YSPDP was purchased from Wuhan Union Hospital (Hubei Medicine Number Z20181033, production batch number: 20220504). Losartan potassium tablet, a commonly used renin-angiotensin system inhibitor for CKD, was chosen as the positive control and obtained from Merck Sharp & Dohme Limited (Hangzhou, China). Both YSPDP and Losartan were ground into powder and mixed with ddH2O sufficiently before gavage.

Metabolomics profiling was supported by Metware Biotechnology Co., Ltd. (Wuhan, China). Liquid nitrogen was used to freeze-dry and crush the YSPDP. Afterward, 20 mg of the lyophilized powder was reconstituted in A 400 μL solution (Methanol: Water = 7:3, V/V). After shaking and centrifugation, the supernatant was filtered for analysis on the UPLC-MS/MS system. A UPLC system (ExionLC AD) coupled with a quadrupole-time-of-flight mass spectrometer (TripleTOF 6,600, AB SCIEX) was used to characterize chemical components in YSPDP. Chromatographic and mass spectrometry acquisition conditions and related data acquisition instrument systems are shown in Supplementary File S1.

Animal studies were conducted following the National Institutes of Health (NIH) Guidelines for the Use and Care of Laboratory Animals and approved by Ethics Committee of Huazhong University of Science and Technology. 50 eight-week-old male Sprague-Dawley rats were purchased from Charles River (Beijing, China). The rats were kept in controlled conditions, with a temperature maintained at 23°C ± 2°C and humidity ranging from 30% to 70%, following a 12-hour dark/light cycle. They were provided ad libitum access to standard mouse chow and tap water. One week after purchase, animals were randomly assigned to 5 groups with 10 in each group: Sham; 5/6 SNx + Vehicle; 5/6 SNx + Losartan; 5/6 SNx + YSPDP-L; 5/6 SNx + YSPSP-H. The 5/6 SNx operation was performed as follows: amputation of the poles of the left kidney at week 1, followed at week 2 by uninephrectomy (Uni-Nx) of the remaining kidney. Sham operations were performed at the same time points. At 2 weeks after surgery, Rats in the Losartan group were gavaged losartan at a dose of 33.3 mg/kg/d for 10 weeks. Rats in the YSPDP-L and YSPDP-H groups were gavaged with YSPDP of 1.5 g/kg/d and 3 g/kg/d, Rats in the Sham and the 5/6 SNx + Vehicle groups were given an equal volume of ddH2O in the same way. 10 weeks after treatment, the animals were sacrificed and the blood, urine, and renal samples were collected for further analysis.

Rat blood samples were allowed to clot at room temperature for 2 h. Subsequently, both blood and urine samples underwent centrifugation at 3,000 rpm for 10 min to collect the supernatant. The serum and urine samples were thereafter stored at −80°C until further analysis. Serum levels of albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), Serum creatinine (Scr), blood urea nitrogen (BUN), and urine protein/creatinine were detected by Automatic Biochemistry Analyzer (Technicon, RA-1640, United States).

Kidney tissues underwent fixation in 4% paraformaldehyde, followed by embedding in paraffin and sectioned at 4 μm thickness. Kidney sections were stained with Periodic acid-Schiff (PAS) and Masson’s trichrome as per the manufacturer’s instructions. Immunohistochemical staining involved overnight incubation at 4°C with primary antibodies, including anti-FN (F3648, Sigma-Aldrich, United States) and anti-Col-III (22734-1-AP, Proteintech). Subsequently, the sections were treated with biotinylated secondary antibodies for 1 h at 37°C, following standard protocols. Post-staining with 3,3′-Diaminobenzidine and counterstaining with hematoxylin, the density of positively stained areas was determined using Image-Pro Plus software.

Total tissue proteins were extracted using RIPA buffer (Beyotime Biotechnology, Shanghai, China). Proteins were separated using SDS-PAGE and transferred to PVDF membranes (Merck Millipore, MA, United States). The membranes were blocked with 5% BSA for 1 h, incubated with primary antibody overnight at 4°C, then incubated with secondary antibodies for 1 h, finally detected using enhanced chemiluminescence solution. The western blot images were analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, United States). The following primary antibodies were used in this study: anti-FN (F3648, Sigma-Aldrich), anti-COl-I (14695-1-AP, Proteintech), anti-α-SMA (14395-1-AP, Proteintech), anti-mTOR (2983T, Cell Signaling Technology), anti-p-mTOR (5536T, Cell Signaling Technology), anti-p-PI3K (4228T, Cell Signaling Technology), anti-PI3K(20584-1-AP, Proteintech), anti-p-AKT (66,444-1- Ig, Proteintech), anti-AKT (9272T, Cell Signaling Technology), and anti-β-actin (20536-1-AP, Proteintech).

Kidney tissue from the Sham, 5/6 SNx + Vehicle, and 5/6 SNx + YSPDP-H groups was used for transcriptomics sequencing. Transcriptomics sequencing was performed by Metware Biotechnology Co., Ltd. (Wuhan, China). We utilized DESeq2 to identify differentially expressed genes, applying the criteria of |log2(FoldChange)| > 1 and padj<0.05. For differential gene enrichment analysis, GOSeq and KOBAS were employed, focusing on corrected p-values < 0.05 for both GO and KEGG enrichment analyses.

All data are presented as Mean ± SEM. Statistical analyses were performed using GraphPad Prism 10.0 software (La Jolla, United States). The unpaired t-test was used to compare the differences between the two groups. Statistical significance was defined as p < 0.05.

Metabolomics was used to identify the chemical components of YSPDP. The total ion flow profiles in negative and positive ion mode for identifying compounds in the alcohol extract of YSPDP were shown in Figures 1A, B. Twenty-four classes of chemicals were identified. Among them, amino acid and its metabolites were the most abundant chemical class in YSPDP, which contained 896 chemicals (Figures 1C, D). Further, we listed the top abundant 20 chemicals in YSPDP, which were shown in Table 1.

To explore the therapeutic mechanism of YSPDP in CKD, network pharmacology was employed to predict its potential targets. The primary herbal ingredients in YSPDP include Rheum officinale, Astragalus membranaceus, Bombyx batryticatus, and Hirudo. Based on a criterion requiring an OB according to Veber’s filter, a DL following Lipinski rule of 5, and a high GI absorption, 127 compounds were determined from YSPDP. Among them, 45 compounds were in Rheum officinale, 30 compounds were in Astragalus membranaceus, 18 compounds were in Bombyx batryticatus, and 25 compounds were in Hirudo. 538 potential targets of these chemicals were obtained using the Swiss Target Prediction website (Figure 2A). The 175 potential targets of YSPDP exhibited significant alterations in the kidney of CKD (Figure 2B). 175 intersection targets were input into STING to construct PPI networks (Figure 2C) depicting YSPDP’s interference with CKD-related target genes. Based on the degree value of nodes, we further selected the top ten targets as the hub genes, including VEGFA, SRC, PIK3CA, AKT1, EGFR, PIK3R1, HRAS, APP, HSP90AA1, and TNF. The top 25 targets in action frequency are shown in Figure 2D. The DAVID tool website were used to analyze GO function annotation and KEGG pathway enrichment. GO enrichment analysis indicated that the biological processes of YSPDP on CKD were related to the negative regulation of the apoptotic process, positive regulation of cell proliferation, response to hypoxia, positive regulation of MAP kinase activity, and positive regulation of cell migration (Figure 2E). KEGG enrichment analysis revealed that the pathways of YSPDP on CKD were predominantly involved in PI3K-AKT, Rap1, HIF-1, FoxO, ErbB and VEGF signaling pathway and focal adhesion (Figure 2F). Finallly, based on these signaling pathway, the network of “compounds-targets-pathways” was constructed to reveal the therapeutic targets and pharmacological mechanisms of YSPDP against CKD (Figure 2G).

To evaluate the effect of YSPDP on CKD, we created a murine model of 5/6 SNx and then treated rats with YSPDP for 10 weeks, starting at 2 weeks after 5/6 SNx. The flowchart of the study is illustrated in Figure 3A. 5/6 SNx caused a decrease in body weight and albumin (Alb), while losartan and YSPDP treatment led to restoration of body weight and Alb (Figures 3B, C). There were no significant differences in the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) of all rats (Figures 3D, E), indicating YSPDP has no side effects on liver function. As shown in Figures 3F–H, serum creatinine (Scr), blood urea nitrogen (BUN), and urine protein/creatinine levels were higher in the 5/6 SNx group than in the Sham group. Both Losartan and YSPDP significantly ameliorated Scr, BUN, and urine protein/creatinine. The efficacy of YSPDP in ameliorating the progression of CKD was comparable to that of losartan. Furthermore, there were no statistically significant differences observed between the YSPDP-high dose (YSPDP-H) and YDSPDP-low dose (YSPDP-L) treated groups. In conclusion, YSPDP effectively improved the renal function of 5/6 SNx-induced CKD rats.

To further evaluate the effect of YSPDP on renal function and structure, we examined renal pathology in these groups using Masson and PAS staining (Figure 4A). From the representative images of Masson staining, the 5/6 SNx group showed tubular degeneration, luminal dilation, and ECM deposition in the renal interstitium. The presence of glomerular sclerosis in the kidneys of the 5/6 SNx group was demonstrated through PAS staining. However, treatment with losartan, YSPDP-L, and YSPDP-H effectively mitigated tubular injury and glomerular sclerosis in 5/6 SNx rats (Figures 4B, C).

Immunohistochemical staining analysis showed that Fibronectin (FN) expression in the glomerulus in 5/6 SNx group was significantly increased, while losartan, YSPDP-L, and YSPDP-H treatment effectively decreased the glomerular deposition of FN in 5/6 SNx rats (Figure 5A). Immunohistochemical staining of Collagen III (COL-III) showed that renal tubulointerstitial COL-III expression in the 5/6 SNx group was significantly enhanced and residual renal tissue staining in the losartan, YSPDP-L, and YSPDP-H treated groups was reduced (Figure 5B). The quantitative analysis of the FN and COL-III staining intensity and area showed that the expression of FN and COL-III was decreased by losartan, YSPDP-L, and YSPDP-H treatment significantly (Figures 5C, D). Western blotting showed that FN, collagen I (COL-I) and α-SMA protein expression in the 5/6 SNx group was significantly increased and that their expression in the losartan, YSPDP-L, and YSPDP-H treated groups was decreased (Figures 5D–G).

To further analyze the underlying mechanism of YSPDP in CKD, the kidney tissues of 5/6 SNx rats treated with vehicle and YSPDP-H were performed with transcriptome sequencing. The OPLS-DA analyses showed the gene expression between the two groups was quite different from each other (Figure 6A). 851 differentially expressed genes (DEGs) were found in the YSPDP group, including 471 upregulated and 380 downregulated genes compared to the 5/6 SNx group (Figures 6B–D). The DEGs were subsequently analyzed using GO and KEGG databases. The enrichment analysis results revealed that YSPDP could regulate several molecular functions, such as collagen fibril organization, oxidative phosphorylation, and oxygen transport, and was involved in important biological processes, including extracellular matrix organization and inflammatory response (Figure 6E). KEGG pathway enrichment analysis further demonstrated that the DEGs were enriched in various inflammation- and apoptosis-related pathways including the PI3K-AKT signaling pathway (Figure 6F).

Based on the network pharmacology and Transcriptomics, we propose that the PI3K/AKT signaling pathway may serve as the primary mechanism by which YSPDP exerts its therapeutic effects in CKD. This pathway is crucial for regulating cell survival, proliferation, and apoptosis, all of which are critical processes in the progression of CKD. Moreover, activation of the PI3K/AKT pathway has been implicated in mediating renal inflammation and fibrosis, which are key features of CKD pathophysiology (Li et al., 2017; Zhang et al., 2021; Wang et al., 2024). To investigate this further, we examined the expression of key proteins in the PI3K/AKT pathway in 5/6 SNx rats. Compared to the sham group, the phosphorylation levels of PI3K, AKT, and mTOR were significantly increased following 5/6 SNx treatment. Importantly, the phosphorylation levels of PI3K, AKT, and mTOR induced by 5/6 SNx were significantly decreased by YSPDH treatment (Figures 7A– D). These results suggest that the therapeutic effects of YSPDP in CKD are closely associated with the modulation of the PI3K/AKT/mTOR pathway.