Renal tissues from 35 patients with CKD (kidney puncture sample) and 20 patients without CDK (paracancerous tissue of renal cancer patients) were retrieved from Yan'an Hospital of Kunming City. Among the patients with CKD who underwent renal puncture biopsy in our hospital from 2013 to 2020, the distribution of renal interstitial fibrosis in the sections obtained accounted for >75% of the total area, and the glomerular filtration rate was less than 15 ml/min/1.73 m2 calculated by EPI formula, that is, patients with stage 5 chronic kidney disease. The kidney tissue was harvested and stored at liquid nitrogen.

Human renal tubular HK2 cells were purchased from Otwo Biotechnology Co., Ltd. and cultured in DMEM/F12 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (MilliporeSigma) at 5% CO₂ and 37°C. HK2 cells treated with 10 ng/ml TGF-β (cat. no. 240-B-002; R&D Systems, Inc.) for 48 h were used to construct a renal fibrosis cell model. To explore the downstream mechanisms, HK2 cells were treated with 5 µM RGFP966 (Selleck Chemicals, cat. No. S7229), a selective HDAC3 inhibitor, or an equivalent volume of vehicle (0.1% DMSO) as a control, at 37°C for 4 h.

The cells were cultured in 24-well plates overnight to reach 70–80% confluence prior to transfection. The fulllength coding sequences of Klotho and ATF3 were amplified by PCR, digested with restriction enzymes, and ligated into the pcDNA3.1(+) vector to generate the overexpression plasmids oe-Klotho and oe-ATF3. Small interfering RNA targeting ATF3 (si-ATF3), Klotho (si-Klotho), and the corresponding negative control siRNA (si-NC) were purchased from Shanghai GenePharma Co., Ltd. The siRNA sequences are listed in Table SI. For transfection, oe-Klotho (0.8 µg/well), oe-ATF3 (0.8 µg/well), si-ATF3 (50 pmol/well), si-Klotho (50 pmol/well), or si-NC (50 pmol/well) were diluted in Opti-MEM reduced serum and mixed with Lipofectamine^® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The transfection mixtures were added to at 37°C in a humidified atmosphere containing 5% CO₂ for 6 h, after which the medium was replaced with fresh complete culture medium. The cells were cultured under the same conditions for an additional 48 h prior to subsequent experimentation. Transfection efficiency was verified by Western blot analysis.

Specific pathogen-free-grade 8-week-old male C57BL/6 mice (20±0.5 g) were obtained from the Animal Experiment Center of Kunming Medical University (Kunming, China). All animals were housed under controlled conditions at 22±2°C with a relative humidity of 50–60%, on a 12-h light/dark cycle, and had ad libitum access to food and water. A total of 50 mice were used in the present study. The animal study was authorized by the Animal Care Committee of Yan'an Hospital Affiliated with Kunming Medical University in conformance with the institutional guidelines (approval no. 2021044). The mouse kidney fibrosis model was established using the unilateral ureteric obstruction (UUO) procedure described previously (25). In the sham-operated group, the ureter was exposed but not ligated. The mice were randomly divided into 5 groups: i) Sham-operated (Sham); ii) renal fibrosis group (UUO); iii) mice injected with si-ATF3 via the tail vein (UUO + si-ATF3, 5 nmol in 100 µl PBS, twice per week for 2 weeks) prior to UUO modeling; iv) mice were renally injected with oe-Klotho (UUO + oeKlotho, genome copy number containing 5×109 virus particles) 1 week prior to UUO modeling; and v) the group in which the mice were renally injected with oeKlotho and oe-ATF3 (UUO + OE-Klotho + oe-ATF3, genome copy number containing 5×109 virus particles each) 1 week prior to UUO modeling. Throughout the experiment, animal health and behaviour were monitored daily. Animals were euthanized immediately upon reaching the predefined humane endpoint: i) >15% weight loss within 1–2 days, or >20% overall weight loss; ii) signs of severe distress, including lethargy, piloerection, hunched posture, dehydration, or reduced responsiveness to external stimuli. No animals reached the humane endpoint during the experiment. The experiment lasted for 14 days after UUO surgery. During this period, a total of 9 mice died during the experiment: 4 due to anesthesia-related complications, 3 due to surgical failure, and 2 due to postoperative infection; these animals were excluded from the final analysis. After UUO surgery for 14 days, the mice were all anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight). Following the loss of consciousness and lack of response to toe pinch, 0.5–1 ml of whole blood was collected from each mouse via cardiac puncture for serum cytokine analysis. The blood samples were stored at room temperature for 30 min to 2 h to allow natural clotting, and serum was separated by centrifugation at 1,000 × g for 10 min at 4°C and stored at −80°C until further use. After blood collection, the kidneys were harvested and stored at −80°C. All procedures were performed in accordance with institutional guidelines.

The whole blood of the mice was stored at room temperature for 30 min to 2 h. After the whole blood had naturally clotted, the serum was aspirated. The expression levels of TNF-α, IL-1β and IL-6 in mouse serum were detected using commercial ELISA kits according to the manufacturer's instructions (cat. no. PT512, PI301 and PI326; all Beyotime Biotechnology). Flow cytometric analysis was performed using a BD FACSCanto II flow cytometer (BD Biosciences).

Total RNA was extracted from tissue or cell samples via TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and the RNA was reverse transcribed into cDNA using a First Strand cDNA synthesis kit (Beijing Genenode Biotech Co., Ltd.). SYBR Green real-time fluorescence qPCR was performed using a SYBR Green reaction mix (Beijing Solarbio Science & Technology Co., Ltd.) on a real-time PCR system (Roche LightCycler^® 480 II) according to the manufacturer's protocol. The thermocycling conditions were as follows: initial denaturation at 95°C for 2 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec, with a final melt curve stage. GAPDH was used as the internal reference gene. The relative expression levels of target genes were calculated using the 2-ΔΔCq method (26). The primer sequences used are listed in Table I.

Total protein was extracted from cells or tissues with RIPA buffer (MilliporeSigma) containing 1% protease inhibitor and phosphatase inhibitor. Protein concentration was determined using a BCA protein assay kit. Equal amounts of protein (30 µg/lane) were separated by 10% SDS-PAGE and transferred to PVDF membranes. After the membranes were blocked with 5% skim milk for 2 h at room temperature, they were incubated with the primary antibody at 4°C overnight, after which they were incubated with an HRP-conjugated secondary antibody (1:2,000; cat. no. ab205718) for 1 h at room temperature. The proteins were visualized via chemiluminescence. All western blotting experiments were repeated at least three times with independent biological samples, and representative images are shown. The protein bands were analyzed using ImageJ v1.8.0 software (National Institutes of Health). The following antibodies were used: Anti-Klotho (1:1,000; cat. no. ab181373), anti-HDAC3 (1:5,000; cat. no. ab32369; Abcam), anti-collagen I (1:1,000; cat. no. ab260043; Abcam), anti-collagen IV (1:1,000; cat. no. ab6586; all Abcam), anti-fibronectin (1:1,000; cat. no. ab2413; Abcam), anti-E-cadherin (1:1,000; cat. no. ab231303; Abcam), anti-α-SMA (1:1,000; cat. no. ab5831; Abcam), anti-ATF3 (1:1,000; cat. no. ab254268; Abcam), anti-Wnt1 (1:1,000; cat. no. ab15251; Abcam), anti-β-catenin (1:5,000; cat. no. ab32572; Abcam), anti-c-MYC (1:2,000; cat. no. ab185656; Abcam), anti-cyclin D1 (1:2,000; cat. no. ab16663; Abcam) and anti-β-actin (1:1,000; cat. no. ab8226; Abcam). The membranes were then incubated with HRP-conjugated goat anti-rabbit IgG (1:2,000; cat. no. ab205718; Abcam) or HRP-conjugated goat anti-mouse IgG (1:2,000; cat. no. ab205719; Abcam).

After the histones in each group were extracted, the acetylation level of total histone H3 was detected by a total histone H3 acetylation detection kit (cat. no. P-4030; EpigenTek Group Inc.) according to the manufacturer's instructions.

After trypsin digestion, the concentrations of the HK2 cells in each group were adjusted using cell culture medium and 20 µl of DCFH-DA solution was added to the samples. The cells were incubated in a cell incubator in the dark for 30 min and shaken every 5 min. The cells were then centrifuged at 1,000 × g for 5 min at 4°C and washed twice with PBS. Serum-free culture medium was added again to resuspend the cells, and the levels of ROS in the cells were measured using a flow cytometer (BD Biosciences).

MDA and SOD levels in HK2 cells were measured using commercial assay kits according to the manufacturers' instructions (cat. no. BC0025; SOD kit; cat. no. BC5165; both, Beijing Solarbio Science & Technology Co., Ltd.). Briefly, the cells were lysed with RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) on ice for 30 min with vortexing every 10 min, and then centrifuged at 10,000 × g for 4 min at 4°C. The supernatants were collected, and the MDA and SOD levels were measured according to the manufacturer's instructions.

In brief, the cells were crosslinked with 1% polyformaldehyde at room temperature for 10 min, and the reaction was quenched with 0.125 M glycine. Then, the cells were spun down, washed, resuspended, lysed and ultrasonicated to obtain chromatin extracts. The fragmented chromatin extract was precultured with protein A/G beads (Thermo Fisher Scientific, Inc.) and incubated overnight with anti-H3K9ac antibody at 4°C (1:50; cat. no. ab4441; Abcam). After thorough elution and reverse cross-linking, DNA was extracted for qPCR analysis.

The cells or tissues were harvested and lysed using IP lysis buffer (Beyotime Biotechnology; cat. no. P0013) containing protease and phosphatase inhibitors on ice for 30 min, followed by centrifugation at 12,000 × g for 15 min at 4°C to collect the supernatants. A total of 500 µg of protein lysate was used per immunoprecipitation reaction. Protein A/G Sepharose beads (Santa Cruz Biotechnology, Inc.; cat. no. sc-2003) were pre-incubated with an anti-HDAC3 antibody (1:20; cat. no. ab32369; Abcam) at 4°C with slow shaking for 60 min and then washed twice with lysis buffer. The antibody-bead complexes were then incubated with the lysates overnight at 4°C with slow shaking. After immunoprecipitation, the beads were collected by centrifugation at 1,000 × g for 5 min at 4°C and washed three times with lysis buffer. The immunoprecipitates were eluted by boiling the beads in 2× SDS-PAGE loading buffer at 95°C for 10 min and then analyzed by Western blotting.

For histological analysis, kidney tissues were fixed in 4% paraformaldehyde at room temperature for 24 h, embedded in paraffin and sectioned at a thickness of 4 µm. Masson's trichrome staining was performed to evaluate collagen deposition and renal fibrosis. Briefly, the sections were deparaffinized, rehydrated and stained using a Masson's trichrome staining kit (Beijing Solarbio Science & Technology Co., Ltd., cat. no. G1340) according to the manufacturer's instructions. The stained sections were imaged under a light microscope (Nikon Corporation). The extent of collagen deposition was assessed based on the blue-stained areas, and the images were analyzed using ImageJ v1.8.0 software (National Institutes of Health, Bethesda, MD, USA).

The cells were seeded in a 24-well plate (2×10⁴ cells/well). After 24 h, the cells were washed twice with PBS (pH 7.4), fixed with 4% paraformaldehyde for 30 min at room temperature, permeabilized with 0.5% Triton X-100 for 10 min and blocked with bovine serum albumin for 1 h at room temperature. Subsequently, the cells were incubated overnight with primary antibodies against p65 (1:100; cat. no. ab32536; Abcam) at 4°C. The next day, the cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:500; cat. no. A-11008; Thermo Fisher Scientific, Inc.) for 1 h at room temperature in the dark, followed by DAPI staining (1:1,000; room temperature; 5 min). Finally, the stained cells were observed and imaged under a fluorescence microscope (Nikon Corporation).

GraphPad Prism version 8 software (Dotmatics) was used to analyze the data, and the data are presented as the mean ± standard deviation. Comparisons between two groups were performed using unpaired two-tailed Student's t-test. For comparisons involving >2 groups, one- or two-way ANOVA was used, followed by Tukey's post hoc test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

Studies have shown that impaired Klotho accelerates the transition of the profibrotic phenotype in renal tubular epithelial cells and strengthens the proliferation of fibroblasts, thereby promoting renal fibrosis (27–29). Accordingly, the present study used kidney tissue samples from 20 non-CKD controls and 35 patients with CKD for verification and found low expression of Klotho and high expression of HDAC3 in CKD samples (Fig. 1A and B). In addition, correlation analysis at the clinical level indicated a negative correlation between HDAC3 and Klotho (Fig. 1C). UUO is a typical model of renal fibrosis, and it has been shown that urine flow obstruction leads to extensive renal tubular lesions and fibrosis (30). After UUO-induced kidney fibrosis was established, low expression of Klotho was observed in the renal tissue of UUO mice (Fig. 2A). Western blot analysis showed decreased Klotho and increased HDAC3 in the renal tissue of UUO mice (Fig. 2B and C). Histone acetylation plays a pivotal role in the control of gene expression and is interrelated with the modulation of several diseases, including tissue fibrosis (31). This modification of histones and several non-histone proteins are under the control of histone acetyltransferase and histone deacetylases (HDACs) (32), including HDAC3. Therefore, we hypothesized that the low expression of Klotho in renal fibrosis was interrelated with HDAC3-mediated histone deacetylation. Compared with those in the sham group, histone H3 acetylation levels were reduced in the UUO group (Fig. 2D). Similarly, decreased Klotho and increased HDAC3 were observed in HK2 cells treated with TGF-β (Fig. 2E and F). Decreased histone H3 acetylation was detected in TGF-β-treated HK2 cells (Fig. 2G). These results indicate that HDAC3-mediated histone deacetylation may be observably interrelated with the abnormal downregulation of Klotho in renal fibrosis.

K9 in histone H3 is a unique target of HDAC3, and HDAC3-mediated deacetylation of H3K9ac is important for H3K9 methylation (33), while increased H3K9ac modification acts as a transcriptional activator and can increase the levels of multiple genes. To further verify the effect of HDAC3 on renal fibrosis via the inhibition of Klotho expression through histone deacetylation, HK2 cells were treated with an HDAC3 selective inhibitor (RGFP966). ChIP-qPCR experiments showed that H3K9ac and Klotho binding was markedly decreased after TGF-β treatment while H3K9ac was increased in the Klotho group after the addition of RGFP966 (Fig. 3A), suggesting that a change in the H3K9ac level of Klotho occurs during renal fibrosis, which is associated with HDAC3. RT-qPCR and western blot analyses demonstrated that TGF-β inhibited the expression of Klotho and that RGFP966 clearly restrained the influence of TGF-β on Klotho (Fig. 3B and C). To examine the function of HDAC3 and Klotho in renal fibrosis, two samples of si-Klotho were designed and transfected into HK2 cells. The knockdown efficiency was detected by western blotting. The results showed that compared with the si-NC group, the expression of Klotho in the si-Klotho-1 and si-Klotho-2 groups decreased, where the si-Klotho-2 group showed significant differences (Fig. 3D). Subsequent experiments were conducted with si-Klotho-2. Further analysis suggested that TGF-β increased the levels of ECM-related proteins, including collagen I, Collagen IV and Fibronectin, and the fibrosis-related protein α-SMA in HK2 cells. The addition of RGFP966 prominently decreased the levels of Collagen I, Collagen IV, Fibronectin and α-SMA, and these effects were reversed by si-Klotho (Fig. 3E). These results imply suppression of HDAC3 can upregulate Klotho expression through histone acetylation, thereby suppressing TGF-β-induced fibrosis in HK2 cells.

ATF3 can bind to HDAC3 without the need for additional proteins (24). The downregulation of Klotho was accompanied by the upregulation and nuclear translocation of ATF3, and ATF3 overexpression restrained the transcriptional activity of Klotho. Therefore, the present study speculated that HDAC3 binds to ATF3 to mediate the transcriptional repression of Klotho in renal fibrosis. Western blot analysis showed that the ATF3 protein level was markedly increased in UUO mice and in TGF-β-treated HK2 cells (Fig. 4A and B). Co-IP indicated that HDAC3 directly interacted with ATF3 both in vitro and in vivo (Fig. 4C and D). ChIP-qPCR analysis revealed that ATF3 was enriched in the Klotho promoter region after TGF-β treatment, while the binding of ATF3 to the Klotho promoter region decreased after the addition of RGFP966 (Fig. 4E). In order to explore the regulatory effect of ATF3 and HDAC3 on Klotho, the present study designed two si-ATF3 and transfected si-ATF3 into HK2 cells. Western blot analysis showed that, compared with the si-NC group, the expression of ATF3 was reduced in the si-ATF3-1 group and the si-ATF3-2 group, and the reduction of ATF3 in the si-ATF3-2 group was significantly different (Fig. 4F). si-ATF3-2 showed improved knockdown efficiency and was used for subsequent experiments. Furthermore, TGF-β inhibited the expression of Klotho in HK2 cells, and silencing ATF3 promoted the expression of Klotho (Fig. 4G and H). Consistently, to determine the ATF3-mediated effects in vivo, ATF3 was knocked down in mouse kidneys, as shown in Fig. S1. Similarly, lower expression of Klotho was detected in the renal tissue of UUO mice, and si-ATF3 increased the expression of Klotho (Fig. 4I). In addition, compared with those in the UOO group, si-ATF3 decreased the levels of Collagen I, Collagen IV, Fibronectin and α-SMA in the renal tissue of the mice, suggesting that ATF3 knockdown can effectively inhibit those proteins (Fig. 4J). Masson's trichrome staining showed glomerular collagen fibril hyperplasia in UUO mice, and collagen fibrils were extensively deposited in the interstitium, ATF3 knockdown decreased collagen deposition (Fig. 4K). These results point toward HDAC3 binding to ATF3 to repress Klotho transcription and promote renal fibrosis.

Blocking Wnt/β-catenin signaling may be beneficial for alleviating renal fibrosis (34–36). To explore the downstream mechanism of Klotho in kidney fibrosis, a Klotho overexpression vector was constructed (Fig. S2A) and used to treat HK2 cells and mice. Western blot analysis showed that the levels of Wnt1, β-catenin, c-MYC and Cyclin D1 were markedly increased in the TGF-β group, while the levels of Wnt1, β-catenin, c-MYC and Cyclin D1 were decreased in the TGF-β+oe-Klotho group; moreover, the overexpression of ATF3 prevented the aforementioned results (Fig. 5A). Compared with that in the TGF-β group, Klotho overexpression inhibited the expression of ECM-related proteins and fibrosis-related proteins, and the ATF3 overexpression vector that was constructed (Fig. S2B) increased the expression of these proteins (Fig. 5B). In addition, increased HDAC3 was detected in TGF-β-induced cells, oe-Klotho had no effect on HDAC3, while overexpression of ATF3 promoted the expression of HDAC3 (Fig. 5C). Similarly, activated Wnt/β-catenin signaling and increased renal fibrosis were observed in UUO mice. To confirm the in vivo effects of Klotho and ATF3, Klotho and ATF3 were overexpressed in kidney tissue (Fig. S1). Overexpression of Klotho inhibited Wnt/β-catenin signaling and the expression of ECM-related proteins and fibrosis-related proteins. Further treatment of overexpression of ATF3 prevented the effect of overexpression of Klotho to some extent (Fig. 5D and E). In addition, increased HDAC3 was detected in the renal tissue of UUO mice. oe-Klotho had no effect on HDAC3, while the overexpression of ATF3 promoted the expression of HDAC3 (Fig. 5F). Masson's trichrome staining showed that glomerular collagen fibrils proliferated and were extensively deposited in the interstitium of UUO mice, and collagen deposition was significantly reduced after Klotho treatment, while overexpression of ATF3 could weaken the effect of Klotho on renal fibrosis (Fig. 5G). The results suggest that the alleviating effect of Klotho on TGF-β-induced fibrosis in HK2 cells and renal fibrosis in UUO mice may be related to the Wnt/β-catenin signaling pathway, and ATF3 prevents the inhibitory effect of Klotho on Wnt/β-catenin signaling pathway.

A previous study showed that Klotho itself has antioxidant and anti-inflammatory effects and that the canonical NF-κB component RelA is one of its targets (37). Therefore, the present study subsequently detected the protein level of NF-κB. Western blot analysis indicated that the level of NF-κB p65 was increased in the TGF-β group, while NF-κB p65 was decreased after the addition of oe-Klotho, and ATF3 promoted the level of NF-κB p65 (Fig. 6A). The immunofluorescence results were consistent with the western blotting results (Fig. 6B). Moreover, TGF-β inhibited SOD levels and increased MDA and ROS levels. Compared with those in the TGF-β group, increased SOD levels and decreased MDA and ROS levels were observed after the addition of oe-Klotho. Furthermore, oe-ATF3 promoted oxidative stress and inflammation (Fig. 6C-E). Similarly, high NF-κB p65 expression was observed in the renal tissue of UUO mice, Klotho inhibited NF-κB p65 expression, and ATF3 prevented the influence of Klotho (Fig. 6F). In addition, inflammatory factor levels in murine serum were measured. UUO mice had increased levels of TNF-α, IL-1β and IL-6 and Klotho decreased the levels of these cytokines. ATF3 overexpression increased the levels of TNF-α, IL-1β and IL-6 (Fig. 6G). The results suggested that the alleviating effect of Klotho on TGF-β-induced fibrosis in HK2 cells and ATF3 prevents the inhibitory effect of Klotho on the NF-κB signaling pathway.