Acetyl‐11‐keto‐β‐boswellic acid ameliorates renal interstitial fibrosis via Klotho/TGF‐β/Smad signalling pathway

Abstract Acetyl‐11‐keto‐β‐boswellic acid (AKBA), an active triterpenoid compound from the extract of Boswellia serrate, has been reported previously in our group to alleviate fibrosis in vascular remodelling. This study aimed to elucidate the in vivo and in vitro efficacy and mechanism of AKBA in renal interstitial fibrosis. The experimental renal fibrosis was produced in C57BL/6 mice via unilateral ureteral obstruction (UUO). Hypoxia‐induced HK‐2 cells were used to imitate the pathological process of renal fibrosis in vitro. Results showed that the treatment of AKBA significantly alleviated UUO‐induced impairment of renal function and improved the renal fibrosis by decreasing the expression of TGF‐β1, α‐SMA, collagen I and collagen IV in UUO kidneys. In hypoxia‐induced HK‐2 cells, AKBA displayed remarkable cell protective effects and anti‐fibrotic properties by increasing the cell viability, decreasing the lactate dehydrogenase (LDH) release and inhibiting fibrotic factor expression. Moreover, in obstructed kidneys and HK‐2 cells, AKBA markedly down‐regulated the expression of TGFβ‐RI, TGFβ‐RII, phosphorylated‐Smad2/3 (p‐Smad2/3) and Smad4 in a dose‐dependent fashion while up‐regulated the expression of Klotho and Smad7 in the same manner. In addition, the effects of AKBA on the Klotho/TGF‐β/Smad signalling were reversed by transfecting with siRNA‐Klotho in HK‐2 cells. In conclusion, our findings provide evidence that AKBA can effectively protect kidney against interstitial fibrosis, and this renoprotective effect involves the Klotho/TGF‐β/Smad signalling pathway. Therefore, AKBA could be considered as a promising candidate drug for renal interstitial fibrosis.

irrespective of the initiating pathology. 1 Inhibiting the progression of interstitial fibrosis is considered as a potential strategy to protect the kidney from the deleterious effects of CKD. Numerous studies have revealed that renal interstitial fibrosis is mediated by multiple mechanisms, and the central cellular event is the accumulation of myofibroblasts, which are primarily responsible for the deposition of ECM at pathologic conditions. 2 Persistent production of ECM decreases the glomerular infiltration rate and renal function, eventually resulting in the acceleration of renal injury. 3 Klotho is a membrane-bound protein abundantly expressed in the kidney, and which was recognized as a promising protein uniquely poised to provide the basis for anti-fibrotic treatment strategies. 4 Numerous studies have indicated that Klotho deficiency induces and promotes tubulointerstitial fibrosis progression, and overexpression of Klotho protects against renal fibrosis. 5,6 In addition, Klotho was reported to inhibit transforming growth factor-β1 (TGF-β1) signalling by directly binding to TGFβ-RII, this is a vital mechanism for Klothomediated prevention of renal fibrosis. 7 TGF-β1 has been identified as the key profibrogenic cytokine via activating downstream mediators, especially Smad proteins, and finally regulating ECM production. 8 Thus, blocking the Klotho/TGF-β/Smad signalling pathway may be promising for preventing renal fibrosis and preserving renal function.
Acetyl 11-keto-β-boswellic acid (AKBA), a pentacyclic triterpenoid compound ( Figure 1A), is one of the most potent active principles within the multi-component mixture of Boswellia serrata resin. Boswellia serrata resin extracts (Boswellic acids) has been reported to have significant anti-fibrosis activity in many conditions, including hepatic fibrosis, 9 colonic fibrosis 10 and pulmonary fibrosis. 11 The anti-renal fibrosis property of pentacyclic triterpenoid has attracted increasing attention recently. For example, betulinic acid ameliorates experimental diabetic renal fibrosis by inhibiting the activation of NF-kB signalling pathway. 12 Oleanolic acid, the similar pentacyclic triterpene compound, exerts its therapeutic effect on tubulointerstitial fibrosis in chronic cyclosporine nephropathy. 13 Another study revealed that Asiatic acid attenuates renal fibrosis by rebalancing TGF-β/Smad signalling pathway. 14 More encouragingly, previous studies of our group have found that AKBA is beneficial for vascular remodelling and fibrosis by blocking TGF-β/Smad pathway. This prompted us to address in greater detail the role and mechanism of AKBA in renal fibrosis.
In this study, we applied in vivo and in vitro renal fibrosis paradigms to analyse the protective properties of AKBA. We hypothesized that AKBA would provide reno-protection against fibrosis in UUO-induced mice and in hypoxia-induced HK-2 cells, and that the protective effects were exerted through inhibiting the Klotho/TGF-β/Smad pathway. to pellet food and water. An acute oral toxicity study of AKBA on normal rats has been conducted in our previous research, and the results demonstrated that AKBA did not show any sign of toxicity or abnormal symptom in the rats of dosing up to 1600 mg/kg. Therefore, the dosage of AKBA in this study is considered to be safe.

| Experimental protocols
After 7 days of acclimatization, the animals were randomly divided into 5 groups (n = 8 in each group): sham-operated group (Sham), UUOoperated group (UUO), low AKBA dosage group (10 mg/kg/d), middle AKBA dosage group (20 mg/kg/d) and high AKBA dosage group (40 mg/ kg/d). The UUO operation was produced as described previously. 15 Briefly, the mice were anaesthetized with 1.5% sodium pentobarbital (50 mg/kg), and then the left ureter was separated and ligated with 4-0 silk sutures. The remnant ureter between the ligatures was cut-off.
Sham group underwent the same procedure and exposured the left ureter without ligation. After completion of modelling, the mice were administered with different dosage of AKBA or equal volumes of sterile saline by gavage. At 14 days after treatment, the mice were weighed and killed. Blood was collected for biochemical examination, and the obstructed kidneys were harvested and weighed for further researches.

| Renal function test
The blood samples were centrifuged to obtain serum for evaluation of serum creatinine (Scr) and blood urea nitrogen (BUN). A Creatinine Assay Kit and Urea Assay Kit (Jiancheng Biotechnology, Nanjing, China) were used for Scr and BUN test according to the manufacturer's instructions.

| Histology and immunohistochemistry
Kidneys were fixed with 4% paraformaldehyde for at least 24 hours, and then they were dehydrated, embedded and cut into 5-μm sec-

| Cell culture and treatment
Human proximal tubular epithelial cell line (HK-2) was obtained from Type Culture Collection of the Chinese Academy of Sciences

| Cell viability
Cell viability was determined at 48 hours post-hypoxia by 2 widely accepted assays, MTT assay and LDH release assay. The experiments were conducted according to the manufacturer's protocol of a cell cytotoxicity assay kit and a LDH assay kit (Jiancheng Biotechnology).

| Western blotting analysis
Total proteins were extracted from the kidneys or HK-2 cells and    Table S1. The PCR cycling conditions were as follows: 94°C for 15 minutes to preincubation and for 10 seconds to denaturation, and at 60°C for 30 seconds for 45 cycles to extension. All the assays were performed in triplicate. Transcript levels were calculated using the 2 ÀΔΔCt method and were normalized to GAPDH.

| Immunofluorescence staining
HK2 cells were fixed in 4% paraformaldehyde and subsequently permeabilized with 0.1% Triton X-100. After blocking with 5% BSA at room temperature for 1 hour, cells were incubated with anti-TGF-β1

| Statistical analysis
The SPSS (IBM, Armonk, New York, USA) 19.0 was used for statistical analysis. Data were presented as the mean ± standard deviation (SD). Statistical significance was determined using the one-way analysis of variance (ANOVA) followed by Tukey's multiple-comparison test. P < 0.05 was considered as statistical significance.

| AKBA alleviated renal function and renal interstitial damage in UUO mice
As shown in Figure 1B Haematoxylin & eosin staining was used to observe the morphology changes in obstructed kidneys ( Figure 1E). Sham-operated kidney presented normal tubule interstitium, while mice in UUO group showed a significant tubulointerstitial injury, including interstitial expansion, epithelial cells atrophy, inflammatory cell infiltration, glomerular sclerosis and ECM deposition. These injuries were ameliorated by incremental doses of AKBA treatment. Renal interstitial fibrosis level was also evaluated by Masson staining. Increased collagen deposition and area of interstitial fibrosis were observed in the UUO group. However, administration of AKBA markedly decreased the degree of interstitial fibrosis ( Figure 1E).

| AKBA decreased the expression of fibrotic factors in UUO mice
Based on the Masson staining above, effect of AKBA on interstitial fibrosis was further valued by detecting the expression of fibrotic factors. TGF-β1 is considered the vital pro-fibrotic growth factor in the pathogenesis of interstitial fibrosis. Myofibroblasts play a critical role in fibrosis, thus as a marker of myofibroblast, the expression of α-SMA was also examined. As shown in Figure 2, compared to the sham group, the expressions of TGF-β1 and α-SMA were significantly increased in the UUO kidneys. While after AKBA treatment, the expressions of TGF-β1 and α-SMA were effectively reduced in a dose-dependent manner. Similar tendency was also observed in collagen I and collagen IV expression, as determined by Western blotting. These results indicated that AKBA could attenuate UUOinduced renal interstitial fibrosis.

| AKBA inhibited the Klotho/TGF-β/Smad signalling pathway in UUO mice
To investigate the possible mechanisms underlying the effects of AKBA, the Klotho/TGF-β/Smad signalling pathway was checked in the obstructed kidney. The immunohistochemistry results showed that the overexpression of α-SMA and TGF-β1 induced by UUO was significantly restrained by AKBA. Smad2/3, the core protein in Klotho/TGF-β/Smad pathway, was up-regulated in obstructed kidneys and decreased after treatment of AKBA ( Figure 3).
Next, the exact mechanism was studied by Western blotting.
Fourteen days after UUO, the expression of Klotho significantly declined in obstructed kidneys, while the level elevated with AKBA treatment. Besides, the expressions of TGFβ-RI, TGFβ-RII, phosphorylated-Smad2/3 (p-Smad2/3) and Smad4 were obviously increased compared to the sham group. After treatment of AKBA in low-tohigh concentration, these elevated protein levels were significantly inhibited. In addition, AKBA dose-dependently increased the downregulation of Smad7 induced by UUO (Figure 4).

| AKBA enhanced the viability and decreased the LDH release in hypoxia-induced HK-2 cells
Emerging evidence has suggested that chronic hypoxia can trigger kidney damage response and cause irreversible pathological change in tubular epithelial cells, including necrosis, apoptosis and phenotype transformation, leading to renal interstitial fibrosis. 16 Therefore, the cell viability of HK-2 cells was firstly evaluated at 48 hours after F I G U R E 2 Effect of AKBA on the expression of fibrotic factors in UUO mice. A, Representative photographs showing the expressions of TGF-β1 collagen I, collagen IV and α-SMA. B, Quantitative analysis of the optical density in part A. Data were presented as mean ± SD for 3 independent experiments (n = 8). ### P < .001 vs sham group; *P < .05, **P < .01, ***P < .001 vs UUO group F I G U R E 3 Effect of AKBA on the expressions of α-SMA, TGF-β1 and Smad2/3. A, Representative photomicrographs of α-SMA, TGF-β1 and Smad2/3 immunohistochemistry on the kidney sections. B, Quantitative analysis of the optical density. Data were presented as mean ± SD (n = 8). ### P < .001 vs sham group; *P < .05, **P < .01, ***P < .001 vs UUO group hypoxia exposure in the absence and presence of AKBA treatment.
The data revealed that the cell viability was significantly decreased and the LDH release was greatly increased in hypoxia-induced HK-2 cells compared with the control group ( Figure 5A-B). When treated with AKBA, the cells MTT value was increased and LDH release was reduced compared with the vehicle treatment under hypoxia condition.

HK-2 cells
To further confirm the anti-fibrosis effect of AKBA on hypoxiainduced HK-2 cells, fibrosis-related factors, including TGF-β1, collagen I, collagen IV and α-SMA, were detected by quantitative realtime RT-PCR. We found that their levels were routinely increased under hypoxic condition but were obviously reduced after AKBA treatment ( Figure 5C).
Likewise, immunofluorescence results manifested that hypoxia could increase the expression of TGF-β1, α-SMA and Collagen IV. In comparison with hypoxia group, a noticeable reduction in TGF-β1, α-SMA and Collagen IV expression was observed in AKBA treated groups ( Figure 6), which suggested that AKBA can effectively relieve fibrosis in hypoxia-induced HK-2 cells.  Figure 8A,B). Also, the knockdown of Klotho reversed the effect of AKBA on the Klotho/TGF-β/Smad signalling pathway in hypoxia-induced HK-2 cells ( Figure 8C). All these results suggested that AKBA ameliorated renal interstitial fibrosis via Klotho/TGF-β/Smad signalling pathway.

| DISCUSSION
In the present study, we investigated the effect of AKBA on UUOinduced renal interstitial fibrosis for the first time. The result showed that AKBA plays a markedly protective role against renal injury via anti-fibrosis effects mediated by the Klotho/TGF-β/Smad pathway. Increasing evidence showed that chronic hypoxia is an important driving factor of renal interstitial fibrosis and leads to end-stage renal failure. 16,18,19 Thus hypoxia-induced HK-2 cell is often used to imitate the pathological process of renal interstitial fibrosis in vitro. 20,21 The data revealed that the cell viability was significantly decreased and the LDH release was greatly increased in hypoxia-induced HK-2 cells as compared to control group. While the AKBA treatment substantially increased the cell viability and reduced LDH release. These results indicated that AKBA is capable of protecting kidneys from progress to interstitial fibrosis.
Klotho is a multifunctional protein that is well known for its antiageing property. Deficiency of Klotho in mice often leads to short lifespan, cognitive dysfunction, osteoporosis, vascular calcification, etc. 22 While overexpression of Klotho could extend lifespan 23 and alleviate renal disease. 24,25 It was reported that full Klotho knockout mice (Klotho −/− ) have a more striking phenotype and serious pathological changes than heterozygous Klotho mice (Klotho +/− ). 4 Compared to the wild-type mice, constitutive overexpression of Klotho in mice could lead to less expression of fibrosis-related proteins, such as α-SMA and collagen I. 25 As expected, the results in our study showed that the levels of Klotho were significantly decreased in obstructed kidneys and hypoxia-induced HK-2 cells. AKBA administration up-regulated the expression of Klotho in a dose-dependent manner. And the renal protective effect of AKBA was reversed with the transfection of siRNA-Klotho.
As a downstream molecule directedly regulated by Klotho, TGF-β1 has been extensively recognized as a critical mediator in interstitial fibrosis. 26 Accumulating evidence has demonstrated that TGF-β1 is excessively produced by fibroblasts in various kidney diseases with interstitial fibrosis. 27,28 The activated TGF-β1 regulates cellular differentiation, proliferation and migration, as well as the protein expression of ECM. 29 Overexpression of TGF-β1 could cause renal fibrosis while inhibition of that would improve renal fibrotic lesions. 30 Our study indicated that the protein expression of TGF-β1 was increased in UUO kidney, and AKBA could attenuate the elevated TGF-β1 level in a dose-dependent manner. In addition, similar effect was observed in HK-2 cells. As the major components of ECM, α-SMA, collagen I and collagen IV are tightly bound to renal fibrosis. The synthesis and degradation of these proteins are in a state of dynamic balance under normal physiological condition. 8 Our The mRNA levels of TGF-β1, collagen I, collagen IV and α-SMA were determined by quantitative RT-PCR. Data were presented as mean ± SD (n = 8). ## P < .01, ### P < .001 vs control group; *P < .05, **P < .01, ***P < .001 vs hypoxia group F I G U R E 6 Effect of AKBA on the expressions of TGF-β1, α-SMA and collagen IV in hypoxia-induced HK-2 cells detected by immunofluorescence. The blue staining refers to the DAPI while the red staining refers to the specific proteins. Scale bar = 20 μm F I G U R E 7 Effect of AKBA on the activation of Klotho/TGF-β/Smad pathway in hypoxia-induced HK-2 cells. The expressions of Klotho, TGFβ-RI, TGFβ-RII, p-Smad2/3, Smad4 and Smad7 were investigated by Western blot. Data were presented as mean ± SD for 3 independent experiments (n = 8). ## P < .01, ### P < .001 vs control group; *P < .05, **P < .01, ***P < .001 vs hypoxia group results showed that the expression of α-SMA, collagen I and collagen IV were evidently increased in obstructed kidneys and hypoxiainduced HK-2 cells. All the elevated expressions were reversed after AKBA treatment, indicating the anti-fibrotic role of AKBA.
Among the downstream signalling pathways involving in TGF-βdependent renal interstitial fibrosis, Smads pathway is considered as the most critical one. 31 TGF-β1 signal is transduced through its cell membrane receptors I and II (TGFβ-RI and TGFβ-RII). Upon TGF-β stimulating, receptors become activated and trigger the phosphorylation of its downstream signalling molecules: Smad2 and Smad3. Phosphorylated Smad2/3 heteroligomerizes with Smad 4, a common partner Smad, and the complexes translocate into the nucleus to regulate the transcription of target genes. 32 In a rat UUO model, TGFβ-RII mRNA significantly increased, and inhibition of TGFβ-RII ameliorated the progression of renal fibrosis. 33 Consistent with the reported data, we also found that TGFβ-RI and RII were highly expressed in the fibrotic kidneys. However, they were markedly inhibited after AKBA treatment. In the context of renal interstitial fibrosis, Smad2/3 was reported to be strongly activated in various CKDs. 34 In addition, deletion of Smad3 in mice or use of Smad3 inhibitor suppressed the progress of renal fibrosis. 35,36 As expected, excessive expression of Smad 2/3 was observed both in obstructive kidneys and hypoxia-insulted HK-2 cells. After AKBA in low-to-high concentrations were given, protein level of Smad2/3 in the treatment group was dose-dependently decreased. Smad7 is one type of inhibitory Smad protein that negatively regulates activation and phosphorylation of Smad2/3. Overexpression of Smad7 could block activation of TGF-β/Smad signalling while reduction of renal Smad7 could result in over-activation of TGF-β/Smad signalling and progression of renal fibrosis. 37 In this study, we found AKBA increased the expression of Smad7 in vivo and in vitro experiments. These results indicated that AKBA has the effect of anti-fibrosis by inhibiting the activation of Smad pathway.
In conclusion, our results revealed for the first time that AKBA could dose-dependently improve renal interstitial fibrosis in vivo and in vitro, and the underlying mechanism may be involved with the inhibition of Klotho/TGF-β/Smad signalling pathway. Our findings indicate that AKBA could be used as a novel candidate drug for renal interstitial fibrosis.

ACKNOWLEDG EMENTS
This work was funded by the National Natural Science Foundation of China (No. 81670655; 81400699).

S U P P O R T I N G I N F O R M A T I O N
Additional supporting information may be found online in the Supporting Information section at the end of the article.
How to cite this article: Liu M, Liu T, Shang P, et al.