Ablation of Gadd45β ameliorates the inflammation and renal fibrosis caused by unilateral ureteral obstruction

Abstract The growth arrest and DNA damage‐inducible beta (Gadd45β) protein have been associated with various cellular functions, but its role in progressive renal disease is currently unknown. Here, we examined the effect of Gadd45β deletion on cell proliferation and apoptosis, inflammation, and renal fibrosis in an early chronic kidney disease (CKD) mouse model following unilateral ureteral obstruction (UUO). Wild‐type (WT) and Gadd45β‐knockout (KO) mice underwent either a sham operation or UUO and the kidneys were sampled eight days later. A histological assay revealed that ablation of Gadd45β ameliorated UUO‐induced renal injury. Cell proliferation was higher in Gadd45β KO mouse kidneys, but apoptosis was similar in both genotypes after UUO. Expression of pro‐inflammatory cytokines after UUO was down‐regulated in the kidneys from Gadd45β KO mice, whereas UUO‐mediated immune cell infiltration remained unchanged. The expression of pro‐inflammatory cytokines in response to LPS stimulation decreased in bone marrow‐derived macrophages from Gadd45β KO mice compared with that in WT mice. Importantly, UUO‐induced renal fibrosis was ameliorated in Gadd45β KO mice unlike in WT mice. Gadd45β was involved in TGF‐β signalling pathway regulation in kidney fibroblasts. Our findings demonstrate that Gadd45β plays a crucial role in renal injury and may be a therapeutic target for the treatment of CKD.


| INTRODUC TI ON
The prevalence and burden of chronic kidney disease (CKD) are rising worldwide, and obstructive renal injury is the most common cause for renal fibrosis. 1,2 Renal fibrosis is the key process underlying the progression of CKD to end-stage renal disease and is the outcome of an excessive amassment of extracellular matrices that occurs in CKD. 2 Glomerulosclerosis, tubulointerstitial fibrosis and excess accumulation of matrices in the interstitial site surrounding tubules and peritubular capillaries are common findings in patients with renal fibrosis. 3 The pathogenesis of CKD is primarily prominent renal hemodynamic and metabolic changes, tubular damage and cell death caused by apoptosis or necrosis, leading to increased infiltration of immune cells into the renal interstitium. 4 Although transforming growth factor-β (TGF-β) is considered the master regulator that culminates in fibrosis and renal parenchymal loss, the importance of genetic and epigenetic factors is accepted.
TGF-β is a prototypic fibrogenic growth molecule that controls various biological actions, including cell growth, differentiation and immunological reactions. 5,6 In renal fibrosis, one of the most important actions of TGF-β is the adjustment of ECM accumulation via inhibiting matrix breakdown by declining the synthesis of metalloproteinases and increasing the synthesis of their inhibitors. 7 In human glomerular diseases, up-regulated TGF-β expression occurs in progressive glomerular diseases, and this expression is strongly correlated with fibrotic areas in biopsy specimens. 8 The expression of TGF-β receptors is also enhanced in diseased glomeruli. 8 The binding of TGF-β to its cognate receptors, such as TGF-β type I and II receptors, results in the stimulation of canonical and non-canonical intracellular signalling pathways. 9,10 Smad2 and Smad3 are the main molecules involved in canonical pathways activated by TGF-β to form a complex with Smad4, which then translocate into the nucleus and regulate the transcription of the target genes. 11 Additionally, Smad6 and Smad7 are known as negative regulators of Smad2/3, and thereby inhibit TGF-β signalling. 12,13 Recently, it was found that Gadd45β is a novel protein that regulates the TGF-β signalling pathway by blocking the Smurfinduced breakdown of TGF-β receptor type 1 in colon tissue and intestinal epithelial cell lines. 14 Gadd45β is a small protein included in the Gadd45 family that is composed of Gadd45α, Gadd45β and Gadd45γ. 15 In addition to the regulation of TGF-β signalling, we and others have previously shown that Gadd45β regulates DNA repair, cell cycle arrest, cell proliferation, apoptosis and inflammation, depending on the cell type. 16,17 However, the roles of Gadd45β in CKD remain unclear.
In the current study, we investigated the effects of Gadd45β ablation on cell survival, apoptosis, inflammation and renal fibrosis in mice. The unilateral ureteral obstruction (UUO) model was used to induce renal fibrosis and immune cell infiltration and to estimate the susceptibility against UUO-induced nephropathy.

| Animals
A whole-body Gadd45β knockout (KO) mouse under a C57BL/6 background was purchased from Jackson Laboratory. All mice were maintained in a room maintained at constant temperature (20-22°C) on a 12:12 hours light:dark cycle with free access to food and water under specific pathogen-free conditions. For the UUO model, Gadd45β wild-type (WT) and KO male mice were anaesthetized with avertin (200 mg/kg) and placed on a hot pad (34°C) to prevent hypothermia. After anaesthesia, the anterior abdominal skin was shaved and wiped with 70% ethanol. The left ureter was exposed and ligated twice using a nylon suture. Sham-operated mice without ligation were prepared as controls. Mice were killed by cervical dislocation, and the kidney tissues were removed at eight days after UUO for molecular analysis and histological examination. All animal experiments were approved by the Institutional Animal Care and Use Committee (KRIBB-AEC-19254) and were performed in accordance with the institutional guidelines of the Korea Research Institute of Bioscience and Biotechnology.

| Cell culture and transfection
Human embryonic kidney HEK293T cells were cultured in

| Cell stimulation with TGF-β1
The cells were transfected with V5-tagged Gadd45β or empty vector for one day, washed with PBS, and subsequently stimulated with 10 ng/mL TGF-β1 for the indicated duration. For the silencing of Gadd45β, cells were plated in a six-well plate (1 × 10 5 ) and transfected with siRNA for the control or Gadd45β for three days.
Silenced cells were further stimulated with or without 10 ng/mL TGF-β1 for the indicated duration.

| Isolation and differentiation of mouse bone marrow cells
Male WT and Gadd45β KO mice were killed by cervical dislocation, and both femurs were removed. Bone marrow was flushed

| Renal histological analysis
The kidney tissues fixed in 10% neutral buffered formalin were embedded in paraffin and cut to 4 μm-thick sections. The sections were deparaffinized and stained with haematoxylin & eosin (H&E), Sirius red and Masson trichrome according to the standard protocol.

| Immunohistochemistry
Kidney slides (with 4-μm thick sections) were deparaffinized in sequential steps of xylene, ethanol, and finally rehydrated in water.

| RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA from kidney tissues were isolated using TRIzol rea-  Table 1. The expression of the target genes was normalized by 18S rRNA.

| Measurement of plasma proinflammatory cytokines
Plasma samples from UUO-mice were harvested at 8 days after UUO and pro-inflammatory cytokines such as interleukin 1 beta (IL-1β), interleukin 6 (IL-6) and tumour necrosis factor alpha (TNFα) were measured using ELISA kit according to the manufacturer's instructions (BD Biosciences), respectively.

| Western blotting
Kidney tissues or cells were lysed in RIPA buffer containing protease inhibitor cocktail (Roche Applied Science). Protein concentrations were quantified by the Bradford's assay method. The equivalent proteins were separated by electrophoresis on a polyacrylamide gel and transferred to a polyvinylidene fluoride membrane. The membranes were incubated with corresponding primary and secondary antibodies. Housekeeping proteins were used as a loading control.

| Statistical analysis
Statistical analyses were performed using GraphPad Prism software.
All data are presented as mean ± SEM. Stained areas were analysed using ImageJ software, and immunoblot analysis results were quantified using Tina 2.0 software and ImageJ. Two-group comparisons were performed using two-tailed Student's t test. A P-value of <.05 was considered statistically significant.

| The effect of Gadd45β deficiency in UUOinduced renal damage
To investigate the involvement of Gadd45β in acute renal injury, the standard UUO experimental model of renal failure was used and the functional contribution of Gadd45β to the development of tubular injury was assessed by estimating glomerular size and tubular injury score in the sections stained with H&E ( Figure 1A). No significant differences in glomerular size between all groups were observed ( Figure 1B). However, tubular injury significantly increased after UUO, as reflected by the presence of dilated tubules, loss of brush borders, and epithelial simplification in the tubulointerstitium in Gadd45β WT and KO mice ( Figure 1C). Interestingly, Gadd45β-KO kidneys showed reduced tubular injury compared with those of Gadd45β WT mice ( Figure 1C), suggesting that Gadd45β is involved in the development of renal injury.

| The effects of Gadd45β deficiency on UUOinduced cell proliferation and apoptosis
Renal injury in the UUO model can lead to an imbalance between tubular epithelial cell apoptosis and proliferation. Therefore, immunohistochemistry of kidney tissues was performed using specific antibodies against Ki-67 or cleaved caspase 3 to determine cell proliferation and apoptosis, respectively. Interestingly, cell proliferation increased by approximately threefold after UUO in Gadd45β WT kidneys and was more prominent in Gadd45β KO  (Figure 2A). Although apoptosis was markedly upregulated after UUO, significant differences were not observed in either genotypes ( Figure 2B). ERK and Akt are major mediators that regulate cell proliferation. Therefore, the activation of these proteins was estimated by using specific antibodies against active phosphorylated residues on these kinases. The total and phosphorylated levels of these kinases increased after UUO compared with the sham operation, but no significant differences were observed between either genotype, suggesting that another pathway might be involved in cell proliferation, mediated by Gadd45β deficiency ( Figure 2C).

| The effects of Gadd45β deficiency against UUO-induced inflammatory cell infiltration
The recruitment of inflammatory cells, such as T-lymphocytes and macrophages, is an early event in progressive renal injury. 19 Therefore, whether Gadd45β deficiency could affect renal immune infiltration in the kidneys after UUO was determined.
Antibodies against the T-cell marker CD3 and macrophage marker F4/80 were applied in IHC analyses, respectively. In line with previous studies, UUO caused immune cell infiltration in the kidneys of Gadd45β WT and KO mice, as estimated by the positive area in the respective kidney samples ( Figure 3A,B).

| The effects of Gadd45β deficiency against UUO-induced inflammatory cytokine expression
To investigate the effect of Gadd45β ablation on inflammatory cytokines in the UUO-kidney, the expression of pro-inflammatory

| Effects of Gadd45β deficiency in UUO-induced renal fibrosis
To investigate the roles of Gadd45β on the fibrotic changes of the kidney after UUO, Sirius red staining was performed and the positive area was quantified to assess the degree of collagen deposition in the kidneys from both genotypes. The sham-operated kidneys of both genotypes expressed only small areas of collagen deposition ( Figure 5A). However, UUO kidneys exhibited a marked increase in Sirius red-positive areas compared with those of the sham controls in the kidneys of both genotypes after UUO, compared with those of the sham-operated mice ( Figure 5B). However, the alteration in UUO was significantly attenuated by the ablation of Gadd45β ( Figure 5B).

Furthermore, Western blotting demonstrated that UUO-induced
αSMA, the putative myofibroblast marker, was significantly reduced by the ablation of Gadd45β ( Figure 5C). These results suggest that Gadd45β is closely involved in the progression of UUO-induced renal fibrosis.

| The roles of Gadd45β on TGF signalling pathways in vitro and in vivo
Previously, 14  and not TGF-β1 stimulation ( Figure 6B). Based on canonical TGF signalling which is initiated by the heterogeneous complexes of Smad2 and Smad3, the phosphorylation of Smad2 in response to TGF-β1 is critical to Gadd45β-mediated regulation of TGF signalling pathway in the UUO condition. We further confirmed the roles of Gadd45β on TGF signalling pathways in vivo. Consistent with the in vitro results, phosphorylation of Smad2 was significantly lower in kidney tissues from Gadd45β KO mice than in those from WT mice ( Figure 6C). These data suggest that the regulation of TGF-β signalling by Gadd45β might be a common pathway in various cell types.
In our previous study, 14 we suggested that Gadd45β is involved in TGF signalling pathways via binding to Smad7, resulting in increased protein levels of TGFβRI in intestinal epithelial cell lines. Therefore, we confirmed the levels of TGFβRI and Smad7 in the UUO-kidneys of both groups. Smad7 protein levels were significantly up-regulated in the UUO-kidney of Gadd45β KO mice compared with those in WT mice, whereas TGFβRI protein levels were not different ( Figure 6C).
These results indicate that Gadd45β can regulate the phosphorylation of Smad2 via modulation of Smad7 stability in kidney after UUO.

| D ISCUSS I ON
The progression of CKD induces renal atrophy, increased leukocyte infiltration and fibrosis in the kidneys. 2 The UUO model is a standard model for renal fibrosis and chronic kidney failure and mimics a human patient with CKD. 20 Gadd45β is a small protein with no enzymatic activity of its own. 15 Instead, its physiological functions are activated by interactions with partner molecules in the nucleus and cytoplasm of cells, and it is involved in cell proliferation, cell death and inflammation. [21][22][23] Recent studies in experimental animals have shown that Gadd45β is involved in the pathogenesis of various diseases. 14,24,25 In the present study, Gadd45β KO mice were used and UUO surgery was carried out to induce early stage CKD. A deficiency of Gadd45β led to the lower susceptibility of UUO-induced nephropathy. The role of Gadd45β on cell proliferation and death,

F I G U R E 6
The involvement of Gadd45β in the TGF signalling pathway. HEK293T cells were treated with Gadd45β siRNA (10 nmol/L) and stimulated with recombinant TGF-β1 (10 ng/mL) in a time-dependent manner. A, Cell lysates were used for Western blotting using specific antibodies against the indicated molecules. β-actin was used as a loading control. B, HEK293T cells were transiently transfected with V5 tagged Gadd45β plasmid (500 ng/well) or empty vector. The downstream molecules of TGF signalling were detected by Western blotting. β-actin was used as a loading control. C, The protein levels and phosphorylation of molecules related to the TGF signalling pathway were determined by Western blot analysis and optical density was expressed by ImageJ program. *P < .05. Data are representative of at least two independent experiments immune cell infiltration and inflammation, and fibrosis was further investigated in mouse kidneys. It was found that cell proliferation increased slightly, but pro-inflammatory cytokines and renal fibrotic markers were reduced in Gadd45β KO mice after UUO.
Gadd45β is a protein that plays a pivotal role in negative growth control, including growth arrest and apoptotic cell death related to cancer. 23 with the WT mice. However, the expression of pro-inflammatory cytokines was lower in the kidneys of Gadd45β KO mice than in those of WT mice. Although we were unable to determine the exact mechanism responsible for the discrepancy between cell infiltration and pro-inflammatory cytokine levels, BMDMs from Gadd45β KO mice and Gadd45β-silenced HK-2 cells showed significantly lower pro-inflammatory cytokine levels than their controls. These data suggest that the accumulated pro-inflammatory cytokines in kidney tissues after UUO originate from infiltrated macrophages.
Renal fibrosis is a major pathological sign of CKD caused by various stresses. 31 TGF-β1 is a key member of the TGF superfamily. 8 Various studies have found that the TGF-β1/Smad signalling pathway contributes to renal fibrosis. 32,33 Importantly, the suppression of the TGF-β signalling pathway by pharmacological or genetic means ameliorated the progression of renal fibrosis in patients or experimental animal models. 33 Recently, we suggested that Gadd45β can bind with Smad7, known as inhibitory Smad, and block the degradation of TGF-β receptor type 1 mediated by Smurf/Smad7 complex in colon tissue and intestinal epithelial cells. 14 However, in the present study, we found that Smad7 protein levels in the UUO-kidney of Gadd45β KO mice were up-regulated, which subsequently reduce the phosphorylation of Smad2, resulting in less renal fibrosis. We previously showed that TGF-β1-induced Smad2 and Smad3 activation was eliminated by silencing Gadd45β, while this effect was reversed by transient up-regulation in Caco2 cells originating from the intestinal epithelium. 14 The current study also showed that Gadd45β regulated TGF-β1-mediated Smad signalling in kidney cell lines, suggesting that the Gadd45β-regulated TGF signalling pathway might be involved in the progression of renal fibrosis.
In summary, Gadd45β plays an important role in the progression of kidney disease. Physiologically, Gadd45β is involved in the regulation of cell proliferation, pro-inflammatory cytokines and renal fibrosis during the progression of CKD. In particular, we found that the TGF signalling pathway is regulated by Gadd45β in kidney fibroblasts. We believe that our novel results contribute to the elucidation of the pathology of CKD, especially in renal fibrosis. Initiative Program. We would like to thank Editage (www.edita ge.co.

ACK N OWLED G EM ENTS
kr) for English language editing.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used in this study are available from the corresponding author on reasonable request.