Renal fibrosis biomarker and application thereof

Abstract

The invention discloses a renal fibrosis biomarker and application thereof, a novel circular RNAhsacirc0008925 is found in renal tubular cells of a renal fibrosis patient, the renal fibrosis is regulated by directly separating the renal tubular cells from urine, and the in-vitro experiment using an HK-2 cell line also proves that the hsacirc0008925 regulates the renal fibrosis; the Hsacirc0008925 is used for promoting the renal fibrosis by up-regulating the expression of the SRSF6 in vitro and in vivo; the targeted hsacirc0008925 / SRSF6 is a promising treatment method for treating the renal fibrosis, and the result of the targeted hsacirc0008925 / SRSF6 is used as a renal fibrosis biomarker.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical molecular biology, and particularly relates to a renal fibrosis biomarker and its application. Background Art

[0002] Chronic kidney disease (CKD) is a major global public health problem, and its prevalence is on the rise globally. The global prevalence of CKD is estimated to be 13.4%, while in China, this figure is approximately 10.8%. Renal fibrosis is a common pathological feature of CKD leading to end-stage renal disease (ESRD). Renal fibrosis is a complex pathological process involving multiple inflammatory molecules and proteins. The detailed mechanism of renal fibrosis remains to be elucidated. Circular RNAs (circRNAs), due to their closed-loop structure, exhibit greater stability than linear RNAs, enabling them to persist in various tissues and body fluids. The expression of circRNAs varies significantly in different disease states, and dysregulation is closely related to the pathogenesis of many diseases. Studies on circular RNAs in kidney diseases have revealed their potential role in regulating pathological processes such as kidney inflammation, apoptosis, and fibrosis. CircRNAs have been shown to be key factors in regulating renal fibrosis in vitro and in vivo.

[0003] Regarding the potential biological indicators of urinary exosomes in renal fibrosis, scholars at home and abroad have made some useful explorations. However, these indicators are only just starting. The pathogenesis of renal fibrosis is the final pathway of the combined action of multiple pathogenic factors. The presence of these factors in urinary exosomes, as well as their significance in disease diagnosis and prognosis evaluation, are still unclear and require a large number of experimental studies to clarify. Therefore, further in-depth research is needed on the biological properties of urinary exosomes and their relationship with the process of renal fibrosis.

[0004] SRSF6 plays an important role in all stages of tumor development and progression by controlling the splicing of oncogenes and tumor suppressor genes. SRSF6 is also involved in other biological processes such as transcriptional regulation and protein stability. Although the role of SRSF6 in various fibrotic diseases is gradually emerging, its specific role in renal fibrosis remains unclear. Summary of the Invention

[0005] The present invention provides a biomarker for diagnosing renal fibrosis, which is down-regulated in urinary exosomes of patients and can effectively classify and diagnose samples.

[0006] Use of a reagent for detecting the expression level of hsa_circ_0008925 in urinary exosomes in the preparation of a diagnostic reagent for renal fibrosis.

[0007] Application of a reagent for detecting the expression level of hsa_circ_0008925 in urinary exosomes in the preparation of a staging diagnostic tool for chronic kidney disease. The expression level of hsa_circ_0008925 in urinary exosomes is positively correlated with the degree of renal tissue fibrosis in chronic kidney disease. The relative expression of hsa_circ_0008925 derived from renal tubular epithelial cells in the moderate-to-severe fibrosis group is significantly higher than that in the non-fibrosis and mild fibrosis groups.

[0008] Hsa_circ_0008925 is involved in the regulation of renal fibrosis through the SRSF6 pathway.

[0009] Hsa_circ_0008925 promotes renal fibrosis by upregulating the expression of SRSF6 in vitro and / or in vivo.

[0010] Application of a reagent for detecting the protein expression level of SRSF6 in the preparation of a renal fibrosis diagnostic tool or in the preparation of a staging diagnostic tool for chronic kidney disease.

[0011] The diagnostic tool is a kit, a chip or a test strip.

[0012] In the said application, it further includes the step of detecting the expression level of hsa_circ_0008925 in urinary exosomes.

[0013] The said expression level refers to the expression level in urinary exosomes.

[0014] It further includes the step of amplifying the reverse transcription product of hsa_circ_0008925 with forward and reverse primers.

[0015] It further includes the step of amplifying the internal reference gene with forward and reverse primers.

[0016] The internal reference gene is U6.

[0017] It further includes the step of extracting circRNA from urinary exosomes.

[0018] A probe for detecting Has_circ_0008925, and the probe is any one of the following:

[0019] Has_circ:0008925-1:5-AGACATATTTTCCCTGATTTTA-3;

[0020] Has_circ_0008925-2:5-TTTTCCCTGTGATTTTATATCCA-3;

[0021] Has_circ_0008925-3:5-GCAAGCAGAAACTATTTTCGTG-3.

[0022] In this invention, through in vivo and in vitro studies, we investigated the correlation between hsa_circ_0008925 derived from renal tubular cells in urine and renal fibrosis, and explored its mechanism of action in renal fibrosis.

[0023] In the UUO model, silencing mmu_circ_0002215 and inhibiting SRSF6 can alleviate renal fibrosis.

[0024] We performed an RNA pull-down experiment to identify the proteins interacting with hsa_circ_0008925. Mass spectrometry analysis showed that SRSF6 is the target protein of hsa_circ_0008925.

[0025] Silencing mmu_circ_0002215 and inhibiting SRSF6 can reduce the expression of renal fibrosis marker proteins.

[0026] A drug for treating renal fibrosis, the drug targets hsa_circ_0008925 / SRSF6.

[0027] This invention mainly studied the pro-fibrotic effect of hsa_circ_0008925, and investigated the potential mechanism of regulating renal fibrosis through cell and animal experiments. We found that the expression of hsa_circ_0008925 increased in renal cells derived from renal tubular epithelial cells of patients with renal fibrosis. Hsa_circ_0008925 promotes renal fibrosis by upregulating the expression of SRSF6 in vitro and in vivo. These findings suggest that targeting hsa_circ_0008925 / SRSF6 may be a promising treatment method for renal fibrosis.

[0028] The chromosomal location of hsa_circ_0008925 is chr6:108222573-108246136, and its gene name is SEC63. We found a new circular RNA in renal tubular cells of patients with renal fibrosis, which regulates renal fibrosis by directly isolating renal tubular cells from urine. In vitro experiments using the HK-2 cell line also confirmed that hsa_circ_0008925 regulates renal fibrosis.

[0029] Beneficial effects

[0030] The present invention discovered a new circular RNA in the renal tubular cells of patients with renal fibrosis, which regulated renal fibrosis by directly isolating renal tubular cells from urine. In vitro experiments using the HK-2 cell line also confirmed that hsa_circ_0008925 regulated renal fibrosis; Hsa_circ_0008925 promoted renal fibrosis by upregulating the expression of SRSF6 in vitro and in vivo; targeting hsa_circ_0008925 / SRSF6 is a promising treatment for renal fibrosis, and its results are used as biomarkers for renal fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Magnetic bead sorting of renal tubular epithelial cells, including: (A) representative flow cytometry images of sorted cells, (B) representative flow cytometry images of sorted cells CD11b-CD13+ The average percentage of cells (n=3), (C) Western blot results show that CD11b-CD13+ AQP-1 is expressed in cells, (D) Quantitative Western blot of AQP-1 (n=3). (*P<0.05; ****P<0.0001).

[0032] Figure 2 Clinical and pathological data of urinary tubular epithelial cells in CKD patients, including: (A) RT-qPCR analysis showed that the expression of hsa_circ_0008925 derived from tubular epithelial cells in the moderate to severe fibrosis group (n=8) was significantly upregulated compared with that in the non-mild fibrosis group (n=18); (B) Correlation analysis between has_circ_0008925 expression and Scr, (*P<0.05).

[0033] Figure 3 TGF-β1 induced an increase in the expression level of hsa_circ_0008925 in HK-2 cells, including: (A) RT-qPCR analysis showing the relative level of hsa_circ_0008925 expression in HK2 cells (n=4); (B) Western blot analysis of type I collagen and α-SMA protein levels in HK2 cells; (C) Quantitative protein blot of type I collagen and α-SMA (n=6); (D) Immunofluorescence images showing α-SMA and collagen in HK2 cells (bar=50μm); (**p<0.01; ***p<0.001; ****p<0.000).

[0034] Figure 4: Silencing of Hsa_circ_0008925 attenuates the expression of type I collagen, α-SMA, and SRSF6 in TGF-β1-induced HK-2 cells; (A) Western blot showed that transfection of siRNA into HK2 cells treated with TGF-β1 inhibited the protein expression of type I collagen, α-SMA, and SRSF6 by silencing hsa_circ_0008925; (B) Quantitative Western blot of type I collagen (n = 3); (C) Quantitative Western blot of α-SMA (n = 3); (D) Quantitative Western blot of SRSF6 (n = 3); (E) RT-qPCR analysis of the relative expression of SRSF6 mRNA in HK2 cells with silenced hsa_circ_0008925 (n = 6); (ns > 0.05; *p < 0.05; **p < 0.01, ***p < 0.001).

[0035] Figure 5 Silencing of Hsa_circ_0008925 attenuates the expression of type I collagen and α-SMA in TGF-β1-induced HK-2 cells; (A) After transfection of siRNA (bar = 50 μm), reduced α-SMA fluorescence was detected in hsa_circ_0008925-silenced HK2 cells by immunofluorescence confocal microscopy; (B) After transfection of siRNA (bar = 50 μm), reduced type I collagen fluorescence was detected in hsa_circ_0008925-silenced HK2 cells by immunofluorescence confocal microscopy.

[0036] Figure 6 Expression of renal fibrosis protein markers after silencing of hsa_circ_0008925 and inhibition of SRSF6; (A) Western blot showed that transfection of siRNA and addition of indacaterol into HK2 cells treated with TGF-β1 inhibited the protein expression of type I collagen, α-SMA, and SRSF6 by silencing hsa_circ_0008925; (B) Quantitative Western blot of type I collagen (n = 4); (C) Quantitative Western blot of α-SMA (n = 4); (D) Quantitative Western blot of SRSF6 (n = 4); (E) Immunofluorescence images showed α-SMA (bar = 50 μm) after transfection of siRNA and addition of indacaterol into HK2 cells treated with TGF-β1; (F) Immunofluorescence images showed type I collagen (bar = 50 μm) after transfection of siRNA and addition of indacaterol into HK2 cells treated with TGF-β1; (*p < 0.05; **p < 0.01; ***p < 0.001). Figure 7Silencing of mmu_circ_0002215 and inhibition of SRSF6 alleviated renal fibrosis in the UUO model; (A) Hematoxylin and eosin (H&E) and Masson-trichrom staining images of mouse kidneys (bar = 20 μm); (B) After transfection of shRNA and addition of indacaterol in mouse kidneys, protein expression of type I collagen, α-SMA, and SRSF6 was inhibited by silencing mmu_circ_0002215; (C) Quantitative western blot of type I collagen (n = 4); (D) Quantitative western blot of α-SMA (n = 4); (E) Quantitative western blot of SRSF6 (n = 4); (F) Representative images of α-SMA protein immunofluorescence in mouse kidneys (bar = 50 μm); (G) Representative images of type I collagen protein immunofluorescence in mouse kidneys (bar = 50 μm); (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

[0037] Figure 8 HK-2 cells were transfected with hsa_circ-0008925 siRNA, and RT-qPCR analysis showed that the siRNA transfected into HK2 cells decreased the relative expression level of hsa_circ_0008925 (n = 3, **p < 0.01; ***p < 0.001).

[0038] Figure 9 Western blot and quantification of SRS6 in TGF-β1-treated HK2 cells. (B) Quantitative western blot of SRSF6 (n = 6, ***p < 0.001)

[0039] Figure 10 (A) Confocal immunofluorescence showed that AAV could be successfully transfected into mouse kidneys by pyelonephritis injection. (B) RT-qPCR showed that all of these inhibited the expression of mmu_circ_0002215 in mouse kidneys, and the transfection efficiency of the AAV-mmu_circ_0001215 shRNA-1W knockout sequence was the highest (n = 3, *p < 0.05).

[0040] Figure 11 Correlation between hsa_circ_0008925 expression in renal tubular epithelial cells and renal function. Detailed implementation

[0041] Through in vivo and in vitro studies, the correlation between tubular cell-derived hsa_circ_0008925 in urine and renal fibrosis was investigated, and its mechanism of action in renal fibrosis was explored.

[0042] 1. Research population

[0043] This study was conducted in the Department of Nephrology, Yijishan Hospital, Wannan Medical College. A total of 26 CKD patients with different stages and pathological types underwent renal biopsy. The selection criteria for CKD patients were as follows: (1) aged between 18 and 80 years old, (2) with complete clinical case data, and (3) not receiving non-steroidal drugs and / or immunosuppressive therapy before biopsy. The exclusion criteria for CKD patients were as follows: (1) acute decline in renal function or history of acute and chronic urinary tract infections, (2) patients with chronic liver disease, cardiovascular disease, malignant tumors, or long-term chemotherapy, and (3) in need of kidney transplantation. All subjects signed the informed consent form. The Ethics Committee of Yijishan Hospital, Wannan Medical College approved this study (Approval number: 2022LS No.77).

[0044] 2. Collection of clinical and pathological data

[0045] Basic demographic and clinical biochemical data of CKD patients were collected, including age, gender, blood pressure, Scr, cystatin C, blood urea nitrogen (BUN), estimated glomerular filtration rate (eGFR), and 24-hour urinary protein. In addition, renal tissue pathological data were also obtained. Paraffin-embedded renal tissues were used to prepare 4-μm sections for Masson staining. Twenty non-overlapping areas per section were randomly selected and photographed at a magnification of 200 times. The tubulointerstitial fibrosis area was graded according to the percentage of the total tissue area: no fibrosis, mild fibrosis (fibrosis area ≤ 25%), and moderate to severe fibrosis (fibrosis area ≥ 26%). Glomerulosclerosis score was performed by calculating the mean ratio of the number of sclerotic glomeruli to the total number of glomeruli in different microscopic fields.

[0046] 3. Cell sorting by magnetic beads

[0047] Early morning urine samples (100 ml) were collected from the enrolled CKD patients. Urinary sediment cells were separated by centrifugation at 4°C and 3000×g for 30 minutes. According to the magnetic bead sorting protocol, the single-cell suspension was resuspended using 0.5% bovine serum albumin buffer (Bioproxx, Germany). PE-conjugated anti-human CD13 antibody (Miltenyi, Germany) was added and incubated in the dark at 4°C for 15 minutes to label the urinary sediment cells. Subsequently, PE-sorted magnetic beads (Miltenyi, Germany) were added and incubated for another 15 minutes. Then the resuspended single-cell suspension was added to the LD column (Milteniy, Germany) in the magnetic field of the MACS separator (Miltenui, Germany), and immunomagnetic bead sorting was performed. Cells were collected by the plunger CD13+ and the positively selected cells were rinsed with magnetic labels. Similarly, CD11b antibody and the corresponding anti-CD11b antibody (Miltenyi, Germany) were added respectively. Finally, CD13+CD11b cells were collected.

[0048] 4. Cell culture, siRNA transfection and treatment

[0049] HK-2 cells purchased from Procell (Pricela, China) were cultured in a humidified incubator at 37 °C and 5% CO2 in complete HK-2 specific medium (Dulbecco's modified Eagle's medium + 10% fetal bovine serum + 1% 100 U / mL penicillin and 100 μg / mL streptomycin; Pricela). According to the manufacturer's protocol, HK-2 cells were transfected with 50 nM hsa_circ_0008925 siRNA or 50 nM negative control siRNA (RiboBio, China) using Lipofectamine 2000 (SignaGen, USA). In addition, SRSF6 was inhibited using 20 μM indacterol (SRSF6 chemical inhibitor from Selleck Chemicals, USA). Then, the cells were cultured for 24 hours. Then, HK-2 cells ( 1×107 ) were exposed to 15 ng / mL recombinant TGF-β1 protein (Sinopharm) for 48 hours. Total RNA or protein was collected from the cells.

[0050] 5. RNA pull-down

[0051] Initially, the collected cells were fixed with 1% formaldehyde (Sigma, Germany) for 10 minutes. Subsequently, the supernatant was collected by centrifugation after adding the lysis mixture (Thermo Fisher Scientific, USA), and then the cells were sonicated until they were completely lysed and clarified. Subsequently, the probe was diluted to 100 μM and mixed with the protein-rich supernatant in an equal volume. Then, 100 μL of streptavidin magnetic beads (Thermo Fisher Scientific, USA) was added to capture the target protein, which was rotated and mixed thoroughly, and then incubated for 4 hours. Then, the magnetic beads and protein complexes were separated by magnetic adsorption (Beyotime, China), the supernatant was discarded, and the magnetic beads were washed to remove the unbound proteins. This washing step was repeated five times to ensure high purity. Next, 100 μL of loading buffer was added, and the mixture was incubated with shaking in a metal bath at 100 °C for 10 minutes. Finally, the supernatant was centrifuged and subjected to mass spectrometry analysis to identify the proteins bound to the probe. The RNA pull-down assay performed by Hibio in Hangzhou, China, detected the proteins bound to hsa_circ_0008925. The binding of the proteins was determined by Western blotting. The RNA pull-down assay performed by Hibio in Hangzhou, China, detected the proteins bound to hsa_circ_0008925. The binding of the proteins was determined by Western blotting. Has_circ_0008925 probe names: Has_circ:0008925-1:5-AGACATATTTTCCCTGATTTTA-3; Has_circ_0008925-2:5-TTTTCCCTGTGATTTTATATCCA-3;

[0052] Has_circ_0008925-3:5-GCAAGCAGAAACTATTTTCGTG-3.

[0053] 6. Animal models and adeno-associated virus (AAV) transfection

[0054] Animal experiments have been approved by the Experimental Animal Welfare and Ethics Committee of Wannan Medical College (Approval No.: LLSC-2022-142). According to previous research (Martínez Klimova E, Aparicio Trejo OE, Tapia E, Pedraza Chaverri J. Unilateral ureteral obstruction as a model for studying fibrosis-reducing therapies. Biomolecules. April 8, 2019; 9(4):141), a UUO model was established using male C57 mice (6-8 weeks old, 25-30 g). Briefly, male C57BL / 6J mice (weighing 20-25 g) were anesthetized intraperitoneally with pentobarbital (35 mg / kg). After exposure, the right ureter was ligated with 5-0 silk thread at two points. The mouse renal pelvis was injected with pAAV-CAG-mCherry-3Xflag-miR30shRNA(mmu_circ_0002215)-WPRE(AAV-mmu_circ_0001215shRNA) to inhibit the expression of mmu_cir_0002215, and the AAV negative control pAAV-CAG-mHerry-3Xfag-miR30s hRNA(NC)-WDRE(AAV-NC) (5×1010 viral genome copies, OBiO) was injected. Seven days later, the right ureter of the surviving mice was ligated to establish the UUO model. Indacaterol was injected intraperitoneally at a concentration of 2.5 mg / kg per day. Seven days later, the mice were euthanized, and the ligated kidneys were removed for subsequent experiments.

[0055] 7. Immunofluorescence staining

[0056] Paraffin sections (4 μm) of kidney tissues or cells containing tubules were fixed with 4% paraformaldehyde and permeabilized with 0.3-0.5% Triton X-100. Primary antibodies against type I collagen (diluted 1:100, Beyotime, China) and α-SMA (diluted 1:100, Proteintech, USA) were used for staining. The secondary antibody used was Alexa Fluor 594-conjugated anti-rabbit IgG (Invitrogen, Carlsbad). DAPI (Beyotime, China) was used to detect cell nuclei. Images were captured using a Leica TCS SP8 confocal laser scanning microscope.

[0057] 8. Renal histopathology

[0058] The kidneys of each mouse were collected after being sacrificed at week 1. Then, the kidneys were removed and wrapped in 4% paraformaldehyde overnight for tissue fixation. Subsequently, the tissues were dehydrated with gradient alcohol and embedded in paraffin. Sections with a thickness of 4 μm were cut from the renal tissue and stained with hematoxylin and eosin (H&E, Solarbio, China) to evaluate the morphological changes of the kidneys. Masson's trichrome staining (Solarbio, China) was used to quantify the degree of renal fibrosis.

[0059] 9. Real-time quantitative PCR (RT-qPCR)

[0060] According to the circBase database, the mouse homologous circRNA of human-derived hsa_circ_0008925 is mmu_circ_0002215. Total RNA was extracted using TRIzol LS reagent (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. cDNA was synthesized using a reverse transcription reagent (PrimeScript RT reagent kit, TaKaRa, Japan) and amplified by RT-qPCR (TBGreen Premix Ex Taq, TaKaRa, Japan) to determine the expression of hsa_circ_0008925, with U6 as an internal reference. The main primers used were as follows:

[0061] hsa_circ_0008925:

[0062] Forward primer: 5-TTATGGCTGTCCTGGGAGTT-3,

[0063] Reverse primer: 5-GGTATTCTCGGTCGTTTGGGAA-3.

[0064] mmu_circ_0002215:

[0065] Forward primer: 5-GGACATAAAATCAGGGAAAAT-3,

[0066] Reverse primer: 5-ACTTCACCTCCTCCTTATCTGG-3.

[0067] Human-SRSF6:

[0068] Forward primer: 5-GCAAGCCTCCACTCTTTC-3,

[0069] Reverse primer: 5-CAAGGTAGACAACCCGCCT-3.

[0070] Human-U6:

[0071] Forward primer: 5'-GCTTCGGCAGCACATACTAAAT-3'

[0072] Reverse primer: 5'-CGCTTCACGAATTTGCGTGTCAT-3'

[0073] Mouse-U6:

[0074] Forward primer: 5'-GCTTCGGCAGCACATACTAAAT-3'

[0075] Reverse primer: 5'-CGCTTCACGAATTTGCGTGTCAT-3'

[0076] 10. Western blot

[0077] Cells or tissues were lysed using a mixture of RIPA lysis buffer and phenylmethylsulfonyl fluoride at a ratio of 100:1. Next, a certain volume of loading buffer was added according to the sample density, and the samples were boiled in a metal bath for 10 minutes. Subsequently, the protein samples were electrophoresed and transferred to a PVDF membrane. Primary antibodies were added, including rabbit type I collagen (diluted 1:1000, Beyotime, China), α-SMA (diluted 1:1500, Proteintech, USA), and SRSF6 (diluted 1:11500, Proteintech, USA), and incubated overnight at 4°C. After several washes, the membrane was incubated with the secondary antibody (goat anti-rabbit IgG (H+L), diluted 1:40000; Beyotime, China) at room temperature for 2 hours. After washing again, the developer was applied to the membrane, and detection was performed using an automatic chemiluminescence image analysis system (Tanon 5200, China).

[0078] 11. Statistical analysis

[0079] The data were analyzed using SPSS 26.0 software. The results of RT-PCR were calculated using 2-ΔΔCt method. Normally distributed measurements were expressed as x±s, while non-normally distributed measurements were expressed as M (P25, P75). A t-test or rank sum test was used for inter-group comparison; categorical count data were expressed as the number of cases, and a χ2 test or Fisher's exact probability method was used for inter-group comparison. Spearman's correlation analysis was used for correlation analysis. A P value < 0.05 was considered statistically significant.

[0080] 2 Results

[0081] 2.1 Magnetic bead sorting of renal tubular epithelial cells

[0082] Renal tubular epithelial cells were isolated from human urine sediment using magnetic bead sorting, and the sorting results were confirmed by flow cytometry and Western blot analysis. Flow cytometry showed that the sorted cells exhibited CD13+ and CD11b - phenotypes ( Figure 1 A). After sorting, CD13+CD11b- the percentage of Figure 1 cells increased (36.7 ± 1.8% vs. 95.3 ± 1.6%, P < 0.0001; Figure 1 B). AQP-1 was used as a marker protein to identify renal tubular epithelial cells. Western blot results showed that AQP-1 was present in +CD11b- cells ( Figure 1 C and D).

[0083] 2.2 Clinical and pathological data of urinary renal tubular epithelial cells in patients with chronic kidney disease

[0084] CKD patients were divided into two groups according to the degree of renal fibrosis: non-fibrosis mild fibrosis group (fibrosis area ≤ 25%, n = 18) and moderate to severe fibrosis group (hepatic fibrosis area ≥ 26%, n = 8). There were no statistically significant differences between the two groups in terms of age, sex, systolic blood pressure or diastolic blood pressure (P > 0.05). The levels of Scr, BUN, cystatin C and 24-hour proteinuria in the moderate to severe fibrosis group were higher than those in the non-fibrosis mild fibrosis group, while the eGFR in the moderate to severe fibrosis group was lower (P < 0.001) (Table 1). The relative expression of hsa_circ_0008925 derived from renal tubular epithelial cells in the moderate to severe fibrosis group [(median expression 1.923 (1.417 - 2.482)] was significantly higher than that in the non-fibrosis mild fibrosis group [0.963 (0.664 - 1.460)] (P = 0.022) ( Figure 2 A). Correlation analysis showed that the expression level of hsa_circ_0008925 was positively correlated with the Scr level ( rs = 0.424, P = 0.031) ( Figure 2 B, Figure 11 ).

[0085] Table 1 Clinical data and pathological information of all subjects participating in the experiment

[0086]

[0087] Among them: Tubulointerstitial fibrosis grading [(-) no fibrosis; (+) mild fibrosis; (++) moderate fibrosis; (+++) severe fibrosis].

[0088] 2.3 TGF-β1 induces an increase in hsa_circ_0008925 expression in HK-2 cells

[0089] After treatment with 15 ng / ml TGF-β1 for 48 hours, circular RNAs and proteins were extracted for further analysis. RT-PCR showed an increased expression level of hsa_circ_0008925 in TGF-β1-treated cells ( Figure 3 of A). In addition, western blot analysis showed elevated levels of fibrosis-related proteins collagen type I and α-SMA after TGF-β1 administration ( Figure 3 of B and Figure 3 of C). Consistent with these findings, immunofluorescence results showed increased expression of collagen type I and α-SMA in TGF-β1-treated HK2 cells ( Figure 3 of D).

[0090] 2.4 Hsa_circ_0008925 silencing attenuates the expression of collagen type I and α-SMA in TGF-β1-induced HK-2 cells

[0091] To investigate the effect of hsa_circ_0008925 silencing on fibrosis, we transfected HK-2 cells with hsa_circ-0008925 siRNA. The effectiveness of hsa_circ:0008925 silencing was confirmed using RT-qPCR. Three siRNA sequences targeting different sites of hsa_circ_0008925 were tested. The most effective sequence CAGGGAAAATAGTTCTGCT ( Figure 8 ) was selected. Western blot ( Figure 4 of A, B and C) and immunofluorescence ( Figure 5 ) results showed that the fibrosis-related marker proteins α-SMA and collagen type I were significantly reduced after silencing hsa_circ_0008925 compared with the TGF-β1 group.

[0092] 2.5 RNA pull-down assay

[0093] We performed an RNA pull-down experiment to identify the proteins that interact with hsa_circ_0008925. Mass spectrometry (MS) analysis indicated that SRSF6 might be the target protein of hsa_circ_0008925, which was further confirmed by western blot ( Figure 9 ).

[0094] Differential protein expression analysis performed after TGF-β1 treatment mainly compared the treated samples with the control samples. Generally, protein species with higher abundance in the treated samples compared with the control samples were highlighted.

[0095] 2.6 Hsa_circ_0008925 silencing inhibits SRSF6 expression

[0096] After treating HK-2 cells with hsa_circ_0008925 siRNA, RT-qPCR ( Figure 4 of E) and Western blot ( Figure 4 of A) showed decreased levels of SRSF6 mRNA and protein.

[0097] 2.7 Expression of renal fibrosis protein markers after hsa_circ_0008925 silencing and SRSF6 inhibition

[0098] Treatment with hsa_circ_0008925 siRNA significantly decreased the expression of collagen type I and α-SMA. Administration of indacaterol not only affected the expression of SRSF6, but also inhibited the expression of collagen type I and α-SMA ( Figure 6 of A, B, C, and D). Immunofluorescence showed that after treatment with hsa_circ_0008925 siRNA and indacaterol, the expression of collagen type I ( Figure 6 of E) and α-SMA ( Figure 6 of F) in HK-2 cells was significantly decreased.

[0099] 2.8 In the UUO model, silencing mmu_circ_0002215 and inhibiting SRSF6 alleviate renal fibrosis

[0100] To further verify that hsa_circ_0008925 is involved in the regulation of renal fibrosis through the SRSF6 pathway, we performed in vivo experiments using a UUO mouse model. According to the circBase database, the mouse homologous circRNA of hsa_circ_0008925 is mmu_circ_0002215. Three AAVs targeting different sites of mmu_circ_0002215 were constructed. After injection of AAV, stable red fluorescence was shown by immunofluorescence in renal tissues. The most effective AAV silencer was selected ( Figure 10) After transfection with AAV-mmu_circ_0002215, a UUO model was established, and indacaterol was intraperitoneally injected at a dose of 2.5 mg / kg per day. After 7 days, the mice were euthanized and the kidneys were collected for analysis. HE staining showed that compared with the control group, the UUO and AAV-NC groups showed obvious inflammatory cell infiltration, atrophy of renal tubular epithelial cells, and lumen dilation in the renal compartment. In contrast, both AAV-mmu_circ_0002215 transfection and indacaterol supplementation could alleviate inflammatory kidney injury. Compared with the UUO group, Masson's trichrome staining showed that the UUO+AVA-mmu_circ_0002215 group, the UUO+indacaterol group, and the UUO+AAV-mmu_cir_0002215+indacaterol group had the least accumulation of blue collagen tissue in the perivascular renal tissue ( Figure 7 A).

[0101] 2.9 Silencing mmu_circ_0002215 and inhibiting SRSF6 can reduce the expression of renal fibrosis marker proteins Western blot analysis and tissue immunofluorescence staining showed that AAV-mmu_circ_0002215 transfection significantly reduced the expression of collagen type I, α-SMA, and SRSF6. In addition, the administration of indacaterol reversed the progression of renal fibrosis and effectively reduced the development of renal interstitial fibrosis ( Figure 7 B-E). Immunofluorescence showed that after AAV-mmu_circ_0002215 transfection and indacaterol administration, the expression of collagen type I and α-SMA in the renal tissue decreased ( Figure 7 F and G).

[0102] This invention focused on studying the pro-fibrotic effect of hsa_circ_0008925 and investigated the potential mechanism of regulating renal fibrosis through cell and animal experiments. We found that the expression of hsa_circ_0008925 increased in renal cells derived from renal tubular epithelial cells of patients with renal fibrosis. Hsa_circ_0008925 promoted renal fibrosis by upregulating the expression of SRSF6 in vitro and in vivo. These findings suggest that targeting hsa_circ_0008925 / SRSF6 may be a promising therapeutic approach for treating renal fibrosis.

[0103] Renal fibrosis is the key pathological process for CKD progression to ESRD. Early diagnosis is crucial for slowing down the progression of CKD. Urine, as a non-invasive source reflecting renal pathological changes, can serve as a biomarker for diagnosing and predicting kidney diseases. The biological information contained in urine can also reflect the pathological and physiological conditions of kidney diseases. Baer et al. (Zheng M, Lv LL, Baer PC, Nockher WA, Haase W, Scherberich JE. Isolation of human renal proximal and distal tubular cells by immunomagnetic separation. Technical note. Kidney International, 1997. 52(5):1321-31.) successfully purified CD13-labeled renal tubular epithelial cells from renal tissue using immunomagnetic bead technology. In the present invention, by sorting urine-derived renal tubular epithelial cells with immunomagnetic beads, we further found that the expression of hsa_circ_0008925 was upregulated in patients with renal fibrosis. These data indicate that hsa_circ_0008925 in renal tubular epithelial cells may be involved in the progression of renal fibrosis.

[0104] CircRNAs are a unique class of RNA molecules with stability and tissue-specific expression patterns due to their unique covalently closed-loop structure. The chromosomal location of hsa_circ_0008925 is chr6:10822253-108246136, and its gene name is SEC63 (Wang S, Zhang K, Tan S, Xin J, Yuan Q, Xu H, Xu X, Liang Q, Christiani DC, Wang M, Liu L, Du M. Circular RNAs in body fluids as cancer biomarkers: a new frontier of liquid biopsy. Cancers. January 11, 2021;20(1):13). No study has reported the relationship between hsa_circ_0008925 and renal fibrosis. We discovered a novel circular RNA in renal tubular cells of patients with renal fibrosis that regulates renal fibrosis by directly isolating renal tubular cells from urine. In vitro experiments using the HK-2 cell line also confirmed that hsa_circ_0008925 regulates renal fibrosis.

[0105] Our research results indicate that hsa_circ_0008925 may contribute to the development of renal fibrosis by interacting with the SRSF6 protein. SRSF6 is a member of the serine / arginine (SR)-rich protein family. In our study, we found that SRSF6 has a profibrotic effect in renal fibrosis, suggesting that it is involved not only in tumors but also in the progression of renal fibrosis. These findings provide new insights into the molecular mechanism of renal fibrosis and can serve as the basis for developing therapeutic approaches targeting SRSF6 for treating renal fibrosis.

Claims

1. Use of a reagent for detecting the expression level of hsa_circ_0008925 in urinary exosomes in the preparation of a diagnostic tool for renal fibrosis.

2. Use of a reagent for detecting the expression level of hsa_circ_0008925 in urinary exosomes in the preparation of a staging diagnostic tool for chronic kidney disease.

3. The application according to claim 1, wherein hsa_circ_0008925 is involved in the regulation of renal fibrosis through the SRSF6 pathway.

4. The application according to claim 3, characterized in that, Hsa_circ_0008925 promotes renal fibrosis by upregulating the expression of SRSF6 in vitro and / or in vivo.

5. Use of a reagent for detecting the protein expression level of SRSF6 in the preparation of a diagnostic tool for renal fibrosis or in the preparation of a staging diagnostic tool for chronic kidney disease.

6. Use of hsa_circ_0008925 in the preparation of a reagent for inhibiting the expression of the SRSF6 gene.

7. The application according to claim 1 or 2 or 5, characterized in that The diagnostic tool is a kit, a chip or a test strip.

8. A drug for treating renal fibrosis, characterized in that, The drug targets hsa_circ_0008925 / SRSF6.

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