Conditional knockout of TGF-βRII /Smad2 signals protects against acute renal injury by alleviating cell necroptosis, apoptosis and inflammation

Rationale: TGF-β/Smad signaling is the central mediator for renal fibrosis, however, its functional role in acute kidney injury (AKI) is not fully understood. We previously showed Smad2 protects against renal fibrosis by limiting Smad3 signaling, but details on its role in acute phase are unclear. Recent evidence showed that TGF-β/Smad3 may be involved in the pathogenesis of AKI, so we hypothesized that Smad2 may play certain roles in AKI due to its potential effect on programmed cell death. Methods: We established a cisplatin-induced AKI mouse model with TGF-β type II receptor or Smad2 specifically deleted from renal tubular epithelial cells (TECs). We also created stable in vitro models with either Smad2 knockdown or overexpression in human HK2 cells. Importantly, we evaluated whether Smad2 could serve as a therapeutic target in both cisplatin- and ischemic/reperfusion (I/R)-induced AKI mouse models by silencing Smad2 in vivo. Results: Results show that disruption of TGF-β type II receptor suppressed Smad2/3 activation and attenuated renal injury in cisplatin nephropathy. Furthermore, we found that conditional knockout of downstream Smad2 in TECs protected against loss of renal function, and alleviated p53-mediated cell apoptosis, RIPK-mediated necroptosis and p65 NF-κB-driven renal inflammation in cisplatin nephropathy. This was further confirmed in cisplatin-treated Smad2 knockdown and overexpression HK2 cells. Additionally, lentivirus-mediated Smad2 knockdown protected against renal injury and inflammation while restoring renal function in established nephrotoxic and ischemic AKI models. Conclusions: These findings show that unlike its protective role in renal fibrosis, Smad2 promoted AKI by inducing programmed cell death and inflammation. This may offer a novel therapeutic target for acute kidney injury.


Introduction
Transforming growth factor-β (TGF-β) plays critical roles in cell growth, differentiation, extracellular matrix deposition and immune response [1,2]. Of note, its function in fibrogenesis has been Ivyspring International Publisher extensively studied [3,4]. When released from LAP (latency-associated peptide) and LTBP (latent TGF-β-binding protein), active TGF-β1 binds with its type II receptor that activates type I receptor and downstream effectors, Smad2 and Smad3, to regulate genes associated with renal fibrosis [3].
Acute kidney injury (AKI) is a clinical syndrome characterized by a sudden decline in renal function [5][6][7]. The common pathological features of AKI are programmed cell death of resident renal cells and excessive inflammation [6,[8][9][10][11]. The role of TGF-β in AKI has drawn significantly more attention recently but is still unclear. Previous studies indicated a protective role for TGF-β in AKI; knockout of TGF-β1 aggravated ischemic kidney injury in mice [12]. And, TGF-β protected against hydrogen peroxide-induced necrosis in tubular epithelial cells (TECs) [13]. However, a recent study with conditional knockout of TGF-β1 type II receptor (TGF-βRII) in mice showed disrupted TGF-β1 signaling attenuated renal damage by reducing tubular apoptosis in mercuric chloride-induced injury [14]. This was further confirmed by a separate study that showed activation of TGF-β signaling by overexpression of TGF-β1 type I receptor (TGF-βRI) induced renal cell apoptosis, necrosis, oxidative stress and interstitial infiltration of inflammatory cells [14,15]. In addition, inhibition of TGF-β1 increased proliferation of kidney tubular epithelial cells (TECs) while promoting repair after renal ischemia/reperfusion (I/R) injury [16]. In this setting, more detailed studies have focused on the functional role of downstream Smads. Interestingly, one group found that knockout of Smad3 attenuated renal damage by limiting renal inflammation in a murine model of ischemic AKI [17]. Our group also found that Smad7 knockout mice suffered more severe renal impairment; Smad7 rescued TGF-β/Smad3/p21/p27-induced G1 cell cycle arrest of TECs [18]. Although we previously reported that Smad2 protected against Smad3-mediated renal fibrosis, no evidence regarding the role of Smad2 in AKI has been reported [19]. In this study, our result showed that conditional knockout of TGF-βRII from TECs attenuated Smad2/3 activation and protected against renal injury caused by cisplatin injection, we further found Smad2 aggravated cisplatin-induced AKI in vivo using Smad2 conditional knockout mice, which was further confirmed in vitro using cisplatin-challenged TECs with Smad2 overexpression (OE) or knockdown (KD). More importantly, the therapeutic potential of Smad2-targeted strategy has been tested in both nephrotoxic and ischemic AKI models.

Generation of TGF-β type II receptor and Smad2 conditional KO Mice
All the animal experiments were approved by the Ethics Committees of the Chinese University of Hong Kong and all procedures were performed under the permission of the Guideline of Animal Care and Use Committee of the Chinese University of Hong Kong. TGF-β type II receptor kidney-specific conditional knockout mice were generated by mating the TGF-βRII FF mouse (C57B/L6) with the KspCre (C57B/L6) mouse as we previously reported [20]. Smad2 kidney-specific conditional knockout mice were generated by mating the Smad2FF mouse (C57B/L6) with the KspCre (C57B/L6) mouse. The Smad2 flox mouse and KspCre transgenic mouse were generated and verified as described previously [19]. Conditional deletion of the Smad2 gene from the kidney TECs was testified as we previously reported [19].

Establishment of cisplatin-induced AKI Model
Animal model of cisplatin-induced AKI was established in groups of TGF-βRII FF and TGF-βRII KspCre mice, Smad2FF and S2FF/KspCre mice (male, 8 weeks of age, 22 to 25 g) by intraperitoneal injection of cisplatin (20 mg/kg) respectively. Mice intraperitoneal injected with saline were used as control. All mice were generated from the genetically identical littermates, and all animals were sacrificed under anesthesia 3 days after injection. Kidney tissue samples were harvested for Periodic acid Schiff (PAS) Staining, immunohistochemistry, Western blot and Real-Time PCR as reported previously [21], and blood samples were harvested for BUN and Creatinine detection in accordance with manufacturer's instructions.

Establishment of I/R-induced AKI Model
Mice were obtained from Laboratory Animal Center of Anhui province. All animal procedures were approved by the Institutional Animal Experimentation Ethics Committee of Anhui Medical University. All animal used were C57BL/6N mice (aged 6-8 weeks). Throughout the surgical procedure, the body temperature was maintained between 35 and 37.5 ℃. Mice were anesthetized, sterilized, and shaved. We performed a midline abdominal incision and a bilateral renal pedicle clipping. Both renal pedicles were clamped for 40 minutes with a microvascular clamp. After removing the clamp, reperfusion was confirmed visually. The abdomen was closed in two layers using standard 6-0 sutures. Sham-operated mice received identical surgical procedures, except that clamps were not applied. Twenty-four hours after IRI, blood and kidney samples from the mice were harvested for further study.

Lentivirus-Mediated Smad2 Knockdown in Mice
Mouse Smad2 shRNA were obtained from GenePharm (Shanghai, China). Lentivirus-mediated Smad2 knockdown in mice was performed as we previously reported [21], then mice were intraperitoneally injected with either 20 mg/ kg cisplatin or the equal volume of saline or ischemia-reperfusion surgery and sham-operation for further analysis.

Transfection of Smad2 small hairpin RNA and Smad2 overexpression plasmid constructs
Smad2 expression was assessed by Western blot and Real-time PCR analysis after transfection of HK2 cells with human-derived Smad2 shRNA or overexpression (OE) plasmid obtained from Genepharma Co., Ltd. Briefly, cells were seeded in 6-well plates and transfected with Smad2 shRNA/Smad2 OE plasmid and control constructs using Lipo3000 transfection reagent (Invitrogen, Carlsbad, CA, USA). Cells were incubated with opti-MEM at 37°C and 5% CO2 for 6 h. Cells were cultured in DMEM containing 5% FBS and selected by puromycin or G418 to establish Smad2 KD and OE stable cell lines.

Cell Culture
Cells were cultured in HyClone™ DMEM-F12 medium-containing 5% FBS at 37℃ and 5% CO 2 . Cells were starved for 12 hours with 0.5% FBS, then treated with cisplatin (20 uM) for 24 hours. Cells were harvested and analyzed for indexes of tubular injury, inflammation, necroptosis, apoptosis, activation of NF-κB, p53, RIPK and Smad3 signaling by Real-Time PCR, Western blot analysis and responsive promoter assay. A minimum of 3 independent experiments were performed.

Renal function detection
Blood samples from mice were used to measure creatinine and blood urea nitrogen (BUN) using Creatinine and BUN Assay Kits as previously described [21].

Flow cytometric analyses
Flow cytometric analysis was performed to evaluate the percentage of apoptotic cells. The stable Smad2 knockdown HK2 cell lines were treated with or without 20uM cisplatin for 24 h. HK2 cells were digested with trypsin for 2 minutes and centrifuged at 1500 rpm for 5 minutes. According to the manufacturer's instructions, the density of cells used was 10 6 cells/ml after addition of 400 ul Annexin V binding fluid. Cells were re-stained with 10ul PI for 5 minutes and lightly placed at 4 °C in dark before being immediately measured with a laser eight-color flow cytometer (FACSVerse, BD, USA) and quantified using FlowJo 7.6 software [21].

Luciferase reporter assay
Luciferase reporter assay were performed according to the manufacturer's instructions as we previously descripted (Promega Corporation, WI, USA) [23]. p53, Smad3, p65 NF-κB luciferase reporters, contained p53, Smad3, p65 NF-κB binding sites in the promoter region of luciferase respectively, were designed and purchased from Genomeditech Co. Ltd (Genomeditech, Shanghai, China). In brief, Smad2 wild type and knockdown Smad2 HK2 cells were transiently transfected with p53, Smad3 or p65-responsive promoter Luc respectively. Cisplatin (20 uM) was added to the cells for 24 hours after transfection and starving for 12 hours. According to the manufacturer's instructions, the p53, Smad3, p65 NF-κB activities were analyzed by luciferase reporter gene assay. Their activities were normalized to promoter activity of normal group. 3-4 independent experiments were performed.

Co-immunoprecipitation
HK2 cells were harvested by 1%NP-40 followed by centrifugation at 3000 rpm for 5 min. Protein was incubated with p53 antibody for 2 h at 4 °C. Then capture the immunocomplex by adding 100 μL of washed Protein A agarose bead slurry (EMD Millipore Corporation, 28820 Single Oak Drive, Temecula, CA 92590, USA). The tagged protein was incubated with the bead for 12 h at 37°C to get the protein-bead complex. Protein-bead complex was washed with three cycles of 1% NP-40. Samples were finally measured by Western blot with Smad2 antibody.

TUNEL assay
Renal apoptosis was examined by TUNEL assay using the One step TUNEL Apoptosis Assay Kit from Beyotime Biotechnology (Beyotime, Jiangsu, China). Briefly, paraffin-embedded renal tissue sections were deparaffinized and added Proteinase K (20 μg/ml) 30 min at 37 ℃. The sections were then exposed to the TUNEL reaction mixture containing TM red-labeled dUTP. TUNEL-positive nuclei were identified by fluorescence microscopy.

Statistical Analysis
The data acquired from this study are presented as the mean ± SEM from 3-4 independent in vitro experiments or 6-8 mice. Statistical analyses were performed using two-tailed unpaired t test or one-way ANOVA, followed by Newman-Keuls post hoc test (Prism 5.0; GraphPad Software, San Diego, CA).

Generation and verification of conditional Smad2 KO mice, conditional TGF-βRII KO mice, stable Smad2 knockdown in vitro and in vivo, and Smad2 overexpression in tubular epithelial cell lines
Smad2 conditional knockout mice (Smad2ff/ KspCre) were generated by mating Smad2 flox/flox mice (Smad2FF) with kidney-specific promoter (Cadherin-16)-driven Cre mice (KspCre). Smad2 was silenced in kidney tubular epithelial cells. Immunohistochemistry show Smad2 and TGF-βRII were depleted from renal tubulars ( Figures 1A and B). Real-time PCR results show Smad2 mRNA significantly decreased in kidneys of conditional Smad2 KO mice ( Figure 1C). Western blot and quantitative data confirm protein levels reduced ( Figure 1D). Results show Smad2 knockdown and overexpression in HK2 cell lines were successfully established ( Figures 1E-H). Finally, Real-time PCR and Western blot results show Smad2 was successfully silenced in mice transfected with lentivirus-packaged Smad2 shRNA plasmid ( Figures  1I and J).  mRNA and suppressed Smad2/3 phosphorylation without altering total Smad2/3 protein in cisplatin nephropathy ( Figure 2D and E). Additionally, disruption of TGF-β signaling suppressed the induction of KIM-1, F4/80+ macrophage infiltration and activation of key signaling molecules regulating programmed cell death ( Figure 2F and G).

Conditional knockout of Smad2 attenuated cisplatin-induced renal injury and programmed cell death in vivo
PAS staining and quantitative data show disruption of Smad2 attenuated kidney damage in cisplatin nephropathy ( Figure 3A). Results of creatinine and BUN assay also show conditional knockout of Smad2 prevented decline in renal function ( Figures 3B and C). We also measured KIM-1 level to evaluate the function of Smad2 in AKI. Immunohistochemical results show KIM-1 significantly lower in Smad2 KspCre mice compared with S2FF mice ( Figure 3D). And, KIM-1 mRNA and protein levels were significantly downregulated after conditional deletion of Smad2 in response to cisplatin ( Figures 3E and F). Western blot results show cleaved caspase-3 and p53 phosphorylation significantly reduced after conditional knockout of Smad2 in cisplatin nephropathy. Additionally, disruption of Smad2 also reduced RIPK1 and RIPK3, the central regulators in necroptosis in cisplatin nephropathy ( Figure 3G).

Results
of immunohistochemistry and quantitative data show that conditional deletion of Smad2 decreased F4/80+ macrophage infiltration by 40% compared with the Smad2FF control. Smad2 conditional knockout mice also had reduced expression of tumor necrosis factor-α (TNF-α). This was further confirmed by Real-time PCR show that mRNA levels of inflammatory indexes TNF-α, interleukin-1β (IL-1β) and chemokine monocyte chemotactic protein-1 (MCP-1) reduced in Smad2 conditional knockout mice (Figures 4A and B). Finally, conditional knockout of Smad2 prevented p65 NF-κB phosphorylation in kidneys of mice with AKI ( Figure 4C).

Knockdown of Smad2 alleviated cisplatin-induced renal injury, inflammation and programmed cell death in vitro
Real-time PCR and Western blot results show KIM-1 mRNA and protein levels significantly reduced in cisplatin-stimulated Smad2 KD cells (Figures 5A  and B). Real-time PCR show mRNA levels of inflammatory indexes TNF-α, IL-1β and MCP-1 significantly reduced after Smad2 knockdown ( Figure  5C). In addition, silence of Smad2 reduced phospho-p65 NF-κB, this was further confirmed by NF-κB luciferase reporter assay (Figures 5D and E). Moreover, loss of Smad2 reduced the programmed cell death of HK2 cells compared with cisplatin-treated group ( Figure 5F). Knockdown of Smad2 reduced cleaved caspase-3, Phospho-p53, RIPK1 and RIPK3 level ( Figure 5G). Additionally, luciferase reporter assay results show disruption of Smad2 largely reduced cisplatin-induced p53 activity compared with control HK2 cells ( Figure 5H). These findings show Smad2 knockdown alleviated cisplatin-induced renal injury, inflammation and programmed cell death in vitro.

Overexpression of Smad2 promoted cisplatin-induced renal injury, inflammation and programmed cell death in vitro
Overexpression of Smad2 enhanced KIM-1 mRNA and protein levels in response to cisplatin compared with Smad2 control vector ( Figures 6A and  B).
Overexpression of Smad2 enhanced cisplatin-induced mRNA levels of inflammatory indexes ( Figure 6C). Mechanistically, we found that overexpression of Smad2 promoted cisplatin-induced p65 NF-κB phosphorylation within 1 hour ( Figure  6D). The interaction between Smad2 and p53 was validated by using co-immunoprecipitation in HK cells ( Figure 6E). Consistently, overexpression of Smad2 increased the key molecules regulating programmed cell death in cisplatin-treated HK2 cells ( Figure 6F).

Lentivirus-mediated Smad2 KD in vivo attenuated cisplatin-and I/R-induced kidney injury, inflammation and apoptosis in established AKI mouse models.
To determine the therapeutic potential of Smad2 in the established AKI mouse model, we silenced Smad2 in vivo by tail vein injection of Lentivirus-packaged Smad2 KD plasmid. PAS staining shows Smad2 KD attenuated kidney damage and restored renal function in cisplatin nephropathy ( Figures 7A-C). This was further supported by results from real-time PCR and immunostaining of KIM-1 ( Figures  7D and E) (Figures 7F and G). We then evaluated therapeutic effect of Smad2 in the second AKI model induced by I/R injury. Our results consistently show that disruption of Smad2 protected against kidney injury, inflammation and apoptosis in established ischemic AKI mouse model (Figures 8).

Disruption of Smad2 enhanced cisplatin-induced phosphorylation of Smad3
We then determined whether loss of Smad2 impacted Smad3 signaling in AKI model. Western blot analysis shows that knockdown of Smad2 upregulated Smad3 phosphorylation, but overexpression of Smad2 suppressed Smad3 phosphorylation without altering total Smad3 protein in vitro (Figures 9A and B). This is consistent with luciferase reporter assay which demonstrated that knockdown of Smad2 enhanced Smad3 activity in cisplatin-treated HK2 cells ( Figure 9C).

Discussion
TGF-β1/Smad signaling plays a central role in mediating renal fibrosis [3,24,25]. Although evidence indicates it may function in AKI, the exact role of TGF-β1 and downstream Smads need to be further understood. Here, we generated TGF-βRII conditional knockout mice, Smad2 conditional knockout mice, Smad2 knockdown and overexpression stable HK2 cell lines. We found that disruption of Smad2 in tubular epithelial cells prevented loss of renal function, cell necroptosis, apoptosis and inflammatory response both in vivo and in vitro. More importantly, lentivirus-mediated Smad2 knockdown protected against renal injury and inflammation while restoring renal function in established nephrotoxic and ischemic AKI models.       In the current study, our result shows that tubular-specific TGF-βRII plays an overall detrimental role in nephrotoxic AKI model. However, some groups found TGF-β1 plays a protective role in acute injury [12,13], and others have shown accumulation of TGF-β1 induces AKI [14,15,26]. This discrepancy may be correlated with TGF-β1 level, its cell-type specific role, different types of AKI models or distinct functions of downstream Smad and non-Smad signaling pathways. Moreover, it is of note that downstream Smads may also function in TGF-β-independent manners [3]. Given our previous data showing Smad3 promoted fibrosis, while Smad2 and Smad7 resolved renal fibrotic response [25,27], the complexity and diversity of downstream Smads may have an impact on the overall effect of TGF-β1 in AKI. It's important to note that global knockout of Smad3 limited ischemic AKI by downregulating the production of inflammatory indexes like MCP-1 and IL-6 [17]. This is consistent with our previous findings that C-reactive protein accelerated AKI by suppressing CDK2/cyclin E in Smad3-dependent mechanisms [28]. We also confirmed that disruption of Smad3 protected against cisplatin-induced renal damage and alleviated renal inflammation and apoptosis (unpublished data). These findings suggest Smad3 may be a therapeutic target for AKI. In a recent study, our group showed that Smad7 attenuated ischemic AKI by limiting Smad3-mediated G1 cell cycle arrest of TECs [18]. Given the role of Smad2 in AKI is not well described, we conditionally knocked out Smad2 from kidney TECs and induced AKI by intraperitoneal injection of cisplatin. Results show creatinine and BUN levels decreased. And, PAS staining show renal damage was alleviated.
We also found that knockout of Smad2 from TECs attenuated cisplatin-induced programmed cell death in mice. Proximal TECs are the primary target of AKI, and programmed cell death of TECs is a common feature for different types of AKI caused by ischemia reperfusion injury and nephrotoxic insult [29,30]. As an important regulator of kidney injury and repair, TGF-β1 is highly involved in apoptosis and proliferation of TECs. Previous studies showed that inhibition of TGF-β1 reduced TECs apoptosis in chronic renal fibrosis [31,32]. Further, TGF-β1 initiated TECs apoptosis via P38/ERK signaling instead of Smad signaling [33,34]. In contrast, other studies showed that Smad2/3 signaling triggers apoptosis by regulating cell cycle repressor elements or Bcl-2 family [35,36]. This indicates TGF-β1 induces apoptosis in both Smad-dependent and Smad-independent mechanisms depending on different disease conditions. Of note, role of Smad2 in nephrotoxic agent-induced apoptosis in AKI is still unknown. In the current study, our results show that knockdown of Smad2 decreased the percentage of apoptotic TECs, cleaved caspase-3 level, p53 phosphorylation and activity. Moreover, overexpression of Smad2 promoted apoptosis in HK2 cells possibly by binding to p53 with the help of co-activators, which was further confirmed in vivo in Smad2 conditional knockout mice. This was consistent with previous study demonstrating that Smad/p53 functional interactions were associated with renal fibrosis [37,38]. We noticed that disruption of Smad2 also alleviated cell necroptosis in cisplatin nephropathy. Necroptosis is the best-known pattern of regulated necrosis, emerging evidence showed RIPK1, RIPK3 and MLKL, central regulators in necroptotic pathway, play significant roles in mediating AKI [39,40]. Our team recently found that by binding to the ATP-binding pocket of RIPK1, wogonin prevents cisplatin-induced necroptosis and renal injury [22]. We also identify hsa-miR-500a-3P directly target the 3'UTR of MLKL, thereby alleviates kidney injury via limiting necroptosis [41]. Compared with apoptosis, necroptosis incurs more severe consequence because cells suffered necroptosis release endogenous pro-inflammatory molecules like damage-associated molecular patterns (DAMPs) and promote renal inflammation [42,43]. In the current study, our results showed that loss of Smad2 prevented, but overexpression of Smad2 promoted, cisplatin-activated necroptotic signaling, this result indicates Smad2 may play an important role in mediating cell necroptosis and consequent necroinflammation.
Additionally, we show disruption of Smad2 decreased cisplatin-induced inflammatory response both in vivo and in vitro. Overproduction of cytokines and recruitment of macrophages are major features of AKI [44], but the role of TGF-β1/Smads in renal inflammation remains disputed. Smad3 confers a chemotactic effect by inducing the transcriptional activity of MCP-1 and macrophage infiltration. In contrast, TGF-β1 and Smad3 exert anti-inflammatory effects on TNF-α-induced renal inflammation [45,46]. We previously found that conditional knockout of TGFβRII or Smad4 increased renal inflammation by abolishing the anti-inflammatory effect of Smad3 [20,45]. In the current study, conditional knockout of Smad2 downregulated cisplatin-induced inflammatory cytokine production and NF-κB activity, this may be led by overactivation of Smad3. Of note, the effect of TGF-β1/Smads on inflammation may be highly dependent on TGF-β1 concentration, disease types and cell types. These factors need to be further examined.
We also investigated the effect of Smad2 deficiency on Smad3 signaling in cisplatin-induced AKI. Although Smad2 and Smad3 have more than 90% homology in amino acid sequence and interact physically, their functions differ in renal fibrosis [19]. We found that disruption of Smad2 induced smad3 phosphorylation and activity in cisplatin-treated TECs. This is consistent with our previous study that showed loss of Smad2 promoted Smad3 signaling in fibrotic kidney [19]. However, unlike the diverse role of Smad2 and Smad3 in renal fibrosis, blocking Smad2 or Smad3 is capable to attenuate AKI. This indicates Smad2 cooperates with Smad3 in mediating programmed cell death and kidney injury.
Finally, we evaluated the therapeutic potential of Smad2 in established AKI mouse models. It is noteworthy that Smad2 knockdown protected against kidney injury, programmed cell death and renal inflammation in both nephrotoxic and ischemic AKI mouse models.
In conclusion, we found that TGF-β promoted renal damage in AKI model, and disruption of Smad2 attenuated cisplatin and I/R-induced kidney injury via reducing programmed cell death of TECs and p65 NF-κB-driven renal inflammation ( Figure 10). Therefore, Smad2 may serve as a potential therapeutic target in AKI treatment.