A novel role of peroxiredoxin 2 in diabetic kidney disease progression by activating the classically activated macrophages

A novel role of peroxiredoxin 2 in diabetic kidney disease progression by activating the classically activated macrophages | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A novel role of peroxiredoxin 2 in diabetic kidney disease progression by activating the classically activated macrophages Xia Li, Hehua Long, Rui Peng, Xue Zou, Siyang Zuo, Yuan Yang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3898778/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted 15 You are reading this latest preprint version Abstract Diabetic kidney disease (DKD) is the main cause of death due to diabetes mellitus (DM). Due to the complexity of the onset, it is difficult to achieve accurate prevention and treatment. The classically activated macrophages (M1) polarization is a crucial proinflammatory mechanism in DKD, while the interaction and cascade effects of oxidative stress and inflammatory response remain to be elucidated. A urine proteomic analysis of DM patients indicated that peroxiredoxin 2 (PRDX2) had the higher abundance in DKD. We recently found that PRDX of parasitic protozoa Entamoeba histolytica , which was similar to human PRDX2 in amino acid sequence and spatial structure, could activate the inflammatory response of macrophages through toll like receptor 4 (TLR4). Hence, our study was designed to explore the role of PRDX2 in chronic inflammation during DKD. Combined with in vivo and in vitro experiments, results showed that the PRDX2 was positively correlated with DKD progression and upregulated by high glucose or recombinant tumor necrosis factor-α in renal tubular epithelial cells; Besides, recombinant PRDX2 could promote macrophages M1 polarization, enhance the migration and phagocytic ability of macrophages through TLR4. In summary, our study has explored the novel role of PRDX2 in DKD to provide basis for further researches on the diagnosis and treatment of DKD. Biological sciences/Cell biology Biological sciences/Immunology Biological sciences/Molecular biology Biological sciences/Physiology Health sciences/Biomarkers Health sciences/Endocrinology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Diabetes mellitus (DM) has become a worldwide public health problem, the number of DM patients in the world has exceeded 425 million, and is expected to reach 629 million in 2045 1 . Diabetic kidney disease (DKD) is the most common microvascular complication of DM, which is characterized by glomerular basement membrane thickening, mesangial dilation, and tuberous sclerosis, leading to decreased renal function, and ultimately end-stage renal disease (ESRD) 2 . Due to the insidious onset and lack of specific treatment targets, DKD has been the main cause of ESRD in adults 3 . Therefore, exploring the detailed pathogenic mechanism of DKD can provide a basis for researches on targeted therapy. The infiltration of inflammatory cells (such as macrophages) is crucial for the occurrence and development of DKD 4 , 5 . Janus kinase (JAK) / signal transducer, activator of transcription (STAT)1 as well as nuclear factor-κB (NF-κB) signaling pathways are key promoters of the inflammation in DKD, which are involved in expression of pro-inflammatory molecules, including interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α (TNF)-α 6 , 7 . Wherein, activation of both STAT1 and NF-κB are closely related to toll like receptors (TLRs), which are overexpressed in DKD renal tissue, with TLR4 playing a crucial role in persistent inflammation 8 – 10 . Macrophages are key to the progression of DKD. Renal biopsy analysis of DM patients confirmed the presence of macrophages in the glomerulus and renal interstitium at all stages of DKD, which is related to the progression of DKD 11 – 14 . Macrophages can be classified into classically activated macrophages (M1) polarization and alternatively activated macrophages (M2) polarization based on their activation mechanisms and functions 15 , with M1 exhibiting a pro-inflammatory phenotype and M2 exhibiting an anti-inflammatory phenotype. In the chronic inflammatory process of DKD, M1 is predominant with expression of characteristic molecules, such as TNF-α, IL-1β, IL-6 and inducible nitric oxide synthase (iNOS), which are associated with inflammation and tissue damage 16 . It was reported that M1 polarization through TLR4 / NF-κB and JAK / STAT1 signaling pathways is one of the key mechanisms on the occurrence and development of DKD 15 , 17 , 18 . However, the key molecules initiating M1 polarization during development of DKD need further exploration. Oxidative stress is the main factor in the progression of DKD, which is characterized by the excessive ROS under hyperglycemia 6 , 19 . ROS can activate various transcription factors, promote the expression of inflammation related genes, resulting in the occurrence and development of diseases 20 . Therefore, various antioxidants have been evolved in cells to eliminate excessive ROS. In addition to superoxide dismutase, catalase and glutathione peroxidase, the role of peroxiredoxin (PRDX) is gradually receiving attention. PRDX is a family of structurally conserved oxidoreductases commonly found in organisms; In addition to functioning as antioxidant enzymes, PRDX also participates as a signal transduction substance in regulating the response of cells to the surrounding environment under stress and non-stress conditions 21 , 22 . Mammalian cells express six PRDX subtypes, namely PRDX1 to PRDX6. PRDX1 and PRDX2 are located in the cytoplasm and nucleus, PRDX3 is located in the mitochondrial matrix, PRDX4 is located in the endoplasmic reticulum and can also be secreted into the extracellular environment, PRDX5 is present in mitochondria, peroxisomes, and cytoplasm, and PRDX6 is present in the cytoplasm 23 . The relevant researches on PRDX in the occurrence and development of DKD have only been reported in recent years: for example, PRDX4 can protect renal podocytes from oxidative damage 24 , and PRDX1 can serve as a downstream molecule regulated by bait receptor 2, participating in the expression of pro-renal fibrosis related factors by regulating the aging of renal tubular epithelial cells 25 . Considering sufficient antioxidant molecules in cells, whether the PRDX family members have functional redundancy and perform other functions besides antioxidant effects has aroused our interest. However, it remains to be elucidated whether PRDX can activate inflammation and lead to DKD progression. The transcriptome data of kidney tissue from DKD patients (Gene Expression Omnibus, GSE142025) 26 showed that the transcription levels of the PRDX family in the kidney tissue of early and middle stage DKD patients were not significantly increased compared to the negative control group. Further urine proteomic analysis of DM patients (ProteomeXchange Datasets, PXD016571) 27 found that only PRDX1, 2, and 5 in PRDX family were detected, wherein PRDX2 had the highest abundance and was 2.6 times higher in DM patients with kidney injury than these without kidney injury. In mammalian cells, PRDX2 exist in the nucleus and cytoplasm 23 . It was reported that PRDX2 can be released into the extracellular space under TNF-α stimulation in HEK293T cells, indicating the relationship of PRDX2 and inflammation 28 . We recently found that PRDX of parasitic protozoa Entamoeba histolytica ( Eh -PRDX), which has a similar amino acid sequence and spatial structure to human PRDX2 (Fig. 7), can be overexpressed by parasites after invasion and then activate the inflammatory response in macrophages through TLR4 29 . Whether mammalian PRDX2 has a similar pro-inflammatory effect, such as promoting M1 polarization of macrophages, has not been reported yet. Briefly, we hypothesized that sustained hyperglycemia followed by excessive ROS in DM promotes expression and secretion of PRDX2 in renal cells, then PRDX2 can induce macrophage M1 polarization through TLR4, leading to the release of inflammatory factors, which in turn promote the expression of more PRDX2 in renal cells, forming a positive feedback loop, and ultimately promoting the progression of DKD. This study aimed to explore the linkage between mammalian PRDX2 up-regulation and macrophage M1 polarization at both animal and cellular levels under DM condition, to clarify the role of PRDX2 in the occurrence and development of DKD, and provide new evidence for the researches on targeted treatment in the future. Results Construction of Mice DKD Model STZ was used for construction of DM mice model. 6–8 w male C57BL/6 mice were injected intraperitoneally with STZ (55 mg/kg) once a day for 5 consecutive days. Then, DM mice (random blood glucose ≥ 16.7 mM) were selected and fed normally. They were anesthetized and executed at 20, 28, and 36 w, respectively (Fig. 1 a). Body weight and kidney weight were measured; Serum and kidney tissue were collected for biochemical and pathological analysis, respectively. The results showed that compared with the control group, the DM group had a significant decrease in body weight ( p < 0.05), while the kidney to body weight ratio (KW:BW) was significantly increased ( p < 0.05) (Fig. 1 b). Serological analysis found that the random blood glucose (BG), blood urea nitrogen (BUN), and blood creatinine (CRE) of DM group mice gradually increased over time, and the differences at each time point were statistically significant ( p < 0.05) (Fig. 1 b). Kidney tissue was fixed with 4% PFA, embedded in paraffin, and sliced for pathological analysis. The results showed that there was significant deposition of renal collagen fibers, which gradually increased over time, and the difference at each time point was statistically significant ( p < 0.05) (Fig. 1 c). In addition, damaged renal tubular brush border microvillus and thicken glomerular basement membrane can be observed at all time points (Fig. 1 c). Both serological and pathological results suggest that sustained DM status can lead to kidney damage. Therefore, this study has successfully constructed a mouse DKD model using STZ. Positive Correlation between the Expression Levels of PRDX2 and M1 Polarization Related Proteins To further confirm M1 polarization of macrophages in DKD kidney tissue, we first detected the distribution of macrophages in mouse kidney tissue through immunohistochemistry. The results showed that there was significant infiltration of macrophages in DKD kidney tissue ( p < 0.05), which was time-dependent (Fig. 2 a). Further detection of the distribution of M1 related inflammatory factors in renal tissues revealed that the distribution area of pro-inflammatory factor IL-1β, IL-6 and TNF-α in the tissue gradually increased with the progression of DKD, and the difference was statistically significant ( p < 0.05) (Fig. 2 a). Further detection by WB revealed a positive correlation between protein expression levels of PRDX2 and M1 polarization, inflammatory factors, or fibrosis related factors as DKD progressed (Fig. 2 b). The results above suggested that PRDX2 could play a role in M1 polarization and DKD progression. Upregulation of PRDX2 in Renal Tubular Epithelial Cells under DM Condition To further investigate the localization of PRDX2 in DKD mice renal tissue, IFA was used to co-stain PRDX2 with α-SMA or AQP1. α-SMA is a marker of renal interstitial tissue, which is highly expressed in vascular smooth muscle cells and myofibroblasts 30 ; AQP1 is a marker of renal tubule 31 . PRDX2 was mainly distributed in renal tubule when observed by laser confocal microscopy with the same laser power; distribution of PRDX2 in the renal interstitial tissue was also observed with the progression of DKD; moreover, the fluorescence intensity of PRDX2 in renal tubule increased during DKD progression. The results suggested that renal tubular epithelial cells were the main source of PRDX2 in renal tissue, and PRDX2 might be secreted into the interstitial tissue to play a role as the disease progresses (Fig. 3 a). To determine whether high glucose (HG) or inflammatory factors (such as TNF-α) can upregulate PRDX2 in renal tubular epithelial cells, this study used HG (30 mM) or TNF-α (50 ng/mL) to treat the renal tubular epithelial cell line NRK52E, and detected the protein expression level of PRDX2 at 6, 12, 24, and 36 h, respectively. It was found that after treatment with HG or TNF-α for 6, 12, and 24 h, the protein expression level of PRDX2 significantly increased (p < 0.05); while there was no statistically significant difference in the PRDX2 expression level between the TNF-α group and its control group at 36 h (Fig. 3 b). To ensure the stability of cellular response, 24 h was selected as the subsequent experimental time point. The above experimental results suggested that under sustained DM conditions, HG or inflammatory factors (such as TNF-α) can stimulate renal tubular epithelial cells to express PRDX2. To investigate whether renal tubular epithelial cells under HG (HG) or inflammatory factor (such as TNF-α) stimulation undergo changes in PRDX2 localization, IFA was used for detection. Compared with the control group, PRDX2 can be transferred from the cell nucleus to the cytoplasm by high glucose (HG) or inflammatory factors (such as TNF-α) stimulation, and the increased fluorescence intensity were observed in the cytoplasm, indicating the linkage between PRDX2 localization and its secretion (Fig. 3 c). Considering that PRDX2 is an antioxidant, we further used DCFH-DA fluorescence probe to detect intracellular ROS levels. The results showed that under high glucose (HG) or inflammatory factor (such as TNF-α) stimulation, there was a significant increase in intracellular ROS levels in renal tubular epithelial cells (Fig. 3 d). The results above suggested that HG or TNF-α could stimulate renal tubular epithelial cells to express PRDX2, which was accompanied by changes in PRDX2 localization and an increase in intracellular ROS levels. Recombinant PRDX2 Can Upregulate M1 Polarization and Inflammatory Related Factors of Macrophages Considering the similarity between PRDX2 and Eh -PRDX in protein sequence and spatial structure (Fig. 7), this study aimed to explore the ability of PRDX2 to promote inflammatory response in macrophages. After stimulation by rPRDX2 for 12 or 24 h, M1 polarization related molecules (marker, iNOS; inflammatory factors, IL-6, TNF-α and IL-1β; M1 polarization signaling pathway-related molecules, NF-κB p65, NF-κB p-p65, STAT1) were significantly increased ( p < 0.05) after 24 h treatment (Fig. 4 a). In addition, the M2 polarization marker Arg-1 slightly increased, but the difference was not statistically significant between treatment group and control group (Fig. 4 a). Considering the similarity between PRDX2 and Eh -PRDX, this study explored whether rPRDX2 showed co-localization with TLR4 on macrophages. Based on the observation that rPRDX2 under electron microscopy appears as 20–30 nm polymer particles (Fig. 8), rPRDX2 attached on the cell membrane will show the bright and coarse fluorescent particles by IFA detection. Therefore, after treatment with rPRDX2 for 24 h, laser confocal microscopy was used to observe macrophages with the same laser power, it was found that resting macrophages have a basal expression of TLR4 and PRDX2, presenting fine and uniform particles distributed evenly in the cell; after addition of rPRDX2, aggregation of TLR4 in the periphery of macrophages was observed and presented as coarse particles, while distribution of PRDX2 was presented as scattered coarse particles over the periphery of macrophages, with scarce rPRDX2 particles observed in cytoplasm; importantly, co-localization of PRDX2 and TLR4 was observed near the surface of macrophage membrane (Fig. 4 b). Results above suggested that rPRDX2 might promote M1 polarization of macrophages through TLR4. M1 Polarization of Macrophages Promoted by PRDX2 via TLR4 To further investigate whether PRDX2 functioned through TLR4, J774A. 1 cells were treated by rPRDX2 and collected for WB detection. After 24 h treatment, M1 polarization related molecules (marker, iNOS; inflammatory factors, IL-6 and TNF-α; M1 polarization signaling pathway-related molecules, NF-κB p65, NF-κB p-p65, STAT1) were significantly increased ( p < 0.05) after 24 h treatment, which could be significantly inhibited by the TLR4 specific inhibitor TAK242 ( p < 0.05) (Fig. 5 a). For further confirming the changes of key molecules localization in macrophages after rPRDX2 stimulation, IFA was used for detection of STAT1 and NF-κB p-p65 in macrophages. Results showed that rPRDX2 can promote relocation of STAT1 and NF-κB p-p65 from cytoplasm to nucleus, which could be suppressed by TLR4 specific inhibitor TAK242 (Fig. 5 b). Results above suggested that PRDX2 was capable of promoting M1 polarization of macrophages through TLR4. Macrophage Migration and Phagocytosis Promoted by PRDX2 via TLR4 The enhanced migration and phagocytic ability of macrophages are characteristic phenotypes of M1 polarization 32 . To investigate whether PRDX2 could enhance macrophage migration, this study conducted scratch experiments. Photos were taken and recorded at the same location in each well during treatment with rPRDX2 at 0 and 24 h. The results showed that rPRDX2 significantly enhanced the migration ability of macrophages, which was significantly inhibited by the TLR4 specific inhibitor TAK242 ( p < 0.05) (Fig. 6 a). Afterwards, green fluorescent microspheres were used for Phagocytosis experiments to investigate whether PRDX2 could enhance macrophage phagocytic ability. It was found that macrophages had a certain phagocytic ability in a resting state, with most single cells capable of phagocytosing 2 to 4 microspheres; After stimulation with rPRDX2, the number of phagocytic microspheres in a single macrophage could reach over 10, and this effect could be significantly inhibited by the TLR4 specific inhibitor TAK242 (Fig. 6 b). These results suggested that PRDX2 can promote macrophage migration and phagocytic ability through TLR4. Discussion Researches on the pathogenesis of DKD mainly focus on oxidative stress 33 , insulin resistance 34 , cell death 35 and gene polymorphism 36 . It is reported that oxidative stress plays an important role in the pathogenesis of DKD. Wherein, PRDX family members are believed to play an important role in the process of oxidative stress 37 . Transcriptomic analysis of kidney tissue from DKD patients revealed that PRDX family members were not significantly upregulated or downregulated at the transcriptional level (Gene Expression Omnibus, GSE142025) 26 ; nevertheless, proteomic analysis of urine from DKD patients found that only PRDX1, 2, and 5 could be detected in urine, with the highest abundance of PRDX2, which was 2.6 times higher than that of DM patients (ProteomeXchange Datasets, PXD016571) 27 . It is suggested that PRDX2 may be related to the occurrence and development of DKD. This study firstly found the role and mechanism of PRDX2 in the progression of DKD. In the kidney tissue of DKD mice, the positive correlation between the protein expression level of PRDX2 and the protein expression levels of renal fibrosis related molecules, macrophage M1 polarization related molecules or proinflammatory factors was detected and analyzed. Then, PRDX2 could be significantly upregulated in renal tubular epithelial cells stimulated by HG or TNF-α, explaining the reason for the upregulation of PRDX2 protein expression level in DKD renal tubule. Moreover, after stimulation with rPRDX2, the protein expression levels of M1 polarization related factors in macrophages were promoted, the migration and phagocytic ability of macrophages were also enhanced, which could be inhibited by TLR4 specific inhibitor TAK242. Results above suggested that PRDX2 was capable of promoting M1 polarization of macrophages through TLR4. Moreover, PRDX2 was up-regulated by TNF-α, and then it could promote production of TNF-α in macrophages, which formed positive feedback to promote persistent inflammation. The role of the PRDX family members is diverse, and in addition to antioxidant activity, they can also function as a molecular chaperone, transduce peroxide signal, protect membranes from damage, and play a role in maintaining iron homeostasis 37 . A recent study found that PRDX2 participated in the cellular response to ROS and activated downstream signaling pathways through excessive oxidation by ROS within the cell. It was an important physiological process in cells and also a sign of cell youth; If a large amount of PRDX2 in the cell was excessively oxidized in a short period of time, it would lose its ability to respond to ROS, leading to aging; Due to the difficulty in reducing excessively oxidized PRDX2, oxidized PRDX2 may be secreted outside the cell as a byproduct in response to ROS 38 . It was consistent with the findings of this study, the increase of ROS levels in renal tubular epithelial cells was induced by HG and TNF-α; Meanwhile, a large amount of PRDX2 could be transferred from the nucleus to the cytoplasm and aggregated into granules, indicating the possibility of PRDX2 secretion after ROS upregulation. In the kidney tissue of DKD mice, it was also found that PRDX2 was distributed in the renal interstitium in addition to the renal tubules, suggesting that PRDX2 could be secreted to the renal interstitium to play a role in the progress of DKD. Therefore, we speculated that PRDX2 might act as a by-product after antagonizing ROS in the progression of DKD, and then function as a proinflammatory factor by activating M1 polarization. However, whether specific knockout of PRDX2 in renal tissue can delay the progression of DKD, as well as the detailed molecular mechanism of PRDX2 upregulation and secretion in renal tubular epithelial cells, all require further exploration, which are also our research interests in future. In conclusion, this study found for the first time that the protein expression level of PRDX2 was positively correlated with the progression of DKD through in vivo animal experiments; Combined with relevant technologies in pathology, immunology, and molecular biology, in vitro cell experiments showed the ability of PRDX2 promoting M1 polarization via TLR4 after its up-regulation by high glucose or TNF-α, which formed positive feedback to promote persistent inflammation. This study has explored new mechanisms for the occurrence and development of DKD, and provided new theoretical basis for research related to the diagnosis and treatment of DKD in the future. Conclusions In this study, we found that PRDX2 could activate M1 polarization via TLR4. In summary, PRDX2 plays an important role in DKD. This study has explored new mechanisms for the occurrence and development of DKD, and provided new theoretical basis for research related to the diagnosis and treatment of DKD in the future. Materials and Methods Ethics Statement All animal experiments in our study were conducted in strict accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (1988.11.1), carried out in compliance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and were approved by the Animal Care Welfare Committee of GuiZhou Medical University (Permit Numbers: 2201399). All efforts were made in our study to minimize the suffering of animals. Animal Model Construction and Analysis SPF grade C57BL/6 mice (6–8 w old, 18–20 g weight) used in this study were purchased from Beijing Charles River Experimental Animal Technology Co., Ltd. Mice in experimental group were given 55 mg/kg Streptozotocin (STZ) (Sigma-Aldrich, 572201) dissolved in 0.1M sodium citrate buffer solution (pH 4.0) via intraperitoneal injection per day for 5 consecutive days, while mice in control group was given the same volume of STZ solvent. After detection of 2 consecutive days, mice with random blood glucose levels > 16.7 mmol/L and positive urine glucose were considered as DM. Then, DM mice were fed normally and executed under anaesthesia at 20, 28, and 36 weeks (w), respectively. Serum was collected to detect renal function related indicators (such as glucose, creatinine and urea nitrogen) by Automatic Biochemical Analyzer. Renal tissue fixed by 4% formaldehyde was used for pathological examination. hematoxylin-eosin staining (HE), Masson and schiff periodic acid shiff (PAS) staining were used to detect morphological changes, collagen fiber deposition, and glomerular basement membrane thickening; macrophage infiltration and expression levels of inflammatory factors were detected through immunohistochemical analysis. Besides, the localization of PRDX2 was detected by immunofluorescence assay (IFA). Moreover, renal tissue frozen at -80 ℃ was used for detection of protein expression levels of PRDX2 and macrophage polarization related molecules. Cell Culture and Treatment Cells were cultured with complete medium (containing 10% (v/v) fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin) in incubator at 37 ℃ with 5% CO2. Specifically, rat renal tubular epithelial cell line (NRK-52E) (SUNNCELL, SNL-027) was cultured in normal glucose DMEM medium containing 5.5 mM glucose (Gibco, 11885092), mice macrophages cell line (J774A.1) (SUNNCELL, SNL-333) was cultured in specialized DMEM medium (SUNNCELL, SNLM-333). Cells at the logarithmic growth phase are collected for experiments. For NRK-52E, cells were equally seeded in 6-well (NEST, 703001) or 12-well plates (NEST, 712001). After fully adherence and stretch, cells were treated with DMEM medium containing 5.5 mM of glucose in normal glucose group (NG group), with DMEM medium containing 30 mM of glucose (Gibco, C11995500BT) in high glucose group (HG group) and with DMEM medium containing 5.5 mM of glucose and 50 ng/mL TNF-α (MCE, HY-P7058) in NG + TNF-α group. After 6, 12, 24 and 36 hours (h), cells were collected for WB analysis of PRDX2 expression levels, respectively; After 24 h treatment, IFA was used to detect the expression and localization of PRDX2, and ROS fluorescence probes DCFH-DA (Solarbio, CA1410) combined with fluorescence microscopy were used to detect ROS levels in living cells. For J774A. 1, cells were equally seeded in 6-well or 12-well plates. After fully adherence and stretch, cells were treated with phosphate buffered saline (PBS) in negative control group (NC group), with 5 µg/mL recombinant PRDX2 (HZbscience, HZF757Mu01) in PRDX2 group, pre-treatment with or without 1 µM TLR4 specific inhibitor TAK242 (Targetmol, TQ0181) for 1 h. After 12 or 24 h, cells were collected for WB analysis to detect the protein expression levels of M1 and M2 polarization related protein; IFA was used to detect the co-localization of PRDX2 and TLR4, as well as localization of NF-κB p-p65 and STAT1. The migration ability of J774A.1 was observed through cell scratch experiments, and the phagocytic ability of J774A.1 was observed using fluorescence microsphere combined with fluorescence microscope. Detection of Protein Levels in Renal Tissue and Cells by WB A mixture of RIPA lysate (Epizyme, PC102) and protease inhibitor cocktail (Epizyme, GRF101) was used for suspension of collected tissue or cells. Ultrasonic Crusher was used for lysates preparation. Then lysates were centrifuged at 4°C at 12000 g for 10 minutes (min), the supernatant was collected and transfered to a clean centrifuge tube, the concentration of the supernatant was measured with a BCA protein concentration assay kit (Epizyme, ZJ101); meanwhile, protein sampling buffer (Epizyme, LT103) was added, samples were boiled at 95°C for 5 min, and cooled on ice. Thereafter, proteins in samples were separated by SDS-PAGE (Epizyme, PG212) and transferred to the PVDF membrane (Millipore, IPVH00010). PVDF membrane was blocked with 5% BSA for 1 h. Primary antibody used in WB including: β-actin (Proteintech, 81115-1-RR), TLR4 (Affinity, AF7017), α-SMA (Bioss, bs-10196R), STAT1 (Cell Signaling Technology, D1K9Y), PRDX2 (Abcam, ab109367), iNOS (Abcam, ab178945), Collagen I (Abcam, ab260043), TNF-α (Abcam, ab1793), IL-1β (Abcam, ab283818), NF-κB p65 (Cell Signaling Technology, D14E12), NF-κB p-p65 (Cell Signaling Technology, 3033S), Arg-1 (Cell Signaling Technology, 93668), IL-6 (Cell Signaling Technology, 12912S); secondary antibody used in WB was Goat Anti-Rabbit IgG H&L (HRP) (Invitrogen, 31460). Image J software was used for grayscale value analysis. Indirect Immunofluorescence Assay (IFA) For paraffin embedded tissue, slides were firstly dewaxed and hydrated by dimethylbenzene and ethanol with concentration gradient, followed by antigen retrieval via thermal repair method with microwave, then endogenous peroxidase blockage using H2O2 for further steps. For cultured cells, discard medium, wash twice with warm PBS, add warm 4% paraformaldehyde (Solarbio, P1110); 30 min later, wash twice with PBS for further steps. Both tissue and cell samples were then treated by 0.2% Triton X-100 (Solarbio, T8200) for 5 min at room temperature to increase cell membrane permeability, by 5% BSA (Solarbio, A8020) for 1 h for blocking, then sequentially by primary antibody and secondary antibody at room temperature for 1 h. Primary antibodies used in this experiment including: PRDX2 (Abcam, ab109367), TLR4 (Protientach, 6635-1-lg), iNOS (Abcam, ab178945), Arg-1 (Cell Signaling Technology, 93668); secondary antibodies used in this experiment including: FITC Goat Anti-Rabbit IgG (H + L) Antibody (APExBIO, K1203), AF594 Goat Anti-Rabbit IgG (H + L) Antibody (ZENBIO, 550043), CoraLite647-conjugated AffiniPure F(ab')2 Fragment Goat Anti-Rabbit IgG (H + L) (Proteintech, SA00014-9), CoraLite647-conjugated AffiniPure F (ab') 2 Fragment Goat Anti-Mouse IgG (H + L) (Proteintech ,SA00014-10); antifading mounting medium with 4',6-diamidino-2-phenylindole (DAPI) (Solarbio, S2110) was used for nuclear staining. Samples were observed and photographed using the Laser Confocal Fluorescence Microscope (Zeiss, LSM710) with same laser power. ROS Fluorescence Probes Assay NRK-52E at the logarithmic growth phase was collected for experiments. Cells were seeded in a 12-well plate (containing a preplaced cell climbing film in each well) with 60% cell density. After fully adherence and stretch, cells were treated with DMEM medium containing 5.5 mM of glucose in NG group, with DMEM medium containing 30 mM of glucose in HG group and with DMEM medium containing 5.5 mM of glucose and 50 ng/mL TNF-α in NG + TNF-α group. After 24 h, cells were used for ROS fluorescence probes assay. Dilute DCFH-DA (Solarbio, CA1410) with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 µM. Remove the cell culture medium and add 500 µL diluted DCFH-DA. Incubate in a 37 ℃ cell incubator for 20 min. Wash the cells three times with serum-free cell culture medium to fully remove DCFH-DA not entering cells. Hoechst33342 (Solarbio, IH0070) was used for nuclear staining. Images were observed and taken using the Laser Confocal Fluorescence Microscope with same laser power. Scratch Assay J774A. 1 at the logarithmic growth phase was collected for experiments. Cells were seeded in a 12-well plate with 80% cell density. After fully adherence and stretch, cells were pre-treated with / without the 1 µM TLR4 specific inhibitor TAK242 for 1 h. Then evenly draw a vertical line with a same pipette tip in each well, discard supernatant, wash twice with PBS, add serum-free DMEM medium with or without rPRDX2 (5 µg/mL). Images were observed and taken at 0 h and 24 h under microscope (Zeiss, Axio Observer) to record the scratch distance. Fluorescent Microsphere Assay J774A.1 at the logarithmic growth phase was collected for experiments. Cells were seeded in a 12-well plate (containing a preplaced cell climbing film in each well) with 60% cell density. After fully adherence and stretch, cells were pre-treated with / without the TLR4 specific inhibitor TAK242 (1 µM) for 1 h. Then diluted (1:500) Latex Beads with green fluorescence (Sigma-Aldrich, L4530) were added into culture medium, with / without rPRDX2 (5 µg/mL). After 24 h treatment, discard the culture medium, wash twice with warm PBS, add warm 4% paraformaldehyde; 30 min later, wash twice with PBS, add 0.2% Triton X-100; 5 min later, wash once with PBS. Antifading mounting medium with DAPI was used for nuclear staining. Images were observed and taken under fluorescent microscopy (Zeiss, Axio Imager A2). Statistical Analysis This study used GraphPad Prism 9 (GraphPad Software, Version 9.00, USA) for statistical analysis of data. All values included in the analysis of this study are continuous random values. All counting data are expressed as mean \(\pm\) standard deviation (Mean ± SD). The Shapiro Wilk method was used for normality testing. Based on the type of data distribution, independent sample t-tests or Wilcoxon tests were used to compare the differences between the two groups. One way ANOVA was used for multiple group comparisons, and p-values less than 0.05 were considered statistically significant. Data availability All data generated or analysed during this study are included in this published article [and its supplementary information files]. Declarations Acknowledgements Not applicable. Author contributions Conceptualization: LRL and BG. Methodology: XL, HHL, RP, XZ, SYZ, YY, MC, HXY, ZYL, and TW. Software: XL and HHL. Validation: RP, XZ, SYZ and QQZ. Writing—original draft preparation: XL, HHL, RP, XZ, SYZ, YY, MC and HXY. Writing—review and editing: LRL and BG. Supervision: LRL and BG. Project administration: LRL. Funding acquisition: XL, QQZ and LRL. All authors have read and agreed to the published version of the manuscript. Funding This work was supported by National Natural Science Foundation of China (82360079), Guizhou Provincial Natural Science Foundation (ZK[2023] general 377), Science and Technology Fund Project of Guizhou Provincial Health Commission (gzwkj2022-263) to XL, and was supported by National Natural Science Foundation of China (32060202) to LRL, Guizhou Provincial Natural Science Foundation (ZK[2023] general 379) to QQZ. Competing interests The authors declare no competing interests. Additional information Supplementary information The following supporting information can be downloaded: Fig. 7: comparison between human PRDX2 and parasitic protozoa Entamoeba histolytica (Eh)- PRDX. Fig. 8: Negative stain of recombinant human PRDX2 under 120 kv electron microscopy. Consent for publication Not applicable. References Lovic, D. et al. The Growing Epidemic of Diabetes Mellitus. Curr Vasc Pharmacol 18, 104–109 (2020). https://doi.org:10.2174/1570161117666190405165911 Tervaert, T. W. et al. Pathologic classification of diabetic nephropathy. J Am Soc Nephrol 21, 556–563 (2010). https://doi.org:10.1681/asn.2010010010 Collins, A. J. et al. 'United States Renal Data System 2011 Annual Data Report: Atlas of chronic kidney disease & end-stage renal disease in the United States. Am J Kidney Dis 59, A7, e1-420 (2012). https://doi.org:10.1053/j.ajkd.2011.11.015 Mima, A. Inflammation and oxidative stress in diabetic nephropathy: new insights on its inhibition as new therapeutic targets. J Diabetes Res 2013, 248563 (2013). https://doi.org:10.1155/2013/248563 Navarro, J. F. & Mora, C. Role of inflammation in diabetic complications. Nephrol Dial Transplant 20, 2601–2604 (2005). https://doi.org:10.1093/ndt/gfi155 Samsu, N. Diabetic Nephropathy: Challenges in Pathogenesis, Diagnosis, and Treatment. Biomed Res Int 2021, 1497449 (2021). https://doi.org:10.1155/2021/1497449 Navarro-González, J. F., Mora-Fernández, C., Muros de Fuentes, M. & García-Pérez, J. Inflammatory molecules and pathways in the pathogenesis of diabetic nephropathy. Nat Rev Nephrol 7, 327–340 (2011). https://doi.org:10.1038/nrneph.2011.51 Sikorski, K., Czerwoniec, A., Bujnicki, J. M., Wesoly, J. & Bluyssen, H. A. STAT1 as a novel therapeutical target in pro-atherogenic signal integration of IFNγ, TLR4 and IL-6 in vascular disease. Cytokine Growth Factor Rev 22, 211–219 (2011). https://doi.org:10.1016/j.cytogfr.2011.06.003 Fan, R., Wang, L., Botchway, B. O. A., Zhang, Y. & Liu, X. Protective role of ethyl pyruvate in spinal cord injury by inhibiting the high mobility group box-1/toll-like receptor4/nuclear factor-kappa B signaling pathway. Front Mol Neurosci 15, 1013033 (2022). https://doi.org:10.3389/fnmol.2022.1013033 Mudaliar, H., Pollock, C. & Panchapakesan, U. Role of Toll-like receptors in diabetic nephropathy. Clin Sci (Lond) 126, 685–694 (2014). https://doi.org:10.1042/cs20130267 Nguyen, D. et al. Macrophage accumulation in human progressive diabetic nephropathy. Nephrology (Carlton) 11, 226–231 (2006). https://doi.org:10.1111/j.1440-1797.2006.00576.x Furuta, T. et al. The role of macrophages in diabetic glomerulosclerosis. Am J Kidney Dis 21, 480–485 (1993). https://doi.org:10.1016/s0272-6386(12)80393-3 Hirata, K. et al. Increased expression of selectins in kidneys of patients with diabetic nephropathy. Diabetologia 41, 185–192 (1998). https://doi.org:10.1007/s001250050888 Bohle, A. et al. The pathogenesis of chronic renal failure in diabetic nephropathy. Investigation of 488 cases of diabetic glomerulosclerosis. Pathol Res Pract 187, 251–259 (1991). https://doi.org:10.1016/s0344-0338(11)80780-6 Wang, N., Liang, H. & Zen, K. Molecular mechanisms that influence the macrophage m1-m2 polarization balance. Front Immunol 5, 614 (2014). https://doi.org:10.3389/fimmu.2014.00614 Lawrence, T. & Natoli, G. Transcriptional regulation of macrophage polarization: enabling diversity with identity. Nat Rev Immunol 11, 750–761 (2011). https://doi.org:10.1038/nri3088 Orihuela, R., McPherson, C. A. & Harry, G. J. Microglial M1/M2 polarization and metabolic states. Br J Pharmacol 173, 649–665 (2016). https://doi.org:10.1111/bph.13139 Zhang, X. et al. Active vitamin D regulates macrophage M1/M2 phenotypes via the STAT-1-TREM-1 pathway in diabetic nephropathy. J Cell Physiol 234, 6917–6926 (2019). https://doi.org:10.1002/jcp.27450 Brezniceanu, M. L. et al. Reactive oxygen species promote caspase-12 expression and tubular apoptosis in diabetic nephropathy. J Am Soc Nephrol 21, 943–954 (2010). https://doi.org:10.1681/asn.2009030242 Hussain, T. et al. Oxidative Stress and Inflammation: What Polyphenols Can Do for Us? Oxid Med Cell Longev 2016, 7432797 (2016). https://doi.org:10.1155/2016/7432797 Latimer, H. R. & Veal, E. A. Peroxiredoxins in Regulation of MAPK Signalling Pathways; Sensors and Barriers to Signal Transduction. Mol Cells 39, 40–45 (2016). https://doi.org:10.14348/molcells.2016.2327 Netto, L. E. & Antunes, F. The Roles of Peroxiredoxin and Thioredoxin in Hydrogen Peroxide Sensing and in Signal Transduction. Mol Cells 39, 65–71 (2016). https://doi.org:10.14348/molcells.2016.2349 Li, Y. R., Zhu, H. & Danelisen, I. Role of Peroxiredoxins in Protecting Against Cardiovascular and Related Disorders. Cardiovasc Toxicol 20, 448–453 (2020). https://doi.org:10.1007/s12012-020-09588-0 Zhang, Q. et al. Sp1-mediated upregulation of Prdx6 expression prevents podocyte injury in diabetic nephropathy via mitigation of oxidative stress and ferroptosis. Life Sci 278, 119529 (2021). https://doi.org:10.1016/j.lfs.2021.119529 Jia, C. et al. Decoy receptor 2 mediation of the senescent phenotype of tubular cells by interacting with peroxiredoxin 1 presents a novel mechanism of renal fibrosis in diabetic nephropathy. Kidney Int 98, 645–662 (2020). https://doi.org:10.1016/j.kint.2020.03.026 Fan, Y. et al. Comparison of Kidney Transcriptomic Profiles of Early and Advanced Diabetic Nephropathy Reveals Potential New Mechanisms for Disease Progression. Diabetes 68, 2301–2314 (2019). https://doi.org:10.2337/db19-0204 Ahn, H. S. et al. Differential Urinary Proteome Analysis for Predicting Prognosis in Type 2 Diabetes Patients with and without Renal Dysfunction. Int J Mol Sci 21 (2020). https://doi.org:10.3390/ijms21124236 Salzano, S. et al. Linkage of inflammation and oxidative stress via release of glutathionylated peroxiredoxin-2, which acts as a danger signal. Proc Natl Acad Sci U S A 111, 12157–12162 (2014). https://doi.org:10.1073/pnas.1401712111 Li, X. et al. A Novel TLR4-Binding Domain of Peroxiredoxin From Entamoeba histolytica Triggers NLRP3 Inflammasome Activation in Macrophages. Front Immunol 12, 758451 (2021). https://doi.org:10.3389/fimmu.2021.758451 Wang, D. et al. TSLP/TSLPR promotes renal fibrosis by activating STAT3 in renal fibroblasts. Int Immunopharmacol 121, 110430 (2023). https://doi.org:10.1016/j.intimp.2023.110430 Seyahian, E. A., Cacciagiu, L., Damiano, A. E. & Zotta, E. AQP1 expression in the proximal tubule of diabetic rat kidney. Heliyon 6, e03192 (2020). https://doi.org:10.1016/j.heliyon.2020.e03192 Xie, L. et al. Tremella fuciformis Polysaccharide Induces Apoptosis of B16 Melanoma Cells via Promoting the M1 Polarization of Macrophages. Molecules 28 (2023). https://doi.org:10.3390/molecules28104018 Darenskaya, M., Kolesnikov, S., Semenova, N. & Kolesnikova, L. Diabetic Nephropathy: Significance of Determining Oxidative Stress and Opportunities for Antioxidant Therapies. Int J Mol Sci 24 (2023). https://doi.org:10.3390/ijms241512378 Smeijer, J. D. et al. Insulin resistance, kidney outcomes and effects of the endothelin receptor antagonist atrasentan in patients with type 2 diabetes and chronic kidney disease. Cardiovasc Diabetol 22, 251 (2023). https://doi.org:10.1186/s12933-023-01964-8 Shen, J. et al. Different types of cell death in diabetic endothelial dysfunction. Biomed Pharmacother 168, 115802 (2023). https://doi.org:10.1016/j.biopha.2023.115802 Abu Seman, N. & Othman, S. H. Recent Progress in Genetics and Epigenetics Research on Diabetic Nephropathy in Malaysia. J Diabetes Res 2023, 9053580 (2023). https://doi.org:10.1155/2023/9053580 de Paula, C. P. et al. Peroxiredoxins in erythrocytes: far beyond the antioxidant role. J Mol Med (Berl) 101, 1335–1353 (2023). https://doi.org:10.1007/s00109-023-02368-7 Meng, J. et al. Redox-stress response resistance (RRR) mediated by hyperoxidation of peroxiredoxin 2 in senescent cells. Sci China Life Sci 66, 2280–2294 (2023). https://doi.org:10.1007/s11427-022-2301-4 Additional Declarations No competing interests reported. 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Institute, Affiliated Hospital of Guizhou Medical University","correspondingAuthor":true,"prefix":"","firstName":"Lirong","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-01-26 02:44:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3898778/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3898778/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-79678-4","type":"published","date":"2024-11-16T15:57:40+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51136508,"identity":"7778ebfe-ecfb-412e-bba0-41b423f7115d","added_by":"auto","created_at":"2024-02-14 18:36:02","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":508409,"visible":true,"origin":"","legend":"\u003cp\u003eMice DKD model was successfully constructed. \u003cstrong\u003e(a)\u003c/strong\u003eModel construction flowchart. DM mice model was constructed by intraperitoneal injection of STZ (55 mg/kg), once per day for 5 consecutive days. Mice in control group was given the same volume of STZ solvent. After detection of 2 consecutive days, mice with random blood glucose level \u0026gt; 16.7 mmol/L and positive urine glucose were considered as DM. Then, DM mice were fed normally and executed under anaesthesia at 20, 28, and 36 w, respectively. Serum and kidney tissue were collected for biochemical and pathological analysis. \u003cstrong\u003e(b)\u003c/strong\u003eSerological analysis. Including random blood glucose (BG), blood urea nitrogen (BUN), and blood creatinine (CRE) in mice; n=4-6, a, b, c, DM vs NC, * \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05. \u003cstrong\u003e(c)\u003c/strong\u003e Pathological analysis. Bars: 50 μm; Black arrows, damaged renal tubular brush border microvillus; Red arrows, thickening of glomerular basement membrane. Quantitative analysis of positive areas of Masson staining was performed using image J software. n=3, * \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3898778/v1/c5bf32b750ff0c17f621140f.jpg"},{"id":51137315,"identity":"26051e07-692d-4584-9011-18292233e80a","added_by":"auto","created_at":"2024-02-14 18:44:01","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":757002,"visible":true,"origin":"","legend":"\u003cp\u003ePRDX2 showed a positive correlation with M1 polarization during DKD progression. \u003cstrong\u003e(a) \u003c/strong\u003eImmunohistochemical analysis. At 20, 28 and 36 w after DM model construction, the infiltration of macrophages (F4/80) and distribution of inflammatory related factors (IL-1β, IL-6 and TNF-α) in the kidney tissue were detected. Positive cell count and positive area analysis were performed by image J software. Data were represented by x ± SD. n=3, *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001. \u003cstrong\u003e(b)\u003c/strong\u003e The expression levels of PRDX2, M1 polarization, inflammatory and fibrosis related factors were detected by WB at 20, 28, and 36 w after DM model construction. Macrophage markers, F4/80; Molecules related to M1 polarization signaling pathway, TLR4, NF-κB p65, NF-κB p-p65, STAT1; Marker of M1 polarization, iNOS. Marker of M2 polarization, Arg-1; Inflammatory factors, IL-6, TNF-α, IL-1β. Grayscale value was quantified by image J software. β-actin was used as an internal reference. Data was represented as x ± SD, n=3. Pearson correlation analysis was performed for the correlation analysis of each line.\u003c/p\u003e","description":"","filename":"floatimage2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3898778/v1/7a2e618fef45c759173d56ab.jpg"},{"id":51137313,"identity":"4107bba9-68e5-4542-8c09-3b579791813f","added_by":"auto","created_at":"2024-02-14 18:44:01","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":467536,"visible":true,"origin":"","legend":"\u003cp\u003eHG and TNF-α can stimulate renal tubular epithelial cells to upregulate PRDX2 under DM condition. \u003cstrong\u003e(a) \u003c/strong\u003eIFA analysis for DKD renal tissue. Purplish red, α- SMA; red, AQP1; green, PRDX2; blue, DAPI. Observation and collection of images were performed by a laser confocal microscope with same laser power. Bars: 50 μm; White arrows, co-localization of PRDX2 and α-SMA. \u003cstrong\u003e(b) \u003c/strong\u003eDetection of the protein expression level of PRDX2 in NRK52E cells. Cells were treated by DMEM with HG (30 mM) or TNF-α (50 ng/mL), DMEM with NG (5.5 mM) was used as a negative control (NC). After treatment for 6, 12, 24, and 36 h, cells were collected and the lysate was subjected to WB analysis. Use image J software for grayscale value detection. β-actin was used as an internal reference for relative expression level analysis, and the data was represented by x ± SD. n=3, ns, not significant, *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001. \u003cstrong\u003e(c)\u003c/strong\u003e Detection of PRDX2 localization in NRK52E cells. Cells (on coverslips) were treated by DMEM with HG (30 mM) or TNF-α (50 ng/mL), DMEM with NG (5.5 mM) was used as a negative control. After 24 h treatment, coverslips were detected by IFA. Observation and collection of images were performed by a laser confocal microscope with same laser power. Green, DCFH-DA; Blue, Hoechst33342; Bars: 20 μm. \u003cstrong\u003e(d) \u003c/strong\u003eDetection of ROS levels in NRK52E cells after treatment with HG and TNF-α. Cells were treated by DMEM with HG (30 mM) or TNF-α (50 ng/mL), DMEM with NG (5.5 mM) was used as a negative control. After 24 h, the ROS level in living cells was detected using a fluorescent probe DCFH-DA. Observation and collection of images were performed by a laser confocal microscope with same laser power. Green, DCFH-DA; Blue, Hoechst33342; Bars: 20 μm.\u003c/p\u003e","description":"","filename":"floatimage3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3898778/v1/8020b32914921af353c91b75.jpg"},{"id":51137840,"identity":"ba07ade4-c621-4677-985f-486136ad0d58","added_by":"auto","created_at":"2024-02-14 18:52:01","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":526400,"visible":true,"origin":"","legend":"\u003cp\u003eM1 polarization stimulated by rPRDX2. \u003cstrong\u003e(a)\u003c/strong\u003eDetection of M1 polarization related protein expression levels by WB. J774A.1 cells were treated by rPRDX2 (5 μg/mL), with equal volume of PBS as a negative control. After 12 and 24 h treatment, cells were collected and the lysate was subjected to WB analysis. Molecules related to M1 polarization signaling pathway, TLR4, NF-κB p65, NF- κB p-p65, STAT1, iNOS. Marker of M2 polarization, Arg-1. Inflammatory factors, IL-6, TNF-α, IL-1β. Grayscale value was quantified by image J software. β-actin was used as an internal reference. Data was represented by x ± SD, n=3, *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001. \u003cstrong\u003e(b)\u003c/strong\u003e Localization of PRDX2 and TLR4 in J774A.1 cells was detected by IFA. Cells (on coverslips) were pre-treated w/ or w/o the TLR4 specific inhibitor TAK242 (1 μM) for 1 h, then cells were treated w/ or w/o rPRDX2 (5 μg/mL), with equal volume PBS as a negative control (NC). After 24 h, coverslips were detected by IFA and immediately observe using a laser confocal microscope with same power. Red, TLR4; Green, PRDX2; Blue, DAPI. Bars: 5 μm. White arrows, co-localization.\u003c/p\u003e","description":"","filename":"floatimage4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3898778/v1/4a28afe35d351d492d781aa6.jpg"},{"id":51136503,"identity":"bae31e9a-cb4e-4e50-a6f4-30d47766ba04","added_by":"auto","created_at":"2024-02-14 18:36:01","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":389719,"visible":true,"origin":"","legend":"\u003cp\u003eM1 polarization of macrophages stimulated by rPRDX2 through TLR4. \u003cstrong\u003e(a)\u003c/strong\u003e M1 polarization in J774A. 1 cells stimulated by rPRDX2 through TLR4. Cells were pre-treated w/ or w/o the TLR4 specific inhibitor TAK242 (1 μM) for 1 h. Afterwards, cells were treated by rPRDX2 (5 μg/mL), with equal volume of PBS as negative control. After 24 h, cells were collected and the lysate was subjected to WB analysis. Molecules related to M1 polarization signaling pathway, NF-κB p65, NF- κB p-p65, STAT1, iNOS. Marker of M2 polarization, Arg-1. Inflammatory factors, IL-6, TNF-α, IL-1β. Grayscale value was quantified by image J software. β-actin was used as an internal reference. Data was represented by x ± SD, n=3, * \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, *** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, **** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001. \u003cstrong\u003e(b)\u003c/strong\u003e The localization of M1 polarization related key molecules was detected by IFA. J774A. 1 cells (on coverslips) were pre-treated w/ or w/o TAK242 (1 μM) for 1 h, then cells were treated w/ or w/o rPRDX2 (5 μg/mL), with equal volume PBS as a negative control (NC). After 24 h, coverslips were detected by IFA and immediately observe using a laser confocal microscope with same laser power. For macrophages polarization markers: green, iNOS; red, Arg-1; blue, DAPI. For molecules in M1 polarization signaling pathway: green, STAT1 or NF-κB p-p65; blue, DAPI. Bars: 5 μm. White arrows, localization in nucleus.\u003c/p\u003e","description":"","filename":"floatimage5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3898778/v1/9e64274c243510f082d7547b.jpg"},{"id":51136506,"identity":"36574be8-d4da-40f1-bb9f-198ac47347d1","added_by":"auto","created_at":"2024-02-14 18:36:01","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":899017,"visible":true,"origin":"","legend":"\u003cp\u003eMacrophage migration and phagocytosis could be enhanced by rPRDX2 through TLR4. \u003cstrong\u003e(a)\u003c/strong\u003e Scratch experiment was used to detect the cell migration ability. Cells were pre-treated w/ or w/o the TLR4 specific inhibitor TAK242 (1 μM) for 1 h. Afterwards, cells were treated by rPRDX2 (5 μg/mL), with equal volume of PBS as negative control. Cells were observed and recorded at the same position of each well at 0 and 24 h. The distance between the red dashed lines represented the width of the scratch. Bars, 20 μm; Data was represented by x ± SD, n=3, ns, not significant, **** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001. \u003cstrong\u003e(b)\u003c/strong\u003eFluorescence microsphere experiment was used to detect macrophage phagocytosis. Cells (on coverslips) were pre-treated w/ or w/o the TLR4 specific inhibitor TAK242 (1 μM) for 1 h. Afterwards, cells were treated by rPRDX2 (5 μg/mL), with equal volume of PBS as negative control. Meanwhile, green fluorescent latex microspheres (1:500) were added into wells. After 24 h treatment, coverslips were fixed with 4% PFA and stained with DAPI, then immediately observed using an upright fluorescence microscope. Green, green latex microspheres; Blue, DAPI; Bars: 20 μm.\u003c/p\u003e","description":"","filename":"floatimage6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3898778/v1/cbef04b8968aaeca1b0fe38e.jpg"},{"id":69286483,"identity":"91a4dd0a-2c6e-4980-8fbe-9cebff2e7022","added_by":"auto","created_at":"2024-11-18 19:35:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4180159,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3898778/v1/b1a5cc78-e678-4825-b6b9-856c0fd02c86.pdf"},{"id":51136510,"identity":"52c1e9e4-c659-45c3-9f05-a323f59dda69","added_by":"auto","created_at":"2024-02-14 18:36:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18077643,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationAnovelroleofperoxiredoxin2indiabetickidneydiseaseprogressionbyactivatingtheclassicallyactivatedmacrophages.docx","url":"https://assets-eu.researchsquare.com/files/rs-3898778/v1/eea7e8417c153fae0eabfc36.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A novel role of peroxiredoxin 2 in diabetic kidney disease progression by activating the classically activated macrophages","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiabetes mellitus (DM) has become a worldwide public health problem, the number of DM patients in the world has exceeded 425\u0026nbsp;million, and is expected to reach 629\u0026nbsp;million in 2045 \u003csup\u003e1\u003c/sup\u003e. Diabetic kidney disease (DKD) is the most common microvascular complication of DM, which is characterized by glomerular basement membrane thickening, mesangial dilation, and tuberous sclerosis, leading to decreased renal function, and ultimately end-stage renal disease (ESRD) \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Due to the insidious onset and lack of specific treatment targets, DKD has been the main cause of ESRD in adults \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Therefore, exploring the detailed pathogenic mechanism of DKD can provide a basis for researches on targeted therapy.\u003c/p\u003e \u003cp\u003eThe infiltration of inflammatory cells (such as macrophages) is crucial for the occurrence and development of DKD \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Janus kinase (JAK) / signal transducer, activator of transcription (STAT)1 as well as nuclear factor-κB (NF-κB) signaling pathways are key promoters of the inflammation in DKD, which are involved in expression of pro-inflammatory molecules, including interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α (TNF)-α \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Wherein, activation of both STAT1 and NF-κB are closely related to toll like receptors (TLRs), which are overexpressed in DKD renal tissue, with TLR4 playing a crucial role in persistent inflammation \u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMacrophages are key to the progression of DKD. Renal biopsy analysis of DM patients confirmed the presence of macrophages in the glomerulus and renal interstitium at all stages of DKD, which is related to the progression of DKD \u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Macrophages can be classified into classically activated macrophages (M1) polarization and alternatively activated macrophages (M2) polarization based on their activation mechanisms and functions \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, with M1 exhibiting a pro-inflammatory phenotype and M2 exhibiting an anti-inflammatory phenotype. In the chronic inflammatory process of DKD, M1 is predominant with expression of characteristic molecules, such as TNF-α, IL-1β, IL-6 and inducible nitric oxide synthase (iNOS), which are associated with inflammation and tissue damage \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. It was reported that M1 polarization through TLR4 / NF-κB and JAK / STAT1 signaling pathways is one of the key mechanisms on the occurrence and development of DKD \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However, the key molecules initiating M1 polarization during development of DKD need further exploration.\u003c/p\u003e \u003cp\u003eOxidative stress is the main factor in the progression of DKD, which is characterized by the excessive ROS under hyperglycemia \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. ROS can activate various transcription factors, promote the expression of inflammation related genes, resulting in the occurrence and development of diseases \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Therefore, various antioxidants have been evolved in cells to eliminate excessive ROS. In addition to superoxide dismutase, catalase and glutathione peroxidase, the role of peroxiredoxin (PRDX) is gradually receiving attention. PRDX is a family of structurally conserved oxidoreductases commonly found in organisms; In addition to functioning as antioxidant enzymes, PRDX also participates as a signal transduction substance in regulating the response of cells to the surrounding environment under stress and non-stress conditions \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Mammalian cells express six PRDX subtypes, namely PRDX1 to PRDX6. PRDX1 and PRDX2 are located in the cytoplasm and nucleus, PRDX3 is located in the mitochondrial matrix, PRDX4 is located in the endoplasmic reticulum and can also be secreted into the extracellular environment, PRDX5 is present in mitochondria, peroxisomes, and cytoplasm, and PRDX6 is present in the cytoplasm \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The relevant researches on PRDX in the occurrence and development of DKD have only been reported in recent years: for example, PRDX4 can protect renal podocytes from oxidative damage \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, and PRDX1 can serve as a downstream molecule regulated by bait receptor 2, participating in the expression of pro-renal fibrosis related factors by regulating the aging of renal tubular epithelial cells \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eConsidering sufficient antioxidant molecules in cells, whether the PRDX family members have functional redundancy and perform other functions besides antioxidant effects has aroused our interest. However, it remains to be elucidated whether PRDX can activate inflammation and lead to DKD progression.\u003c/p\u003e \u003cp\u003eThe transcriptome data of kidney tissue from DKD patients (Gene Expression Omnibus, GSE142025) \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e showed that the transcription levels of the PRDX family in the kidney tissue of early and middle stage DKD patients were not significantly increased compared to the negative control group. Further urine proteomic analysis of DM patients (ProteomeXchange Datasets, PXD016571) \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e found that only PRDX1, 2, and 5 in PRDX family were detected, wherein PRDX2 had the highest abundance and was 2.6 times higher in DM patients with kidney injury than these without kidney injury. In mammalian cells, PRDX2 exist in the nucleus and cytoplasm \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. It was reported that PRDX2 can be released into the extracellular space under TNF-α stimulation in HEK293T cells, indicating the relationship of PRDX2 and inflammation \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. We recently found that PRDX of parasitic protozoa \u003cem\u003eEntamoeba histolytica\u003c/em\u003e (\u003cem\u003eEh\u003c/em\u003e-PRDX), which has a similar amino acid sequence and spatial structure to human PRDX2 (Fig.\u0026nbsp;7), can be overexpressed by parasites after invasion and then activate the inflammatory response in macrophages through TLR4 \u003csup\u003e29\u003c/sup\u003e. Whether mammalian PRDX2 has a similar pro-inflammatory effect, such as promoting M1 polarization of macrophages, has not been reported yet.\u003c/p\u003e \u003cp\u003eBriefly, we hypothesized that sustained hyperglycemia followed by excessive ROS in DM promotes expression and secretion of PRDX2 in renal cells, then PRDX2 can induce macrophage M1 polarization through TLR4, leading to the release of inflammatory factors, which in turn promote the expression of more PRDX2 in renal cells, forming a positive feedback loop, and ultimately promoting the progression of DKD. This study aimed to explore the linkage between mammalian PRDX2 up-regulation and macrophage M1 polarization at both animal and cellular levels under DM condition, to clarify the role of PRDX2 in the occurrence and development of DKD, and provide new evidence for the researches on targeted treatment in the future.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of Mice DKD Model\u003c/h2\u003e \u003cp\u003eSTZ was used for construction of DM mice model. 6\u0026ndash;8 w male C57BL/6 mice were injected intraperitoneally with STZ (55 mg/kg) once a day for 5 consecutive days. Then, DM mice (random blood glucose\u0026thinsp;\u0026ge;\u0026thinsp;16.7 mM) were selected and fed normally. They were anesthetized and executed at 20, 28, and 36 w, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Body weight and kidney weight were measured; Serum and kidney tissue were collected for biochemical and pathological analysis, respectively. The results showed that compared with the control group, the DM group had a significant decrease in body weight (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the kidney to body weight ratio (KW:BW) was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Serological analysis found that the random blood glucose (BG), blood urea nitrogen (BUN), and blood creatinine (CRE) of DM group mice gradually increased over time, and the differences at each time point were statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Kidney tissue was fixed with 4% PFA, embedded in paraffin, and sliced for pathological analysis. The results showed that there was significant deposition of renal collagen fibers, which gradually increased over time, and the difference at each time point was statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). In addition, damaged renal tubular brush border microvillus and thicken glomerular basement membrane can be observed at all time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Both serological and pathological results suggest that sustained DM status can lead to kidney damage. Therefore, this study has successfully constructed a mouse DKD model using STZ.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePositive Correlation between the Expression Levels of PRDX2 and M1 Polarization Related Proteins\u003c/h2\u003e \u003cp\u003eTo further confirm M1 polarization of macrophages in DKD kidney tissue, we first detected the distribution of macrophages in mouse kidney tissue through immunohistochemistry. The results showed that there was significant infiltration of macrophages in DKD kidney tissue (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), which was time-dependent (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Further detection of the distribution of M1 related inflammatory factors in renal tissues revealed that the distribution area of pro-inflammatory factor IL-1β, IL-6 and TNF-α in the tissue gradually increased with the progression of DKD, and the difference was statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Further detection by WB revealed a positive correlation between protein expression levels of PRDX2 and M1 polarization, inflammatory factors, or fibrosis related factors as DKD progressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The results above suggested that PRDX2 could play a role in M1 polarization and DKD progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eUpregulation of PRDX2 in Renal Tubular Epithelial Cells under DM Condition\u003c/h2\u003e \u003cp\u003eTo further investigate the localization of PRDX2 in DKD mice renal tissue, IFA was used to co-stain PRDX2 with α-SMA or AQP1. α-SMA is a marker of renal interstitial tissue, which is highly expressed in vascular smooth muscle cells and myofibroblasts \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e; AQP1 is a marker of renal tubule \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. PRDX2 was mainly distributed in renal tubule when observed by laser confocal microscopy with the same laser power; distribution of PRDX2 in the renal interstitial tissue was also observed with the progression of DKD; moreover, the fluorescence intensity of PRDX2 in renal tubule increased during DKD progression. The results suggested that renal tubular epithelial cells were the main source of PRDX2 in renal tissue, and PRDX2 might be secreted into the interstitial tissue to play a role as the disease progresses (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine whether high glucose (HG) or inflammatory factors (such as TNF-α) can upregulate PRDX2 in renal tubular epithelial cells, this study used HG (30 mM) or TNF-α (50 ng/mL) to treat the renal tubular epithelial cell line NRK52E, and detected the protein expression level of PRDX2 at 6, 12, 24, and 36 h, respectively. It was found that after treatment with HG or TNF-α for 6, 12, and 24 h, the protein expression level of PRDX2 significantly increased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05); while there was no statistically significant difference in the PRDX2 expression level between the TNF-α group and its control group at 36 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). To ensure the stability of cellular response, 24 h was selected as the subsequent experimental time point. The above experimental results suggested that under sustained DM conditions, HG or inflammatory factors (such as TNF-α) can stimulate renal tubular epithelial cells to express PRDX2. To investigate whether renal tubular epithelial cells under HG (HG) or inflammatory factor (such as TNF-α) stimulation undergo changes in PRDX2 localization, IFA was used for detection. Compared with the control group, PRDX2 can be transferred from the cell nucleus to the cytoplasm by high glucose (HG) or inflammatory factors (such as TNF-α) stimulation, and the increased fluorescence intensity were observed in the cytoplasm, indicating the linkage between PRDX2 localization and its secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Considering that PRDX2 is an antioxidant, we further used DCFH-DA fluorescence probe to detect intracellular ROS levels. The results showed that under high glucose (HG) or inflammatory factor (such as TNF-α) stimulation, there was a significant increase in intracellular ROS levels in renal tubular epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). The results above suggested that HG or TNF-α could stimulate renal tubular epithelial cells to express PRDX2, which was accompanied by changes in PRDX2 localization and an increase in intracellular ROS levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eRecombinant PRDX2 Can Upregulate M1 Polarization and Inflammatory Related Factors of Macrophages\u003c/h2\u003e \u003cp\u003eConsidering the similarity between PRDX2 and \u003cem\u003eEh\u003c/em\u003e-PRDX in protein sequence and spatial structure (Fig.\u0026nbsp;7), this study aimed to explore the ability of PRDX2 to promote inflammatory response in macrophages. After stimulation by rPRDX2 for 12 or 24 h, M1 polarization related molecules (marker, iNOS; inflammatory factors, IL-6, TNF-α and IL-1β; M1 polarization signaling pathway-related molecules, NF-κB p65, NF-κB p-p65, STAT1) were significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) after 24 h treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In addition, the M2 polarization marker Arg-1 slightly increased, but the difference was not statistically significant between treatment group and control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Considering the similarity between PRDX2 and \u003cem\u003eEh\u003c/em\u003e-PRDX, this study explored whether rPRDX2 showed co-localization with TLR4 on macrophages. Based on the observation that rPRDX2 under electron microscopy appears as 20\u0026ndash;30 nm polymer particles (Fig.\u0026nbsp;8), rPRDX2 attached on the cell membrane will show the bright and coarse fluorescent particles by IFA detection. Therefore, after treatment with rPRDX2 for 24 h, laser confocal microscopy was used to observe macrophages with the same laser power, it was found that resting macrophages have a basal expression of TLR4 and PRDX2, presenting fine and uniform particles distributed evenly in the cell; after addition of rPRDX2, aggregation of TLR4 in the periphery of macrophages was observed and presented as coarse particles, while distribution of PRDX2 was presented as scattered coarse particles over the periphery of macrophages, with scarce rPRDX2 particles observed in cytoplasm; importantly, co-localization of PRDX2 and TLR4 was observed near the surface of macrophage membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Results above suggested that rPRDX2 might promote M1 polarization of macrophages through TLR4.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eM1 Polarization of Macrophages Promoted by PRDX2 via TLR4\u003c/h2\u003e \u003cp\u003eTo further investigate whether PRDX2 functioned through TLR4, J774A. 1 cells were treated by rPRDX2 and collected for WB detection. After 24 h treatment, M1 polarization related molecules (marker, iNOS; inflammatory factors, IL-6 and TNF-α; M1 polarization signaling pathway-related molecules, NF-κB p65, NF-κB p-p65, STAT1) were significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) after 24 h treatment, which could be significantly inhibited by the TLR4 specific inhibitor TAK242 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor further confirming the changes of key molecules localization in macrophages after rPRDX2 stimulation, IFA was used for detection of STAT1 and NF-κB p-p65 in macrophages. Results showed that rPRDX2 can promote relocation of STAT1 and NF-κB p-p65 from cytoplasm to nucleus, which could be suppressed by TLR4 specific inhibitor TAK242 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Results above suggested that PRDX2 was capable of promoting M1 polarization of macrophages through TLR4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMacrophage Migration and Phagocytosis Promoted by PRDX2 via TLR4\u003c/h2\u003e \u003cp\u003eThe enhanced migration and phagocytic ability of macrophages are characteristic phenotypes of M1 polarization \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. To investigate whether PRDX2 could enhance macrophage migration, this study conducted scratch experiments. Photos were taken and recorded at the same location in each well during treatment with rPRDX2 at 0 and 24 h. The results showed that rPRDX2 significantly enhanced the migration ability of macrophages, which was significantly inhibited by the TLR4 specific inhibitor TAK242 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Afterwards, green fluorescent microspheres were used for Phagocytosis experiments to investigate whether PRDX2 could enhance macrophage phagocytic ability. It was found that macrophages had a certain phagocytic ability in a resting state, with most single cells capable of phagocytosing 2 to 4 microspheres; After stimulation with rPRDX2, the number of phagocytic microspheres in a single macrophage could reach over 10, and this effect could be significantly inhibited by the TLR4 specific inhibitor TAK242 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). These results suggested that PRDX2 can promote macrophage migration and phagocytic ability through TLR4.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eResearches on the pathogenesis of DKD mainly focus on oxidative stress \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, insulin resistance \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, cell death \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e and gene polymorphism \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. It is reported that oxidative stress plays an important role in the pathogenesis of DKD. Wherein, PRDX family members are believed to play an important role in the process of oxidative stress \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Transcriptomic analysis of kidney tissue from DKD patients revealed that PRDX family members were not significantly upregulated or downregulated at the transcriptional level (Gene Expression Omnibus, GSE142025) \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e; nevertheless, proteomic analysis of urine from DKD patients found that only PRDX1, 2, and 5 could be detected in urine, with the highest abundance of PRDX2, which was 2.6 times higher than that of DM patients (ProteomeXchange Datasets, PXD016571) \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. It is suggested that PRDX2 may be related to the occurrence and development of DKD.\u003c/p\u003e \u003cp\u003eThis study firstly found the role and mechanism of PRDX2 in the progression of DKD. In the kidney tissue of DKD mice, the positive correlation between the protein expression level of PRDX2 and the protein expression levels of renal fibrosis related molecules, macrophage M1 polarization related molecules or proinflammatory factors was detected and analyzed. Then, PRDX2 could be significantly upregulated in renal tubular epithelial cells stimulated by HG or TNF-α, explaining the reason for the upregulation of PRDX2 protein expression level in DKD renal tubule. Moreover, after stimulation with rPRDX2, the protein expression levels of M1 polarization related factors in macrophages were promoted, the migration and phagocytic ability of macrophages were also enhanced, which could be inhibited by TLR4 specific inhibitor TAK242. Results above suggested that PRDX2 was capable of promoting M1 polarization of macrophages through TLR4. Moreover, PRDX2 was up-regulated by TNF-α, and then it could promote production of TNF-α in macrophages, which formed positive feedback to promote persistent inflammation.\u003c/p\u003e \u003cp\u003eThe role of the PRDX family members is diverse, and in addition to antioxidant activity, they can also function as a molecular chaperone, transduce peroxide signal, protect membranes from damage, and play a role in maintaining iron homeostasis \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. A recent study found that PRDX2 participated in the cellular response to ROS and activated downstream signaling pathways through excessive oxidation by ROS within the cell. It was an important physiological process in cells and also a sign of cell youth; If a large amount of PRDX2 in the cell was excessively oxidized in a short period of time, it would lose its ability to respond to ROS, leading to aging; Due to the difficulty in reducing excessively oxidized PRDX2, oxidized PRDX2 may be secreted outside the cell as a byproduct in response to ROS \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. It was consistent with the findings of this study, the increase of ROS levels in renal tubular epithelial cells was induced by HG and TNF-α; Meanwhile, a large amount of PRDX2 could be transferred from the nucleus to the cytoplasm and aggregated into granules, indicating the possibility of PRDX2 secretion after ROS upregulation. In the kidney tissue of DKD mice, it was also found that PRDX2 was distributed in the renal interstitium in addition to the renal tubules, suggesting that PRDX2 could be secreted to the renal interstitium to play a role in the progress of DKD. Therefore, we speculated that PRDX2 might act as a by-product after antagonizing ROS in the progression of DKD, and then function as a proinflammatory factor by activating M1 polarization. However, whether specific knockout of PRDX2 in renal tissue can delay the progression of DKD, as well as the detailed molecular mechanism of PRDX2 upregulation and secretion in renal tubular epithelial cells, all require further exploration, which are also our research interests in future.\u003c/p\u003e \u003cp\u003eIn conclusion, this study found for the first time that the protein expression level of PRDX2 was positively correlated with the progression of DKD through \u003cem\u003ein vivo\u003c/em\u003e animal experiments; Combined with relevant technologies in pathology, immunology, and molecular biology, \u003cem\u003ein vitro cell\u003c/em\u003e experiments showed the ability of PRDX2 promoting M1 polarization via TLR4 after its up-regulation by high glucose or TNF-α, which formed positive feedback to promote persistent inflammation. This study has explored new mechanisms for the occurrence and development of DKD, and provided new theoretical basis for research related to the diagnosis and treatment of DKD in the future.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we found that PRDX2 could activate M1 polarization via TLR4. In summary, PRDX2 plays an important role in DKD. This study has explored new mechanisms for the occurrence and development of DKD, and provided new theoretical basis for research related to the diagnosis and treatment of DKD in the future.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEthics Statement\u003c/h2\u003e \u003cp\u003eAll animal experiments in our study were conducted in strict accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (1988.11.1), carried out in compliance with the ARRIVE (Animal Research: Reporting of \u003cem\u003eIn Vivo\u003c/em\u003e Experiments) guidelines and were approved by the Animal Care Welfare Committee of GuiZhou Medical University (Permit Numbers: 2201399). All efforts were made in our study to minimize the suffering of animals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAnimal Model Construction and Analysis\u003c/h2\u003e \u003cp\u003eSPF grade C57BL/6 mice (6\u0026ndash;8 w old, 18\u0026ndash;20 g weight) used in this study were purchased from Beijing Charles River Experimental Animal Technology Co., Ltd. Mice in experimental group were given 55 mg/kg Streptozotocin (STZ) (Sigma-Aldrich, 572201) dissolved in 0.1M sodium citrate buffer solution (pH 4.0) via intraperitoneal injection per day for 5 consecutive days, while mice in control group was given the same volume of STZ solvent. After detection of 2 consecutive days, mice with random blood glucose levels\u0026thinsp;\u0026gt;\u0026thinsp;16.7 mmol/L and positive urine glucose were considered as DM. Then, DM mice were fed normally and executed under anaesthesia at 20, 28, and 36 weeks (w), respectively.\u003c/p\u003e \u003cp\u003eSerum was collected to detect renal function related indicators (such as glucose, creatinine and urea nitrogen) by Automatic Biochemical Analyzer. Renal tissue fixed by 4% formaldehyde was used for pathological examination. hematoxylin-eosin staining (HE), Masson and schiff periodic acid shiff (PAS) staining were used to detect morphological changes, collagen fiber deposition, and glomerular basement membrane thickening; macrophage infiltration and expression levels of inflammatory factors were detected through immunohistochemical analysis. Besides, the localization of PRDX2 was detected by immunofluorescence assay (IFA). Moreover, renal tissue frozen at -80 ℃ was used for detection of protein expression levels of PRDX2 and macrophage polarization related molecules.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture and Treatment\u003c/h2\u003e \u003cp\u003eCells were cultured with complete medium (containing 10% (v/v) fetal bovine serum, 100 U/mL penicillin and 100 \u0026micro;g/mL streptomycin) in incubator at 37 ℃ with 5% CO2. Specifically, rat renal tubular epithelial cell line (NRK-52E) (SUNNCELL, SNL-027) was cultured in normal glucose DMEM medium containing 5.5 mM glucose (Gibco, 11885092), mice macrophages cell line (J774A.1) (SUNNCELL, SNL-333) was cultured in specialized DMEM medium (SUNNCELL, SNLM-333). Cells at the logarithmic growth phase are collected for experiments.\u003c/p\u003e \u003cp\u003eFor NRK-52E, cells were equally seeded in 6-well (NEST, 703001) or 12-well plates (NEST, 712001). After fully adherence and stretch, cells were treated with DMEM medium containing 5.5 mM of glucose in normal glucose group (NG group), with DMEM medium containing 30 mM of glucose (Gibco, C11995500BT) in high glucose group (HG group) and with DMEM medium containing 5.5 mM of glucose and 50 ng/mL TNF-α (MCE, HY-P7058) in NG\u0026thinsp;+\u0026thinsp;TNF-α group. After 6, 12, 24 and 36 hours (h), cells were collected for WB analysis of PRDX2 expression levels, respectively; After 24 h treatment, IFA was used to detect the expression and localization of PRDX2, and ROS fluorescence probes DCFH-DA (Solarbio, CA1410) combined with fluorescence microscopy were used to detect ROS levels in living cells.\u003c/p\u003e \u003cp\u003eFor J774A. 1, cells were equally seeded in 6-well or 12-well plates. After fully adherence and stretch, cells were treated with phosphate buffered saline (PBS) in negative control group (NC group), with 5 \u0026micro;g/mL recombinant PRDX2 (HZbscience, HZF757Mu01) in PRDX2 group, pre-treatment with or without 1 \u0026micro;M TLR4 specific inhibitor TAK242 (Targetmol, TQ0181) for 1 h. After 12 or 24 h, cells were collected for WB analysis to detect the protein expression levels of M1 and M2 polarization related protein; IFA was used to detect the co-localization of PRDX2 and TLR4, as well as localization of NF-κB p-p65 and STAT1. The migration ability of J774A.1 was observed through cell scratch experiments, and the phagocytic ability of J774A.1 was observed using fluorescence microsphere combined with fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDetection of Protein Levels in Renal Tissue and Cells by WB\u003c/h2\u003e \u003cp\u003eA mixture of RIPA lysate (Epizyme, PC102) and protease inhibitor cocktail (Epizyme, GRF101) was used for suspension of collected tissue or cells. Ultrasonic Crusher was used for lysates preparation. Then lysates were centrifuged at 4\u0026deg;C at 12000 g for 10 minutes (min), the supernatant was collected and transfered to a clean centrifuge tube, the concentration of the supernatant was measured with a BCA protein concentration assay kit (Epizyme, ZJ101); meanwhile, protein sampling buffer (Epizyme, LT103) was added, samples were boiled at 95\u0026deg;C for 5 min, and cooled on ice. Thereafter, proteins in samples were separated by SDS-PAGE (Epizyme, PG212) and transferred to the PVDF membrane (Millipore, IPVH00010). PVDF membrane was blocked with 5% BSA for 1 h. Primary antibody used in WB including: β-actin (Proteintech, 81115-1-RR), TLR4 (Affinity, AF7017), α-SMA (Bioss, bs-10196R), STAT1 (Cell Signaling Technology, D1K9Y), PRDX2 (Abcam, ab109367), iNOS (Abcam, ab178945), Collagen I (Abcam, ab260043), TNF-α (Abcam, ab1793), IL-1β (Abcam, ab283818), NF-κB p65 (Cell Signaling Technology, D14E12), NF-κB p-p65 (Cell Signaling Technology, 3033S), Arg-1 (Cell Signaling Technology, 93668), IL-6 (Cell Signaling Technology, 12912S); secondary antibody used in WB was Goat Anti-Rabbit IgG H\u0026amp;L (HRP) (Invitrogen, 31460). Image J software was used for grayscale value analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eIndirect Immunofluorescence Assay (IFA)\u003c/h2\u003e \u003cp\u003eFor paraffin embedded tissue, slides were firstly dewaxed and hydrated by dimethylbenzene and ethanol with concentration gradient, followed by antigen retrieval via thermal repair method with microwave, then endogenous peroxidase blockage using H2O2 for further steps. For cultured cells, discard medium, wash twice with warm PBS, add warm 4% paraformaldehyde (Solarbio, P1110); 30 min later, wash twice with PBS for further steps. Both tissue and cell samples were then treated by 0.2% Triton X-100 (Solarbio, T8200) for 5 min at room temperature to increase cell membrane permeability, by 5% BSA (Solarbio, A8020) for 1 h for blocking, then sequentially by primary antibody and secondary antibody at room temperature for 1 h. Primary antibodies used in this experiment including: PRDX2 (Abcam, ab109367), TLR4 (Protientach, 6635-1-lg), iNOS (Abcam, ab178945), Arg-1 (Cell Signaling Technology, 93668); secondary antibodies used in this experiment including: FITC Goat Anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) Antibody (APExBIO, K1203), AF594 Goat Anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) Antibody (ZENBIO, 550043), CoraLite647-conjugated AffiniPure F(ab')2 Fragment Goat Anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) (Proteintech, SA00014-9), CoraLite647-conjugated AffiniPure F (ab') 2 Fragment Goat Anti-Mouse IgG (H\u0026thinsp;+\u0026thinsp;L) (Proteintech ,SA00014-10); antifading mounting medium with 4',6-diamidino-2-phenylindole (DAPI) (Solarbio, S2110) was used for nuclear staining. Samples were observed and photographed using the Laser Confocal Fluorescence Microscope (Zeiss, LSM710) with same laser power.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eROS Fluorescence Probes Assay\u003c/h2\u003e \u003cp\u003eNRK-52E at the logarithmic growth phase was collected for experiments. Cells were seeded in a 12-well plate (containing a preplaced cell climbing film in each well) with 60% cell density. After fully adherence and stretch, cells were treated with DMEM medium containing 5.5 mM of glucose in NG group, with DMEM medium containing 30 mM of glucose in HG group and with DMEM medium containing 5.5 mM of glucose and 50 ng/mL TNF-α in NG\u0026thinsp;+\u0026thinsp;TNF-α group. After 24 h, cells were used for ROS fluorescence probes assay.\u003c/p\u003e \u003cp\u003eDilute DCFH-DA (Solarbio, CA1410) with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 \u0026micro;M. Remove the cell culture medium and add 500 \u0026micro;L diluted DCFH-DA. Incubate in a 37 ℃ cell incubator for 20 min. Wash the cells three times with serum-free cell culture medium to fully remove DCFH-DA not entering cells. Hoechst33342 (Solarbio, IH0070) was used for nuclear staining. Images were observed and taken using the Laser Confocal Fluorescence Microscope with same laser power.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eScratch Assay\u003c/h2\u003e \u003cp\u003eJ774A. 1 at the logarithmic growth phase was collected for experiments. Cells were seeded in a 12-well plate with 80% cell density. After fully adherence and stretch, cells were pre-treated with / without the 1 \u0026micro;M TLR4 specific inhibitor TAK242 for 1 h. Then evenly draw a vertical line with a same pipette tip in each well, discard supernatant, wash twice with PBS, add serum-free DMEM medium with or without rPRDX2 (5 \u0026micro;g/mL). Images were observed and taken at 0 h and 24 h under microscope (Zeiss, Axio Observer) to record the scratch distance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eFluorescent Microsphere Assay\u003c/h2\u003e \u003cp\u003eJ774A.1 at the logarithmic growth phase was collected for experiments. Cells were seeded in a 12-well plate (containing a preplaced cell climbing film in each well) with 60% cell density. After fully adherence and stretch, cells were pre-treated with / without the TLR4 specific inhibitor TAK242 (1 \u0026micro;M) for 1 h. Then diluted (1:500) Latex Beads with green fluorescence (Sigma-Aldrich, L4530) were added into culture medium, with / without rPRDX2 (5 \u0026micro;g/mL). After 24 h treatment, discard the culture medium, wash twice with warm PBS, add warm 4% paraformaldehyde; 30 min later, wash twice with PBS, add 0.2% Triton X-100; 5 min later, wash once with PBS. Antifading mounting medium with DAPI was used for nuclear staining. Images were observed and taken under fluorescent microscopy (Zeiss, Axio Imager A2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThis study used GraphPad Prism 9 (GraphPad Software, Version 9.00, USA) for statistical analysis of data. All values included in the analysis of this study are continuous random values. All counting data are expressed as mean \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\pm\\)\u003c/span\u003e\u003c/span\u003e standard deviation (Mean \u0026plusmn; SD). The Shapiro Wilk method was used for normality testing. Based on the type of data distribution, independent sample t-tests or Wilcoxon tests were used to compare the differences between the two groups. One way ANOVA was used for multiple group comparisons, and p-values less than 0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eAll data generated or analysed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: LRL and BG. Methodology: XL, HHL, RP, XZ, SYZ, YY, MC, HXY, ZYL, and TW. Software: XL and HHL. Validation: RP, XZ, SYZ and QQZ. Writing\u0026mdash;original draft preparation: XL, HHL, RP, XZ, SYZ, YY, MC and HXY. Writing\u0026mdash;review and editing: LRL and BG. Supervision: LRL and BG. Project administration: LRL. Funding acquisition: XL, QQZ and LRL. All authors have read and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China (82360079), Guizhou Provincial Natural Science Foundation (ZK[2023] general 377), Science and Technology Fund Project of Guizhou Provincial Health Commission (gzwkj2022-263) to XL, and was supported by National Natural Science Foundation of China (32060202) to LRL, Guizhou Provincial Natural Science Foundation (ZK[2023] general 379) to QQZ.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003einformation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe following supporting information can be downloaded: Fig. 7:\u0026nbsp;comparison between human PRDX2 and\u0026nbsp;parasitic protozoa\u003cem\u003e\u0026nbsp;Entamoeba histolytica (Eh)-\u003c/em\u003ePRDX. Fig. 8: Negative stain of recombinant human PRDX2 under 120 kv electron microscopy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLovic, D. \u003cem\u003eet al.\u003c/em\u003e The Growing Epidemic of Diabetes Mellitus. Curr Vasc Pharmacol 18, 104\u0026ndash;109 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.2174/1570161117666190405165911\u003c/span\u003e\u003cspan address=\"https://doi.org:10.2174/1570161117666190405165911\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTervaert, T. W. \u003cem\u003eet al.\u003c/em\u003e Pathologic classification of diabetic nephropathy. J Am Soc Nephrol 21, 556\u0026ndash;563 (2010). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1681/asn.2010010010\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1681/asn.2010010010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCollins, A. J. \u003cem\u003eet al.\u003c/em\u003e 'United States Renal Data System 2011 Annual Data Report: Atlas of chronic kidney disease \u0026amp; end-stage renal disease in the United States. Am J Kidney Dis 59, A7, e1-420 (2012). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1053/j.ajkd.2011.11.015\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1053/j.ajkd.2011.11.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMima, A. Inflammation and oxidative stress in diabetic nephropathy: new insights on its inhibition as new therapeutic targets. \u003cem\u003eJ Diabetes Res\u003c/em\u003e 2013, 248563 (2013). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1155/2013/248563\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1155/2013/248563\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNavarro, J. F. \u0026amp; Mora, C. Role of inflammation in diabetic complications. Nephrol Dial Transplant 20, 2601\u0026ndash;2604 (2005). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1093/ndt/gfi155\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1093/ndt/gfi155\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSamsu, N. Diabetic Nephropathy: Challenges in Pathogenesis, Diagnosis, and Treatment. \u003cem\u003eBiomed Res Int\u003c/em\u003e 2021, 1497449 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1155/2021/1497449\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1155/2021/1497449\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNavarro-Gonz\u0026aacute;lez, J. F., Mora-Fern\u0026aacute;ndez, C., Muros de Fuentes, M. \u0026amp; Garc\u0026iacute;a-P\u0026eacute;rez, J. Inflammatory molecules and pathways in the pathogenesis of diabetic nephropathy. Nat Rev Nephrol 7, 327\u0026ndash;340 (2011). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/nrneph.2011.51\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/nrneph.2011.51\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSikorski, K., Czerwoniec, A., Bujnicki, J. M., Wesoly, J. \u0026amp; Bluyssen, H. A. STAT1 as a novel therapeutical target in pro-atherogenic signal integration of IFNγ, TLR4 and IL-6 in vascular disease. Cytokine Growth Factor Rev 22, 211\u0026ndash;219 (2011). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.cytogfr.2011.06.003\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.cytogfr.2011.06.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan, R., Wang, L., Botchway, B. O. A., Zhang, Y. \u0026amp; Liu, X. Protective role of ethyl pyruvate in spinal cord injury by inhibiting the high mobility group box-1/toll-like receptor4/nuclear factor-kappa B signaling pathway. Front Mol Neurosci 15, 1013033 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.3389/fnmol.2022.1013033\u003c/span\u003e\u003cspan address=\"https://doi.org:10.3389/fnmol.2022.1013033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMudaliar, H., Pollock, C. \u0026amp; Panchapakesan, U. Role of Toll-like receptors in diabetic nephropathy. Clin Sci (Lond) 126, 685\u0026ndash;694 (2014). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1042/cs20130267\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1042/cs20130267\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen, D. \u003cem\u003eet al.\u003c/em\u003e Macrophage accumulation in human progressive diabetic nephropathy. Nephrology (Carlton) 11, 226\u0026ndash;231 (2006). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1111/j.1440-1797.2006.00576.x\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1111/j.1440-1797.2006.00576.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFuruta, T. \u003cem\u003eet al.\u003c/em\u003e The role of macrophages in diabetic glomerulosclerosis. Am J Kidney Dis 21, 480\u0026ndash;485 (1993). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/s0272-6386(12)80393-3\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/s0272-6386(12)80393-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHirata, K. \u003cem\u003eet al.\u003c/em\u003e Increased expression of selectins in kidneys of patients with diabetic nephropathy. Diabetologia 41, 185\u0026ndash;192 (1998). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1007/s001250050888\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1007/s001250050888\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBohle, A. \u003cem\u003eet al.\u003c/em\u003e The pathogenesis of chronic renal failure in diabetic nephropathy. Investigation of 488 cases of diabetic glomerulosclerosis. Pathol Res Pract 187, 251\u0026ndash;259 (1991). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/s0344-0338(11)80780-6\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/s0344-0338(11)80780-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, N., Liang, H. \u0026amp; Zen, K. Molecular mechanisms that influence the macrophage m1-m2 polarization balance. Front Immunol 5, 614 (2014). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.3389/fimmu.2014.00614\u003c/span\u003e\u003cspan address=\"https://doi.org:10.3389/fimmu.2014.00614\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLawrence, T. \u0026amp; Natoli, G. Transcriptional regulation of macrophage polarization: enabling diversity with identity. Nat Rev Immunol 11, 750\u0026ndash;761 (2011). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1038/nri3088\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1038/nri3088\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrihuela, R., McPherson, C. A. \u0026amp; Harry, G. J. Microglial M1/M2 polarization and metabolic states. Br J Pharmacol 173, 649\u0026ndash;665 (2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1111/bph.13139\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1111/bph.13139\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, X. \u003cem\u003eet al.\u003c/em\u003e Active vitamin D regulates macrophage M1/M2 phenotypes via the STAT-1-TREM-1 pathway in diabetic nephropathy. J Cell Physiol 234, 6917\u0026ndash;6926 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1002/jcp.27450\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1002/jcp.27450\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrezniceanu, M. L. \u003cem\u003eet al.\u003c/em\u003e Reactive oxygen species promote caspase-12 expression and tubular apoptosis in diabetic nephropathy. J Am Soc Nephrol 21, 943\u0026ndash;954 (2010). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1681/asn.2009030242\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1681/asn.2009030242\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHussain, T. \u003cem\u003eet al.\u003c/em\u003e Oxidative Stress and Inflammation: What Polyphenols Can Do for Us? \u003cem\u003eOxid Med Cell Longev\u003c/em\u003e 2016, 7432797 (2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1155/2016/7432797\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1155/2016/7432797\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLatimer, H. R. \u0026amp; Veal, E. A. Peroxiredoxins in Regulation of MAPK Signalling Pathways; Sensors and Barriers to Signal Transduction. Mol Cells 39, 40\u0026ndash;45 (2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.14348/molcells.2016.2327\u003c/span\u003e\u003cspan address=\"https://doi.org:10.14348/molcells.2016.2327\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNetto, L. E. \u0026amp; Antunes, F. The Roles of Peroxiredoxin and Thioredoxin in Hydrogen Peroxide Sensing and in Signal Transduction. Mol Cells 39, 65\u0026ndash;71 (2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.14348/molcells.2016.2349\u003c/span\u003e\u003cspan address=\"https://doi.org:10.14348/molcells.2016.2349\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, Y. R., Zhu, H. \u0026amp; Danelisen, I. Role of Peroxiredoxins in Protecting Against Cardiovascular and Related Disorders. Cardiovasc Toxicol 20, 448\u0026ndash;453 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1007/s12012-020-09588-0\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1007/s12012-020-09588-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, Q. \u003cem\u003eet al.\u003c/em\u003e Sp1-mediated upregulation of Prdx6 expression prevents podocyte injury in diabetic nephropathy via mitigation of oxidative stress and ferroptosis. Life Sci 278, 119529 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.lfs.2021.119529\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.lfs.2021.119529\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJia, C. \u003cem\u003eet al.\u003c/em\u003e Decoy receptor 2 mediation of the senescent phenotype of tubular cells by interacting with peroxiredoxin 1 presents a novel mechanism of renal fibrosis in diabetic nephropathy. Kidney Int 98, 645\u0026ndash;662 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.kint.2020.03.026\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.kint.2020.03.026\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan, Y. \u003cem\u003eet al.\u003c/em\u003e Comparison of Kidney Transcriptomic Profiles of Early and Advanced Diabetic Nephropathy Reveals Potential New Mechanisms for Disease Progression. Diabetes 68, 2301\u0026ndash;2314 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.2337/db19-0204\u003c/span\u003e\u003cspan address=\"https://doi.org:10.2337/db19-0204\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhn, H. S. \u003cem\u003eet al.\u003c/em\u003e Differential Urinary Proteome Analysis for Predicting Prognosis in Type 2 Diabetes Patients with and without Renal Dysfunction. Int J Mol Sci 21 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.3390/ijms21124236\u003c/span\u003e\u003cspan address=\"https://doi.org:10.3390/ijms21124236\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalzano, S. \u003cem\u003eet al.\u003c/em\u003e Linkage of inflammation and oxidative stress via release of glutathionylated peroxiredoxin-2, which acts as a danger signal. Proc Natl Acad Sci U S A 111, 12157\u0026ndash;12162 (2014). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1073/pnas.1401712111\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1073/pnas.1401712111\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, X. \u003cem\u003eet al.\u003c/em\u003e A Novel TLR4-Binding Domain of Peroxiredoxin From Entamoeba histolytica Triggers NLRP3 Inflammasome Activation in Macrophages. Front Immunol 12, 758451 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.3389/fimmu.2021.758451\u003c/span\u003e\u003cspan address=\"https://doi.org:10.3389/fimmu.2021.758451\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, D. \u003cem\u003eet al.\u003c/em\u003e TSLP/TSLPR promotes renal fibrosis by activating STAT3 in renal fibroblasts. Int Immunopharmacol 121, 110430 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.intimp.2023.110430\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.intimp.2023.110430\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeyahian, E. A., Cacciagiu, L., Damiano, A. E. \u0026amp; Zotta, E. AQP1 expression in the proximal tubule of diabetic rat kidney. Heliyon 6, e03192 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.heliyon.2020.e03192\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.heliyon.2020.e03192\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie, L. \u003cem\u003eet al.\u003c/em\u003e Tremella fuciformis Polysaccharide Induces Apoptosis of B16 Melanoma Cells via Promoting the M1 Polarization of Macrophages. Molecules 28 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.3390/molecules28104018\u003c/span\u003e\u003cspan address=\"https://doi.org:10.3390/molecules28104018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDarenskaya, M., Kolesnikov, S., Semenova, N. \u0026amp; Kolesnikova, L. Diabetic Nephropathy: Significance of Determining Oxidative Stress and Opportunities for Antioxidant Therapies. Int J Mol Sci 24 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.3390/ijms241512378\u003c/span\u003e\u003cspan address=\"https://doi.org:10.3390/ijms241512378\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmeijer, J. D. \u003cem\u003eet al.\u003c/em\u003e Insulin resistance, kidney outcomes and effects of the endothelin receptor antagonist atrasentan in patients with type 2 diabetes and chronic kidney disease. Cardiovasc Diabetol 22, 251 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1186/s12933-023-01964-8\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1186/s12933-023-01964-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen, J. \u003cem\u003eet al.\u003c/em\u003e Different types of cell death in diabetic endothelial dysfunction. Biomed Pharmacother 168, 115802 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.biopha.2023.115802\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.biopha.2023.115802\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbu Seman, N. \u0026amp; Othman, S. H. Recent Progress in Genetics and Epigenetics Research on Diabetic Nephropathy in Malaysia. \u003cem\u003eJ Diabetes Res\u003c/em\u003e 2023, 9053580 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1155/2023/9053580\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1155/2023/9053580\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Paula, C. P. \u003cem\u003eet al.\u003c/em\u003e Peroxiredoxins in erythrocytes: far beyond the antioxidant role. J Mol Med (Berl) 101, 1335\u0026ndash;1353 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1007/s00109-023-02368-7\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1007/s00109-023-02368-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeng, J. \u003cem\u003eet al.\u003c/em\u003e Redox-stress response resistance (RRR) mediated by hyperoxidation of peroxiredoxin 2 in senescent cells. Sci China Life Sci 66, 2280\u0026ndash;2294 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1007/s11427-022-2301-4\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1007/s11427-022-2301-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3898778/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3898778/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDiabetic kidney disease (DKD) is the main cause of death due to diabetes mellitus (DM). Due to the complexity of the onset, it is difficult to achieve accurate prevention and treatment. The classically activated macrophages (M1) polarization is a crucial proinflammatory mechanism in DKD, while the interaction and cascade effects of oxidative stress and inflammatory response remain to be elucidated. A urine proteomic analysis of DM patients indicated that peroxiredoxin 2 (PRDX2) had the higher abundance in DKD. We recently found that PRDX of parasitic protozoa \u003cem\u003eEntamoeba histolytica\u003c/em\u003e, which was similar to human PRDX2 in amino acid sequence and spatial structure, could activate the inflammatory response of macrophages through toll like receptor 4 (TLR4). Hence, our study was designed to explore the role of PRDX2 in chronic inflammation during DKD. Combined with \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments, results showed that the PRDX2 was positively correlated with DKD progression and upregulated by high glucose or recombinant tumor necrosis factor-α in renal tubular epithelial cells; Besides, recombinant PRDX2 could promote macrophages M1 polarization, enhance the migration and phagocytic ability of macrophages through TLR4. In summary, our study has explored the novel role of PRDX2 in DKD to provide basis for further researches on the diagnosis and treatment of DKD.\u003c/p\u003e","manuscriptTitle":"A novel role of peroxiredoxin 2 in diabetic kidney disease progression by activating the classically activated macrophages","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-14 18:35:56","doi":"10.21203/rs.3.rs-3898778/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-29T09:21:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-16T02:44:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"185147431854687514108420497983365639756","date":"2024-07-06T01:22:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-01T05:49:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-01T05:48:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"68486262183932687203085358351374202999","date":"2024-06-19T01:29:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-04T06:48:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-28T15:07:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"31fe6eca-f834-42e1-ae45-d662d97bf62b","date":"2024-02-20T12:46:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"fadaaea4-42ff-4c69-8968-4f4bff6982e4","date":"2024-02-18T23:46:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-18T15:34:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-13T06:51:29+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-02-13T06:49:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-13T06:48:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-01-26T02:40:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"84e65b5d-6f38-46ad-a036-a1dbfca05906","owner":[],"postedDate":"February 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28743703,"name":"Biological sciences/Cell biology"},{"id":28743704,"name":"Biological sciences/Immunology"},{"id":28743705,"name":"Biological sciences/Molecular biology"},{"id":28743706,"name":"Biological sciences/Physiology"},{"id":28743707,"name":"Health sciences/Biomarkers"},{"id":28743708,"name":"Health sciences/Endocrinology"}],"tags":[],"updatedAt":"2024-11-18T19:35:49+00:00","versionOfRecord":{"articleIdentity":"rs-3898778","link":"https://doi.org/10.1038/s41598-024-79678-4","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-11-16 15:57:40","publishedOnDateReadable":"November 16th, 2024"},"versionCreatedAt":"2024-02-14 18:35:56","video":"","vorDoi":"10.1038/s41598-024-79678-4","vorDoiUrl":"https://doi.org/10.1038/s41598-024-79678-4","workflowStages":[]},"version":"v1","identity":"rs-3898778","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3898778","identity":"rs-3898778","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}