Wild-type male C57BL/6 mice (body weight 20–25 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd., and housed in a specific pathogen-free-grade animal facility in the Shanghai University of Traditional Chinese Medicine under the local regulations. Shanghai University of Traditional Chinese Medicine has approved the animal experiments (PZSHUTCM18111601).

Mice were anesthetized with pentobarbital sodium (P3761; Sigma-Aldrich Co., St. Louis, MO, USA) at a dose of 100 mg/kg via intraperitoneal (i.p.) injection before surgery. The UUO surgery was performed by ligating the left ureter twice using 4-0 nylon sutures. Mice were randomly divided into four groups: (1) Sham/vehicle (n = 6), (2) Sham/triptolide (n = 7), (3) UUO/vehicle (n = 7), and (4) UUO/triptolide (n = 7). Triptolide (Topscience, T2179, Shanghai, China) was dissolved in dimethylsulfoxide (DMSO) and diluted with normal saline to prepare a working solution containing 1% DMSO. Sham or UUO mice were treated with vehicle or 0.25 mg/kg triptolide daily by i.p. injection for 10 days starting from day 0. The triptolide dosage (0.25 mg/kg/day) was selected based on a previous study that demonstrated its protective effect against renal disease in mice [20]. Mice were sacrificed on day 10. Mice were euthanized by cervical dislocation under anesthesia with 100 mg/kg pentobarbital sodium at the experimental endpoint for kidney collection.

Renal proximal tubular epithelial (HK2) cells were obtained from the Cell Bank of Shanghai Institute of Biological Sciences (Chinese Academy of Science, Cat. BFN60700259). The cell line used in this study was identified by short tandem repeat profiling and tested negative for mycoplasma. HK2 cells were seeded in 6-well plates to 40%–50% confluence and starved overnight with DMEM/F12 medium (GNM12400, GENOM, China) containing 0.5% fetal bovine serum. The following day, the medium was replaced with fresh medium containing 0.5% fetal bovine serum (04-001-1ACS, Biological Industries, Israel), and the cells were exposed to 2.5 ng/mL TGF-$\beta$ (Peprotech, Rocky Hill, NJ, USA) for 48 h in the presence of different concentrations of triptolide. The triptolide concentration range (10–100 nM) was determined based on a previous study reporting a protective effect of triptolide on renal cells within this range [21]. In specific experiments, cells were treated with either 1 mM PCK1 inhibitor 3-mercaptopicolinic acid (3MPA, T12929, TargetMol, Shanghai, China) or 20 µM PGC1$\alpha$ inhibitor SR-18292 (T4353, TargetMol, Shanghai, China).

Mouse kidneys were sliced, fixed, and embedded in paraffin, and cut into 4 µm-thick sections. Paraffin-embedded kidney sections were stained with hematoxylin, followed by ponceau red liquid dye acid complex, and then incubated in phosphomolybdic acid solution. Finally, the tissues were stained with aniline blue liquid and acetic acid. Images were acquired using a microscope (Nikon 80i, Tokyo, Japan).

Cell or kidney proteins were extracted using lysis buffer (P0013; Beyotime Biotech, Nantong, Jiangsu, China). The BCA Protein Assay Kit (P0012S, Beyotime Biotech, Nantong, Jiangsu, China) was used to quantify the protein concentration. Protein samples were dissolved in 5$\times$ SDS-PAGE loading buffer (P0015L; Beyotime Biotech, Nantong, Jiangsu, China) and subjected to SDS-PAGE. After electrophoresis, the proteins were electro-transferred to a polyvinylidene difluoride (PVDF) membrane (ISEQ00010; Merck Millipore, Darmstadt, Germany). Unspecific binding on the PVDF membrane was blocked by incubating with the blocking buffer (5% non-fat milk, 20 mM Tris-HCl, 150 mM NaCl, pH = 8.0, 0.01% Tween 20) for 1 h at room temperature, followed by incubation with primary antibodies, including anti-fibronectin (1:1000, ab23750, Abcam), anti-collagen-I (1:500, sc-293182, Santa Cruz), anti-$\alpha$-smooth muscle actin ($\alpha$-SMA) (1:1000, ET1607-53, HUABIO), anti-$\beta$-actin (1:50,000, 66009-1-Ig, proteintech), anti-N-cadherin (1:1000, sc-59887, Santa Cruz), anti-vimentin (1:1000, R1308-6, HUABIO), anti-Snail (1:1000, A11794, ABclonal), anti-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:5000, 60004-1-lg, Proteintech), PCK1 (A2036, Abclonal, Port Talbot, UK, 1:1000), FBP1 (1:1000, 14405406, Raybiotech), PGC1$\alpha$ (1:1000, 144-62296-100, Raybiotech), and FOXO1 (1:1000, 144-60502-200, Raybiotech) overnight at 4 °C. Binding of the primary antibody was detected using an enhanced chemiluminescence method (SuperSignal™ West Femto, 34094, Thermo Fisher Scientific) with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG, 1:1000, A0208, Beyotime or goat anti-mouse IgG, 1:1000, A0216, Beyotime).

The renal cortex was collected for protein extraction. Protein samples were isolated using 10$\times$ normal saline via mechanical disruption. Lactate content was measured using a lactate test kit (A019-2-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) by measuring the optical density (OD) at 530 nm. Renal glucose concentration was determined by measuring OD at 505 nm using a glucose assay kit (F006-1-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). All measurements were performed according to the manufacturer’s instructions and standardized by protein concentration.

The supernatant was collected from HK2 cells and centrifuged to remove cell debris. Glucose and lactate concentrations were determined using commercial kits, as described previously.

Paraffin-embedded tissue sections were dewaxed and immersed in a retrieval vessel containing citrate-based antigen retrieval buffer (pH 6.0, RC03; Shanghai Huilan Biotech) for high-pressure antigen retrieval. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. The sections were incubated overnight at 4 °C with primary antibodies: Histone H4 (Lys12) (PTM-1411RM, PTM bio, Hangzhou, Zhejiang, China, 1:100), F4/80 (HA721745, HUABIO, Hangzhou, Zhejiang, China, 1:100), or interleukin-1 beta (IL-1$\beta$; A1112, ABclonal, Wuhan, Hubei, China, 1:100). Subsequently, the sections were incubated with a secondary antibody (sheep anti-rabbit HRP, RCA054/RC0080RM, Shanghai Huilan Biotech) at 37 °C for 30 min, followed by 3,3^′-diaminobenzidine (DAB) color development (RCD002; Shanghai Huilan Biotech) for nuclear staining. Kidney sections were counterstained with hematoxylin, dehydrated sequentially, mounted with neutral gum, and examined under a microscope. Images were acquired using a scanner system (KF-PRO-005; KFBIO Technology Co., Ltd., Ningbo, Zhejiang, China).

Non-targeted metabolomics analysis was performed by Beijing Biomarker Technologies Co., Ltd. (Beijing, China) on six mouse kidney samples using LC-QTOF. Metabolites were extracted and analyzed, and the data were processed using Progenesis QI for peak alignment and identification against METLIN, HMDB, LIPID MAPS, and in-house databases. The quality control sample reproducibility was high (correlation $>$0.8). PCA and OPLS-DA were used to assess group separation and identify differential metabolites (FC $\ge$1, VIP $\ge$1, p 0.05). Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis was performed to identify perturbed pathways. All analyses were performed using the BMKCloud platform.

The results are presented as mean $\pm$ standard deviation (SD). Differences among multiple groups were analyzed using one-way analysis of variance (ANOVA), and comparisons between two groups were performed using the unpaired Student’s t-test using GraphPad Prism (Version 8, GraphPad Software, San Diego, CA, USA). Statistical significance was set at p 0.05.

The effect of triptolide on renal tubulointerstitial fibrosis was assessed using a UUO mouse model. Masson staining revealed extensive interstitial collagen deposition in the kidneys of UUO mice at 10 days postoperation. Notably, triptolide therapy significantly reduced collagen deposition (Fig. 1A,B). Western blotting analysis further demonstrated that ECM markers, including collagen-I and fibronectin, were upregulated in UUO kidneys, whereas triptolide treatment significantly decreased their levels (Fig. 1C,D). Importantly, triptolide treatment did not significantly affect the expression of these ECM markers in Sham-operated kidneys (Fig. 1C,D).

Fig. 1.

Triptolide inhibits tubulointerstitial fibrosis in obstructive mouse kidneys and renal cells. Sham (-) or UUO (+) operation was performed on wild-type C57BL/6 mice, followed by 10 days of treatment with DMSO or triptolide. (A,B) Renal fibrosis was assessed using Masson’s trichrome staining and quantified. Scale bar = 100 µm. (C,D) Expression levels of fibronectin and collagen-I were analyzed using Western blotting and quantified. Data are presented as mean $\pm$ SD. (E,F) Expression levels of vimentin, $\alpha$-SMA, and Snail were analyzed using Western blotting and quantified. Representative results of at least three independent experiments are presented. ns indicates not significant. *p 0.05. **p 0.01. ***p 0.001. Differences among multiple groups were analyzed using one-way ANOVA, and comparisons between two groups were performed using an unpaired Student’s t-test. UUO, unilateral ureteral obstruction; $\alpha$-SMA, $\alpha$-smooth muscle actin; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.

Furthermore, we evaluated the effect of triptolide on EMT in the fibrotic kidneys. The expression levels of three key EMT markers, vimentin, $\alpha$-smooth muscle actin ($\alpha$-SMA), and Snail, were elevated in UUO kidneys, and triptolide treatment significantly reversed this upregulation (Fig. 1E,F).

Collectively, these findings demonstrate that triptolide exhibits anti-fibrotic effects in mice with obstructive kidney injury.

To explore the mechanism underlying the renoprotective effects of triptolide in the fibrotic kidneys, we performed untargeted metabolomics analysis. A total of 260 differentially expressed metabolites (comprising 85 upregulated and 175 downregulated) were identified between the kidneys of UUO mice treated with vehicle and those treated with triptolide (Fig. 2A). KEGG enrichment analysis revealed that glucose metabolism, including gluconeogenesis and glycolysis, was among the most significantly dysregulated pathways affected by triptolide (Fig. 2B).

Fig. 2.

Renal gluconeogenesis is promoted by triptolide in fibrotic kidneys. (A) Volcano plots of differentially expressed metabolites in mouse kidneys between UUO/DMSO and UUO/triptolide groups based on untargeted metabolomics analysis. (B) KEGG pathway enrichment analysis of triptolide-targeted metabolites by untargeted metabolomics. (C,D) Expression levels of PCK1 and FBP1 in Sham (-) or UUO (+) kidneys were analyzed using Western blotting and quantified. (E) Lactate levels in whole kidney samples were determined. Data from at least three independent experiments are presented as one sample. Data are presented as mean $\pm$ SD. ns represents not significant. *p 0.05. **p 0.01. ***p 0.001. Differences among multiple groups were assessed using one-way ANOVA, and comparison between two groups was performed using an unpaired Student’s t-test. UUO, unilateral ureteral obstruction; PCK1, phosphoenolpyruvate carboxykinase 1; FBP1, fructose-1,6-bisphosphatase 1.

Subsequently, we validated the effects of triptolide on renal gluconeogenesis in fibrotic kidneys. The expression levels of PCK1 and FBP1, two rate-limiting enzymes involved in renal gluconeogenesis, were significantly downregulated in UUO kidneys compared to those in Sham-operated kidneys (Fig. 2C,D). Notably, triptolide treatment significantly increased the expression of these two enzymes in UUO kidneys; however, it failed to fully restore PCK1 and FBP1 expression in UUO kidneys to the levels observed in Sham-operated kidneys (Fig. 2C,D). Importantly, triptolide exhibited no significant effect on the expression of these two enzymes in Sham-operated kidneys (Fig. 2C,D).

Furthermore, renal lactate concentration was higher in UUO kidneys than in Sham kidneys (Fig. 2E), and triptolide treatment reduced lactate levels in UUO kidneys (Fig. 2E). However, triptolide did not completely reverse the UUO-induced increase in renal lactate to the baseline levels of Sham-operated kidneys (Fig. 2E). No significant difference in renal glucose concentration was observed between Sham and UUO kidneys (data not presented).

Collectively, these findings demonstrate that triptolide promotes renal gluconeogenesis in the fibrotic kidneys.

The anti-fibrotic effect of triptolide was validated in vitro using TGF-$\beta$-stimulated HK2 cells. Triptolide inhibited the expression of fibronectin and collagen-I in TGF-$\beta$-stimulated HK2 cells in a dose-dependent manner at concentrations ranging from 10 to 100 nM. The maximum inhibitory effect on both proteins was observed at 100 nM (Fig. 3A,B). Similarly, triptolide decreased the expression of EMT markers (N-cadherin, vimentin, and Snail) in TGF-$\beta$-stimulated HK2 cells in a dose-dependent manner (10–100 nM), with the most significant inhibition observed at 100 nM (Fig. 3C,D).

Fig. 3.

Triptolide prevents fibrotic changes and promotes glucose metabolism in TGF-$\beta$-stimulated HK2 cells. HK2 cells were starved overnight, stimulated with 2.5 ng/mL TGF-$\beta$, and treated with different concentrations of triptolide for 48 h. Cell lysates were then extracted. (A,B) Expression levels of fibronectin and collagen-I were examined using Western blotting and quantified. (C,D) Expression levels of N-cadherin, vimentin, and Snail were assessed using Western blotting and quantified. (E,F) Glucose and lactate concentrations in the supernatant were measured. (G,H) PCK1 expression was analyzed using Western blotting and quantified. Data from at least three independent experiments are presented as one sample. Data are presented as mean $\pm$ SD. NS represents not significant. *p 0.05. **p 0.01. ***p 0.001. Differences among multiple groups were analyzed using one-way ANOVA, and comparison between two groups was performed using an unpaired Student’s t-test. TGF-$\beta$, Transforming growth factor beta; PCK1, phosphoenolpyruvate carboxykinase 1; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.

We further observed that TGF-$\beta$ stimulation decreased glucose levels and increased lactate levels in HK2 cells, whereas treatment with 100 nM triptolide reversed these metabolic changes (Fig. 3E,F). These changes in gluconeogenesis were associated with increased PCK1 expression in TGF-$\beta$-stimulated HK2 cells following triptolide therapy (Fig. 3G,H).

To determine whether the anti-fibrotic effects of triptolide depend on gluconeogenesis, we treated TGF-$\beta$-stimulated HK2 cells with triptolide, followed by 3MPA, a selective PCK1 inhibitor. 3MPA reversed the triptolide-induced increase in glucose production and decrease in lactate levels (Fig. 4A,B). Furthermore, 3MPA abrogated the triptolide-mediated decrease in the expression of fibrotic markers (fibronectin and vimentin) (Fig. 4C,D). These results suggest that triptolide inhibits renal fibrosis by promoting PCK1-dependent gluconeogenesis.

Fig. 4.

Triptolide inhibits renal fibrosis by promoting gluconeogenesis. (A,B) Glucose and lactate levels in HK2 cells treated with (+) or without (-) 1 mM PCK1 inhibitor 3MPA were measured. (C,D) Western blotting was performed to evaluate the expression of fibronectin and vimentin in HK2 cells treated with (+) or without (-) the PCK1 inhibitor 3MPA and triptolide. The results were quantified. Data are presented as mean $\pm$ SD. **p 0.01. ***p 0.001. Differences among multiple groups were analyzed using one-way ANOVA, and comparison between two groups was performed using an unpaired Student’s t-test. 3MPA, 3-Mercaptopicolinic acid; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.

We subsequently investigated the expression levels of PGC1$\alpha$ and FOXO1, two key regulators of gluconeogenesis, in fibrotic kidneys. UUO surgery significantly downregulated the expression of PGC1$\alpha$ and FOXO1 (Fig. 5A,B). Triptolide treatment slightly increased FOXO1 expression and significantly upregulated PGC1$\alpha$ expression in UUO kidneys (Fig. 5A,B). We further investigated the effect of triptolide on PGC1$\alpha$ expression in vitro. TGF-$\beta$-induced downregulation of PGC1$\alpha$ in HK2 cells was reversed by triptolide (Fig. 5C,D). In vitro experiments further confirmed that SR-18292 (a PGC1$\alpha$ inhibitor) reversed triptolide-induced increase in glucose concentration and decrease in lactate concentration in TGF-$\beta$-stimulated HK2 cells (Fig. 5E,F). Furthermore, SR-18292 suppressed the triptolide-mediated upregulation of PCK1 (Fig. 5G,H) and reversed the suppressive effect of triptolide on fibronectin and vimentin expression (Fig. 5I,J).

Fig. 5.

Triptolide promotes glucose metabolism through PGC1$\alpha$ in renal fibrosis. (A,B) Expression levels of PGC1$\alpha$ and FOXO1 in Sham (-) or UUO (+) kidneys were analyzed using Western blotting and quantified. (C,D) HK2 cells were stimulated with TGF-$\beta$ and treated with (+) or without (-) 100 nM of triptolide for 48 h. PGC1$\alpha$ expression was analyzed using Western blotting and quantified. (E,F) HK2 cells were starved overnight, stimulated with TGF-$\beta$, and treated with (+) or without (-) 20 µM of PGC1$\alpha$ inhibitor SR-18292 and 100 nM of triptolide for 48 h. Glucose and lactate concentrations in the supernatant were measured. (G,H) Western blotting analysis and quantification of PCK1 expression in HK2 cells treated with (+) or without (-) the PGC1$\alpha$ inhibitor SR-18292 and triptolide. (I,J) Western blotting was performed to analyze the expression of fibronectin and vimentin in HK2 cells treated with (+) or without (-) PGC1$\alpha$ inhibitor SR-18292 and triptolide. The results were subsequently quantified. Data from at least three independent experiments are represented as one sample. Data are presented as mean $\pm$ SD. *p 0.05. **p 0.01. ***p 0.001. Differences among multiple groups were analyzed using one-way ANOVA, and comparison between two groups was performed using an unpaired Student’s t-test. UUO, unilateral ureteral obstruction; TGF-$\beta$, transforming growth factor beta; FOXO1, Forkhead box protein O1; PGC-1$\alpha$, peroxisome proliferator-activated receptor-gamma co-activator 1 alpha; PCK1, phosphoenolpyruvate carboxykinase 1; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.

Collectively, these findings suggest that PGC1$\alpha$ mediates the pro-gluconeogenic effect of triptolide in fibrotic kidneys.

A recent study has demonstrated that lactate, previously considered only a metabolic waste product, promotes renal fibrosis by inducing H4K12 histone lactylation-mediated inflammatory responses in tubular cells [22]. Therefore, we investigated whether triptolide inhibits renal fibrosis by suppressing histone lactylation-mediated inflammation, a mechanism that lies further downstream of gluconeogenesis.

IHC staining revealed elevated H4K12 lactylation in the renal tubular cells of fibrotic kidneys (Fig. 6A,B). This elevation was associated with increased macrophage infiltration, as indicated by F4/80 staining (Fig. 6C,D), and upregulated the expression of inflammatory factors such as IL-1$\beta$ (Fig. 6E,F). Triptolide treatment reduced H4K12 lactylation levels and attenuated these inflammatory responses, as indicated by decreased F4/80 and IL-1$\beta$ staining (Fig. 6A–F).

Fig. 6.

Triptolide inhibits histone lactylation and inflammatory responses in UUO kidneys. (A,B) ICH analysis and quantification of H4K12 in sham (-) or UUO (+) mouse kidneys with (+) or without (-) triptolide treatment. Scale bar = 400 µm or 100 µm. (C,D) ICH analysis and quantification of F4/80 in the kidneys of triptolide-treated UUO mice. Scale bar = 400 µm or 100 µm. (E,F) ICH analysis and quantification of IL-1$\beta$ in the kidneys of triptolide-treated UUO mice. Scale bar = 400 µm or 100 µm. (G) Schematic summary of the molecular mechanism underlying triptolide-induced recovery of renal gluconeogenesis in CKD. “↑” stands for up-regulation/activation; “↓” stands for down-regulation/inactivation. The schematic diagram was generated with BioGDP.com. Data from at least three independent experiments are represented as one sample. Data are presented as mean $\pm$ SD. ***p 0.001. Differences among multiple groups were analyzed using one-way ANOVA, and comparison between two groups was performed using an unpaired Student’s t-test. UUO, unilateral ureteral obstruction; H4K12, histone H4 Lys12; IL-1$\beta$, interleukin-1 beta.

These findings imply that the anti-fibrotic effect of triptolide involves the suppression of histone lactylation, a pathway that contributes to inflammation in fibrotic kidneys.