CXCL16 deficiency attenuates diabetic nephropathy through decreasing oxidative stress and inflammation

Accepted Manuscript CXCL16 deficiency attenuates diabetic nephropathy through decreasing hepatic oxidative stress and inflammation Yanna Ye, Qingzhen Chen, Jinmeng Li, Leigang Jin, Jujia Zheng, Xiaokun Li, Zhuofeng Lin, Fanghua Gong PII:

S0006-291X(17)30859-8

DOI:

10.1016/j.bbrc.2017.05.013

Reference:

YBBRC 37728

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 28 April 2017 Accepted Date: 2 May 2017

Please cite this article as: Y. Ye, Q. Chen, J. Li, L. Jin, J. Zheng, X. Li, Z. Lin, F. Gong, CXCL16 deficiency attenuates diabetic nephropathy through decreasing hepatic oxidative stress and inflammation, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/ j.bbrc.2017.05.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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CXCL16 deficiency attenuates diabetic nephropathy through decreasing hepatic oxidative stress and inflammation

Li Xiaokun 1, 2*, Lin Zhuofeng 1*, Gong Fanghua 1*

School of Pharmacy, Wenzhou Medical University, Chashan College

2

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Park, Wenzhou, Zhejiang, China；

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1

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Ye Yanna 1#, Chen Qingzhen 2#, Li Jinmeng1, Jin Leigang 1, Zheng Jujia 1,

College of Life and Environmental Science, Wenzhou University,

Chashan College Park, Wenzhou, Zhejiang, China.

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*Author for correspondence:

Address: School of Pharmacy, Wenzhou Medical University, Chashan College Park, Wenzhou325035, People’s Republic of China. E-mail:

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[email protected] (F. Gong); [email protected] (X. Li);

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[email protected] (Z. Lin)

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Abstract

Soluble C-X-C chemokine ligand 16 (CXCL16) is related to the

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inflammatory response in liver injury and involved in the pathogenesis of renal dysfunction in diabetes patients. However, the exact role of elevated CXCL16 in diabetic nephropathy (DN) remains unclear. In this study, we

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investigated the role of CXCL16 in streptozcin (STZ)-induced diabetic

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nephropathy (DN) in mice. The results showed that fasting blood glucose (FBG) and 24 h urinary protein, triglyceride, and cholesterol levels increased in diabetic mice, and these changes were partially ameliorated in CXCL16 KO mice. Meanwhile, the results also showed that ROS

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generation was suppressed and the expression levels of inflammatory factors and infiltration factors were inhibited both in vivo and in vitro using DN models. In addition, the total AKT protein and p-AKT levels

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were decreased in CXCL16-depleted HK-2 cells that were treated with

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LPS. These findings suggest that the CXCL16 gene product promotes inflammatory factors and cell infiltration factors, and inhibits the expression of antioxidant factors to accelerate the development of DN, and CXCL16 deficiency attenuates DN may be involved in the AKT signaling pathway. Key words: CXCL16; diabetic nephropathy; inflammatory factors; infiltration factors AKT signaling pathway

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Introduction

Diabetic nephropathy (DN) is a progressive kidney disease and a

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well-known complication of long-standing diabetes, and the leading cause of end-stage renal disease (ESRD) [1, 2]. DN of Diabetes mellitus (DM) accompanied with glomerulosclerosis, renal arteriosclerosis, and

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pyelonephritis [3, 4]. Previous studies revealed that approximately in

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many Western countries, 50% of ESRD patients who require dialysis are also diabetic and are highly susceptible to macrovascular complications. CXCL16

was

characterized

as

a

scavenger

receptor

for

phosphatidylserine and oxidized low density lipoprotein (ox-LDL) based

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on atherogenesis studies [5, 6]. Our previous work showed that serum CXCL16 levels in DN were higher than those of chronic kidney disease (CKD) patients without DN [7], and the serum CXCL16 may be an

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indicator of renal injury in subjects with Type 2 Diabetes Mellitus [8].

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However, the detailed mechanism of the effects of CXCL16 on the onset and development of DN remains ambiguous. To explore the physiological and pathological characteristics of CXCL16 in DN, 8-week old CXCL16 knockout (C16 KO) mice were treated with streptozcin (STZ) for a diabetes model. Additionally, the human renal tubular epithelial cell line HK-2 was stimulated with lipopolysaccharide (LPS) and used for a cellular model. Our data demonstrated that CXCL16 deficiency obviously

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inhibited the development of STZ-induced DN in mice, and CXCL16 deficiency attenuated DN through the AKT signaling pathway.

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Materials and Methods Animals

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CXCL16 KO mice with a C57BL/6 background were a generous gift

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from Professor Du Jie (Beijing Anzhen Hospital of the Capital Medical University), and the wild-type (WT) male C57B/6J mice were purchased from the Vital River Laboratory Animal Technology Co. Ltd. Mice were maintained in the animal care facility of Wenzhou Medical University

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and had ad libitum access to food and water. All animal procedures were performed in accordance with national and international animal care and ethical guidelines and were approved by the Institutional Animal Care

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and Usage Committee at Wenzhou Medical University. The type 2

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diabetic mice model was established by intraperitoneal injection of STZ. The body weight and blood glucose level of mice were measured every week, and the fasting blood glucose and 24 h proteinuria levels were measured 12 weeks after STZ injection [9]. After the mice were sacrificed, the plasma and kidney were collected and the triglyceride and cholesterol levels were measured using an automatic biochemical detector.

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Cell culture and transfection

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Normal renal tube HK-2 cells were purchased from the Shanghai Cell Collection (Shanghai, China). The HK-2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal

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bovine serum (FBS; GIBCO) at 37 °C in a humidified atmosphere with 5%

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CO2. The CXCL16 (GenBank accession number: NM_022059) shRNA lentiviral plasmid (target sequent: CCACCAGAAGCATTTACTT) and a nontargeting negative control plasmid were, respectively, constructed by cloning the appropriate sequences into the Age I/EcoR I sites of the

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GV248 vector. Preparation and transduction of the recombinant lentiviral particles were performed as described previously [10]. HK-2 cells were transfected with the shRNA lentivirus against CXCL16 or the universal

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instructions.

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nonsilencing control shRNA lentivirus according to the manufacturer’s

RNA Extraction and Real-Time PCR

Total RNA was extracted from kidney or HK-2 cells with Trizol reagent (TAKARA), and complementary DNA was synthesized from 1 µg total RNA by reverse transcription using an M-MLV first strand kit

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(Invitrogen). Quantitative real-time PCR was carried out using the SYBR Green QPCR system (QIAGEN) with specific primers (Tables 1 and 2). Expression levels determined by real-time PCR were determined using

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Histological Analysis and Immunoblotting

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the 2-∆∆Ct method.

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Kidney specimens were fixed in 10% formalin solution (Sigma) and embedded in paraffin. Kidney tissue sections were stained with H&E (×400) via standard procedures. All slides were examined using a NIKON biological microscope, and images were captured with an

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Olympus DP72 color digital camera. Proteins were extracted from HK-2 cells in the presence of a protease inhibitor cocktail (Roche Applied Science), resolved by SDS-PAGE, and then transferred to PVDF

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membranes. The membranes were then probed with primary antibodies

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against AKT (Cell Signaling Technology), phospho-AKT (Cell Signaling Technology),

CXCL16

(Abcam),

and

GAPDH

(Santa

Cruz

Biotechnology).

Statistical Analysis

Experiments were performed with seven or eight mice per group. All

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analyses were performed with the Statistical Package for Social Sciences version 14.0 (SPSS, Chicago. IL). Data were expressed as mean ± SEM. Statistical significance was determined by Student’s t test. In all statistical

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comparisons, a p value <0.05 was considered a statistically significant difference.

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Results

Effects of CXCL16 deficiency on body weight, fasting blood glucose, 24 h urinary protein, triglyceride, and cholesterol levels in diabetic

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mice

Three days after induction of insulin resistance by administration of intraperitoneal STZ, diabetic mice FBG levels exhibited marked

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hyperglycemia (>16.7 mmol/L, data not shown). As shown in Table 3,

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the FBG levels were significantly increased in both the WT diabetic mice (22.6 ± 2.30 mmol/L) and CXCL16 KO diabetic mice (17.8 ± 1.60 mmol/L) compared to mice without STZ treatment, and CXCL16 deficiency correlated with decreased FBG level compared with WT diabetic mice. Concomitantly, the results revealed no significant changes in body weight between the WT (30.2 ± 0.6 g) and CXCL16 KO (31.2 ± 0.3 g) mice without STZ treatment. The body weight were significantly

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reduced both in WT (25.8 ± 0.7 g) and CXCL16 KO (26.3 ± 0.6 g) diabetic mice, but there was no obvious difference between the two groups. As expected, the results showed that the 24 h urine protein levels

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were increased about 37-fold both in CXCL16 KO and WT diabetic mice. In addition, the results also demonstrated that the 24 h urine protein levels of CXCL16 KO diabetic mice (9.36 ± 0.03 µmol/24h) were lower than

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those of WT diabetic mice (11.32 ± 0.09 µmol/24h). The triglyceride and

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cholesterol levels were compared after 12 weeks and there were no significant changes between CXCL16 deficiency mice and WT mice.

CXCL16 deficiency attenuated kidney dysfunction and oxidative

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stress in diabetic mice

As shown in Fig. 1A, HE analysis of kidney sections demonstrated no

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kidney injury in WT and CXCL16 KO control mice. The STZ-induced

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diabetic mice exhibited typical diabetic nephropathy symptoms, including glomerulus hypertrophy, thickened basement membrane, and dissolved renal

mesangial.

The

PCR

results

further

indicated

that

the

malonaldehyde (MDA) level in the kidney was significantly increased 12.81-fold in WT diabetic mice and 4.39-fold in CXCL16 KO diabetic mice (Fig. 1B). In addition, the mRNA expression levels of the ROS-producing aconitase (Aco) gene were obviously increased in the

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kidneys of diabetic mice, but there were no significant changes between CXCL16 KO and WT diabetic mice (Fig. 1C). Furthermore, the expression levels of antioxidant factor superoxide dismutase 2 (Sod-2) in

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kidneys were increased 1.56-fold in WT diabetic mice and 3.5-fold in CXCL16 KO diabetic mice (Fig. 1D).

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CXCL16 deficiency reduced expression of inflammation factors and

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adhesion molecules both in vivo and in vitro

Inflammation is a major cause of DN, so we next measured the inflammation factors of kidney by Q-PCR. The results showed that both

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the tumor necrosis factor α (TNFα) and transforming growth factor β (TGFβ) expression levels were increased obviously in the kidneys of diabetic mice. The mRNA expression levels of TNFα kidney were

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significantly increased 22.31-fold in STZ-treated WT mice and 7.29-fold

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in CXCL16 KO diabetic mice (Fig. 1E). Analogously, the mRNA expression levels of TGFβ in the kidney were significantly increased 13.61-fold in WT diabetic mice and 6.37-fold in CXCL16 KO diabetic mice (Fig. 1F). Previous studies demonstrated elevated levels of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecular-1 (ICAM-1) in patients with diabetes mellitus. Here, our data showed that both the VCAM-1 and ICAM-1 expression levels were

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significantly increased in the kidneys of diabetic mice. More precisely, the mRNA expression levels of VCAM-1 kidney were significantly increased 10.47-fold in WT diabetic mice and 2.41-fold in CXCL16 KO

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diabetic mice (Fig. 1G). Similarly, the mRNA expression levels of ICAM-1 in the kidney were significantly increased 6.16-fold in WT

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diabetic mice and 3.16-fold in CXCL16 KO diabetic mice (Fig. 1H).

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The recombinant lentiviral particles were packaged in 293T cells, and the titer of the lentivirus was measured at 1×108 TU/ml. The protein level of CXCL16 was reduced 2.71-fold in HK-2 cells transfected with shRNA lentivirus (Lv-cxcl16) compared with those transfected with the control

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shRNA lentivirus (Lv-con) (Fig. 2A, 2C). Previous work demonstrated that LPS could induce acute renal injury in diabetes mice. Here, the data showed that the CXCL16 protein expression levels were increased

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4.15-fold in control cells and 2.26-fold in CXCL16-inhibited cells after

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LPS treatment (Fig. 2B, 2D). Furthermore, we measured the mRNA expression levels of Aco, Sod-2, TNF-α, TGF-β, VCAM-1, and ICAM-1 in LPS-induced HK-2 cells, and the results were consistent with those observed for diabetic mice (Fig. 3).

CXCL16 deficiency attenuates diabetic nephropathy involves in the AKT signaling pathway

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To explore how CXCL16 deficiency attenuates diabetic nephropathy, AKT and p-AKT protein levels were examined by Western blotting (Fig.

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4A) in HK-2 cells that were transfected with shRNA lentivirus for 72 h and then treated with LPS for 1 h. The results showed that the expression levels of AKT and p-AKT were decreased significantly in LPS-treated

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CXCL16- suppressing HK-2 cells compared to cells that did not receive

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LPS treatment (Fig. 4b, 4d).

Discussion

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CXCL16, a scavenger receptor for oxidized low density lipoprotein, was reported to be associated with long-term mortality after adjustment for risk factor in patients with acute coronary syndrome [11]. In addition,

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CXCL16 was characterized as a scavenger receptor for ox-LDL in the

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pathology of glomerular kidney diseases, particularly membranous nephropathy [12]. Furthermore, our previous studies found that serum CXCL16 levels are significantly increased in subjects with CKD and gout and are independently involved in renal function change in patients [7, 12]. Based on these results, we proposed that serum CXCL16 may be a novel marker of renal injury in type 2 diabetes mellitus [1]. In the present study, the H&E results indicated that the area of kidney damage in

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CXCL16 KO diabetic mice was significantly less than the amount of kidney damage in WT diabetic mice. These data suggest that reduced CXCL16 expression levels in diabetic mice are related to renal damage.

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Currently, many studies have demonstrated an important role for oxidative stress in the origin and development of DN [13, 14]. The main cause of oxidative stress is the presence of reactive oxygen species (ROS),

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which serve as a marker of the level of oxidative stress [15]. MDA is the

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end-product of lipid peroxidation, and thus can be used to estimate the level of oxidative stress and the severity of DN damage [16]. Here, our data revealed a significantly increased level of MDA in the kidney tissues of mice with DN compared with that of normal mice. The MDA level

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was reduced in CXCL16-deficient diabetes mice compared with that of diabetic mice. In addition, PCR analysis of diabetic mice kidney showed increased mRNA expression of the oxidation factor Aco, which promotes

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ROS generation. Analogously, the mRNA expression level of the

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antioxidant factor Sod-2 was markedly increased in diabetic mice, but was reduced in CXCL16-deficient diabetes mice. Similar results also were observed in HK-2 cells that were treated with LPS. These results suggest that CXCL16 deficiency attenuates diabetic nephropathy related to oxidative stress. Recently, multiple pieces of data have emphasized a key role of inflammation in the pathogenesis of DN [17] TNF-α is an inflammatory

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cytokine with many inflammatory response activities in several tissues and pleiotropic effects [18]. The TGF-β transcription factor is involved in the development of renal damage by promoting renal fibrosis [19, 20]. In

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the present study, we demonstrated significantly increased TNF-α and TGF-β mRNA expression levels in the kidney tissues of mice with DN and LPS-stimulated cells compared with that of normal mice and cells,

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and reduced TNF-α and TGF-β levels in CXCL16-deficient diabetes mice

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compared with those of control mice and cells. These data suggest that CXCL16 deficiency may attenuate diabetic nephropathy by reducing inflammation. Similarly, cellular infiltration was found to play an important role in the pathogenesis of DN, as ICAM-1 and VCAM-1

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protein expression levels were increased in the kidneys of DN rats [21] Here, we also found significantly increased ICAM-1 and VCAM-1 mRNA expression levels in the kidney tissues of mice with DN and

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LPS-stimulated cells compared with those of normal mice and cells, and

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reduced levels in CXCL16-deficient diabetes mice. These results suggest that cellular infiltration reduction is a possible mechanism for the attenuation of diabetic nephropathy due to CXCL16 deficiency. Taken together, these results suggest that the CXCL16 gene participates in the generation of ROS in STZ-induced diabetic mice, promotes inflammatory factors, enriches cell infiltration factors, and inhibits the expression of antioxidant factors to accelerate the development of diabetic nephropathy.

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Importantly, in the present study, we demonstrated that the activity of AKT was decreased in CXCL16-suppressing cells that were treated by LPS, suggesting that the AKT signaling pathway may be involved in the

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attenuation of diabetic nephropathy by CXCL16 deficiency.

Acknowledgments

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This research was supported by Zhejiang Provincial Natural Science

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Foundation of China (Grant No. LQ16H160021), the Opening Project of Zhejiang Provincial Top Key Discipline of Pharmaceutical Sciences (Grant No. YKFJ2-010), and the Introduced Talented Persons of

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Wenzhou medical University (No. 89214033).

Conflict of interest

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The authors declare no conflict of interest

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Ethical approval

This article does not contain any studies with human participants

performed by any of the authors. And, all of the experiments were conducted under our institutional guidelines for the humane treatment of laboratory animals.

References

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[2] H.J. Oh, M. Kato, S. Deshpande, et al, Inhibition of the processing of miR-25 by HIPK2-Phosphorylated-MeCP2 induces NOX4 in early diabetic nephropathy, Sci Rep 6 (2016):38789

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[3] Y. Shen, R. Cai, J. Sun, et al, Diabetes mellitus as a risk factor for

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incident chronic kidney disease and end-stage renal disease in women compared with men: a systematic review and meta-analysis, Endocrine 55 (2017): 66-76

[4] V. Jha, G. Garcia-Garcia, K. Iseki, et al. Chronic kidney disease:

260-272

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[6] M. Minami, N. Kume, T. Shimaoka, et al, Expression of SR-PSOX, a novel cell-surface scavenger receptor for phosphatidylserine and oxidized LDL in human atherosclerotic lesions, Arterioscler Thromb Vasc Biol 21.11 (2001): 1796-1800

[7] Z. Lin, Q. Gong, Z. Zhou, Increased plasma CXCL16 levels in patients with chronic kidney diseases, Eur J Clin Invest 41.8 (2011): 836-845

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[8] L. Zhao, F. Wu, L. Jin, et al, Serum CXCL16 as a novel marker of renal injury in type 2 diabetes mellitus, PloS one 9.1 (2014): e87786 [9] Z. Lin, H. Tian, K.S.L. Lam, Adiponectin mediates the metabolic

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(2015): 1-9

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pathway in esophageal squamous cell carcinoma, Med Oncol 32.2

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(2009): 3181-3188

[12] Q. Gong, F. Wu, X. Pan, et al, Soluble CXC chemokine ligand 16 levels are increased in gout patients, Clin Biochem 45.16 (2012):

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[13] D.K. Singh, P. Winocour, K. Farrington, Oxidative stress in early diabetic nephropathy: fueling the fire, Nat Rev Endocrinol 7.3 (2011): 176-184

[14] M. Kumar Arora, U. Kumar Singh, Oxidative stress: meeting multiple targets in pathogenesis of diabetic nephropathy, Curr Drug Targets, 15.5 (2014): 531-538 [15] Y. Zhou, Q. Liao, Y. Luo, Rosalaevigata Michx extract inhibits

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oxidative stress in diabetic nephropathy by activating Nrf2/ARE signaling, Int J Clin Exp Med 9.2 (2016): 2831-2839 [16] J.M. Chang, M.C. Kuo, H.T. Kuo, et al, Increased glomerular and

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extracellular malondialdehyde levels in patients and rats with diabetic nephropathy, J Lab Clin Med 146.4 (2005): 210-215

[17] A. Mima, Inflammation and oxidative stress in diabetic nephropathy:

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new insights on its inhibition as new therapeutic targets, J Diabetes

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Res 2013 (2013): 248563

[18] M.B. Duran-Salgado, A.F. Rubio-Guerra, Diabetic nephropathy and inflammation, World J Diabetes 5.3 (2014): 393-398 [19] A.P. Sanchez, K. Sharma, Transcription factors in the pathogenesis

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of diabetic nephropathy, Expert Rev Mol Med 11 (2009): e13 [20] H.O. E.l. Mesallamy, H.H. Ahmed, A.A. Bassyouni, et al, Clinical significance of inflammatory and fibrogenic cytokines in diabetic

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nephropathy, Clin Biochem 45.9 (2012): 646-650

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[21] L. Tang, W. Ni, M. Cai, Renoprotective effects of berberine and its potential effect on the expression of β-arrestins and intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in streptozocin-diabetic nephropathy rats, J Diabetes 8.5 (2016): 693-700

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Table

Table 1 Primers sequences (mouse)

Forward primer(5’-3’)

Reverse primer(5’-3’)

Aco

TAACTTCCTCACTCGAAGCCA

AGTTCCATGACCCATCTCTGTC

Sod-2

CAGACCTGCCTTACGACTATGG

CTCGGTGGCGTTGAGATTGTT

TNF-α

TGATCCGCGACGTGGAA

ACCGCCTGGAGTTCTGGAA

TGF-β

ATGTCACGGTTAGGGGCTC

GGCTTGCATACTGTGCTGTATAG

CTCCGTGGGGAGGAGATACT

TGGCCTCGGAGACATTAGAG

AGTTGGGGATTCGGTTGTTCT

CCCCTCATTCCTTACCACCC

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VCAM-1

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ICAM-1

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Genes (mouse)

β-actin

CATCACTATTGGCAACGAGC

TACGGATGTCAACGTCACAC

Table 2 Primers sequences (human)

Genes (Human)

Forward primer(5’-3’)

Reverse primer(5’-3’)

Aco

GGAACTCACCTTCGAGGCTTG

TTCCCCTTAGTGATGAGCTGG

Sod-2

GCTCCGGTTTTGGGGTATCTG

GCGTTGATGTGAGGTTCCAG

ACCEPTED MANUSCRIPT CGCGTTCATGTCGTAATAGTT

CGGGCCGATTGATCTCAGC

TGF-β

CTAATGGTGGAAACCCACAACG

TATCGCCAGGAATTGTTGCTG

ICAM-1

GTATGAACTGAGCAATGTGCAAG

GTTCCACCCGTTCTGGAGTC

VCAM-1

GGGAAGATGGTCGTGATCCTT

TCTGGGGTGGTCTCGATTTTA

β-actin

GTTGCGTTACACCCTTTCTTGAC

CTCGGCCACATTGTGAACTTTG

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TNF-α

Table 3 The changes of body weight, FBS, 24 h urinary protein, TG,

Animal

FBG

(g)

(mmol/L)

24 h urinary protein (µmol/24h)

TG

TC

(mmol/L)

(mmol/L)

Vehicle

8

30.2±0.6

4.8±1.00

0.30±0.06

0.36±0.28

1.78±0.29

STZ

7

25.8±0.7*

22.6±2.30*

11.32±0.09*

1.26±0.45*

2.85±0.68*

Vehicle

8

31.2±0.3

4.6±1.10

0.25±0.03

0.35±0.21

1.66±0.27

STZ

8

26.3±0.6*

17.8±1.60*#

9.36±0.03*#

1.17±0.38*

2.55±0.57*

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C16 KO

body weight

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WT

n

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TC among WT and CXCL16 KO mice treated with or without STZ

* P<0.05 vs same genotype mice; # P<0.05 vs WT mice

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Figure legends

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Fig.1 CXCL16 deficiency attenuated kidney dysfunction and oxidative stress in vivo. (a) H&E staining analysis of kidney sections in wild type and CXCL16 knockout mice after STZ or vehicle injection (×400). The levels of MDA (b) and mRNA expression of Aco (c), Sod-2 (d), TNF-α (e), TGF-β (f), VCAM-1 (g), and ICAM-1 (h) in kidney. * P<0.05, ** P<0.01, *** P<0.001.

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Fig.2 The expression level of CXCL16 protein was enhanced by LPS. GAPDH was used as a control. (a, c) Inhibition of CXCL16 protein expression in HK-2 cells by shRNA lentivirus; (b, d) CXCL16 protein

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P<0.001.

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expression in HK-2 cells was induced by LPS. * P<0.05, ** P<0.01, ***

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Fig.3 CXCL16 deficiency attenuated kidney dysfunction and oxidative stress in vitro. The levels of mRNA expression of Aco (a), Sod-2 (b), TNF-α (c), TGF-β (d), VCAM-1 (e), and ICAM-1 (f) in kidney. * P<0.05, ** P<0.01, *** P<0.001.

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Fig.4 The expression levels of AKT and p-AKT were decreased in

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CXCL16-suppressing HK-2 cells. GAPDH was used as a control. (a) The expression levels of AKT and p-AKT proteins were examined by Western blotting in CXCL16-suppressing HK-2 cells and the control cells with or

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without LPS treatment, respectively. (b, c and d) Semi-quantitated values of

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Western blotting experiments (a) were statistically analyzed. ** P<0.01.