Eighteen healthy male SD rats with body weight (200 ± 20 g), purchased from Beijing Viton Lever Laboratory Animal Co. The animal production license number is SCXK (Jing) 2016–0006, and the animal quality certificate is No.110011200110536318. After 1 week of adaptive feeding, rats were randomly divided into sham group, unilateral ureteral obstruction (UUO) group, and UUO hirudin treatment group, with six rats in each group. Except for the sham group, the rats in all groups underwent left unilateral ureteral obstruction according to Martínez’s method (Martínez-Klimova et al., 2019). The rats were fasted the night before surgery and the left unilateral ureteral obstruction was performed under aseptic conditions. We first injected 10% chloral hydrate (3 ml/kg) intraperitoneally, and after the rats were anesthetized, they were fixed in prone position, and a longitudinal incision of about 1.5 cm in length was made on the left side of the spine, about 0.5 cm below the lumbar point of the ribs, and the left ureter were isolated, ligated and disconnected layer by layer. The rats in the sham group were treated as the UUO group except that they were not ligated and the ureter was not disconnected. The corresponding drug was given to each group of rats on the same day after the operation. The dose of the drug administered was determined according to the animal/human surface area method of conversion and combined with the results of the previous experiments (Yang K. et al., 2020). UUO hirudin treatment group with a dose of 40 IU/kg/d by tail vein injection, and the UUO group as well as Sham group with stroke-physiological saline solution (SPSS) by tail vein injection too. This study was approved by the Animal Protection and Utilization Committee of the first teaching hospital of Tianjin university of traditional Chinese medicine (Tianjin, China), in accordance with our institutional regulations.

Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (3 ml/kg) after 14 days of continuous tail vein administration. Subsequently, the rats were executed by cervical dislocation and the left kidney was removed. we divided the left kidney of each rat in each group into two parts along the coronal plane of the longitudinal axis of the kidney. A fresh half of the left kidney from all six rats in each group was fixed in 4% paraformaldehyde at room temperature for 24 h, while the other half of the left kidney from each group was rapidly frozen in liquid nitrogen and stored at −80°C until use.

After the rats were executed, the left side of the obstructed kidney tissue was left, and the kidney was incised along the sagittal plane and fixed in 10% neutral formalin solution for 24 h, followed by gradient dehydration, hyalinization, paraffin immersion, paraffin bedding, hematoxylin and eosin (HE) staining. Histological sections of the kidney were dewaxed and dehydrated, after that stained with an acidic complex of hematoxylin and Ponceau red liquid dye, followed by direct staining of the histological sections. Then soaked in 1% phosphomolybdic acid solution and stained directly with aniline blue solution and 1% acetic acid. The observed collagen is represented as a blue area for the degree of fibrosis, and the interstitial collagen deposition in Masson staining sections was observed under optical microscope.

Normal rat kidney epithelial cell line (NRK-52E) was purchased from Pronosai Life Sciences Co. (Wuhan, China). Cells were cultured in 90% high glucose DMEM (Thermo Fisher Science, Inc., United States) and 10% fetal bovine serum (FBS) at 37°C and 5% CO² concentration. Then, NRK-52E cells were treated with 5 ng/ml TGF-β1 for 48 h to establish an in vitro fibrosis model.

NRK-52E cells in excellent growth condition were prepared in cell suspension, and the cell density was adjusted to 8×10⁴ cells/mL at 100 μL per well and inoculated in 96-well plates. The cells were divided into control group and experimental group, and the experimental group was added with different concentrations (2.5, 5,7.5,10,12.5.15,17.5, and 20 IU/ml) of hirudin (patent no. ZL03113566.8,100 IU/vial, Canton Xike Kang Biotechnology Co. Ltd.,Guangxi, China), while the control group was added with fresh medium, and three replicate wells were set up for each group. After incubation for 24 and 48 h, 10 μL of CCK-8 reagent (Bioworld, BD0079-1) was added to each well and incubated for another 4 h. The 96-well plates were placed under an enzyme marker to determine the OD value at 450 nm, and the cell viability was calculated based on the OD value to determine the appropriate concentration of hirudin to continue the experiment. All samples were assayed at least three times.

Total protein was extracted from obstructed kidney tissues or cells using 2×SDS lysate, and the protein concentration was determined by BCA protein assay kit (Solarbio life sciences Co., Beijing, China); 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel was used for electrophoretic separation, and the separation was transferred to PVDF membrane (Pierce Corporation, United States). The PVDF membrane was immersed in QuickBlock™ Western blocking solution (Beyotime Biotechnology, P0252) for 30 min at room temperature then incubated overnight at 4°C with anti-FN(1:2000, Proteintech, 15613-1-AP), anti-Col1 (1:1,000,Abcam, ab184993), anti-Col3 (1:1,000,Abcam, ab7778), anti- E-Cad (1:5,000, Proteintech, 20874-1-AP) , anti- PI3K (1:1,000, CST, 4,249), anti- Akt (1:1,000, CST, 4,691), anti-p-Akt (1:2000, CST, 4,060), anti-mTOR (1:1,000, CST, 1983), anti-Beclin1 (1:2000, Bioworld, AP0768),anti-P62 (1:500, Wanleibio,WL02385)and anti-GADPH (1:10,000, Proteintech, 10494-1-AP) respectively. After washing the membrane 3 times with TBST, the membrane was incubated with HRP-conjugated AffiniPure goat anti-mouse IgG (1:10,000, Proteintech, SA00001-1) or anti-rabbit IgG (1:10,000, Proteintech, SA00001-2) for 1 h at room temperature and washed 3 times again with TBST. Lastly, the membranes were added with enhanced chemiluminescence system (ECL) detection kit (Boster, China), ImageJ software was used for grayscale analysis of the image bands, and GAPDH was used as an internal reference for semi-quantitative analysis of the target proteins.

Total real-time polymerase chain reaction RNA was extracted using Trizol reagent (Invitgen, Carlsad, CA, United States), and the concentration of RNA for each sample was measured using an ND5000 ultra-micro UV spectrophotometer and reverse transcribed using the All-in-One™ First-Strand cDNA Synthesis Kit (GeneCopoeia Cat. No. AORT-0050) for reverse transcription. Target genes were analyzed by RT-qPCR according to the manufacturer’s instructions (Applied Biosystems Inc., Foster City, CA, United States). GAPDH forward primer is 5′-TCC​TGC​ACC​ACC​AAC​TGC​TTA​G-3'; reverse primer is 5′-AGT​GGC​AGT​GAT​GGC​ATG​GAC​T-3'; FN primer forward is 5 ′-ATG​AGA​AGC​CTG​GAT​CCC​CT-3′ and the reverse is 5′-GGA​AGG​GTA​ACC​AGT​TGG​GG-3'; COL1 is forward:5′- GGG​CAA​CAG​CAG​ATT​CAC​CTA​CAC-3′ and reverse:5′-CAAGGAATGGCAGGCGAGATGG-3'; for COL3, forward:5′- AGT​GGC​CAT​AAT​GGG​GAA​CG-3′ and reverse:5′-CACCTTTGTCACCTCGTGGA-3'. GAPDH was used as an internal control. Expression differences were assessed by the 2^−ΔΔCT method.

Similar to HE staining, kidney tissues were fixed in paraffin and next cut into paraffin-embedded kidney sections using a microtome. The tissue sections were then dewaxed, rehydrated and subjected to antigen retrieval in 0.01 M citrate buffer (pH 6.0). After washing twice with PBS (0.01 M, pH 7.4), sections were prepared for blocking with primary antibodies against α-SMA (1:1,500, Proteintech, 14395-1-AP) and PI3K (1:300, Proteintech, 20584-1-AP) and incubated overnight, followed by biotin labeled secondary antibodies. Bound antibodies were visualized with diaminobenzidine (DAB) staining and imaged (magnification, × 200) using a light microscope (TE 2000, Nikon, Japan). Brown granule staining in the cytoplasm and/or nucleus represents positive expression of α-SMA and PI3K.

NRK-52E cells were crawled, then treated with TGF-β1 and hirudin for 24 h, fixed with 4% paraformaldehyde, followed by the addition of 0.1% Triton X-100 to break the membrane for 10 min, and blocked with 10% FBS to eliminate non-characteristic fluorescent color development. After treatment with rabbit anti-rat LC3 (1:300, Proteintech, 14600-1-AP) and Beclin-1 (1:100, Bioworld, AP0768), and DyLight 488/594-labeled secondary antibody, the nuclei were stained with DAPI and sealed with anti-quenching blocker, and finally placed under a fluorescent microscope and photographed. The nuclei were stained with DAPI and blocked with anti-quenching blocker. The expression levels of the corresponding proteins were expressed as the integrated absorbance values.

High quality RNA was used for library construction and high-throughput sequencing. The RNA sequencing libraries were performed using the NEBNext Ultra RNA Library Prep Kit according to the manufacturer’s protocol. The library was subsequently sequenced by CaptalBio technology Co. Ltd. (Beijing, China) on a Novaseq6000 platform (Illumina, Chicago, IL, United States). Transcriptome analysis was performed using the rat reference genome-based read mapping. Gene expression levels were estimated using Fragments Per kilobase of an exon model per Million mapped fragments (FPKM) values.

All experiments were performed at least three times. SPSS 26.0 and GraphPad prism8 software were used for statistical analysis of the data, and all data were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was used for comparison between groups. Tukey’s test was used to compare multiple methods. p < 0.05 was considered significant.

We initially investigated the effects of resveratrol on interstitial fibrosis in the kidneys of UUO models, which is a typical model of RIF (Song et al., 2014). There were no rats died during the experiment. The extent of kidney injury in the 14 days rats was evaluated by HE staining (Figure 1A). Briefly, the kidney tissue structure of the control group was normal. On the 14 days, the renal tubular structure of the UUO group rats was severely damaged, with massive necrosis of epithelial cells, atrophy and collapse of interstitium, thickening or disappearance of basement membrane, and obvious proliferation of renal interstitial fibrous tissue with a large number of monocytes and macrophages infiltration. The above pathological damage was significantly reduced after hirudin treatment compared with the UUO group. Masson trichrome staining was performed at 14 days to assess the extent of fibrosis in the rat kidney tissue (Figure 1B). No significant fibrosis was seen in the kidney tissues of the control rats. The model kidney disturbed cell arrangement, disrupted tubular structure and obvious proliferation of fibrous connective tissue; compared with the UUO group, the kidney fibrosis was reduced in the hirudin-treated group.

Next, we examined the protein levels of fibrosis markers in the kidney tissues of each group. The expression of proteins COL1, COL3, and FN, which are closely related to fibrosis formation, was upregulated in the UUO group compared with the Sham group, and the expression was reversed after hirudin treatment (Figures 2A,B). Based on the biological importance of α-SMA in mediating RIF, we next assessed the protein levels of α-SMA in rat kidney tissues by immunohistochemistry (IHC). Compared with the Sham group, α-SMA positive expression was increased in the UUO group rats and was downregulated by hirudin treatment (Figures 2C,D). The expression trends of COL1, COL3, and FN mRNA were further confirmed by RT-qPCR to be consistent with protein expression (Figure 2E), and thus the transcript levels were statistically significant between the groups.

We performed a comparative analysis of transcriptome analysis and gene expression between the Sham group, the UUO group and the UUO hirudin-treated group. In total, the RNA-Seq results included 30,408 differential expressed genes (DEGs, | log2FoldChange | ≥1). Among them, the Sham and UUO groups had 15,112 DEGs, while there were 15,296 DEGs between Sham and UUO hirudin-treated group. In which, 2013 genes were up-regulated and 2,255 genes were down-regulated in the UUO group compared to the Sham group (Figure 3A). 708 genes were changed, of which 419 genes were up-regulated and 289 genes were down-regulated in the UUO hirudin-treated group compared with the UUO group (Figure 3B). We then mapped the differentially expressed genes from the above two results to obtain 322 identical differential expressed genes (IDEs), which were the potential target genes for hirudin against kidney fibrosis (Figure 3C). Of these genes, 169 common genes were consistently upregulated and 153 common genes were consistently downregulated.

To further discover the potential functional pathways of hirudin for CKD treatment, we performed GSEA analysis on IDEs. A total of 115 signaling pathways were enriched, as shown in Figure 4A, AMPK, JAK-STAT and PI3K-Akt signaling pathways were the important signaling pathways enriched. PI3K/Akt signaling pathway ranking five was priorly chosen to be verified in the following experiments. To explore the biological functions of IDEs, we performed Gene Ontology (GO) pathway annotation, and the results showed that cellular amino acid metabolic process was the most significantly enriched pathway in biological processes (Figure 4B). For molecular functional enrichment analysis, IDEs were mainly enriched in coenzyme binding, pyridoxal phosphate binding and other pathways (Figure 4C). In addition, microbody is the most obvious pathway for cellular components (Figure 4D).

Based on the above results, PI3K, Akt, mTOR were selected as potential targets and their expressions were further examined by RT-qPCR. Figure 5E shows the mRNA expression trends of PI3K, p-Akt, and mTOR, and thus the transcript levels were statistically significant between the groups. In addition, using the same remaining samples for Western blot analysis, the expression of PI3K, p-Akt, and mTOR was significantly elevated in the UUO group of rats, whereas it was significantly suppressed in the UUO hirudin-treated group (Figures 5A–D). These results suggest that hirudin can inhibit the PI3K/Akt signaling pathway, which is involved in the construction of the UUO rats and hirudin treatment of RIF.

To avoid the potential toxic effects of hirudin on NRK-52E cells and to explore the effective and safe dose of hirudin, we treated NRK-52E cells at 0,2.5,5,7.5,10,12.5,15, 17.5 and 20 IU/ml concentrations of hirudin for 24 and 48 h, respectively. Hirudin was found to significantly reduce the increase in cell viability induced by 10 ng/ml TGF-β1, and this inhibition showed a dose-dependent effect. In addition, cell viability was significantly inhibited at hirudin concentrations >10 IU/ml for 24 h and at hirudin concentrations >5 IU/ml for 48 h (Figure 6A). Therefore, we concluded that intervention with hirudin had no significant inhibitory effect on the cell viability of NRK-52E cells at a concentration of 10 IU/ml for 24 h. Thereby, we used 10 IU/ml of hirudin in the following experiments. To assess whether hirudin has antifibrotic properties in vitro, we analyzed the expression of fibrosis-associated proteins, which are reliable markers of TGF-β1-induced fibrosis after activation. Treatment with TGF-β1 significantly induced the expression of COL1, FN but decreased E-Cad expression.

We observed that treatment of NRK-52E with 10 IU/ml of hirudin for 24 h had an inhibitory effect on COL1 and FN expression in TGF-β1-induced NRK-52E cells, while promoting E-Cad expression (Figures 6B,C). This result was consistent with the expression of COL1, FN, and E-Cad at the mRNA level (Figure 6D).

We further examined the effect of hirudin on regulating the PI3K/Akt pathway in NRK-52E. The expression of PI3K, p-Akt and mTOR was significantly upregulated after TGF-β1 treatment compared to the control group. However, hirudin treatment inhibited their increased expression (Figures 7A,B). Furthermore, we treated NRK-52E cells in the TGF-β1 group using the PI3K inhibitor wortmannin to investigate whether attenuating renal fibrosis in the TGF-β1 group was caused by inhibition of the PI3K/Akt pathway. As shown in Figures 7C,D, wortmannin treatment partially reversed the TGF-β1-induced increase in PI3K compared to cells in the TGF-β1 group. In addition, p-Akt and mTOR were correspondingly reduced by wortmannin treatment (p < 0.01). These data confirm that renal fibrosis is inhibited and restored by blocking the PI3K/Akt pathway.

Changes in autophagy are important for the physiological function of the kidney and the course of disease development (Kaushal et al., 2020; Tang et al., 2020). And PI3K/Akt signaling pathway is the classical pathway of autophagy (Reedquist et al., 2006; Jung et al., 2010). Therefore we then tested the effect of TGF-β1 on the autophagic capacity of NRK-52E cells. Western blotting results showed that the expression of P62 and Beclin-1 was increased in the TGF-β1 group compared with the Control group; the expression of P62 and Beclin-1 was significantly decreased in the TGF-β1+Hirudin group compared with the TGF-β1 group (Figures 7A,B). Immunofluorescence assay results showed that intracellular autophagy marker proteins LC3 and Beclin-1 were significantly increased in the TGF-β1 group compared with the Control group; intracellular LC3 and Beclin-1 were significantly decreased in the TGF-β1+Hirudin group compared with the TGF-β1 group (Figures 8, 9). In conclusion, hirudin could attenuate TGF-β1-induced autophagy impairment in NRK-52E cells.