Background
Acute kidney injury (AKI) is a common clinical problem in hospitalized patients worldwide, and it is one of the most common acute and critical illnesses in clinical departments. AKI not only affects the patient survival rate, long-term prognosis and quality of life but also places a huge economic burden on the patient’s family and society. AKI confers formidable morbidity and mortality in its acute phase, and among survivors of AKI, the long-term outcomes are far from benign. Patients who recover from AKI have a 25% increase in the risk of chronic kidney disease (CKD) and a 50% increase in mortality after a follow-up of approximately 10 years [
1‐
3]. Although clinical observations describe a clear association, the underlying mechanism of AKI progression to CKD needs to be explored.
Bone-derived fibroblast growth factor-23 (FGF-23) is an important endocrine regulator of mineral homeostasis, and its effects are transduced by cognate FGF receptor (FGFR)1-α-Klotho complexes [
3,
4]. Circulating FGF-23 levels increase precipitously in patients with kidney diseases and indicate worse renal and cardiovascular outcomes [
5,
6]. FGF-23 levels increase early in CKD [
7], predominantly as intact protein, and generally precede changes in other mineral metabolites [
8]. Similarly, circulating FGF-23 concentrations increase rapidly in AKI, also preceding changes in other mineral markers and conventional measures of renal function [
9].
Renal fibrosis is the pathological basis of CKD [
10]. The canonical TGF-β1/smad3 signalling pathway mediates the transdifferentiation of renal tubular epithelial cells and plays an important role in the progression of renal fibrosis [
11]. Animal studies of ureteral obstruction (UUO) have shown that tubule-derived FGF-23 can enhance the activity of myofibroblasts during AKI, possibly promoting the signalling cascade of renal fibrosis by activating TGF-β channels. [
12,
13] The Wnt/β-catenin signalling pathway is closely related to the occurrence and development of renal interstitial fibrosis. Persistent activation of the Wnt/β-catenin pathway plays an important role in promoting the development of AKI to CKD [
14].
In this study, we examined whether the increase in circulating FGF-23 was associated with CKD development after AKI. Then, we explored the potential molecular mechanism of FGF-23 in the progression of AKI to CKD. Our study aimed to uncover a novel mechanism of the progression of AKI to CKD and provides a potential therapeutic target for preventing and improving the prognosis of AKI.
Materials and methods
AKI patients
We selected 94 AKI patients who were admitted to the Nephrology Intensive Care Unit (ICU) of the National Kidney Disease Clinical Research Center and included 60 healthy humans in the same period as the control group. AKI diagnosis and staging criteria were based on the AKI guidelines of the Kidney Disease Improvement Global Prognosis Organization (KDIGO) [
15]. The diagnostic criteria for CKD were a basal eGFR of less than 60 ml/(min 1.73 m
2) and a duration of more than 3 months.
Animals
C57BL/6 mice (male, 12 weeks old) were purchased from Gem Pharmatech at Nanjing. The mice were housed individually at 23 °C with a 12:12-h light-dark cycle and were maintained on water and food.
Surgery protocols
Briefly, the mice were anaesthetized with pentobarbital (50 mg/ml, i.p.), and the body temperature was maintained at 36.8–37.5 °C during surgery with a temperature-controlled operating table. The renal pedicle was carefully dissected and clamped with a silver clip for 35 min. After releasing the clip, the wound was sutured. Sham-operated animals without clamping served as controls.
Treatment with PD173074
The pan-FGF receptor blocker PD173074 (Sigma‒Aldrich, Inc.) was dissolved in PBS. The mice (sham and IRI, twelve-week-old male C57BL/6 mice) were intraperitoneally injected with PBS or PD173074 (1 mg/kg) once daily for the same duration. After 2 weeks, the animals were sacrificed, and blood and kidney tissues were collected.
Serum biochemistry
Blood samples (~ 200 µl/each) were centrifuged at 3,000 rpm for 10 min at 4 °C to separate the serum (~ 100 µl/each). Blood urea nitrogen and creatinine were measured by Servicebio (
https://www.servicebio.cn/).
FGF23 concentration assay
The active intact FGF23 (iFGF23) enzyme-linked immunosorbent assay (ELISA) kit (catalog no. CY-4000; Kainos Laboratories, Tokyo, Japan) was used. It is a two-point ELISA kit for the determination of iFGF23 in serum.
Morphological analysis of mouse tissues
After blood collection, the animals were sacrificed. The kidneys were isolated and prepared for molecular and histological analyses. Kidneys tissues were stained with Masson dye to observe fibrosis.
HK2 cell culture and treatments
The HK2 cell line was purchased from ATCC, and the cells were cultured in F12 (Gibco, Inc.) supplemented with 10% foetal bovine serum (Gibco, Inc.) in an atmosphere of 5% (v/v) CO2 in air at 37 °C. The cells were treated with 25 ng/ml FGF-23 (Research & Diagnostics Systems, Inc.).
Immunofluorescence
After being treated, cultured HK2 cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS, followed by blocking with 5% goat serum in PBS. Rabbit monoclonal antibodies against p-smad3 (catalogue C25A9; Cell Signalling Technologies, Inc.) were used at 1:1000. A rabbit monoclonal antibody against β-catenin (catalogue 610,154; BD Biosciences, Inc.) was used at 1:2000. Cy3-conjugated goat anti-mouse (catalogue A0521; Beyotime Biotechnology, Inc.) was used as a secondary antibody at 1:500. To visualize nuclei, the fixed cells were incubated with DAPI (400 ng/mL in PBS) for 10 min. Immunofluorescence images were taken with a DM5000B microscope (Leica). The myocyte cross-sectional area was measured by ImageJ software (
http://rsbweb.nih.gov/ij/).
RNA extraction and quantification.
Cultured HK2 cells and mouse kidney tissues were subjected to total RNA extraction with an isolation kit (Thermo Fisher Scientific, Inc.). In brief, this extraction method is based on the ability of glass fibers to bind nucleic acids in concentrated chaotropic salt solutions. Samples are disrupted in a typical high concentration guanidinium salt solution that simultaneously lyses cells and inactivates endogenous RNases. The lysate is diluted with an ethanol solution to make the RNA competent for binding to the glass fiber filter in the RNAqueous Filter Cartridge. This solution is passed through the filter pad where RNA binds and most other cellular contents flow through. The Filter Cartridge is washed 3 times to remove contaminants, and the RNA is eluted in a very low ionic strength solution. Reverse transcription of 300 ng of RNA to cDNA using the Reverse Transcription Kit (Takara Biomedical Technology (Beijing) Co., Ltd.). 2 reverse transcription primers, Random 6 mers and Oligo dT Primer, are included in the kit to synthesize cDNA suitable for Real Time PCR. mRNA sample was quantified by qPCR using a kit from TaKaRa (TaKaRa Bio, Inc.). The primers used are summarized in Table
1. For Q-PCR experiments, annealing temperature is set at 55–60 °C. Relative mRNA expression was evaluated with the 2
–ΔΔCT method using 18 S for normalization.
Table 1
The primers used in this study
COL1A1 | GCTCCTCTTAGGGGCCACT | ATTGGGGACCCTTAGGCCAT |
COL4A1 | TCCGGGAGAGATTGGTTTCC | CTGGCCTATAAGCCCTGGT |
18s | TTTCTCGATTCCGTGGGTGG | AGCATGCCAGAGTCTCGTTC |
Protein extraction and Western blot analysis
Proteins were extracted from kidney tissues with RIPA buffer (Beyotime Biotechnology, Inc.) according to the manufacturer’s instructions. Antibodies against GAPDH (catalogue BS65529; Bioworld Technology, Inc.), α-SMA (catalogue ab5694; Abcam, Inc.), β-catenin (catalogue 610,154; BD Biosciences, Inc.) and p-smad3 (catalogue C25A9; Cell Signalling Technologies, Inc.), E-cad (catalogue 20874-1-AP; Proteintech, Inc.), and NGAL (catalogue ab216462; Abcam, Inc.) were used as primary antibodies, and horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse (Beyotime Biotechnology, Inc.) secondary antibodies were used. Image analysis with image J software.
Statistical analysis
The data were tested for normal distribution. If the measures were normally distributed, they were expressed as mean ± SD, and the t-test was used for comparison between two groups, and Pearson correlation was used for correlation analysis. The Kruskal-Wallis test was used for comparison between two or more groups, and the Spearman’s rank correlation test was used for correlation analysis. The chi-square test was used for comparison of the sex and diabetes ratio. The correlation between AKI patients and FGF-23 was analysed by logistic regression analysis. P < 0.05 was considered statistically significant.
Discussion
AKI is an increasing health burden with high morbidity and mortality rates worldwide. AKI is a risk factor for CKD development. The understanding of AKI developing into CKD was derived from retrospective clinical observations. AKI is now considered to be an independent risk factor for the development of CKD [
17]. However, there are limited experimental data directly addressing the AKI-to-CKD transition. Thus, we explored the potential profibrotic role of FGF-23 after AKI and described its molecular mechanism.
Bone is the major source of circulating FGF-23. FGF-23 acts on the kidneys through fibroblast growth factor receptors (FGFRs) and the coreceptor Klotho to promote phosphaturia by downregulating phosphate transporters, as well as controlling vitamin D metabolizing enzymes to reduce blood 1,25-dihydroxyvitamin D [
18]. Intact FGF-23 (iFGF-23) can be cleaved into N-terminal FGF-23 and C-terminal FGF-23 (cFGF-23) by Furin or plasminogen activators [
19]. According to the literature, circulating FGF-23 is nearly all iFGF-23 in haemodialysis patients due to impaired FGF-23 cleavage [
20], and approximately 80% of circulating FGF-23 is iFGF-23 in predialysis patients [
21]. In this study, we selected iFGF-23 concentration detection in human and mouse serum. Several studies have shown that when present in excess, FGF-23 can produce off-target effects beyond classical endocrine mineral processing. This effect is thought to occur in the hearts of CKD patients, possibly driving hypertrophic and fibrotic signalling programs through the inappropriate activation of cells that do not express their physiological coreceptor Klotho [
22]. In our previous study, we demonstrated that FGF-23 was elevated in the serum of CKD rats, which led to myocardial hypertrophy and promoted cardiomyocyte fibrosis [
23]. Most notably, it has been shown that FGF-23 enhances the profibrotic signalling cascade in injury-induced renal fibroblasts by activating FGFR4 and upregulating the calcium transporter transient receptor potential cation channel 6 [
24‐
26].
In this study, we found that circulating FGF-23 levels were significantly increased in AKI patients, and FGF-23 levels in AKI patients were closely related to the recovery of renal function; in other words, the higher the FGF-23 level was, the higher the possibility of progression to CKD (Fig.
1). Moreover, after ischaemia‒reperfusion, continuous increases in serum FGF-23 were observed in IRI mice (Fig.
2). Based on these results, we hypothesized that FGF-23 could mediate CKD progression.
Several pathways are thought to be involved in FGF-23-mediated promotion of CKD. For instance, hyperphosphatemia is closely related to the degree of tubulointerstitial damage [
26]. FGF23 is associated with the inflammatory response [
27], endothelial injury [
22], and sympathetic and RAAS activation [
28]. Most notably, renal fibrosis is the pathological basis of CKD. In recent years, studies have revealed that the canonical TGF-β1/smad3 signalling pathway plays an important role in the progression of renal fibrosis, and persistent activation of the Wnt/β-catenin pathway is involved in promoting the development of AKI to CKD. Coincidently, our previous study showed that TGF-β and Wnt/β-catenin pathway inhibition could reverse renal tubular fibrosis [
29]. According to these results, we hypothesized that FGF-23 accelerated AKI-CKD via the TGF-β and Wnt/β-catenin pathways. Through in vivo and in vitro experiments, we suggest that FGF-23 could not only lead to renal tubular fibrosis directly but could also activate the fibrotic TGFβ/Smad and Wnt/β-catenin pathways (Figs.
2 and
3). In addition, PD173074, a blocker of FGFR, inhibited this effect both in vivo and in vitro. (Figures
2 and
4). On the other hand, myofibroblasts are the main source of extracellular matrix (ECM) during kidney fibrosis [
30], and genetic fate-tracing data in mice and histological analyses of human tissue suggested that epithelial, endothelial, haematopoietic and resident mesenchymal cells all contribute to fibrosis [
31]. Does FGF-23 lead to myofibroblast activation and eventually cause renal fibrosis by damaging renal tubular epithelial cells? This needs to be confirmed by further studies.
In summary, we extended our understanding of FGF-23 in AKI-CKD. We showed a sustained increase in circulating FGF-23 in AKI patients and IRI mice, which may be related to AKI progression to CKD. We further confirmed that the upregulation of FGF-23 facilitates the activation of TGF-β and Wnt/β-catenin signalling. Moreover, FGF-23 upregulation may result in renal fibrosis via TGF-β and Wnt/β-catenin activation. We believe these data not only uncover a novel mechanism of AKI-CKD but also hint at a potential therapeutic target for AKI treatment.
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