You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


Introduction
============

Heterogeneous disorders affecting kidney structure and function, chronic kidney disease has been treated as a threat to the health of the people all over the world ([@b1-ijmm-41-06-3577]). Due to the recurrent hospitalization, accelerated death and morbidity, increasing research has been devoted to investigate the underlying molecular mechanism. As the final manifestation of chronic kidney disease, renal fibrosis is the common pathway for chronic kidney disease towards kidney failure ([@b2-ijmm-41-06-3577],[@b3-ijmm-41-06-3577]). It is characterized by an excessive accumulation and deposition of extracellular matrix components. Fibrosis formation could be stimulated by chronic glomerulonephritis, chronic pyelonephritis, obstructive nephropathy, systemic lupus; hereditary kidney disease, diabetic nephropathy, hypertensive kidney disease, drug-induced kidney disease, hepatitis B or the HIV caused kidney disease and renal transplantation ([@b4-ijmm-41-06-3577]--[@b7-ijmm-41-06-3577]). Among these inducements, with the development of economy and the improvement of people\'s living standard, eating habits caused kidney disease has been widely received attention by scholars. In previous reports, diet-induced increase in plasma oxidized LDL could promote early fibrosis in a renal porcine auto-transplantation model ([@b8-ijmm-41-06-3577],[@b9-ijmm-41-06-3577]). Moreover, high fat diet-induced obesity would change kidney-related gene expression and lead to chronic kidney disease formation ([@b10-ijmm-41-06-3577],[@b11-ijmm-41-06-3577]). These changes alter normal physiological activity and promote type II diabetes, in turn; diabetes enhancement will increase kidney injury development and fibrosis formation ([@b12-ijmm-41-06-3577]). However, though the medical substantial progress has expanded our knowledge of kidney related diseases, there has been little in the way of new therapies. Hence, the additional intervention, including treatment with chemoprevention from natural products, for the treatment of kidney injury.

To our knowledge, in the development of kidney inflammation, TGF-β1/Smads pathway has been regarded as the most significant in the promotion for fibrosis formation and kidney injury ([@b13-ijmm-41-06-3577]). High fat diet-induced kidney damage also has been thought to be associated with inflammation development. As previously reports, kidney injury as a typical inflammatory response, nuclear factor-κB (NF-κB) signaling pathway was directly involved in the developmental process of lung injury ([@b14-ijmm-41-06-3577],[@b15-ijmm-41-06-3577]). Thus, how to inhibit the activation of NF-κB pathway may be the key point for the treatment of renal inflammation. CX3CL1-CX3CR1 interaction has been indicated as important in many inflammation-related disorder, including hepatitis and fibrosis, nerve damage and tumors ([@b16-ijmm-41-06-3577],[@b17-ijmm-41-06-3577]). CX3CL1-CX3CR1 axis has the ability to enhance the levels of phosphorylated NF-κB, which in turn, could continually upregulate CX3CL1-CX3CR1 interaction. Crosstalk of CX3CL1-CX3CR1 axis and NF-κB pathways play a critical role in the inflammatory formation and development ([@b15-ijmm-41-06-3577],[@b17-ijmm-41-06-3577]). Hence, we investigated whether CX3CR1 knock out inhibits high fructose-stimulated acute kidney injury by interfering with CX3CL1-CX3CR1 axis expression and NF-κB pathways activation. Furthermore, podocyte cells were used as a model to evaluate the effects of CX3CR1 on inflammatory responses.

Materials and methods
=====================

Reagents and animals
--------------------

Fructose (CAS: 53188-23-1, HPLC analysis ≥99%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Interleukin-1β (IL-1β) (freeze-dried powder) was obtained from Nanjing Biohelper Co., Ltd. (Nanjing, China) and prepared in phosphate-buffered saline (PBS) at the concentration of 100 *µ*g/ml. The antibodies used in this study were obtained from CST Technology Co. (Danvers, MA, USA). The mouse enzyme-linked immunosorbent assay (ELISA) kits were purchased from BioLegend (San Diego, CA, USA). Male C57BL/6 mice with CX3CR1 gene-knock out were purchased from Beijing Biocytogen Co., Ltd. (Beijing, China). The mice weighting 20--25 g were housed in a temperature and humidity-controlled environment (25±2°C, 50±10% humidity) with a standard 12 h light/dark cycle with food and water. This study was approved by the Ethics Committee on Animal Research at Department of Nephrology, Navy General Hospital of Chinese PLA. The mice were randomly divided into 3 groups: i) wild-type (WT); ii) Fructose-fed wild-type (Fru-WT); iii) Fructose-fed CX3CR1-knock out mice (Fru-CX3CR1-KO). Fru-WT and Fru-CX3CR1-KO group mice were orally administered with 30% fructose water/day; and the WT mice were orally fed with equal amount of PBS. The entire experimental period was 56 days. All animal experiments were performed in accordance with the guide for the Care and Use of Laboratory Animals established by the US National Institutes of Health.

Cell culture
------------

The conditionally immortalized mouse podocyte cell line was obtained from Institute of Biochemistry and Cell Biology, SIBS, CAS (Shanghai, China). Cells were grown and maintained at 33°C in RPMI-1640 medium containing 10% fetal bovine serum (FBS), recombinant interferon-γ (R&D Systems, Minneapolis, MN, USA) and 1% penicillin/streptomycin. Recombinant lent viral particles encoding CX3CR1 and short hairpin RNA targeting CX3CR1 were produced, concentrated, and titrated. The short hairpin RNA oligonucleotide sequences of CX3CR1 were described earlier ([@b18-ijmm-41-06-3577]). Cells were seeded at 3×10^4^ cells/ml and were infected with recombinant lent virus twice with an interval of 12 h and incubated for 24 h. After 24 h, the medium was refreshed and the cells were cultured for another 24 h. Cells were serum starved for 36 h and stimulated with IL-1β (75 ng/ml) for the indicated time periods.

Biochemical analysis
--------------------

At the end of all experiments, rats were sacrificed by decapitation at 9:30--10:30 a.m. after a 16-h fast in order to avoid the fluctuation of hormone levels due to circadian rhythms. Before this, the insulin tolerance test (ITT) and oral glucose tolerance test (OGTT) were performed in accordance with a perviously described method ([@b20-ijmm-41-06-3577]).

Inflammatory cell counts of kidney tissue
-----------------------------------------

The kidney samples were centrifuged (4°C, 3,000 rpm for 10 min) to pellet the cells. The cell pellets were resuspended in PBS for the total cell counts using a hemacytometer, and cytospins were prepared for differential cell counts by staining with the Wright-Giemsa staining method.

Histopathological examination of kidney tissues and flow cytometry analysis
---------------------------------------------------------------------------

Histopathological evaluation was performed by Nanjing Biohelper Co., Ltd. (Nanjing, China). Kidneys were fixed with 10% buffered formalin, imbedded in paraffin, and sliced. After hematoxylin and eosin (H&E) staining, Red Oil O, Masson and EM pathological changes of tissues were observed under a light microscope or electron microscope. Some samples were subjected to immunohistochemical staining (CX3CL1, CX3CR1 and NF-κB) according to CST technology Co. introduction and performed by Shanghai Zhenda Biotechnology, Co., Ltd. (Shanghai, China). For flow cytometry analysis, the cells were obtained through shearing kidney tissue, separating by collagenase type II (Invitrogen, Carlsbad, CA, USA) digestion and suspended in RPMI-1640 medium (Gibco-BRL, Grand Island, NY, USA). Cell suspensions were centrifuged at 1,000 rpm for 5 min to remove cellular debris and impurities. Then, the entire cells were harvested and washed twice in Hank\'s buffer (Gibco-BRL). According to the protocol of R&D kit systems (R&D Systems) for flow cytometry, add anti-CD45 FITC and anti-CD80 FITC antibody to the flow cytometry tube containing single-cell suspension, and these tubes were analyzed by Cytomics™ FC 500 MCL of Beckman Coulter (Brea, CA, USA).

ELISA measurement and biochemical analysis
------------------------------------------

At the end of the experiments, blood were extracted from the eyeball, the different serum concentration of the inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), IL-2, IL-1β and IL-6 were measured using ELISA kits according to the manufacturer\'s instructions (R&D Systems). The other indicators shown in [Table I](#tI-ijmm-41-06-3577){ref-type="table"} were also performed by Shanghai Zhenda Biotechnology, Co., Ltd.

Western blotting and quantitative RT-PCR (qRT-PCR) analysis
-----------------------------------------------------------

Proteins were extracted from the kidney using T-PER Tissue Protein Extraction Reagent kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer\'s instructions. Protein concentrations were determined by BCA protein assay kit, and equal amounts of protein were loaded per well on a 10% sodium dodecyl sulphate-polyacrylamide gel. Subsequently, proteins were transferred onto polyvinylidene difluoride membrane. The resulting membrane was blocked with Tris-buffered saline containing 0.05% Tween-20 (TBS-T), supplemented with 5% skim milk (Sigma, St. Louis, MO, USA) at room temperature for 2 h on a rotary shaker, and followed by TBS-T washing. The specific primary antibody, diluted in TBST, was incubated with the membrane at 4°C overnight. Subsequently, the membrane was washed with TBS-T followed by incubation with the peroxidase-conjugated secondary antibody at room temperature for 1 h. The immunoactive proteins were detected by using an enhanced chemiluminescence western blotting detection kit. Western blot bands were observed using GE Healthcare ECL western blotting analysis system and exposed to Kodak X-ray film. For qRT-PCR experiments, quantitative RT-PCR (qRT-PCR) and western blot analysis were performed as described previously ([@b17-ijmm-41-06-3577]).

Statistical analysis
--------------------

Data are expressed as means ± SEM. Treated cells, tissues and the corresponding controls were compared using GraphPad Prism (version 6.0; GraphPad Software, Inc., La Jolla, CA, USA) by one-way ANOVA with Dunn\'s least significant difference tests. Differences between groups were considered significant at p\<0.05.

Results
=======

CX3CR1 deficiency ameliorates high fructose-induced metabolic disorder responses and inflammatory cell counts
-------------------------------------------------------------------------------------------------------------

We investigated that the effect of CX3CR1 deficiency mice on fructose-stimulated kidney injury. As shown in [Table I](#tI-ijmm-41-06-3577){ref-type="table"}, major kidney injury-related indicators in serum such as uric acid, blood urea nitrogen and serum total cholesterol, were significantly increased with the fructose administration, compared to WT group. In contrast, in CX3CR1-KO with fructose treatment mice, these were downregulated, compared with the model. Also, as shown in [Fig. 1A and B](#f1-ijmm-41-06-3577){ref-type="fig"}, the serum glucose levels tested by ITT and OGTT were increased in WT+Fru group, compared with CX3CR1-KO. Thus, high fructose is capable of changing metabolic activities and weaken renal function. [Fig. 1C](#f1-ijmm-41-06-3577){ref-type="fig"} shows that inflammation-related cytokines in WT+Fru were significantly upregulated with the increase of treatment in a time-independent manner. Of note, The data of [Fig. 2A-E](#f2-ijmm-41-06-3577){ref-type="fig"} indicated that CX3CR1-KO may suppress the inflammation-related cells in kidneys, including neutrophils and macrophages caused by fructose administration-induced renal injury. These basic biomarkers suggested that CX3CR1 deficiency is capable of suppressing kidney damage with fructose administration.

Influence of CX3CR1 deficiency on inflammation-related cytokines expression in fructose-induced kidney injury
-------------------------------------------------------------------------------------------------------------

As indicated in [Fig. 2F-N](#f2-ijmm-41-06-3577){ref-type="fig"}, the fructose administration significantly enhance renal injury levels compared to PBS group. Moreover, CX3CR1^−/−^ mice had a lower inflammatory cytokine expression than fructose-fed WT mice. We further investigated the mRNA level expression of inflammatory cytokines. Data in [Fig. 3A](#f3-ijmm-41-06-3577){ref-type="fig"} illustrate that the administration of fructose can increase inflammatory cytokine expression in mRNA level, and downregulate in CX3CR1 deficiency mice. [Fig. 3B](#f3-ijmm-41-06-3577){ref-type="fig"} shows that fructose significantly enhance the ratio of CD45 and CD80 positive cells. In contrast, in CX3CR1-KO mice a lower ratio of positive cells in CD45 and CD80 than fructose-fed WT was seen. The histopathologic examination of kidney tissues in [Fig. 3C](#f3-ijmm-41-06-3577){ref-type="fig"} show the effects of fructose on renal injury. The results indicated that fructose caused lipid accumulation, inflammatory cell infiltration, a certain degree of fibrosis and damage of the tissues structure.

Effects of CX3CR1-KO on CX3CL1/CX3CR1 axis-stimulated NF-κB signaling pathway
-----------------------------------------------------------------------------

As showed in [Fig. 4A](#f4-ijmm-41-06-3577){ref-type="fig"}, the IHC analysis given the AKT and NF-κB pathways were significantly activated with the administration of fructose. Also, in CX3CR1^−/−^ mice, the phosphorylation of NF-κB is lower than it in WT group. In addition, the western blot data ([Fig. 4B](#f4-ijmm-41-06-3577){ref-type="fig"}) showed that AKT-stimulated NF-κB signaling pathway in CX3CR1^−/−^ mice with a significant difference compared with WT. These findings demonstrated that AKT pathway could be a link in the development of fructose-induced inflammation of renal injury, which was directly activated by CX3CL1-CX3CR1 axis. The AKT-stimulated NF-κB signaling pathway activation was downregulated when the CX3CR1^−/−^ mice were challenged with fructose administration.

CX3CR1 deficiency inhibits inflammatory response through NF-κB pathway in podocyte cells
----------------------------------------------------------------------------------------

Research has demonstrated that the activation of the NF-κB pathway is involved in inflammation-related diseases both in experimental models and in humans. Moreover, CX3CL1-CX3CR1 axis has been shown to regulate the NF-κB pathway in multiple cell systems. Hence, we investigated the effect of CX3CL1-CX3CR1 axis on NF-κB pathway and the induced inflammatory responses with fructose administration. As showed in [Fig. 5A, B and D](#f5-ijmm-41-06-3577){ref-type="fig"}, the IκBα degradation level was significantly upregulated in CX3CR1 deficiency, compared with WT mice. The increase of IκBα phosphorylation level results in the upregulation of NF-κB pathway activation, compared to CX3CR1 deficiency mice. The activity of NF-κB is also downregulated in the deficiency mice. Of note, in podocyte cells with CX3CR1 deficiency, the inflammatory cytokines have a lower release levels than WT group ([Fig. 5C](#f5-ijmm-41-06-3577){ref-type="fig"}). Thus, these findings demonstrated that CX3CR1 deficiency has a key role in the inhibitory effect of fructose-induced inflammatory responses.

Discussion
==========

Kidney injury syndrome consisting of chronic inflammatory responses, has been emphasized as a serious health threat world-wide ([@b1-ijmm-41-06-3577],[@b19-ijmm-41-06-3577]). Moreover, as a serious syndrome, the formation and development of kidney diseases are associated with high morbidity and mortality. It is characterized by an excessive accumulation and deposition of extracellular matrix components. Kidney injury formation could be stimulated by chronic glomerulonephritis, chronic pyelonephritis, obstructive nephropathy, systemic lupus; hereditary kidney disease, diabetic nephropathy, hypertensive kidney disease, drug-induced kidney disease, hepatitis B or the HIV caused kidney disease and renal transplantation. However, the molecular mechanism of high fructose intake-induced chronic kidney disease, even kidney fibrosis is not absolutely explained and reported. In previous studies, renal damage as a typical inflammatory response, NF-κB signaling pathway was directly involved in the developmental process of injury ([@b19-ijmm-41-06-3577],[@b20-ijmm-41-06-3577]). Thus, how to inhibit the activation of NF-κB pathway may be the key point for the treatment of renal damage. CX3CL1-CX3CR1 interaction has been treated as important in inflammation-related disorders, including hepatitis and fibrosis, nerve damage and tumor ([@b21-ijmm-41-06-3577]). CX3CL1-CX3CR1 axis has the ability to enhance the levels of phosphorylated NF-κB; in turn, NF-κB activation could continually upregulate CX3CL1-CX3CR1 interaction ([@b17-ijmm-41-06-3577],[@b18-ijmm-41-06-3577]). Crosstalk of CX3CL1-CX3CR1 axis and NF-κB pathways play a critical role in the inflammation and development ([@b17-ijmm-41-06-3577]). Hence, we investigated whether CX3CR1-KO inhibits fructose-stimulated kidney injury by interfering CX3CL1-CX3CR1 axis expression and NF-κB pathways activation.

We used C57BL/6 mice to construct fructose-induced renal injury model in order to investigate the effects of CX3CL1-CX3CR1 axis. The results of our data indicated that fructose administration could cause inflammatory cytokine releases including TNF-α, IL-1β, IL-6, TGF-β and IL-18 in serum. Moreover, the inflammatory cell counts of fructose-induced renal tissues were significantly enhanced. Furthermore, we tested the crucial role of CX3CL1/CX3CR1 signaling pathway in fructose-induced kidney injury. The results showed that CX3CL1/CX3CR1 axis is involved in the process of renal damage and could be activated via fructose administration. The activation of CX3CL1/CX3CR1 axis will further enhance the NF-κB pathway phosphorylation levels. As previous reported the increase of NF-κB pathway phosphorylation leads to inflammatory responses and cytokines production. Researchers have indicated that the activation of CX3CL1/CX3CR1 axis was associated with AKT pathway **(ref. ?)**. Thus, we further assessed the relationship of AKT pathway and CX3CL1/CX3CR1 axis. The data demonstrated that fructose administration has the ability to activate AKT pathway and increase the levels of phosphorylated AKT expression. Phosphorylated AKT expression could significantly enhance the activation of CX3CL1/CX3CR1 pathway and increase the NF-κB pathway activation.

To study the possible role of CX3CL1/CX3CR1 axis in fructose-induced kidney injury of mice, we used CX3CR1-knock out mice as model to investigate the effects of crosstalk of CX3CL1-CX3CR1 axis and NF-κB pathways. IHC analysis of IκB of fructose administration showed that IκB was involved in the development of renal injury. Compared with WT group, the CX3CR1^−/−^ mice have a lower IκB activation when challenged with fructose. As the inducer of CX3CR1, we tested the CX3CL1 expression between WT and CX3CR1^−/−^ mice by IHC. The results indicated that CX3CR1 knockout directly impacts the expression of CX3CL1. The AKT signaling pathway may directly or indirectly associate with fructose-induced kidney injury. In knockout mice, the p-AKT expression was determined to be involved in the process of injury. The data indicated AKT as an important upstream of CX3CL1-CX3CR1 axis may impact CX3CL1-mediated inflammatory responses.

We investigated fructose-induced kidney injury model from the point of CX3CL1-CX3CR1 axis and its-related indicators. We found that fructose as functional food additive has the ability to cause inflammation by enhancing CX3CL1-CX3CR1 axis and NF-κB activation. Also, the phosphorylated AKT could be significantly activated in fructose-induced renal injury via CX3CL1-CX3CR1 axis. We further investigated the role of CX3CL1-CX3CR1 pathway in lung injury. CX3CR1 expression between WT and CX3CR1^−/−^ mice were tested to establish their relationship to injury. Our results indicated that CX3CR1 may be the central and major indicator in the process of kidney injury, which mediate the CX3CL1 to activate AKT pathway and further enhance the NF-κB activation.

**Competing interests**

Authors declare that they have no competing interest.

![CX3CR1 deficiency affects metabolism and kidney function in fructose-fed mice. (A) Insulin tolerance test of rats. (B) Oral glucose tolerance test of rats. (C) Real-time quantitative PCR analysis of renal fibrosis related gene expression in different experimental periods. Data are expressed as the mean ± SEM. +P\<0.05, ^++^P\<0.01 and ^+++^P\<0.001 vs. WT; ^\#^P\<0.05, ^\#\#^P\<0.01 and ^\#\#\#^P\<0.001 vs. Fructose (Fru); ns, no significances.](IJMM-41-06-3577-g00){#f1-ijmm-41-06-3577}

![CX3CR1 deficiency ameliorates high fructose-induced metabolic disorder responses and inflammatory cell counts. (A-E) The inflammation-related cells counts in fructose-induced renal tissues. (F-N) Enzyme-linked immunosorbent assay (ELISA) analysis was used to show the inflammatory cytokine expression in fructose-induced kidney injury. Data are expressed as the mean ± SEM. ^+^P\<0.05, ^++^P\<0.01 and ^+++^P\<0.001 vs. WT; ^\#^P\<0.05, ^\#\#^P\<0.01 and ^\#\#\#^P\<0.001 vs. Fructose (Fru); ns, no significance.](IJMM-41-06-3577-g01){#f2-ijmm-41-06-3577}

![Influence of CX3CR1 deficiency on inflammation-related cytokine expression in fructose-induced kidney injury. (A) Quantitative RT-PCR analysis showed the inflammatory cytokines in mRNA expression. (B) Flow cytometry analysis showed the major maker expression of renal injury-CD45 and CD80 in fructose-induced WT mice and CX3CR1-KO mice. Data are expressed as the mean ± SEM. ^\#^P\<0.05, ^\#\#^P\<0.01 and ^\#\#\#^P\<0.001 vs. WT; ^\*^P\<0.05, ^\*\*^P\<0.01 and \*\*\*P\<0.001 vs. Fru-WT.](IJMM-41-06-3577-g02){#f3-ijmm-41-06-3577}

![Effects of CX3CR1 deficiency on CX3CL1/CX3CR1 axis-stimulated nuclear factor-κB (NF-κB) signaling pathway. (A) IHC analysis of CX3CL1, CX3CR1 and phosphorylated NF-κB expression in fructose-induced renal injury. (B) Western blot analysis showed AKT pathway and NF-κB signaling pathway activation. Data are expressed as the mean ± SEM. ^\#^P\<0.05, ^\#\#^P\<0.01 and ^\#\#\#^P\<0.001 vs. WT; ^\*^P\<0.05, ^\*\*^P\<0.01 and ^\*\*\*^P\<0.001 vs. Fru-WT.](IJMM-41-06-3577-g03){#f4-ijmm-41-06-3577}

![CX3CR1 deficiency inhibits inflammatory response through nuclear factor-κB (NF-κB) pathway in podocyte cells. (A) Western blot analysis for the phosphorylated IκB protein expression in interleukin-1β (IL-1β) induced podocyte cells between WT and CX3CR1-KO mice. (B) Relative NF-κB activity in WT and CX3CR1-KO mice. (C) Enzyme-linked immunosorbent assay (ELISA) and qPCR analysis for the inflammatory cytokines expression in IL-1β-induced cells between WT and CX3CR1-KO mice. (D) IHC analysis for the phosphorylated IκB protein expression in IL-1β induced podocyte cells between WT and CX3CR1-KO mice. Data are expressed as the mean ± SEM. ^\*^P\<0.05, ^\*\*^P\<0.01 and ^\*\*\*^P\<0.001 vs. CX3CR1-KO mice; ^\#^P\<0.05, ^\#\#^P\<0.01 and ^\#\#\#^P\<0.001 vs. WT.](IJMM-41-06-3577-g04){#f5-ijmm-41-06-3577}

###### 

Effects of fructose on physiological indexes and biochemical parameters in serum of CX3CR1-KO mice.

  Items                                        WT            Fru-WT                                                       Fru-CX3CR1-KO
  -------------------------------------------- ------------- ------------------------------------------------------------ ------------------------------------------------------------
  Serum uric acid (mg/dl)                      3.28±70.79    6.91±71.50[a](#tfn2-ijmm-41-06-3577){ref-type="table-fn"}    4.73±70.19[d](#tfn4-ijmm-41-06-3577){ref-type="table-fn"}
  Urinary uric acid (mg/dl)                    41.77±71.52   32.15±73.79^b^                                               40.70±72.34[e](#tfn5-ijmm-41-06-3577){ref-type="table-fn"}
  Serum creatinine (mg/dl)                     0.96±70.24    1.94±70.26[c](#tfn3-ijmm-41-06-3577){ref-type="table-fn"}    0.84±71.18[e](#tfn5-ijmm-41-06-3577){ref-type="table-fn"}
  Urinary creatinine (mg/dl)                   37.53±72.89   27.41±73.76[a](#tfn2-ijmm-41-06-3577){ref-type="table-fn"}   34.97±71.96[d](#tfn4-ijmm-41-06-3577){ref-type="table-fn"}
  Blood urea nitrogen (mg/dl)                  32.95±71.61   42.47±72.74[a](#tfn2-ijmm-41-06-3577){ref-type="table-fn"}   32.93±74.38[d](#tfn4-ijmm-41-06-3577){ref-type="table-fn"}
  FEUA (%)                                     19.17±70.34   12.37±70.25^b^                                               16.85±71.30[e](#tfn5-ijmm-41-06-3577){ref-type="table-fn"}
  Serum triglyceride (mg/dl)                   119.84±5.68   175.30±4.58[a](#tfn2-ijmm-41-06-3577){ref-type="table-fn"}   125.17±6.96[d](#tfn4-ijmm-41-06-3577){ref-type="table-fn"}
  Serum total cholesterol (mg/dl)              72.09±1.21    85.36±1.87[a](#tfn2-ijmm-41-06-3577){ref-type="table-fn"}    72.80±1.21[d](#tfn4-ijmm-41-06-3577){ref-type="table-fn"}
  Serum very-low density lipoprotein (μg/ml)   93.42±4.98    137.97±6.62[c](#tfn3-ijmm-41-06-3577){ref-type="table-fn"}   95.98±6.56[d](#tfn4-ijmm-41-06-3577){ref-type="table-fn"}
  Serum insulin (*µ*g/ml)                      2.33±0.19     3.90±0.46[c](#tfn3-ijmm-41-06-3577){ref-type="table-fn"}     2.02±0.23[d](#tfn4-ijmm-41-06-3577){ref-type="table-fn"}
  Serum leptin (*µ*g/l)                        0.48±0.02     0.64±0.03[a](#tfn2-ijmm-41-06-3577){ref-type="table-fn"}     0.46±0.05[e](#tfn5-ijmm-41-06-3577){ref-type="table-fn"}

These results are expressed as mean ± SEM, n=7.

P\<0.01 compared with WT group;

P\<0.001 compared with WT group;

P\<0.01 compared with fructose-fed group;

P\<0.05 compared with fructose-fed group;

P\<0.001 compared with fructose-fed group. WT, wild-type; Fru-WT, Fructose-fed wild-type.

[^1]: Contributed equally