Carbohydrate response element binding protein (ChREBP) modulates the inflammatory response of mesangial cells in response to glucose

Diabetic nephropathy (DN) is one of the most devastating complications of diabetes mellitus. Carbohydrate response element binding protein (ChREBP) is a basic helix–loop–helix leucine zipper transcription factor that primarily mediates glucose homeostasis in the body. The present study investigated the role of ChREBP in the pathogenesis of DN. The expression of ChREBP was detected in patients with type 2 diabetes mellitus (T2DM), diabetic mice, and mesangial cells. ELISA was used to measure cytokine production in mesangial cells. Flow cytometry analysis was performed to detect the apoptosis of mesangial cells in the presence of high glucose. The expression levels of ChREBP and several cytokines (TNF-α, IL-1β, and IL-6) were up-regulated in T2DM patients. The mRNA and protein levels of ChREBP were also significantly elevated in the kidneys of diabetic mice. Moreover, glucose treatment promoted mRNA levels of TNF-α, IL-1β, and IL-6 in mesangial cells. Glucose stimulation induced significant apoptosis of SV40 MES 13 cells. In addition, transfection with ChREBP siRNA significantly inhibited ChREBP expression. Consequently, the inflammatory responses and apoptosis were inhibited in SV40 MES 13 cells. These results demonstrated that ChREBP could mediate the inflammatory response and apoptosis of mesangial cells, suggesting that ChREBP may be involved in the pathogenesis of DN.


Introduction
Diabetic nephropathy (DN) is one of the most devastating complications of diabetes mellitus and is characterized by macroalbuminuria, hypertension, and decreased glomerular filtration rate (GFR) [1]. Recently, DN has become an important cause of mortality in patients with diabetes mellitus. There is a cause link between glucose control and the development of DN in diabetes patients. In addition, effective control of glucose leads to an obvious reduction in the risk of albuminuria of DN [2,3]. However, little is known about the molecular pathogenesis of DN.
Carbohydrate response element binding protein (ChREBP) is a basic helix-loop-helix leucine zipper transcription factor that primarily mediates glucose homeostasis in the body. After forming a heterodimeric complex in the nucleus, this protein activates carbohydrate response element motifs for transcriptional regulation of its target genes in a glucose-dependent manner [4,5]. Amounting evidence shows that ChREBP and its targets genes play critical roles in a wide range of physiological and pathological processes in the body [6]. In the liver, the production and transcriptional activity of ChREBP are controlled by fasting and feeding [7]. Moreover, the expression of ChREBP is markedly elevated in the liver of diabetic mice [8]. Conversely, knockdown of ChREBP in obese mice causes obvious metabolic disorders, such as glucose intolerance, insulin resistance, and liver steatosis [9].

Western blot
Whole cells were lysed in 1× SDS sample buffer and resolved by electrophoresis using SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with primary antibodies overnight, and then incubated with appropriate horseradish peroxide-conjugated secondary antibodies for 3 h followed by detection with a Super Signal Enhanced Chemiluminescence kit (Pierce, Rockford, IL). For sequential blotting, the membranes were stripped with Stripping Buffer (Pierce) and re-probed with proper antibodies.

Statistical analysis
The results were calculated as the mean + − the standard derivation (SD). Significances between groups were evaluated using Student's t-test and one-way ANOVA. Values with P<0.05 were considered statistically significant.

Clinical and biochemical characteristics
Main metabolic and biochemical characteristics of all subjects are summarized in Table 1 Table 1.

Serum ChREBP was up-regulated in T2DM patients and was correlated with pro-inflammatory cytokines
To investigate the inflammatory reaction to the high glucose in T2DM subjects, we then examined the serum levels of pre-inflammatory cytokines, such as TNFα, IL-1β, and IL-6, in T2DM patients and in control subjects. It was demonstrated that the serum levels of TNFα, IL-1β, and IL-6 were up-regulated in T2DM patients, compared with the control subjects (P<0.05) ( Figure 1A-C). ChREBP was also increased in T2DM patients, compared with the control subjects (P<0.05) ( Figure 1D). Additionally, there was a positive correlation between ChREBP and TNFα, IL-1β, or IL-6 ( Figure 2A-C).

ChREBP were increased in diabetic mice and mesangial cells
C57BL/6 mice with diabetic models were induced by administration with streptozotocin (STZ). Consequently, we found that the mRNA and protein levels of ChREBP were significantly elevated in the kidneys of diabetic mice ( Figure  3A,B). Moreover, mRNA expression of its target genes, L-PK, FASN and SCD-1, were also up-regulated ( Figure 3C). In addition, we detected the expression of ChREBP and its target genes in mesangial cells response to glucose. As expected, we found that high glucose treatment led to an elevated expression of ChREBP and its target genes, L-PK, FASN and SCD-1, in mesangial cells ( Figure 4A-C). Taken together, these results suggested that ChREBP were increased in diabetic mice and glucose-treated mesangial cells.

High glucose induced pro-inflammatory cytokines and apoptosis in mesangial cells
To confirm the inflammatory reaction to the high glucose in mesangial cells, we detected the expression of several pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in SV40 MES 13 cells. Results showed that glucose treatment promoted mRNA levels of TNF-α, IL-1β, and IL-6 ( Figure 5A). Moreover, flow cytometry analysis showed that glucose stimulation induced significant apoptosis of SV40 MES 13 cells ( Figure 5B).

ChREBP mediated inflammatory reactions in mesangial cells induced by glucose administration
To explore the role of ChREBP in the inflammatory response, we manipulated the ChREBP level in mesangial cells using RNAi method. We found that transfection with ChREBP siRNA significantly inhibited the mRNA and protein expression of ChREBP in SV40 MES 13 cells ( Figure 6A,B). As a result, the expression levels of TNF-α ( Figure 6C), IL-1β ( Figure 6D), and IL-6 ( Figure 6E) were also significantly reduced in mesangial cells after transfection with ChREBP siRNA.

Discussion
ChREBP is a critical transcription factor that primarily mediates glucose homeostasis [10]. Recently, a study investigated the role of ChREBP in mesangial cells in DN. They found that treatment with high glucose increased cellular O-GlcNAc and O-GlcNAcylated ChREBP in mesangial cells, which in turn augmented the protein stability, transcriptional activity, and nuclear translocation of ChREBP [11,12]. In the present study, we observed the elevated expression of ChREBP in T2DM patients, high glucose-treated mesangial cells, and diabetic mice. Additionally, the target genes of ChREBP, L-PK, FASN, and SCD-1 were also up-regulated. Therefore, these results demonstrate that ChREBP might play a critical role in the development of DN. It is widely considered that DN is a chronic low-grade inflammatory disease [13][14][15]. Many inflammatory cytokines are shown to be involved in the pathogenesis and clinical outcome of DN, such as TNF-α, IL-1β, and IL-6 [16][17][18]. Therefore, targeting cytokine production many be an effective therapeutic strategy against DN. Actually, the administration of anti-inflammatory activities is able to reduce the renal dysfunction and damage induced by DN [19]. In our study, the serum levels of TNFα, IL-1β, and IL-6 were up-regulated in T2DM patients and mesangial cells. In addition, there was a positive correlation between ChREBP and TNFα, IL-1β, or IL-6. High glucose administration can increase the generation of ROS, which correlates with ATP production and apoptosis in mesangial cells [20][21][22]. Consistently, we observed that glucose stimulation induced significant apoptosis of SV40 MES 13 cells. To further confirm the role of ChREBP in inflammatory responses, we manipulated the ChREBP level in mesangial cells using RNAi method. Consequently, down-regulation of ChREBP suppressed the expression levels of TNF-α, IL-1β, and IL-6 in SV40 MES 13 cells.
There are several limitations of this study. First, we detected the expression of ChREBP in a relatively small sample size, which should be further validated in a larger cohort population. Second, the knockdown of ChREBP was only performed in renal mesangial cells, and thus the in vivo data of depletion of ChREBP were absent. In our future study, we will knockdown ChREBP to validate its role in the rat DN model. At last, the specific underlying mechanism by which ChREBP exerts its biological role in diabetes is not fully elucidated.
In summary, the present study reveals that ChREBP is elevated by the glucose in vivo or in vitro, in an association with an elevated inflammatory response. Moreover, the strategy of ChREBP inhibition reduced the inflammatory cytokines in response to high glucose. The present study demonstrates that ChREBP may be developed as a novel therapeutic target for controlling the progression of DN.