Male BKS db/db diabetic mice and age-matched db/m mice (Nanjing University Model Animal Research Center) were used in the study. The mice were 8 weeks old and housed in a specific pathogen-free (SPF) environment at our animal center. Diabetic db/db mice with random blood glucose levels exceeding 16.7 mmol/L were included in the observational studies. Subsequently, the mice were randomly assigned to three groups (n = 6 each): db/m group, db/db group, and db/db + KXC group. KXC, provided by Guangzhou Chen Li Ji Pharmaceutical Co. Ltd (Guangzhou, China), was dissolved in 0.5% carboxymethylcellulose sodium (CMC-Na) to increase its solubility before use. According to clinical practice and previous literature (18), the db/db + KXC group received intragastric administration of KXC (234 mg/kg/day). The db/m and db/db groups were administered the same volume of vehicle (0.5% CMC-Na) by gavage. Samples of spot urine were collected every 4 weeks during the treatment and stored at −80°C. After 8 weeks of treatment, fasting blood glucose (FBG) was measured in each group using a glucometer (LifeScan Milpitas, CA, United States) with blood samples obtained from the mouse tail vein. Subsequently, the mice were sacrificed, and blood samples, as well as kidney tissues, were harvested.

The Ethics Committee on Animal Experiments of Nanjing Drum Tower Hospital (Nanjing, China) approved all experimental protocols in accordance with the guidelines established by the Committee on Laboratory Animals.

Albumin and creatinine levels in urine were measured using the Albumin ELISA Kit (GeneTex) and QuantiChrom™ Creatinine Assay Kit (BioAssay Systems), respectively. Blood specimens were collected via cardiac puncture. The levels of serum creatinine (Scr), alanine transaminase (ALT), and aspartate transaminase (AST) were analyzed using an automatic biochemical analyzer (Hitachi).

The renal tissues were excised, fixed in 4% PFA and embedded in paraffin wax. Subsequently, renal tissues were sectioned into 4-μm slices. To assess glomerular matrix accumulation, slides were stained with periodic acid-Schiff (PAS). For immunohistochemical staining, slides were deparaffinized, and then blocked with 10% BAS at room temperature. Tissues were incubated overnight at 4°C with primary antibodies against WT1 (Abcam Cat# ab89901, 1:100), ZO-1 (GeneTex Cat# GTX636399, 1:100), Col1a1 (Santa Cruz Ca# sc-25,974, 1:100), β-catenin (Abcam Ca# ab32572, 1:100), and snail1 (Abcam Ca# ab53519; 1:100). The sections were then incubated with secondary antibodies for 1 h. Color development was achieved using diaminobenzidine (DAB). Histological changes in the tissue were examined using a light microscope (Nikon E800 microscope).

Transmission electron microscopy (TEM) was performed to examine the ultrastructural changes in podocytes. The renal cortex was sectioned into 1 mm³ pieces, which were then fixed in 3.75% glutaraldehyde for 4 h. Subsequently, the specimens were treated with 1% osmic acid for 2 h. After gradual dehydration of tissues in acetone and ethanol, the specimens were embedded in epoxy resin (SPI, Indianapolis, IN, United States). Ultrathin slices were stained with uranyl acetate and lead citrate. GBM and podocytes were examined using transmission electron microscopy (Hitachi 7,500, Tokyo, Japan). The thickness of the GBM and the width of the podocyte foot process were measured and calculated as previously described in the literature (19).

Cortical kidney specimens from mice were placed on ice and lysed using RIPA buffer. Protein concentrations were determined using the bicinchoninic acid protein assay kit (Pierce Thermo-Scientific, Rockford, IL). Samples with equal amounts of total protein (50 μg/mL) were separated by 12% SDS–PAGE under reducing conditions and subjected to Western blot (WB) analysis. The membranes were incubated overnight at 4°C with primary antibodies against WT1 (Abcam Cat# ab89901; 1:1000), ZO-1 (GeneTex Cat# GTX636399; 1:1000), Col1a1 (Cell Signaling Technology Cat# 72026; 1:2000), snail1 (Abcam Cat# ab53519; 1:1000), β-catenin (Abcam Ca# ab32572; 1:1000) and α-tubulin (Abcam ca# ab7291; 1:2000), Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h. Finally, the immunoreactive bands were visualized using an ECL kit (Amersham Biosciences, UK).

Total RNA from renal cortex tissues was isolated using TRIzol reagent (Invitrogen cat# 15596018). Subsequently, the extracted RNA was reverse-transcribed into cDNA using a commercial kit (Vazyme Cat# R111-02). Gene expression levels were detected by real-time qRT‒PCR (Vazyme Cat# Q141–02) and an ABI7300 Sequence Detection System (Applied Biosystems, CA). The relative amount of mRNA, normalized against β-actin mRNA, was calculated as 2^-∆∆CT. The primer sequences are listed in Table 1.

All data are presented as the means ± SDs, comparisons of continuous variables between two groups were tested by using Student’s t tests, and comparisons of continuous variables among multiple groups were tested by using one-way ANOVA. Statistical analysis was executed using SPSS software version 22.0, and p < 0.05 was defined as statistically significant.

We specifically chose 8-week-old db/db mice because they exhibit significant metabolic disturbances and increased albuminuria at this stage (20). Confirming our expectations, the db/db mice exhibited considerably higher levels of FBG and urinary UACR than the db/m group. During the 4-week treatment period, administration of KXC resulted in a marked reduction in UACR, and this therapeutic effect became more pronounced with extended treatment duration (Figure 1A). Interestingly, there were no noticeable differences in blood glucose levels between the KXC group and the untreated db/db group at the end of the treatment period (Figure 1B). Furthermore, we observed no remarkable variations in the levels of Scr (Figure 1C), ALT (Figure 1D), and AST (Figure 1E) between the KXC group and the untreated db/db group. These findings indicate that KXC administration effectively mitigated albuminuria in db/db mice with DKD, without affecting blood glucose levels or causing notable hepatotoxicity or renotoxicity.

Histological analysis of kidney sections stained with PAS revealed a remarkable expansion of the mesangial matrix in db/db mice compared to db/m mice, indicative of DKD. However, administration of KXC resulted in a marked amelioration of glomerular mesangial expansion, surpassing the observed levels in untreated db/db mice (Figures 2A, B). Furthermore, TEM examination of podocyte ultrastructure revealed notable thickening of the GBM and effacement of podocyte foot processes in db/db mice compared to db/m mice (Figures 2C, D). Interestingly, treatment with KXC prominently attenuated these histological changes, indicating its protective effects on podocyte structure and function. These findings hightlight the potential of KXC as a therapeutic intervention for DKD, specifically targeting mesangial matrix expansion and podocyte ultrastructural alterations.

Previous studies have consistently emphasized the pivotal role of podocyte EMT in the development of podocyte malfunction and proteinuria in DKD (21–26). To evaluate the effect of KXC on podocyte EMT, we utilized a comprehensive approach involving qPCR, WB analysis, and immunohistochemistry to assess the expression of key markers, including WT1, ZO-1, and Col1a1. Immunostaining revealed a significant decrease in the expression of WT1 and ZO-1 in db/db mice compared to db/m mice, indicating podocyte dysfunction in the diabetic state. Remarkably, KXC treatment led to a clear reversal of these decreases, suggesting its potential in restoring podocyte integrity and function. Moreover, we observed a notable increase in the expression of Col1a1 in db/db mice compared to db/m mice. However, this increase was significantly attenuated following KXC administration (Figures 3A–D). The immunohistochemical staining findings were further supported by WB and qPCR analyses, which consistently demonstrated alterations in the protein (Figures 3E–H) and mRNA (Figures 3I–K) expression levels of Col1a1, WT1, and ZO-1. Collectively, these results provide compelling evidence for the therapeutic potential of KXC in modulating podocyte EMT in DKD.

Previous studies have extensively demonstrated the pivotal role of β-catenin signaling activation in mediating podocyte EMT and proteinuria (27–30). To assess the impact of KXC on β-catenin signaling, we evaluated the expression levels of β-catenin and snail1. Western blot (WB) analysis revealed a significant increase in β-catenin and snail1 protein expression in db/db mice. Notably, following KXC treatment, we observed a marked decrease in the expression of these proteins (Figures 4D–F). Furthermore, immunohistochemical staining provided additional confirmation of our results, illustrating the changes in β-catenin and snail1 expression (Figures 4A–C). This comprehensive analysis supported the notion that KXC treatment exerts a beneficial effect on β-catenin signaling, which may contribute to the preservation of podocyte integrity and the amelioration of proteinuria in DKD.