Drug preparation and administration

A&P concentrate granules (30 g of Astragalus propinquus Schischkin, 10 g of Panax notoginseng, 30 g of Angelica sinensis, 30 g of Achyranthes bidentata, and 6 g of Rheum palmatum) were obtained from the Preparation Department of the Affiliated Traditional Chinese Medicine Hospital at Southwest Medical University. The drugs were dissolved in physiological saline and administered via oral gavage twice daily at a dose of 4.5 mg/kg per administration (calculated based on the conversion formula from experimental animals to human dosage). The control group received an equivalent volume of physiological saline at the same frequency via oral gavage. Different concentrations of A&P (5%, 10%, 20%, 25%, and 30% ) enriched serum were prepared using rats to facilitate in vitro cell experiments intervention.

Extraction of pTECs and establishment of Fibrosis Model in vitro

For this study, C57BL/6 male mice below 23 days of age were selected. The cortex and medulla of the kidneys were separated, and the cortex was finely minced to extract cells. The cell suspension was repeatedly pipetted through a pipette tip several times. A new culture dish with a 70 μm mesh sieve was used to filter the cells. The 70 μm sieve and kidney tissue fragments were repeatedly washed with a stopping solution to terminate digestion, followed by centrifugation of the filtrate. The supernatant was removed, and the cell pellet in the centrifuge tube was resuspended in a preheated basic culture medium. After 1–2 min of settling, the supernatant was discarded, and a fresh primary culture medium pre-equilibrated at 37 °C was added. The cells were placed in a 37 °C, 5% CO2 incubator, and the medium was changed after 72 h. Treatment was initiated when cell confluence reached approximately 80%. Subsequently, cells were harvested for flow cytometry analysis. Cells with Anti-Cytokeratin 18 > 90% and Anti-Podocin < 5% were considered qualified for experiments. The cell model group received an addition of 5 ng/ml TGF-β1 for model construction.

Modulation of LncRNA A33 expression in pTECs

The downregulation and overexpression of LncRNA A33 in primary tubular epithelial cells (pTECs) were achieved using Lipofectamine RNAiMAX reagent (Invitrogen, USA). siRNA-A33 was transfected into pTECs using the following sequence: Forward, 5’-GCGUAGCUUGCUCAUAUT-3’; Reverse, 5′-AUAUAGAGCAGAGUACCGCTT (Synthesized by Sangon Biotech, China). Additionally, the pcDNA3.1-A33 plasmid (constructed by FulenGen Corporation) was transfected into pTECs using Lipofectamine 3000 reagent to induce A33 overexpression.

Animal model establishment and grouping

A total of 30 male c57BL/6 mice at the age of 6 weeks were purchased from Chongqing Tengxin biology Co.,ltd. After 12 h of fasting and water deprivation, the mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg). The mice were subjected to ureteral ligation near the renal hilum, followed by a second ligation away from the renal hilum. The mice were randomly divided into the following groups (n = 10): Ctrl group (normal control), UUO group (model control), UUO with lncRNA A33 knockdown group (UUOsh), UUO with empty vector group (UUOep), and UUO with A&P group (UUO + L or H). Gene organ-targeting delivery of lncRNA A33 knockdown.

Gene organ-targeting delivery of lncRNA A33 knockdown: A 32G needle was used to inject 30 µg of lncRNA A33 shRNA knockdown plasmid into the renal pelvis. In the control group, an empty vector was injected. Subsequently, the kidney was clamped using a specialized electroporation instrument. The kidney was subjected to 100 V voltage, 50 ms pulses, for a total of 6 times. After 7 days, the mice were anesthetized with isofurane and sacrifcedand, the ligated kidneys were collected for further analysis. All animal experiments were conducted according to the guidelines of the Ethics Committee of Southwest Medical University (No 20211126-033). Animal care and procedures comply with the ethical guidelines issued by the International Scientifc Committee on Experimental Animals (ICLAS).

Pathological examination

HE staining: Deparaffinization was performed using xylene, followed by a series of ethanol washes until rehydration was achieved. The sections were stained with hematoxylin and eosin (HE), rinsed with tap water, differentiated with hydrochloric acid-ethanol solution, and counterstained with eosin for 2 min. After a final rinse with running water, the samples were dehydrated in ethanol and cleared with xylene before being mounted with a neutral mounting medium.

Masson’s staining: Deparaffinization was performed using xylene, followed by a series of ethanol washes until rehydration was achieved. The sections were then incubated with potassium dichromate overnight, rinsed with running water, stained with Weigert’s iron hematoxylin, differentiated with 1% hydrochloric acid alcohol, and reblued. They were then stained with ponceau acid fuchsin, differentiated with phosphomolybdic acid, rinsed with 1% acetic acid, dehydrated in ethanol, and cleared with xylene before being mounted with neutral resin. All sections were examined under the Leica DM4B upright digital research microscope (Leica, DMC6200, Germany).

Immunohistochemistry

Paraffin sections of kidney tissue were deparaffinized and rehydrated with water. Antigen retrieval was performed using a sodium citrate buffer, followed by cooling to room temperature and PBS washes. After treatment with hydrogen peroxide solution at room temperature and a subsequent PBS wash, the sections were blocked for 1 h. Primary antibodies (α-SMA, Fn, Collagen1, KIM1, etc.) were then added and incubated overnight at 4 °C. Counterstaining was performed with hematoxylin. Brown particles were observed under an optical microscope. A negative control was prepared by omitting the primary antibody treatment, and the positivity rate (PR) was calculated as the percentage of positive area or cells relative to the total area or cell count.

Real-time PCR

Reverse transcription reactions were performed using the cDNA Synthesis Kit (Vazyme) according to the manufacturer’s instructions. Expression levels of various RNA molecules (α-SMA, Fn, lncRNA A330074K22Rik, GPX4, COX2, etc.) in each group were determined using the protocols provided in the fluorescent real-time quantitative PCR reagent kit instructions.

Protein extraction and western blotting

Total protein from kidney tissue was extracted using a conventional method. The protein concentration was determined using the Bio-Rad DC protein assay reagent. An appropriate volume of protein samples was loaded onto an SDS-PAGE gel, followed by electrophoresis. Subsequently, the proteins were transferred to a membrane, blocked, and incubated with primary antibodies (α-SMA, Fn, KIM1, GPX4, COX2, etc.) overnight at 4 °C. After washing with TBST, the membrane was incubated with appropriate secondary antibodies. The protein bands were then visualized using an ECL detection system.Statistical analysis was performed with GraphPad Prism 9.0 software(GraphPad Software Inc CA, USA). The p value < 0.05 was statistically significant.