expression levels of WNT1, nuclear β-catenin, and p-β-catenin(Y654) after AA treatment. Results of Western blot showed that WNT1, nuclear β-catenin, and p-β-catenin(Y654) increased significantly after AA stimulation(Additional file 6a). Besides, immunofluorescence staining revealed the nuclear translocation of β-catenin (Additional file 6b), which suggests that AA stimulation can activate the WNT/β-catenin signaling pathway. Considering β-catenin is the central component of the WNT/β-catenin pathway, we wonder whether regulation of β-catenin regulates AA-induced HK2 cells injury. Thus, we stably knocked down β-catenin by shRNA lentivirus in HK2 cells(Additional file 6c). And we observed that β-catenin knockdown impaired the AA-induced Caspasel, IL-1β, Vimentin, PAI-1, and MMP9 expression and promoted E-cadherin expression(Fig.6a, b). Moreover,β-catenin shRNA cells stimulated with AA exhibited significantly decreased migration compared with the β-catenin control cells stimulated with AA alone(Fig.6c). Taken together, these findings suggested that the WNT/β-catenin pathway was involved in AA-induced HK2 cells injury.

Fig.6 WNT/B-catenin signaling pathway is involved in AA-induced HK2cells injury.HK2 cells were treated with B-catenin shRNA or Scramble shRNA in the presence of 10 umol/LAA.real-time PCR analysis of Caspase1.L-1B.E-cadherin.Vimentin, PA/-1.and MMP9 mRNA levels in HK2 cells. b Representative immunofluorescence images of E-cadherin and Vimentin in HK2cells. Scale bar=50 um.c Representative migration results of HK2 cells. Scale bar=50 um. Data are shown as mean±s.d.*P<0.05 vs Control, "P<0.05 vs AA. All experiments were performed in triplicate

A WNT/β-catenin signaling pathway is required for SIK1(Salt Inducible Kinase 1) mediated HK2 cells injury induced by AA

To study whether SIK1(Salt Inducible Kinase 1) regulated AA-induced HK2 cells injury through the WNT/β-catenin signaling pathway, β-catenin was further decreased in SIK1(Salt Inducible Kinase 1) knockdown HK2 cells. Compared with SIK1 knockdown cells, further knockdown of β-catenin decreased the mRNA levels of Caspasel, COLl, and Vimentin, while increasing the mRNA levels of E-cadherin(Fig. 7a). The results of West-ern blot were consistent with real-time PCR, showing silencing of β-catenin and SIK1 reversed the downregulation of E-cadherin, upregulation of Caspasel/p20/p10 and Vimentin induced by SIK1(Salt Inducible Kinase 1) knockdown (Fig. 7b).

Collectively, these results indicated that inhibition of β-catenin reversed inflammatory response, EMT, and fibrosis progression mediated by SIK1(Salt Inducible Kinase 1) knockdown, which strongly reveals that WNT/β-catenin pathway was required for SIK1(Salt Inducible Kinase 1) mediated HK2 cells injury induced by AA.

Fig.7 WNT/β-catenin pathway is required for SIK1 mediated HK2 cells injury induced by AA. HK-2 cells were co-transfected with SIK1 and B-catenin shRNA or transfected with SIK1 orβ-catenin shRNA alone, and then treated with 10 umol/L AA for 72 h.a real-time PCR analysis of Caspase1, COL1.E-cadherin, and Vimentin expression. b. Western blot analysis of Caspase1/p20/p10, E-cadherin, and Vimentin expression.Data are shown as mean±s.d.*P<0.05 vs Control,p<0.05 vs SIK1 shRNA, &P<0.05vs β-catenin shRNA. All experiments were performed in triplicate

The Role of Twist1 in AA-induced HK2 cells injury

Acting as EMT-TFs, Twistl can promote EMT and kidney fibrosis. In this study, we found AA promoted the protein and mRNA expression of Snail and Twist1 while knockdown β-catenin inhibited the expression of Snail and Twist1 induced by AA(Fig.8a), indicating Snail and Twist1 located in the downstream of β-catenin and played a role in AA-induced HK2 cells injury. To verify our hypothesis, we knockdown Twist1 by siRNA(Additional file 7) and carried out real-time PCR and Western blot analysis. As expected, when compared with control cells stimulated with AA alone, the expression of Vimentin, and COLI was reduced while ZO-1 was increased in Twist1 siRNA cells in the presence of AA, suggesting silence of Twist1 alleviated the occurrence of EMT and the progression of renal fibrosis induced by AA(Fig. 8b and c).

Fig.8 The role of Twist in AA-induced HK2 cells injury. a Western blot and real-time PCR analysis of Snail andTwist1levels in HK2 cells. b Real-time PCR analysis of Twist1, COL1, ZO-1, and Vimentin levels in HK2 cells treated with Twist1 siRNA or NC siRNA.c. Western blot analysis of Twist1, ZO-1, and vimentin levels in HK2 cells treated with Twist1 siRNA or NC siRNA. Data are shown as mean±s.d.*P<0.05 ys Control. *P<0.05 ys AA. All experiments were performed in triplicate

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