Male C57BL/6 mice weighing approximately 22–25 g were purchased from Vital River Laboratory Animal Technology (Beijing, China), and maintained in the animal facility (temperature: 22 ± 2°C, humidity: 60 ± 5%, 12h dark/light cycle) at Southern Medical University (Guangzhou, China) with water and food ad libitum. Animal studies were approved by the Animal Ethical and Welfare Committee at Southern Medical University. All animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals.

The induction of AKI and CKD in mice was described in our previous studies (Xiao et al., 2016; Zhou et al., 2018). Bilateral renal ischemia/reperfusion injury (BIRI) has been widely used to induce AKI in mice, whereas it increases mortality during the progression of AKI to CKD. Therefore, unilateral renal ischemia/reperfusion injury (UIRI) is used for the research on the progression of AKI to CKD. Briefly, a midline abdominal incision was made in mouse under general anesthesia, and bilateral or left renal pedicles were clipped for 30 min using microaneurysm clamps (item no. 18051-35; Fine Science Tools, Cambridge, United Kingdom). During the surgery, body temperature was maintained approximately 37–38°C using a temperature-controlled heating system.

To evaluate the preventive effect of Wnt/β-catenin activation on AKI, mice were injected with 1 mg/kg hemagglutinin (HA)-tagged Wnt1 expression vector (pHA-Wnt1, Upstate Biotechnology) or empty vector (pcDNA3) 2 days before BIRI using the hydrodynamic-based gene transfer technique as reported previously (Xiao et al., 2016). Blood and kidney tissues were collected 1 day after renal BIRI.

To evaluate the effect of early Wnt/β-catenin activation on the progression of AKI to CKD, mice were either subjected to daily hydrodynamic tail vein injection of Wnt1 expression plasmids at 1 mg/kg or daily intraperitoneal injection of ICG-001 (847591-62-2, Chembest, Shanghai, China) at 5 mg/kg for 3 days before UIRI. Blood and kidney tissues were collected 11 days after UIRI. In our preliminary study, 2 days of Wnt1 injection could induce the expression of Wnt1 in AKI mice (data not shown). It should be noted that 3 days of Wnt1 injection was chosen to ensure and lengthen the expression of Wnt1 during the progression of AKI to CKD.

Human proximal tubular epithelial cell line (HKC-8) was provided by Dr. L. Racusen at the Johns Hopkins University (Baltimore, MD, United States). Cell culture were performed as previously described (Zhou et al., 2013). HKC-8 cells were treated with human recombinant Wnt1 (SRP4754; Sigma-Aldrich, St. Louis, MO, United States) at 100 ng/mL or ICG-001 (847591-62-2, Chembest, Shanghai, China) at 10 μM for 4 h. Cells were then incubated in a hypoxia workstation (X3-CK, Biospherix) at 1% pO2 for 48 h. Next, cells were re-oxygenated for 2 h before collection for various analyses.

Cells were trypsinized, collected, and washed with PBS. PE Annexin V staining was performed according to the manufacturer protocol. A cytometric analysis was performed with a flow cytometer (BD FACSCanto II, San Jose, CA, United States) to measure the apoptosis rates by detecting the relative amount of PE Annexin V positive and 7-AAD negative cells. Each assay was performed in triplicate.

Creatinine levels in serum and urine as well as blood urea nitrogen (BUN) levels were measured using an automatic biochemical analyzer (AU480, Beckman-Coulter Inc., Brea, CA, United States).

Kidney sections and immunohistochemical staining were performed as previously described (Liu et al., 2020). Primary antibodies included rabbit polyclonal anti–Wnt1 (ab15251; Abcam, Inc.), rabbit polyclonal anti-β-catenin (ab15180; Abcam, Inc.), and rabbit polyclonal anti-fibronectin (F3648; Sigma-Aldrich).

Western blot analysis was performed as described previously (Zhou et al., 2019). Primary antibodies included rabbit polyclonal anti-fibronectin (F3648; Sigma-Aldrich), mouse monoclonal anti-β-catenin antibody (610154; BD Transduction Laboratories), mouse monoclonal anti-α-SMA antibody (A2547; Sigma-Aldrich), rabbit polyclonal anti-Wnt1 (ab15251; Abcam), mouse anti-α-tubulin (T9026; Sigma-Aldrich), mouse anti-PAI-1 antibody (AF3828;R&D Systems), anti-active β-catenin (#05–665; EMD Millipore), anti-Fas ligand (FasL) (SC -6237; Santa Cruz, CA, United States), p53 (#2524S; CST), Bax (SC-20067; Santa Cruz), p65 (#8242S; CST), p-p65 (#3033S; CST), PCNA (#2586S; CST).

Total RNA isolation and qPCR were carried out as previously described. The mRNA levels of various genes were normalized with β-actin. The primer sequences of the genes are listed in Supplementary Table 1.

All data were expressed as mean ± SEM. The data were statistically analyzed using Sigma Stat software (Jandel Scientific Software, San Rafael, CA, United States). Comparison between groups was made using one-way ANOVA, followed by the Student–Newman–Keuls test. P < 0.05 was considered significant.

To determine the role of Wnt/β-catenin activation in AKI prevention, mice were administered with either HA-tagged Wnt1 expression vector (pHA-Wnt1) or empty vector (pcDNA3) via hydrodynamic tail vein injection at 2 days before BIRI (Figure 1A). Serum levels of creatinine and BUN, two biomarkers of kidney injury, were significantly increased in mice 1 day after BIRI, suggesting the existence of AKI (Figures 1B,C). Consistently, prominent kidney morphologic lesions, such as tubular injuries in the corticomedullary junction, were observed in AKI mice (Figure 1D). Notably, these alterations in serum kidney injury biomarkers and morphologic lesions in AKI mice were markedly attenuated by exogenous Wnt1. Western blot analyses revealed that exogenous Wnt1 promoted the expression of renal β-catenin in AKI mice (Figures 1E,F). Taken together, these findings suggest that pre-treatment of exogenous Wnt1 activates renal β-catenin and prevents AKI in mice.

To explore whether regulation of apoptosis is the mechanism underlying the protective effect of exogenous Wnt1 on AKI, TUNEL staining was performed to identify apoptosis in kidney tissue of AKI mice. Compared with Sham mice, IRI markedly increased the apoptosis rate as well as the levels of apoptosis-related proteins, such as FasL, p53 and Bax, in kidneys of mice (Figures 2A–F). Such alterations in kidneys of AKI mice were markedly reduced by in vivo expression of exogenous Wnt1 before IR. Nuclear factor-kappa B (NF-κB), particularly its heterodimer form p65/p50, plays a critical role in regulating the inflammatory response in kidneys of AKI mice. Western blot analysis showed that both p65 and its phosphorylated form (p-p65) were markedly increased in kidneys of mice after IRI, whereas such alterations were prevented by exogenous Wnt1 (Figure 2G). These findings suggest that in vivo expression of exogenous Wnt1 before IR prevents apoptosis and inhibits NF-κB activation in kidneys of AKI mice.

We next examined whether activation of Wnt/β-catenin before IR can prevent the progression of AKI to CKD. As shown in Figure 3A, mice underwent either hydrodynamic tail vein injection of Wnt1 expression plasmid (pHA-Wnt1) or intraperitoneal injection of ICG-001 at 3 days before UIRI. Both serum creatinine and BUN levels were increased in mice at 11 days after UIRI (Figures 3B,C). Additionally, Masson trichrome staining revealed prominent renal collagen deposition and fibrotic lesions in mice at 11 days after UIRI (Figure 3D). These observations indicate the progression of AKI to CKD in mice after UIRI. Notably, the observed alterations of serum kidney injury biomarkers and renal fibrosis in CKD mice were almost completely prevented by pre-treatment of exogenous Wnt1, but not ICG-001. Therefore, in vivo expression of exogenous Wnt1 before IR prevents the progression of AKI to CKD in mice.

We further investigated the effect of exogenous Wnt1 on endogenous Wnt/β-catenin signaling in kidneys of CKD mice. As shown in Figure 4A, immunohistochemical staining revealed that both endogenous Wnt1 and β-catenin were markedly up-regulated in kidneys of mice at 11 days after IRI. Their protein levels were greatly reduced by pre-treatment of exogenous Wnt1, but not ICG-001. Western blot analyses confirmed the inhibitory effect of exogenous Wnt1, but not ICG-001, on endogenous expression of Wnt1, β-catenin and active β-catenin in kidneys of CKD mice (Figures 4B–E). The Wnt/β-catenin and TGF-β signaling pathways crosstalk in renal fibrosis. The mRNA expression of TGF-β was induced in kidneys of CKD mice, and such induction was diminished by exogenous Wnt1, but not ICG-001 (Figure 4F). Therefore, pre-treatment of exogenous Wnt1 inhibits endogenous renal Wnt/β-catenin signaling in CKD mice.

Compared with Sham mice, the mRNA expression of PAI-1 and MMP-7, two direct downstream targets of Wnt/β-catenin signaling, was significantly up-regulated in kidneys of mice at 11 days after UIRI (Figures 5A,B). Pre-treatment of exogenous Wnt1, but not ICG-001, inhibited PAI-1 and MMP-7 mRNA expression in CKD mice. Western blot analysis showed that PAI-1 protein levels were increased in kidneys of CKD mice, where were abolished by pretreatment of exogenous Wnt1, but not ICG-001 (Figures 5C,D). Klotho is an endogenous Wnt antagonist by binding and sequestering Wnt ligands. Compared with Sham mice, both mRNA and protein levels of Klotho were decreased in kidneys of mice at 11 days after UIR (Figures 5E,F). Pre-treatment of exogenous Wnt1, but not ICG-001, almost completely restored Klotho mRNA and protein expression in kidneys of CKD mice. These findings further suggest the inhibitory effect of exogenous Wnt1 on endogenous Wnt/β-catenin signaling in kidneys of CKD mice.

We next investigated the effect of Wnt/β-catenin activation before IR on renal matrix genes and fibrotic lesions in CKD mice. Compared with Sham mice, the mRNA expression of fibronectin (FN), collagen I, collagen III, and α-SMA was markedly induced in kidneys of mice at 11 days after UIR (Figures 6A–D). Such alterations were diminished by pre-treatment of exogenous Wnt1, but not ICG-001. Western blot analysis showed that the protein levels of FN and α-SMA were significantly increased in kidneys of CKD mice, and such alterations were abolished by pre-treatment of exogenous Wnt1, but not ICG-001 (Figures 6E–G). The effects of exogenous Wnt1 and ICG-001 on FN protein expression in CKD mice were validated by kidney immunostaining with FN antibody (Figure 6H). These findings suggest that pre-treatment of exogenous Wnt1 prevents renal fibrogenesis in IR-induced CKD mice.

Compared with Sham mice, the mRNA expression of inflammatory genes, such as IL-1, IL-6, and TNF-α, was significantly up-regulated in kidneys of mice at 11 days after UIR (Figures 7A–C). Such alterations were almost completely abolished by pre-treatment of exogenous Wnt1, but not ICG-001. Western blot analysis showed that the protein levels of both P65 and its phosphorylated form (p-p65) were markedly increased in kidneys of mice at 11 days after UIRI, indicating the activation of NF-κB signaling in CKD mice (Figures 7D–F). Notably, pre-treatment of exogenous Wnt1, but not ICG-001, significantly reduced p65 and p-p65 protein levels in kidney of CKD mice. These findings indicate that pre-treatment of exogenous Wnt1 prevents renal inflammatory responses in CKD mice.

To further pinpoint the role of Wnt1 in AKI, we established HKC-8 tubular cell models of AKI using hypoxia-reoxygenation (H/R) treatment. Flow cytometry was employed to detect the apoptosis, and revealed the elevation of apoptosis rate in HCK-8 cells after H/R treatment (Figures 8A,B). Exogenous Wnt1 markedly reduced apoptosis in HCK-8 cells after H/R treatment. Western blot analyses showed that H/R treatment significantly increased the levels of apoptosis-related proteins, such as FasL, p54, Bax, and Parp-1 in HCK-8 cells (Figures 8C–G). Such alterations were diminished by exogenous Wnt1. These data suggest that exogenous Wnt1 protects against H/R-induced apoptosis in tubular cells.