Exosomal lincRNA-p21/AIF-1 promoted inflammation of human tubular epithelial cell via CMPK2/NLRP3 pathway in urate nephropathy

doi:10.21203/rs.3.rs-3905410/v1

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Research Article

Exosomal lincRNA-p21/AIF-1 promoted inflammation of human tubular epithelial cell via CMPK2/NLRP3 pathway in urate nephropathy

https://doi.org/10.21203/rs.3.rs-3905410/v1

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Background and hypothesis

Urate nephropathy, a common complication of hyperuricemia, has garnered increasing attention worldwide. However, the exact pathogenesis of this condition remains unclear. Currently, inflammation is widely accepted as the key factor in urate nephropathy. Therefore, the aim of this study was to elucidate the mechanism of exosomal lincRNA-p21/AIF-1 in urate nephropathy.

Methods

This study evaluated the effects of exosomes using clinical data collected from patients with urate nephropathy and human renal tubular epithelial cells (HK2) cultured with different concentrations of urate.

Results

In the clinical research section, the level of exosomal lincRNA-p21/AIF-1 in the urine of patients with hyperuricemia or urate nephropathy was found to be increased, particularly in patients with urate nephropathy. In vitro study section, the levels of exosomes, inflammation, autophagy, and apoptosis were increased in HK2 cells induced by urate. Additionally, the expressions of lincRNA-p21, AIF-1, CMPK2, and NLRP3 were upregulated in exosomes and HK2 cells. Furthermore, manipulating the activity of lincRNA-p21, AIF-1, CMPK2, and NLRP3 through overexpression or interference vectors promoted inflammation, autophagy, and apoptosis in HK2 cells induced by urate.

Conclusions: In conclusion, the aforementioned results suggested that exosomal lincRNA-p21/AIF-1 induces inflammation via the CMPK2/NLRP3 pathway, thereby promoting autophagy and apoptosis in renal tubular epithelial cells induced by urate.

urate nephropathy

exosome

long intergenic noncoding RNA-p21

allograft inflammatory factor 1

inflammation

Increasing evidence has confirmed that hyperuricemia is a global metabolic disease and an independent risk factor for chronic kidney disease [1]. The accumulation of urate in the kidneys is known as urate nephropathy [2]. Several retrospective analyses have indicated that the risk of renal injury increases by 7–11%, and the risk of renal deterioration increases by 14% with every 1mg/dL increase in serum uric acid concentration [3, 4, 5]. However, the pathogenesis of urate nephropathy remains unclear. In recent decades, it has become evident that inflammation, which is not only crucial for fighting infections but also contributes to various non-communicable diseases, plays a central role in urate nephropathy [6, 7]. Studies using human kidney biopsy samples have demonstrated a positive association between serum uric acid concentration and inflammation and fibrosis in the tubulointerstitium [8, 9]. While inflammation is recognized as the fundamental pathological process in urate nephropathy, there have been no significant breakthroughs in understanding how the inflammatory signal is activated, regulated, and transmitted among different renal cells. In our preliminary experiment, we observed upregulated expression of lincRNA-p21, AIF-1, CMPK2, and NLRP3 in human renal tubular cells cultured with urate. Reviewing the literature, we found that these molecules are associated with inflammation. However, their exact roles in urate nephropathy remain unclear.

To begin with, long intergenic non-coding RNA p21 (lincRNA-p21) is a non-coding RNA located approximately 15KB upstream of the cell cycle regulation gene P21, with a length of approximately 3.0KB. It regulates processes such as proliferation, apoptosis, and DNA damage progression through RNA-protein interactions, "recruiting" or "guiding" protein complexes that bind to specific regulatory regions in the genome. Numerous studies have demonstrated the important role of lincRNA-p21 in chronic inflammatory diseases. For example, overexpression of lincRNA-p21 promotes hepatocyte apoptosis and fibrosis in hepatic tissues [10], as well as vascular smooth muscle cell apoptosis in thoracic aortic aneurysms [11]. Down-regulation of lincRNA-p21 has been shown to alleviate extracellular matrix induced by high glucose and plays an important role in diabetic nephropathy [12]. Deletion of lincRNA-p21 in renal tubular epithelial cells has been found to attenuate kidney injury caused by persistent obesity and hyperlipidemia through inflammation, apoptosis, and endoplasmic reticulum stress [13]. Spurlock et al. have confirmed that administration of methotrexate upregulates the expression of lincRNA-p21 and inhibits the activity of NF-κB [14]. These findings suggest a close relationship between lincRNA-p21 and inflammatory response, although its role in urate-related inflammation has not been reported. Therefore, it is necessary to clarify the effect of lincRNA-p21 in urate nephropathy.

On the other hand, allograft inflammatory factor 1 (AIF-1) is a calcium-binding cytoplasmic scaffold protein observed in several inflammatory diseases, including atherosclerosis, rheumatoid arthritis, renal tubulointerstitial fibrosis, vascular calcification, and diabetic kidney disease [15, 16, 17, 18, 19]. Our group has conducted a series of studies on AIF-1 and confirmed its role in promoting inflammation and fibrosis in the renal tubulointerstitium. However, the upstream and downstream regulatory mechanisms of AIF-1 are still unknown.

Furthermore, the inflammasome, an important component of inflammation [20], has been implicated in various diseases. Among the best-characterized inflammasomes, NLR family pyrin domain containing 3 (NLRP3) has been shown to activate inflammation in numerous diseases. NLRP3 is a cytoplasmic signal complex that activates inflammatory cytokines, serving as a major sensor of sterile inflammatory signaling and a key inducer of chronic inflammation [21]. Animal and clinical studies have demonstrated the association of NLRP3 with various inflammatory diseases, including NLRP3-dependent autoimmune diseases [22], metabolic disorders [23], diseases caused by crystal formation [24, 25], and acute tissue damage or fibrosis resulting from chronic inflammation. Crystal-induced chronic or acute inflammatory responses through the NLRP3 pathway have been implicated in various diseases, such as gout attacks triggered by urate crystals [26, 27], atherosclerosis caused by cholesterol crystals [28], and renal function decline associated with calcium oxalate crystals [29]. Additionally, it has recently been discovered that mitochondrial nucleotide phosphokinase 2 (CMPK2) is an upstream factor affecting NLRP3, predominantly located in cell mitochondria and closely related to NLRP3 activation [30]. Luo et al. proposed that CMPK2 is essential for mitochondrial DNA synthesis and NLRP3 activity, promoting a series of inflammatory responses in liver ischemia and reperfusion injury [31]. These findings suggest that the CMPK2/NLRP3 axis directly contributes to inflammation, although the mechanism of inflammatory signal transmission and activation needs to be elucidated. Moreover, exosomes, small vesicles measuring 30-100nm in diameter, produced and released by living cells into the extracellular matrix, play a crucial role in regulating cellular functions such as proliferation, differentiation, apoptosis, and migration through autocrine and paracrine mechanisms [32]. Currently, there is a lack of specific biomarkers and treatment strategies for urate nephropathy, making it difficult to detect and treat early. The discovery of extracellular vesicles holds promise for elucidating the pathogenesis of urate nephropathy and identifying new biomarkers.

Based on these observations, we hypothesized that lincRNA-p21, AIF-1, CMPK2, and NLRP3 were involved in the pathogenesis of inflammation in urate nephropathy. The aim of this study was to elucidate the mechanisms underlying the roles of lincRNA-p21, AIF-1, CMPK2, and NLRP3 via exosome pathway in urate nephropathy.

Patients

In accordance with the clinical study program approved by the ethics committee of Southern University of Science and Technology Hospital and the ethical standards of the 1964 Helsinki Declaration, 20 asymptomatic hyperuricemia patients who were treated in Southern University of Science and Technology Hospital during the period from January 1, 2022 to December 31, 2023 were selected. The patients were divided to group 1 (asymptomatic hyperuricemia with renal injury) and group 2 (asymptomatic hyperuricemia only). The inclusion criteria of group 1 were used as follows: ⑴ age between 18 and 40 years old; ⑵ serum uric acid ≥ 6 mg/dl; ⑶ with markers of tubulointerstitial lesion (KIM-1, NAG enzyme and β₂-microglobulin in urine) above normal values; ⑷ serum creatinine ≥ 1.1 mg/dl, eGFR ≤ 90 ml/min, serum cystatin C ≤ 1.07 mg/l, uACR ≤ 30 mg/g; ⑸ without any glomerular or tubulointerstitial diseases; ⑹ without any chronic underlying diseases such as hypertension, diabetes, coronary heart diseases and ischemic cerebrovascular diseases and so on; ⑺ without abnormal physiological and biochemical indexes; no history of renal damaging drugs treatment such as nonsteroidal anti-inflammatory drugs and so on. However, the inclusion criteria of group 2 were the same as group 1 except ⑶ and ⑷ which were opposite to group 1. In addition, 10 healthy people without any underlying disease served as the control group. 100 mL urine specimens were collected, centrifugated and stored at -80℃ for further examination. Serum uric acid ≥ 6 mg/dl was defined as hyperuricemia. What’s more, urate nephropathy was defined as hyperuricemia with serum creatinine ≥ 1.1 mg/dl, eGFR ≤ 90 ml/min, serum cystatin C ≤ 1.07 mg/l, abnormal markers of tubulointerstitial lesion, normal markers of glomerular injury excluding other kidney diseases. All volunteers had signed the consent for use of urine specimens and biochemical data.

Clinical index measurement

General biochemical indicators of all volunteers were collected from medical records. The levels of uric acid, creatinine, and cystatin C in serum were measured by commercial assay kits (Roche Diagnostics, Basel, Switzerland) on an automatic blood chemistry analyzer (Roche-Hitachi 7180, Roche Diagnostics, Basel, Switzerland). Estimated glomerular infiltration rate (eGFR) was calculated using the 2009 Chronic Kidney Disease Epidemiology Collaboration Equation. In addition, the levels of creatine, albumin, β₂-microglobulin, specific gravity, kidney injury molecule-1, N-acetyl-beta-d-glucosaminidase, in urine were measured by automatic biochemical analyser or immunonephelometric assay. Moreover, exosomes extracted from urine samples were quantitated and detected. The levels of lincRNA-p21 and AIF-1 in urinary exosomes were detected by qPCR and ELISA. The final results were standardized with urinary creatinine.

Cell culture

Human renal tubular epithelial cell lines (HK2) were purchased from ATCC and used in all cell experiments. After recovery, the cells were cultured with DMEM/F12 medium containing 10% fetal bovine serum at 37°C and 5% CO₂. The medium was changed every 2–3 days. When cells were 80% − 90% fusion, trypsin was added for digestion and passage. The cells were used for further experiment after 12 h synchronization cultured with serum-free DMEM/F12 medium. First, HK2 cells were cultured with different concentrations of urate (300 µM, 500 µM and 700 µM) for 24 h, and collected for follow-up experiments. Second, HK-2 cells were transfected with lincRNA-p21, AIF-1 and CMPK2 overexpression or interference vector. After transfection for 6 h − 8 h, cells were treated with 500 µM urate for 24 h, and collected for follow-up experiments. Third, HK2 cells were treated with 10 µM INF39 or Nigericin. After treatment for 12 h, the cells were treated with 500 µM urate for 24 h, and collected for follow-up experiments. Finally, the morphological changes of cells were observed under an inverted microscope. Moreover, the levels of inflammation and autophagy in cells and exosomes were detected.

Plasmid construction

In order to construct an overexpressed plasmid, the sequences of lincRNA-p21 (GenBank: KU881769.1, 2882 bp), AIF-1 (NM_001318970.2, CDS 282 bp) or CMPK2 (NM_207315.4, CDS 1350 bp) were amplified and connected to pcDNA3.1 (generalbiol, China). The cleavage site is BamHI/HindII. siRNA of lincRNA-p21, AIF-1 or CMPK2 was designed and synthesized by JST biological technology co, China. Each siRNA was designed with three types as Table 1.

Cell transfection

According to experimental groups, the cells were inoculated into 6-well plates, 4 × 10⁵ per well, cultured in an incubator at 37℃ and 5% CO₂, and transfected 24 hours later. To prepare the transfection reagent, taken one of the 6-well plates as an example: solution 1, 125 µl of opti-MEM medium (Thermo Scientific) + 7.5 µl of lipo3000 (Invitrogen); solution 2, 125 µl of opti-MEM medium (Thermo Scientific) + 2.5 µg plasmid + 5 µl P3000 (auxiliary transfection reagent, Invitrogen) or 75 pmol sequence. Solution 1 and 2 were mixed and placed at room temperature for 15 min.

The mixture of solution 1 and solution 2 was added into 6-well plates, which were gently shaken and mixed, and continued to culture for 24 h.

Fluorescence In Situ Hybridization (FISH)

The expression of linRNA-p21 was detected by RNA FISH kit (Shanghai GenePharma Co, China). According to the manufacturer's operation, the process was described as follows. Experimental cells were fixed with 4% paraformaldehyde for 10 min, and soaked with PBS for 5 min × 3 times. Added triton X-100 to completely cover cells and incubate at 4℃ for 15 min, and soaked with PBS for 5 min × 3 times. Added 100 µl sealing solution per well and incubate at 37℃ for 30 min, 100 µl 2× Buffer C at 37ºC for 30 min. Preparation of probe working liquid: for example, in a 10 µl system, 1 µmol/L biotin-probe 1 µl + 1 µmol/L SA-Cy3 1 µl + PBS 8 µl, and incubated at 37ºC for 30 min. Mix the 10 µl probe working solution with 90 µl Buffer E. 100 µl denatured probe mixture was added to each well, and hybridized in a culture box at 37ºC overnight after taking measures to avoid light. On the next day of hybridization, samples were removed from the incubator at 37ºC, the probe mixture was sucked away, and 100 µl 0.1% Buffer F was added to each well and washed at 37ºC for 10 min. Removed 0.1% Buffer F, added 100µl of 2 × Buffer C to each well, and washed at 60ºC for 10min × 3 times, at 37℃ for 10 min × 3 times. Removed 2 × Buffer C, added 100 µl diluted DAPI working liquid to each well, and stained away from light for 5 min. Removed DAPI working liquid, and washed with PBS twice, 5 min each time. Added anti-fluorescence attenuation sealer, covered with the cover glass, and observed the sealer under a fluorescence microscope. The staining effect was observed and taken pictures under fluorescence microscope.

Extraction of exosomes

Exosomes of urine or culture supernate were extracted by ultrafast centrifugation method. Samples were melted at 37℃, moved to a new centrifuge tube and centrifuged at 4℃ and 2000 rpm for 30 min. The supernatant was carefully moved into a new centrifuge tube and centrifuged again at 4℃ 10,000 rpm for 45 min to remove large vesicles. Taken the supernatant and filtered by 0.45 µm filter membrane. And the filtrate was centrifuged at 100,000 rpm for 70 min. After the supernatant was removed and re-suspended with 10 mL pre-cooled PBS, and centrifuged again at 100,000 rpm for 70 min. After supernatant was removed, the exosomes were re-suspended with 100 µL pre-cooled PBS preserved at -80℃.

Transmission electron microscopy

Exosome samples were observed by transmission electron microscopy. Taken 10 µL of exosomes and added into the copper net to precipitate for 1 min. 10 µL of uranyl acetate added into the copper net and precipitate for 1 min. The float was absorbed by filter paper. Exosome samples was dried at room temperature for a few minutes, examined and taken pictures by transmission microscopy (HT-7700, Hitachi) at 100 kv.

Particle size analysis of exosome

The concentration of exosomes was detected by nanoflow meter, and the purity of exosomes was detected by nanoparticle tracking analyzer (NTA). 10µL of exosome samples were diluted to 30 µL. After the instrument performance test with standard products is qualified, the exosome samples were loaded. It should be noted that gradient dilution should be carried out to avoid sample clogging of the injection needle. The particle size and concentration information of exosomes were detected by nanoparticle tracking analyzer (N30E, NanoFCM).

qPCR analysis (SYBR method)

Total RNAs of urine or HK2 were extracted using the total RNAs Isolation Kit, and the concentration of RNA was determined using ultraviolet spectrophotometer NANO 2000. Obtained RNA samples were reversely transcribed synthesized to cDNA by MMLV reverse transcription kit, according to the manufacturer’s protocols. 20 µl cDNA sample was stored at -20℃ or used for downstream experiments. The relative expression of RNA was amplified and determined by Exicycler 96 (BIONEER, South Korea), as described by the manufacturer. The primer sequences were used as Table 2. β-actin was used as an endogenous reference gene for normalization of the data. The reaction system was 20 µL (cDNA template 1 µL, SYBR GREEN mastermix 10 µL, upstream primers 0.5 µL, downstream primers 0.5 µL, ddH₂O 8 µL). And the reaction conditions were as follows: incubated at 95℃ for 5 min, 95℃ for 10s, 60℃ for 10s, 72℃ for 15s, after then went to line 2 for 40 cycles: incubated at 72℃ for 90s, incubate at 40℃ for 1 min, melted from 60℃ to 94℃, every 1℃ for 1s, incubated at 25℃ for 1 min. The method of 2^−△△CT was used in this experiment.

Apoptosis analysis

Apoptotic cells were detected and quantified by flow cytometry using Annexin V-FITC/PI apoptosis Kit (wanleibio, China). The specific detection process was as follows. Experimental cells were collected and washed twice with PBS. And then the cells were suspended with 500 µL binding buffer to adjust the concentration to 10⁶/ml. 5 µL AnnexinV-FITC and 10 µL Propidium Iodide was added and mixed which was incubated at room temperature for 15 min away from light. Cells suspension was filtered by mesh screen, and detected by flow cytometry (NovoCyte, Agilent, USA). In the two-parameter scatter plot, the living cells were shown in the lower left quadrant (Annexin V FITC⁻/PI⁻); the non-living cells namely the late apoptotic cells were shown in the upper right quadrant (Annexin V FITC⁺/PI⁺); the early apoptotic cells were shown in the lower right quadrant (Annexin V FITC⁺/PI⁻).

Western blot analyses

Total protein was extracted with total protein extraction kit (wanleibio, China). The protein concentration was detected by the BCA protein concentration determination kit (wanleibio, China). For electrophoresis, 8%, 12% or 15% SDS-PAGE gels were made respectively. 40 µg of total protein were loaded onto SDS-PAGE gels and separated by double vertical protein electrophoresis apparatus (DYCZ-24DN, Liuyi Biotechnology, China). After electrophoresis, the separated proteins were transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked in Western Blocking Buffer for 1 h at room temperature and then incubated with primary antibodies against AIF-1(1:500, wanleibio), CMPK2 (1:3000, Proteintech), NLRP3 (1:500, wanleibio), p53 (1:500, wanleibio), p62 (1:500, wanleibio), LC3BII/I (1:500, wanleibio), β-actin (1:1000, wanleibio) and CD63 (1:500, Affinity Biosciences) for at 4℃ overnight. Membranes were then incubated with goat anti-rabbit IgG-HRP (1:5000, wanleibio) at 37℃ for 45 min. After incubation with second antibody, the PVDF membrane was immersed in TBST, shaken for 5 min, 6 times. When the water of PVDF was drained with filter paper, and evenly sprinkled the ECL luminescent solution, at room temperature for 5 min. Finally, PVDF was transferred to a dark box, and exposed in the dark room. The films were scanned and the optical density value of the target strip was analyzed with the Gel image processing system (GEL-Pro-Analyzer software, China). Protein levels were quantified and normalized to β-actin level.

The levels of lincRNA-p21/AIF-1 in exosome of urine were related to urate nephropathy

The study analyzed the levels of lincRNA-p21 and AIF-1 in urine exosomes of individuals with urate nephropathy, hyperuricemia, and healthy individuals. The study included 30 male volunteers, with 10 individuals in each group. Several biochemical indicators were measured, including age, eGFR (estimated glomerular filtration rate), serum cystatin C, uACR (urine albumin-to-creatinine ratio), urine specific gravity, serum uric acid, serum creatinine, uβ2-MG (urine β2-microglobulin), uKim-1 (urine kidney injury molecule-1), and uNAG (urine N-acetyl-β-D-glucosaminidase). The results as shown in table 3 indicated that there were no significant differences in age, eGFR, serum cystatin C, uACR, and urine specific gravity between the healthy individuals, hyperuricemia group, and urate nephropathy group. However, patients with urate nephropathy had significantly increased levels of serum uric acid, serum creatinine, uβ2-MG, uKim-1, and uNAG compared to the other two groups. Furthermore, the levels of lincRNA-p21 and AIF-1 in urine exosomes were also increased in both the hyperuricemia and urate nephropathy groups. This suggests that urine exosomal lincRNA-p21/AIF-1 may be involved in the kidney injury induced by urate.

Urate induced inflammation, autophagy and apoptosis of HK2 cells

It is well-known that tubulointerstitial fibrosis is a major complication of hyperuricemia. Therefore, it is crucial to investigate the mechanism of renal tubular epithelial cell injury induced by urate in order to understand urate nephropathy. In this study, HK2 cells were cultured with different concentrations of urate for 24 hours. The results shown in Fig. 1 indicated that compared to the control group, there were significant differences in the expression of lincRNA-p21, inflammatory proteins (AIF-1, CMPK2, and NLRP3), autophagy-related proteins (p53, p62, and LC3BII/I), and the level of apoptosis. These differences were positively correlated with the concentration of urate, with the most significant variation observed in the 700 µM urate group. However, it was worth noting that the apoptosis of HK2 cells was also highest in the 700 µM urate group. Therefore, a concentration of 500 µM urate was chosen for subsequent experiments.

Urate increased the level of exosome in HK2 cells

Exosomes play a crucial role in kidney diseases by serving as carriers for intercellular communication within the kidney. To investigate the impact of exosomes on renal tubular epithelial cell injury induced by urate, exosomes were extracted and analyzed from the cell supernatant of HK2 cells. As depicted in Fig. 2, the average particle size of the exosomes was measured to be 80 nm, and their concentration was found to increase in a concentration-dependent manner in response to urate stimulation. Furthermore, compared to the control group, the levels of inflammatory proteins (AIF-1, CMPK2, and NLRP3) and autophagy-related proteins (p53, p62, and LC3BII/I) within the exosomes were significantly elevated. These findings provide compelling evidence that exosomes are involved in the inflammation and autophagy processes in HK2 cells induced by urate.

LincRNA-p21 promoted inflammation, autophagy and apoptosis of HK2 cells induced by urate

LincRNA-p21, a non-coding RNA, plays a role in regulating inflammation and other cellular processes. To assess the impact of lincRNA-p21 on inflammation, autophagy, and apoptosis in HK2 cells induced by urate, overexpression or interference vectors of lincRNA-p21 were transfected into HK2 cells. As depicted in Fig. 3, the expression of lincRNA-p21 was significantly increased in the overexpression group and significantly decreased in the interference group. Compared to the control group, lincRNA-p21 notably enhanced the expression of inflammatory proteins (AIF-1, CMPK2, and NLRP3) and autophagy-related proteins (p53, p62, and LC3BII/I) in both HK2 cells and exosomes. Additionally, lincRNA-p21 induced apoptosis in HK2 cells. Conversely, the siRNA targeting lincRNA-p21 had the opposite effects. Based on these research findings, it could be concluded that lincRNA-p21 promoted autophagy and apoptosis in HK2 cells induced by urate via the AIF-1/CMPK2/NLRP3 pathway

AIF-1 promoted autophagy and apoptosis of HK2 cells induced by urate via CMPK2/NLRP3

Numerous studies have provided evidence supporting the involvement of AIF-1 in renal tubulointerstitial inflammation and fibrosis. To investigate the impact of AIF-1 on inflammation, autophagy, and apoptosis in HK2 cells induced by urate, overexpression or interference vectors of AIF-1 were transfected into HK2 cells. As depicted in Fig. 4, the expression of AIF-1 mRNA and protein was significantly increased in the overexpression group and significantly decreased in the siRNA group. Compared to the control group, the expression of lincRNA-p21 did not show a significant effect in either the AIF-1 overexpression or interference group. However, AIF-1 significantly promoted cell apoptosis and the expression of inflammatory proteins (CMPK2 and NLRP3) and autophagy-related proteins (p53, p62, and LC3BII/I) in both HK2 cells and exosomes. Conversely, the siRNA targeting AIF-1 had the opposite effects. Therefore, these results suggested that, in addition to lincRNA-p21, AIF-1 could promote autophagy and apoptosis in HK2 cells induced by urate via the CMPK2/NLRP3 pathway.

The effect of CMPK2 on inflammation, autophagy and apoptosis of HK2 cells induced by urate

To investigate the impact of CMPK2 on inflammation, autophagy, and apoptosis in HK2 cells induced by urate, overexpression or interference vectors of CMPK2 were transfected into HK2 cells. As depicted in Fig. 5, the expression of CMPK2 mRNA and protein was significantly increased in the overexpression group and significantly decreased in the siRNA group. Compared to the control group, CMPK2 overexpression significantly promoted cell apoptosis and the expression of inflammatory proteins (NLRP3) and autophagy-related proteins (p53, p62, and LC3BII/I) in both HK2 cells and exosomes. Conversely, the siRNA targeting CMPK2 had the opposite effects. Therefore, these results suggested that CMPK2 can promote autophagy and apoptosis in HK2 cells induced by urate via the NLRP3 pathway.

The effect of NLPR3 on inflammation, autophagy and apoptosis of HK2 cells induced by urate

To investigate the effect of NLRP3 on inflammation, autophagy, and apoptosis in HK2 cells induced by urate, HK2 cells were treated with an activator or inhibitor of NLRP3. As shown in Fig. 6, compared to the control group, the NLRP3 activator (Nigericin) significantly promoted cell apoptosis and the expression of inflammatory proteins (CMPK2) and autophagy-related proteins (p53, p62, and LC3BII/I) in both HK2 cells and exosomes. On the other hand, the NLRP3 inhibitor (INF39) had the opposite effects. Therefore, these results suggested that NLRP3 interacts with CMPK2, leading to the promotion of autophagy and apoptosis in HK2 cells via inflammation.

Urate nephropathy, the main complication of hyperuricemia, contributes to chronic kidney disease (CKD) [33, 34]. The pathogenesis of urate nephropathy involves crystal deposition of monosodium urate, oxidative stress, endothelial dysfunction, renal tubule interstitial fibrosis, and inflammation [35, 36]. Inflammatory and fibrotic lesions are the primary features of urate nephropathy, as indicated by renal biopsy findings. Our previous study suggested that lincRNA-p21, AIF-1, CMPK2, and NLRP3 were involved in urate nephropathy, but the exact mechanism remains unclear. In this study, we confirmed that exosome-mediated lincRNA-p21, AIF-1, CMPK2, and NLRP3 activation promoted inflammation, autophagy, and apoptosis in human tubular epithelial cells induced by urate. Additionally, there was an interaction between lincRNA-p21, AIF-1, CMPK2, and NLRP3.

LincRNA-p21, a non-coding RNA, plays pro-inflammatory roles in various diseases. For example, it has been observed that overexpression of lincRNA-p21 promotes the proinflammatory phenotype transformation of microglia in neuroinflammatory models [37]. In this study, we also found that lincRNA-p21 activated inflammation, autophagy, and apoptosis in renal tubular epithelial cells induced by urate. Moreover, previous studies have suggested that lincRNA-p21 was a crucial regulator of cell proliferation, apoptosis, and DNA damage responses. Wu et al. confirmed that lincRNA-p21 inhibited cell proliferation and induced apoptosis in vascular smooth muscle cells and macrophages in vitro and in vivo via the p53 pathway [38]. Additionally, Zhou et al. suggested that lincRNA-p21 promoted the proliferation of lung fibroblasts, leading to pulmonary fibrosis [39]. Therefore, we speculated that lincRNA-p21 served as the initiator of cellular inflammation induced by urate. Furthermore, our previous study confirmed that AIF-1 contributed to renal tubulointerstitial inflammation and fibrosis. However, whether there was a mutual effect of lincRNA-p21 and AIF-1 in urate-induced inflammation was unknown. In this study, we suggested that the pro-inflammatory role of lincRNA-p21 was mediated by AIF-1, and lincRNA-p21 promotes inflammation via AIF-1. To further clarify the mechanism of AIF-1 in inflammation, the expression of AIF-1 was upregulated or downregulated in vitro. The results of this study showed that AIF-1 exerted pro-inflammatory effects via the CMPK2/NLRP3 pathway.

Previous studies have indicated that the CMPK2/NLRP3 axis plays a crucial role in inflammation. CMPK2 induces inflammation through NLRP3, and in turn, NLRP3 influences the pro-inflammatory role of CMPK2. Emerging evidence suggests that the activity of the CMPK2/NLRP3 axis is a critical process in the inflammatory reaction [40, 41]. Furthermore, in this study, we confirmed that the activity of CMPK2 or NLRP3 resulted in inflammation, autophagy, and apoptosis of renal tubular epithelial cells. Autophagy and apoptosis are the main pathological processes observed in renal tubular epithelial cells induced by urate. Consequently, we proposed that lincRNA-p21 promoted inflammation in renal tubular epithelial cells via the AIF-1/CMPK2/NLRP3 pathway. However, due to the poor stability of lincRNA-p21, AIF-1, CMPK2, and NLRP3 outside the cell, it was necessary to determine how these molecules were transmitted between different cells.

Exosomes, as carriers of information transfer between cells, are involved in the pathogenesis of various diseases. In this study, we had demonstrated that the levels of lincRNA-p21, AIF-1, CMPK2, and NLRP3 in exosomes correspond to their intracellular levels. This suggested that exosomes protected these contents from destruction and degradation, facilitating the interaction between different cells and potentially serving as markers of pathological processes [42, 43]. Chen et al. demonstrated that urinary exosome tsRNAs served as biomarkers for the diagnosis and prediction of nephritis in systemic lupus erythematosus (SLE) [44]. Therefore, we believe that urine exosome lincRNA-p21 and AIF-1 might serve as novel biomarkers for renal tubulointerstitial inflammation and fibrosis in patients with hyperuricemia.

In conclusion, based on the aforementioned results, we proposed that exosome-mediated lincRNA-p21/AIF-1 promoted inflammation via the CMPK2/NLRP3 pathway, leading to autophagy and apoptosis of renal tubular epithelial cells induced by urate. Additionally, exosomal lincRNA-p21/AIF-1 might serve as a novel marker for urate nephropathy. However, the function and value of exosomal lincRNA-p21/AIF-1 in urate nephropathy require further research and exploration.

Data availability statement

The data underlying this article will be shared on reasonable request to the corresponding author.

Acknowledgements

We would also like to thank the relevant institutions and teams of Southern University of Science and Technology Hospital for their support and collaboration. Finally, we extend our gratitude to all individuals who have contributed to this study, including members of the research team and volunteers involved in the experiments. Their efforts and dedication have been crucial to the success of this research.

Funding

The study was received funding from the Key Project of Sub-item Funding for Technology Research and Creative Design in Nanshan District, Shenzhen (Project No. NS006), the High-level Project A Cultivation Program of the Chief Research Fund of the Southern University of Science and Technology Hospital (Project No. 2021-A05), and theSanming Project of Medicine in Shenzhen nanshan (Project No. SZSM202103002).

Authors contributions

The study's overall design and research objectives were conceptualized by the HJB and HLR. WSY, GXY, GXJ, YK and CRH collected clinical data from patients with urate nephropathy and conducted experiments. HJB, GXY and HLR performed data analysis, including statistical analysis and interpretation of the results obtained from the clinical data and in vitro experiments. HJB, WSY, GXY, GXJ, YK and CRH wrote the manuscript, including the introduction, methods, results, and discussion sections, based on the study findings and relevant literature. HJB and HLR. WSY, GXY, GXJ, YK and CRH conducted experiments in the laboratory, including the measurement of exosomal lincRNA-p21/AIF-1 levels, evaluation of inflammation, autophagy, and apoptosis in HK2 cells, and manipulation of lincRNA-p21, AIF-1, CMPK2, and NLRP3 expressions. HJB and HLR interpreted the experimental results and discussed their implications in the context of urate nephropathy pathogenesis. HJB and HLR critically reviewed and revised the manuscript based on feedback from co-authors and reviewers, ensuring the accuracy and clarity of the scientific content.

Conflict of interest statement

There is no conflict between all authors.

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Tables 1-3 is available in the Supplementary Files section.

No competing interests reported.

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Exosomal lincRNA-p21/AIF-1 promoted inflammation of human tubular epithelial cell via CMPK2/NLRP3 pathway in urate nephropathy

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Abstract

Figures

Introduction

Materials and Methods

Patients

Clinical index measurement

Cell culture

Plasmid construction

Cell transfection

Fluorescence In Situ Hybridization (FISH)

Extraction of exosomes

Transmission electron microscopy

Particle size analysis of exosome

qPCR analysis (SYBR method)

Apoptosis analysis

Western blot analyses

Results

The levels of lincRNA-p21/AIF-1 in exosome of urine were related to urate nephropathy

Urate induced inflammation, autophagy and apoptosis of HK2 cells

Urate increased the level of exosome in HK2 cells

LincRNA-p21 promoted inflammation, autophagy and apoptosis of HK2 cells induced by urate

AIF-1 promoted autophagy and apoptosis of HK2 cells induced by urate via CMPK2/NLRP3

The effect of CMPK2 on inflammation, autophagy and apoptosis of HK2 cells induced by urate

The effect of NLPR3 on inflammation, autophagy and apoptosis of HK2 cells induced by urate

Discussion

Declarations

References

Tables

Additional Declarations

Supplementary Files

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