Col4a5-R471X mice (Cat. NO. NM-KI-200183) were purchased from Shanghai Model Organisms Center, Inc (Shanghai, China). A total of 20 female mice were used in this study. This included 10 wild-type (WT) mice and 10 Col4a5 (X + X-) Alport syndrome (AS) mice on a similar genetic background (C57BL/6J). All mice were aged 4–6weeks at the start of the experiment.

The mice were randomly assigned into the following four groups (n = 5 per group): WT Group: Wild-type mice treated with normal saline. WT + M Group: Wild-type mice treated with Minnelide (Shanghai SCR-Biotech Co., ltd, Shanghai, China). AS Group: Alport syndrome mice treated with normal saline. AS + M Group: Alport syndrome mice treated with Minnelide. Mice in the WT + M and AS + M groups received intraperitoneal (i.p.) injections of Minnelide at a dose of 200 μg/kg, administered twice weekly (biw). Mice in the WT and AS control groups received i.p. injections of an equal volume of normal saline on the same schedule. The treatment period lasted for 3 months.

Adriamycin nephropathy (AN) mouse model was established in 6–8 weeks C57BL/6 female mice by tail vein injection of 25 mg/kg Adriamycin (ADR) (Sigma-Aldrich, St. Louis, MO, USA, D1515). The mice were divided into three groups, the Control group, the ADR group, and the ADR + Minnelide group, with five mice in each group. Mice in the Control group were injected into the same dose of saline intraperitoneally and in the tail vein, mice in the ADR group were injected into the tail vein with ADR and intraperitoneally with an equal dose of saline, and mice in the ADR + Minnelide group were injected intraperitoneally with 200 ug/kg/d of Minnelide in addition to the tail vein injection of ADR. After 1 week, the kidneys were removed and used for RAN-seq and WB experiments. The detailed experimental protocol for mice can be found in our group’s previous articles (Ji et al., 2023a; Ji et al., 2023b; Ji et al., 2023c).

All mice were housed under standard specific pathogen-free (SPF) conditions with a 12-h light/dark cycle and had free access to food and water. All mouse experiments were approved by the Ethics Committee of Fudan University (approval number: IDM2024056a).

Total RNA was extracted from kidney tissues or podocytes using the Total RNA Extractor (Trizol) kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s protocol. The renal tissue RNA-seq data analyzed in this study were generated from the same samples as described in our previous work (Ji et al., 2023a). While the sequencing data source is shared, the present analysis is novel and focused on a distinct set of genes relevant to glomerular basement membrane integrity in Alport syndrome (e.g., Col4a3, Col4a4, Col4a5, Lama4, Lama5, Lamb1, Lamb3, Hspg2). RNA integrity was evaluated by agarose gel electrophoresis, and RNA concentration and purity were measured using a NanoPhotometer spectrophotometer (IMPLEN, CA, USA) and a Qubit 2.0 Fluorometer (Invitrogen, Waltham, MA, USA).

Library preparation and sequencing: Sequencing libraries were prepared using the VAHTSTM mRNA-seq V2 Library Prep Kit for Illumina (Vazyme Biotech, Nanjing, China). Briefly, mRNA was enriched using poly-T oligo-attached magnetic beads, fragmented, and reverse-transcribed into cDNA. After adapter ligation and PCR amplification, libraries were purified and quantified using an Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). Paired-end sequencing was performed on an Illumina NovaSeq platform (Illumina, San Diego, CA, USA) with an average depth of 40 million reads per sample.

Data processing and alignment: Raw reads were quality-assessed using FastQC (version 0.11.2) and trimmed with Trimmomatic (version 0.36). Clean reads were aligned to the mouse reference genome (GRCm38/mm10) using HISAT2 (version 2.0). Alignment quality was evaluated with RSeQC (version 2.6.1) and Qualimap (version 2.2.1).

Differential expression analysis: Gene expression levels were quantified using StringTie (version 1.3.3b) and reported as TPM (Transcripts Per Million). Differential expression analysis was performed using DESeq2 (version 1.12.4). Genes with an absolute fold change ≥2 (Figure 4), ≥1.3 (Figure 5) and a q-value (FDR) ≤ 0.001 were considered significantly differentially expressed. Functional enrichment analyses of Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were conducted using a hypergeometric test, with q-value (FDR) < 0.05 considered significant.

Protein extraction and quantification: Proteins from kidney tissues and podocytes were extracted using RIPA lysis buffer (GB15544, Servicebio, Wuhan, China) supplemented with protease and phosphatase inhibitor cocktails. Protein concentration was determined using a BCA Protein Assay Kit (P0012, Beyotime, Shanghai, China) following the manufacturer’s instructions.

Electrophoresis and transfer: Equal amounts of protein (20 μg per lane) were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 120 V for 90 min and then transferred onto NC membranes (10,600,002, Amersham, USA) using a wet transfer system at 300 mA for 60 min.

Immunoblotting: Membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: GAPDH (GB15004, Servicebio, Wuhan, China); β-actin antibodies (GB12001, Servicebio, Wuhan, China); COL4A5 (ab231957; Abcam, Cambridge, UK; GTX56030; GeneTex, Irvine, CA, USA); PERK (GB11510, Servicebio, Wuhan, China); phospho-eIF2α (#9721; Cell Signaling Technology, Danvers, MA, USA); eIF2α (GB15544, Servicebio, Wuhan, China) and BiP (GB15098, Servicebio, Wuhan, China). After washing three times with TBST (10 min each), membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (GB23301, GB23303, Servicebio, Wuhan, China) for 1 h at room temperature.

Detection and quantification: Protein bands were visualized using enhanced chemiluminescence (ECL) substrate (G2020, Servicebio, Wuhan, China) and imaged with a Tanon 5,200 Multi Chemiluminescent Imaging System (Tanon Science and Technology, Shanghai, China). Band intensities were quantified using ImageJ software (National Institutes of Health, USA). Molecular weight markers were included in all gels for reference.

The paraffin sections of kidney tissue were deparaffinized and then antigenically repaired. The serum was closed to draw circles and titrated with primary antibodies for α-SMA (1:300, Servicebio, Wuhan, China, GB111364) incubated overnight at 4 °C. Secondary antibodies were added and incubated for 50 min at room temperature. The nuclei were stained with DAPI under a microscope (FV3000, Olympus) for observation.

Following deparaffinization, kidney sections were subjected to Masson’s trichrome staining using a commercial kit (G1006, Servicebio, Wuhan, China) according to the manufacturer’s instructions. Finally, the stained sections were mounted and imaged under an Olympus FV3000 microscope.

Primary podocytes (Col4a5+/−) were isolated from the kidneys of an Alport syndrome mouse model. For experiments, the cells were treated with Triptolide (TCI, Tokyo, Japan, T2899) at a concentration of 10 ng/mL for 30 min. Subsequently, the cells were harvested for Western blot analysis.

The MPC-5 was purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology CO., Ltd (Shanghai, China). Cells were cultured at 37 °C in a mixture of DMEM+10% FBS+1% P/S medium. Exposure to 0.4ug/ml ADR (Sigma-Aldrich, St. Louis, MO, USA, D1515) for 24 h was used to induce podocyte injury, and 10 ng/mL Triptolide was pretreated 30 min before the exposure to treat podocyte injury.

MPC-5 cells of four–eight generations were used, and when the cells reached 30% fusion, the serum-free medium was replaced and incubated for half an hour in serum diluted medium with Triptolide (10 ng/mL). For transfection studies, MPC-5 cells were transferred into six-well plates. The cells were then transfected using Lipofectamine 2000 transfection reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and four ul of si-Col4a5 mixture (Shanghai GenePharma Co., Ltd., Shanghai, China). Cells were cultured in an antibiotic-free medium with Lipofectamine and anti-si-Col4a5 complex for 6 h, then the original medium was replaced and the cells were cultured for another 48 h. At the end of the incubation, proteins were extracted according to the manufacturer’s instructions and used for subsequent WB experiments.

Data were analyzed using GraphPad Prism 9.0.0 (GraphPad Software, San Diego, CA, USA). All quantitative data are expressed as mean ± standard deviation (SD) unless otherwise noted in figure legends. Normality of data distribution was assessed using the Shapiro–Wilk test. Homogeneity of variances was verified with Brown–Forsythe or Bartlett’s test where appropriate.

Specific statistical tests applied were as follows:

For in vivo studies involving two independent variables (genotype and treatment): Data in Figures 1–3 were analyzed by two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test for multiple comparisons. The urinary albumin-to-creatinine ratio (UACR) presented in Figure 1A represents a single endpoint measurement after 3 months of treatment and was analyzed accordingly.

For in vitro studies with one independent variable across multiple groups: Data in Figures 4–6 were analyzed by one-way ANOVA followed by Tukey’s post-hoc test.

Statistical details for each figure panel (including the test used, number of replicates, and post-hoc correction) are provided in the respective figure legends. A p-value <0.05 was considered statistically significant.

To evaluate the therapeutic efficacy of Minnelide, we treated Col4a5 (X + X-) Alport syndrome (AS) mice and their wild-type (WT) with either Minnelide (200 μg/kg, biw) or vehicle for 3 months. As anticipated, vehicle-treated AS mice developed severe proteinuria, as evidenced by a significantly elevated urinary albumin-to-creatinine ratio (UACR) compared to WT controls (Figure 1A). Notably, Minnelide treatment markedly attenuated renal albumin leakage in AS mice, resulting in an approximately 64.2% reduction in UACR (Figure 1A). Due to the absence of significant renal function decline in this Alport mouse model at 4 months of age, parameters including albumin (ALB), blood urea nitrogen (UREA), and serum creatinine (CRE) showed no notable differences between the AS and AS + M groups (Figures 1B–D).

We next assessed the glomerular ultrastructural pathology by transmission electron microscopy (TEM). Representative images revealed the characteristic lesions of Alport syndrome in vehicle-treated AS mice, including profound thinning of the glomerular basement membrane (GBM) with interspersed irregular thickening and lamellation (Figures 1E,F, AS-1 and AS-2). In stark contrast, Minnelide-treated AS mice showed a considerable preservation of GBM architecture, with a more uniform thickness and a significant reduction in pathological alterations (Figures 1E,F, AS + M-1 and AS + M-2). Together, these data demonstrate that Minnelide treatment effectively mitigates functional renal impairment and structural podocyte injury in this murine model of Alport syndrome.

Further investigation revealed that Minnelide significantly increased the protein expression of Col4a5 in the renal tissue of Alport mice (Figures 2A,B). Consistent with this in vivo observation, triptolide also enhanced Col4a5 protein expression in Col4a5+/− podocytes in vitro (Figures 2C,D). These results collectively suggest that the therapeutic benefits of Minnelide, including the alleviation of proteinuria and structural improvements, may be mediated, at least in part, through the upregulation of Col4a5 expression. This restoration of the defective collagen IV α5 chain is a plausible mechanism underpinning the preservation of the glomerular basement membrane integrity and the amelioration of disease progression in Alport syndrome.

To elucidate the molecular mechanisms underlying the renoprotective effects of Minnelide, we first examined its impact on the key pathogenic defect in Alport syndrome–the deficiency of the collagen IV α5 chain (Col4a5) – and on the associated endoplasmic reticulum (ER) stress pathway in mouse kidney tissues. The expression of key ER stress markers, including the molecular chaperone BIP and the phosphorylated form of eukaryotic initiation factor 2α (p-eIF2α), was markedly elevated in the AS group, indicating activated ER stress. Minnelide treatment substantially attenuated this response, as demonstrated by the significant downregulation of both BIP and p-eIF2α (Figures 3A–C).

We next sought to determine whether the active moiety of Minnelide, triptolide, could exert a direct effect on Col4a5-deficient podocytes. We utilized a primary podocyte cell line derived from Col4a5+/− mice. Consistent with our in vivo findings, triptolide treatment effectively suppressed the baseline levels of ER stress and reduced the expression of p-eIF2α (Figures 3D,E).

Also, Minnelide treatment significantly attenuated renal fibrosis in Alport syndrome mice. Masson’s trichrome staining revealed elevated collagen deposition in AS mice, which was markedly reduced by Minnelide (Figure 3F). Consistently, immunofluorescence for α-smooth muscle actin (α-SMA) showed intensified staining in AS kidneys, indicating myofibroblast activation, and this was similarly suppressed with Minnelide therapy (Figure 3G). These results demonstrate the anti-fibrotic efficacy of Minnelide in this model.

In a previous study exploring the specific mechanism by which minnelide protects mice with AN (Ji et al., 2023a), RNA-seq found that in renal tissues of AN mice after minnelide treatment, a significant increase in the expression of Col4a3/4/5, Lama4, Lama5, Lamb1, Lamb3, and Hspg2 genes, which encode type IV collagen, Laminin, and Perlecan respectively, the main constitutive proteins of GBM (Figure 4A). It was surprising to note that the expression of these genes was upregulated 2-7-fold after minnelide intervention (Table 1). Next, we conducted the current study, and it was verified that WB illustrated a significant increase in Col4a5 protein expression after minnelide intervention (Figures 4B,C).

In renal tissues, podocytes are the sole cells that produce and secrete Col4a3/4/5 proteins. To verify whether triptolide (a metabolite of minnelide) still enhances the expression of Col4a3/4/5 genes in podocytes, we carried out an RNA-seq of podocytes.

The volcano plot shows that triptolide still enhances Col4a4/5 gene expression in podocytes, with a surprising 1.3 to 1.5-fold upregulation (Figures 5A,B; Table 2). Next, WB showed that protein expression of Col4a5 was significantly increased after treatment with triptolide (Figures 5C,D).

To further verify whether triptolide could still upregulate Col4a5 expression under si-RNA knockdown of the Col4a5 gene, we carried out relevant experiments. The Col4a5 protein expression was upregulated after triptolide treatment (Figures 6A,C). Similarly, ERS was induced to emerge after si-Col4a5 knockdown of Col4a5, and ERS was alleviated after triptolide treatment (Figures 6B–E).

Although siRNA knockdown reduced Col4a5 mRNA levels, Triptolide treatment partially restored Col4a5 protein expression, likely through a combination of modest transcriptional upregulation and enhanced translational efficiency or protein stability under reduced ER stress.

Given the documented time- and dose-dependent hepatotoxicity and reproductive toxicity associated with triptolide, we carefully evaluated the safety profile of Minnelide (200 μg/kg, biw). Histopathological examination of ovarian and liver tissues via H&E staining revealed no significant morphological alterations in the Minnelide-treated groups (WT + M) compared to their respective vehicle-treated controls (Figures 7A,B).