Recombinant human FGF2, Tert-Butyl hydroperoxide (TBHP) solution, LY294002 (selective PI3K inhibitor), and U0126 (selective MKK1/2 inhibitor) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, United States). Fetal bovine serum (FBS), Dulbecco’s Modified Eagle Medium (DMEM) and 4′, 6-Diamidino-2-phenylindole (DAPI) were purchased from Invitrogen/Gibco Corp. (Carlsbad, CA, United States). Antibodies against cleaved Caspase-3 (catalog number: 9661), Akt (catalog number: 4685), phospho-Akt (catalog number: 4060), and phospho-ERK1/2 (catalog number: 9101) were purchased from Cell Signaling Technology (CST) Inc. Anti-ERK1/2 antibody (catalog number: 82380) was purchased from Thermo Fisher Scientific (Sunnyvale, CA, United States). Antibodies against CHOP (catalog number: ab11419), GRP78 (catalog number: ab21685), ATF-6 (catalog number: ab203119), XBP1 (catalog number: ab37152), β-actin (catalog number: ab179467), and GAPDH (catalog number: ab9485) were purchased from Abcam Inc. (Cambridge, MA, United States). The secondary antibodies were purchased from Cell Signaling Technology (CST) Inc. (Danvers, MA, United States) or Santa Cruz Biotechnology (Santa Cruz, CA, United States). The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) Assay Kit was purchased from Abcam, Inc. (Cambridge, MA, United States).

A renal I/R injury model was established in rats to clarify the protective effect of FGF2 against I/R injury in kidney. Male Sprague-Dawley (SD) rats (8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and were housed in an Specific-pathogen-free (SPF) facility. The experimental protocol in the present study was approved by the Institutional Animal Ethical and Use Committee of Wenzhou Medical University. The renal I/R injury model was created as we described in previous studies (Tan et al., 2013, 2017). Briefly, SD rats were anesthetized with an intraperitoneal (ip) injection of 25 mg/kg of Pentobarbital sodium and placed on a thermostatic surgical pad. The right kidney was carefully liberated from surrounding tissue, and nephrectomy was performed. The left kidney was exposed after a midline incision, and the renal artery was occluded with a vascular clamp for 45 min, after which renal blood flow was re-established. For measurement of renal function, serum creatinine (Cr) was measured at 6, 24, and 72 h after renal ischemia-reperfusion in the hospital medicine biochemical laboratory (the First Affiliated Hospital of Wenzhou Medical University). Kidneys were harvested and stored in a cryogenic refrigerator for further experiments. Rats were randomly divided into four groups in the present study. (a) Sham group: 0.5 ml saline was given to rats by ip injection half an hour before sham operation; (b) FGF2 group: 0.5 mg/kg FGF2, dissolved in equivalent amount of sterile saline, was given to rats by ip half an hour before sham operation; (c) I/R group: kidneys were subjected to 45 min of ischemia through clipped renal artery followed by reperfusion; (d) I/R-FGF2 group: 0.5 mg/kg FGF2 was given to rats by ip half an hour before renal ischemia operation.

Cell culture was performed to further clarify the role of the PI3K/AKT and MEK-ERK1/2 signaling pathways in the protective effect of FGF2. NRK-52E, a rat renal tubular epithelial cell line, was used. The NRK-52E cell line was purchased from the American Type Culture Collection (Manassas, VA, United States) and maintained in DMEM supplemented with 10% FBS and incubated under 37°C, 95% air, and 5% CO₂. NRK52E cells were plated at 2 × 10⁵ in each 35-mm culture dish for 24 h, and then serum starvation was performed for 12 h in DMEM containing 1% FBS. Serum stimulation was performed by switching the culture medium to 10% FBS after serum starvation. TBHP is an oxidant for I/R injury in various cultured cells, including renal epithelial cells (Paller and Manivel, 1992; Wu et al., 2017; Chen et al., 2018; Shen et al., 2019). In order to determine the effect of FGF2 on TBHP-induced ER stress, NRK-52E cells were divided into six groups randomly: (a) Vehicle group: NRK-52E cells were cultured in complete medium without any supplement; (b) FGF2 group: NRK-52E cells were cultured in complete medium and treated with recombinant human FGF2 (100 ng/ml) for 2 h; (c) TBHP group: NRK-52E cells were cultured in complete medium, and then TBHP (200 μmol/L) was added for an additional 12 h; (d) TBHP-FGF2 group: NRK-52E cells were pretreated with recombinant human FGF2 (100 ng/ml) for 2 h, and then TBHP (200 μmol/L) was added for an additional 12 h; (e) LY294002 group: NRK-52E cells were pretreated for 2 h with specific Akt pathway inhibitor LY294002 (20 μmol/L), and then cells were treated as in the TBHP + FGF2 group. (f) U0126 group: NRK-52E cells were pretreated for 2 h with specific ERK1/2 pathway inhibitor U0126 (20 μmol/L), and then cells were treated as in the TBHP-FGF2 group. All experiments were repeated three times.

The regulation role of FGF2 on ER stress and apoptosis was assessed by analyzing the expression of proteins by Western blot. For protein analysis of renal tissue in different groups, kidney tissues (containing both cortex and medulla but not containing renal fibrous capsule) were homogenized, and total proteins were extracted using tissue lysis buffer. For protein analysis of in vitro samples, NRK-52E cultured in a petri dish was rinsed with PBS buffer three times, and total proteins were extracted using cell lysis buffer. An equivalent of 100 μg protein extracted from kidney tissues (or 30 μg protein extracted from NRK-52E cells) was separated by SDS-PAGE gels and then transferred onto PVDF membrane for immunoblot analysis with the primary antibodies directed against the relevant proteins. Primary antibodies against cleaved Caspase-3 (1:1000), Bax (1:3000), Bcl-2 (1:1000), GRP78 (1:1000), CHOP (1:5000), XBP-1 (1:1000), ATF-6 (1:2000), Akt (1:1000), phosphor-Akt (1:1000), ERK1/2 (1:1000), and phosphor-ERK1/2 (1:1000) were used in the present study. GAPDH (1:2500) or β-actin (1:2000) were used to quantify the protein expression levels. The signals were visualized with the ChemiDoc^TM XRS + Imaging System (Bio-Rad Laboratories). The band densities were quantified with Multi Gauge Software of Science Lab 2006 (FUJIFILM Corporation, Tokyo, Japan).

Immunohistochemistry and immunofluorescence staining were performed in order to observe the expression and location of ER stress- and apoptosis-relevant proteins in kidney. Kidneys were excised and harvested 6, 24, or 72 h following I/R injury. Renal tissue embedded in paraffin was cut into 5-μm sections and then incubated with antibodies against cleaved Capase-3, GRP78, and CHOP at 4°C overnight. After being incubated with primary antibodies, the slides were then incubated with secondary antibodies for 1 h at room temperature. Cell apoptosis in the kidney was detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay kit (Roche, Mannheim, Germany). The apoptosis index was analyzed based on five randomly selected images from each group. All images were captured under a Laser confocal microscope (Nikon, Ti-E&A1 plus).

In order to evaluate the degree of renal histopathology damage, sections were stained with hematoxylin and eosin (H&E). Images were captured under a light microscope (Nikon ECLIPSE 80i). The degree of renal histopathology damage was evaluated based on intraluminal necrotic cells, cell swelling, interstitial congestion, edema, and protein casts. The following 5-point scoring system was utilized to assess renal damage: 0 points - normal renal morphology, 1 point - damage of kidney tissue ≤ 10%, 2 points - damage of kidney tissue 11–25%, 3 points - damage of kidney tissue 26–45%, 4 points - damage of kidney tissue 46–75%, and 5 points - damage of kidney tissue ≥ 76%. The pathologists assessing the images were blinded to allocation group.

The statistical evaluation of the data was performed using one-way Analysis of Variance (ANOVA), and when more than two groups were compared, the statistical evaluation of the data was performed using two-way ANOVA. Values of ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 were considered statistically significant.

In the present study, a rat model of renal I/R injury was established to assess the protective effect of FGF2 on AKI. As shown in Figure 1A, renal histomorphology changes were assessed by H&E staining at 6, 24, and 72 h after reperfusion. No obvious renal pathological damage was observed in the kidney tissues of either the Sham group or the FGF2 group (Figure 1A a–f), whereas kidneys of the I/R group rapidly exhibited typical features of AKI (Figure 1A g–i). Renal interstitial congestion proteinaceous casts and edema (shown as asterisks) were observed at 6, 24, and 72 h after reperfusion, and large masses of necrotic cellular debris (shown as arrows) were detected, especially at 24 h after reperfusion. The extent of renal damage of I/R rats at 72 h was reduced compared to at 24 h, which indicates self-recovery of kidney after I/R injury. As shown in Figure 1A j–l, administration of FGF2 significantly attenuated the degree of renal damage and largely preserved renal integrity. As shown in Figure 1B, the degree of tubular injury was quantified based on H&E staining. FGF2 treatment significantly ameliorated kidney damage after I/R injury. There is no significant difference in the renal tubular injury between the I/R-FGF2 group and the Sham group. Serum creatinine (Cr) levels were separately measured at 6, 24, and 72 h after reperfusion to assess renal function. As expected, the level of serum Cr was markedly elevated in the I/R group compared to the Sham group, whereas FGF2 significantly reduced the level of serum Cr in I/R-FGF2 rats compared to that of I/R rats at 24 h and 72 h after reperfusion (Figure 1C).

We further examined the activation of FGFR by immunohistochemistry staining with phosphor-FGFR antibody. FGF family members perform their diverse biological functions through binding and activating FGFR (Beenken and Mohammadi, 2009). As shown in Figure 1D, few phospho-FGFR-weakly positive tubular epithelial cells were detected in kidney tissues of the Sham group. The number of phospho-FGFR-positive epithelial cells was increased in the kidney tissues of the FGF2 group and I/R group. Significantly, the phosphorylation of FGFR was substantially increased in the kidney tissue of the I/R-FGFR group compared to the I/R group. Activation of FGFR in the present study was similar to that in our previous study (Tan et al., 2017). Furthermore, we observed that most of the phospho-FGFR-positive epithelial cells were present in distal tubule. Based on these results, we speculated that FGF2 is responsible for the protective effect on renal I/R injury via the activation of FGFR in distal tubular epithelial cells.

On the basis of observation of renal histological damage and detection of serum Cr at different time points after reperfusion, we generally find that the extent of renal damage peaked at 24 h after reperfusion. Therefore, we used TUNEL staining to assess apoptosis and necrosis of renal cells 24 h after reperfusion in kidney tissues of each group. As shown in Figure 2A, few TUNEL-positive cells were observed in the kidney tissues of the Sham group and FGF2 group, whereas the proportion of TUNEL-positive cells was dramatically increased in I/R rats. Notably, the proportion of TUNEL-positive cells was much lower in the I/R-FGF2 group compared to the I/R group. Quantification of the proportion of TUNEL-positive cells in kidney revealed that the average percentage of apoptotic cells was 1.18% in the Sham group, 0.89% in the FGF2 group, 28.89% in the I/R group, and 8.15% in the I/R-FGF2 group (Figure 2B). FGF2 treatment dramatically decreased the proportion of TUNEL-positive cells in kidney tissues 24 h after reperfusion.

As TUNEL staining can be used in the identification of both apoptosis and necrosis, expression of key apoptotic proteins, including cleaved Caspase-3, Bax, and Bcl-2, were also detected to distinguish the protective effect of FGF2 against I/R injury. Caspase-3, also known as CPP32, is synthesized as an inactive proenzyme that is processed to the active form (cleaved Caspase-3) in cells undergoing apoptosis (Fernandes-Alnemri et al., 1994). In the present study, increased production of cleaved Caspase-3 was observed in renal tubular epithelial cells of kidney tissues in I/R rats on the basis of immunohistochemistry staining, whereas the expression of cleaved Caspase-3 was strikingly decreased in kidney tissues of the I/R-FGF2 group (Figure 3A). We further used Western blotting to quantify the expression of cleaved Caspase-3, Bax, and Bcl-2 at 6, 24, and 72 h after reperfusion (Figures 3B–D). The production of Bax and cleaved Caspase-3 was markedly increased after I/R injury, whereas the expression of Bcl-2 was decreased. FGF2 can substantially inhibit the expression/activation of Bax and cleaved Caspase-3. Together, the present results indicate that FGF2 administration protects kidneys by preventing apoptosis of renal tubular epithelial cells after I/R injury.

Endoplasmic reticulum stress is generally considered to be an important pathological mechanism in cell survival in various diseases. To investigate the association between the renoprotective effect of FGF2 and ER stress, we examined the expression of ER stress-relevant proteins CHOP, GRP78, XBP-1, and ATF-6, which could participate in the regulation of the unfolded protein response and thus contribute to cellular homeostasis in kidney. As shown in Figures 4A–E, the expression of ER stress-relevant proteins in kidney tissues of I/R rats was dramatically increased, whereas FGF2 treatment significantly reduced their expression. To further investigate the relationship between the protective effect of FGF2 against apoptosis and ER stress, immunofluorescence double-staining was performed at 24 h after reperfusion. As shown in Figure 4F, most TUNEL-positive cells simultaneously exhibit a high expression of CHOP. The results of GRP78 and TUNEL double-staining is similar to the result of CHOP. These results suggest that the administration of FGF2 can effectively attenuate tubular cell apoptosis through inhibition of excessive ER stress induced by I/R injury.

Several studies have revealed the role of ERK1/2 signaling pathways in the resistance of apoptosis via down-regulation of ER stress (Guo et al., 2012; Wang et al., 2012; Bright et al., 2018; Kim and Woo, 2018). Given that FGF2 elicits the regulatory functions by high-affinity binding to FGF receptors and activating downstream signal pathways, we examined the phosphorylation of Akt and ERK1/2 in kidney tissues after I/R injury. As shown in Figures 5A–C, I/R injury caused a transient increase in the phosphorylation of Akt and ERK1/2 compared to the Sham group at 6 h after reperfusion, which indicates the activation of the PI3K/Akt and MEK-ERK1/2 signaling pathways in the self-recovery of kidney tissues after reperfusion. There was no significant difference between the I/R group and the Sham group at 24 and 72 h. FGF2 administration substantially improved the phosphorylation of Akt and ERK1/2 compared to the I/R group at 24 and 72 h after reperfusion. However, there is no significant difference in the activation of the Akt and ERK1/2 pathways between the Sham group and the FGF2 group. Based on these results, we may preliminarily conclude that the role of FGF2 in the regulation of ER stress after renal I/R injury is associated with the activation of the PI3K/Akt and ERK1/2 signaling pathways.

To further clarify the role of FGF2 in the regulation of ER stress and the resistance to apoptosis, we determined the protective effect of FGF2 on NRK-52E cells induced by TBHP. The expressions of cleaved Caspase-3, Bax, and Bcl2 were detected by Western blotting, as described (Figure 6A). The results indicate that FGF2 treatment effectively reduced TBHP-induced cleaved Caspase-3 and Bax. Figures 6B,C the expression levels of CHOP, GRP78, ATF-6, and XBP-1 were also examined by Western blotting (Figure 6A). TBHP remarkably increased the expression of CHOP, GRP78, ATF-6, and XBP-1 in NRK-52E cells (Figures 6D–G). Similar to the results of the in vivo experiment, FGF2 significantly reduced the expression of ER stress-relevant proteins in NRK-52E cells treated with TBHP. Furthermore, the phosphorylation of AKT and ERK1/2 was significantly increased in the TBHP-FGF2 group compared to the TBHP group (Figures 6H,I). Different from the results of FGF2 treatment in vivo, FGF2 could active the Akt and ERK1/2 pathways in NRK-52E cells cultured with normal medium. There may be many reasons for the discrepancy, but the strong binding of FGF2 to heparan sulfate (HS) is likely one of the main causes. Many studies have reported that HS in the tissue can stabilize the biological functions of canonical paracrine/autocrine FGFs (Kan et al., 1988, 1991; Li et al., 2016). Tissue matrix HS acts as an FGF depot and limits the access of FGFs to cellular membrane FGFR except when needed (McKeehan et al., 1998).

To further clarify whether PI3K/Akt or ERK1/2 pathways are involved in the protective effect of FGF2, we examined the phosphorylation of Akt and ERK1/2 in vitro using selective PI3K inhibitor (LY294002) and selective MEK inhibitor (U0126). As shown in Figure 7, FGF2 treatment significantly activated the phosphorylation of Akt and ERK1/2 compared to the TBHP group, which is consistent with the in vivo results. As expected, pre-addition of LY294002 and U0126 respectively largely abolished the effect of FGF2 on the activation of the PI3K/Akt and MEK-ERK1/2 signaling pathways in NRK-52E cells induced by TBHP.

To clarify the role of the PI3K/Akt and ERK1/2 pathways in the regulation of ER stress, the expression of ER stress-relevant proteins CHOP, GRP78, ATF-6, and XBP-1 was detected by Western blotting (Figures 8A–E). The results indicated that FGF2 significantly reduced the production of CHOP, GRP78, ATF-6, and XBP-1, whereas both LY294002 and U0126 inhibited the role of FGF2, individually. To clarify the role of the PI3K/Akt and ERK1/2 pathways in apoptosis, the expression of cleaved Caspase-3, Bax, and Bcl2 was detected by Western blotting (Figures 8A,F,G). The results indicated that FGF2 treatment effectively reduced the activation of Caspase-3 and the expression of Bax, whereas either LY294002 or U0126 abolished the protective effect of FGF2. These results strongly suggest that FGF2 could prevent excessive ER stress-mediated apoptosis in kidney tissues after I/R injury. The activation of the PI3K/Akt and MEK-ERK1/2 signaling pathways plays a crucial role in the protective effect of FGF2.