Peroxiredoxin 1 aggravates acute kidney injury by promoting inflammation through Mincle/Syk/NF-κB signaling

Damage-associated molecular patterns (DAMPs) are a cause of acute kidney injury (AKI). Our knowledge of these DAMPs remains incomplete. Here, we report serum peroxiredoxin 1 (Prdx1) as a novel DAMP for AKI. Lipopolysaccharide (LPS) and kidney ischemia/reperfusion injury instigated AKI with concurrent increases in serum Prdx1 and reductions of Prdx1 expression in kidney tubular epithelial cells. Genetic knockout of Prdx1 or use of a Prdx1–neutralizing antibody protected mice from AKI and this protection was impaired by introduction of recombinant Prdx1 (rPrdx1). Mechanistically, lipopolysaccharide increased serum and kidney proinflammatory cytokines, macrophage infiltration, and the content of M1 macrophages. All these events were suppressed in Prdx1-/- mice and renewed upon introduction of rPrdx1. In primary peritoneal macrophages, rPrdx1 induced M1 polarization, activated macrophage-inducible C-type lectin (Mincle) signaling, and enhanced proinflammatory cytokine production. Prdx1 interacted with Mincle to initiate acute kidney inflammation. Of note, rPrdx1 upregulated Mincle and the spleen tyrosine kinase Syk system in the primary peritoneal macrophages, while knockdown of Mincle abolished the increase in activated Syk. Additionally, rPrdx1 treatment enhanced the downstream events of Syk, including transcription factor NF-κB signaling pathways. Furthermore, serum Prdx1 was found to be increased in patients with AKI; the increase of which was associated with kidney function decline and inflammatory biomarkers in patient serum. Thus, kidney-derived serum Prdx1 contributes to AKI at least in part by activating Mincle signaling and downstream pathways. Damage-associated molecular patterns (DAMPs) are a cause of acute kidney injury (AKI). Our knowledge of these DAMPs remains incomplete. Here, we report serum peroxiredoxin 1 (Prdx1) as a novel DAMP for AKI. Lipopolysaccharide (LPS) and kidney ischemia/reperfusion injury instigated AKI with concurrent increases in serum Prdx1 and reductions of Prdx1 expression in kidney tubular epithelial cells. Genetic knockout of Prdx1 or use of a Prdx1–neutralizing antibody protected mice from AKI and this protection was impaired by introduction of recombinant Prdx1 (rPrdx1). Mechanistically, lipopolysaccharide increased serum and kidney proinflammatory cytokines, macrophage infiltration, and the content of M1 macrophages. All these events were suppressed in Prdx1-/- mice and renewed upon introduction of rPrdx1. In primary peritoneal macrophages, rPrdx1 induced M1 polarization, activated macrophage-inducible C-type lectin (Mincle) signaling, and enhanced proinflammatory cytokine production. Prdx1 interacted with Mincle to initiate acute kidney inflammation. Of note, rPrdx1 upregulated Mincle and the spleen tyrosine kinase Syk system in the primary peritoneal macrophages, while knockdown of Mincle abolished the increase in activated Syk. Additionally, rPrdx1 treatment enhanced the downstream events of Syk, including transcription factor NF-κB signaling pathways. Furthermore, serum Prdx1 was found to be increased in patients with AKI; the increase of which was associated with kidney function decline and inflammatory biomarkers in patient serum. Thus, kidney-derived serum Prdx1 contributes to AKI at least in part by activating Mincle signaling and downstream pathways. Translational StatementAcute kidney injury (AKI) remains a major health issue. As a disease characterized by inflammatory response, AKI is exacerbated by damage-associated molecular patterns (DAMPs). Advances in our understanding of AKI-associated DAMPs are a vital aspect of improving the clinical management of AKI. We here have demonstrated serum peroxiredoxin 1 (Prdx1) as a novel DAMP for AKI. Although serum Prdx1 contributes to AKI, reduction of Prdx1 in the circulation, using a Prdx1-neutralizing antibody or via genetic knockout of Prdx1, protects mice from AKI. This study reveals the therapeutic potential of managing AKI via reduction of circulating Prdx1. Acute kidney injury (AKI) remains a major health issue. As a disease characterized by inflammatory response, AKI is exacerbated by damage-associated molecular patterns (DAMPs). Advances in our understanding of AKI-associated DAMPs are a vital aspect of improving the clinical management of AKI. We here have demonstrated serum peroxiredoxin 1 (Prdx1) as a novel DAMP for AKI. Although serum Prdx1 contributes to AKI, reduction of Prdx1 in the circulation, using a Prdx1-neutralizing antibody or via genetic knockout of Prdx1, protects mice from AKI. This study reveals the therapeutic potential of managing AKI via reduction of circulating Prdx1. Acute kidney injury (AKI) is a major health issue, with 13.3 million annual cases and 1.7 million mortalities worldwide every year.1Mehta R.L. Cerdá J. Burdmann E.A. et al.International Society of Nephrology's 0by25 initiative for acute kidney injury (zero preventable deaths by 2025): a human rights case for nephrology.Lancet. 2015; 385: 2616-2643Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar The disease is characterized by a rapid loss of kidney function.2Ronco C. Bellomo R. Kellum J.A. Acute kidney injury.Lancet. 2019; 394: 1949-1964Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar AKI develops in 10%–15% of hospitalized patients, and up to 50% of patients are admitted to the intensive care unit, with a morbidity rate reaching 5.7%.3Moore P.K. Hsu R.K. Liu K.D. Management of acute kidney injury: core curriculum 2018.Am J Kidney Dis. 2018; 72: 136-148Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 4Al-Jaghbeer M. 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DAMPening sterile inflammation of the kidney.Kidney Int. 2019; 95: 489-491Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar DAMPs include high-mobility group box-1 (HMGB1), heat shock proteins (HSPs), and other molecules.15Kurts C. Panzer U. Anders H.J. et al.The immune system and kidney disease: basic concepts and clinical implications.Nat Rev Immunol. 2013; 13: 738-753Crossref PubMed Scopus (456) Google Scholar, 16Rosin D.L. Okusa M.D. Dangers within: DAMP responses to damage and cell death in kidney disease.J Am Soc Nephrol. 2011; 22: 416-425Crossref PubMed Scopus (214) Google Scholar, 17Anders H.J. Toll-like receptors and danger signaling in kidney injury.J Am Soc Nephrol. 2010; 21: 1270-1274Crossref PubMed Scopus (107) Google Scholar, 18Wu H. Ma J. Wang P. et al.HMGB1 contributes to kidney ischemia reperfusion injury.J Am Soc Nephrol. 2010; 21: 1878-1890Crossref PubMed Scopus (285) Google Scholar An important area of research is uncovering the contributions of individual DAMPs to AKI. Peroxiredoxin 1 (Prdx1) is a typical 2-Cys antioxidant protein belonging to the peroxiredoxin family; the protein is commonly expressed in the cytosol and plays important roles in maintaining cellular redox homeostasis by reducing peroxide levels.19Rhee S.G. Kil I.S. Multiple functions and regulation of mammalian peroxiredoxins.Annu Rev Biochem. 2017; 86: 749-775Crossref PubMed Scopus (164) Google Scholar,20Kim Y. Jang H.H. Role of cytosolic 2-Cys Prx1 and Prx2 in redox signaling.Antioxidants (Basel). 2019; 8: 169Crossref PubMed Scopus (30) Google Scholar Recent studies also revealed the presence of Prdx1 in the extracellular space, which contributes to inflammation as a novel DAMP by binding to PRRs, including Toll-like receptor 2/4.21Shichita T. Hasegawa E. Kimura A. et al.Peroxiredoxin family proteins are key initiators of post-ischemic inflammation in the brain.Nat Med. 2012; 18: 911-917Crossref PubMed Scopus (333) Google Scholar,22Riddell J.R. Wang X.Y. Minderman H. et al.Peroxiredoxin 1 stimulates secretion of proinflammatory cytokines by binding to TLR4.J Immunol. 2010; 184: 1022-1030Crossref PubMed Scopus (167) Google Scholar Extracellular Prdx1 released from necrotic brain cells plays a key role in postischemic inflammation in brain ischemia–reperfusion injury (IRI).21Shichita T. Hasegawa E. Kimura A. et al.Peroxiredoxin family proteins are key initiators of post-ischemic inflammation in the brain.Nat Med. 2012; 18: 911-917Crossref PubMed Scopus (333) Google Scholar A significant elevation of the level of circulating Prdx1 was confirmed in both patients and mice with acute liver injury.23He Y. Li S. Tang D. et al.Circulating peroxiredoxin-1 is a novel damage-associated molecular pattern and aggravates acute liver injury via promoting inflammation.Free Radic Biol Med. 2019; 137: 24-36Crossref PubMed Scopus (40) Google Scholar In supporting the DAMP property of serum Prdx1 in tissue injury, we report circulating Prdx1 as a novel DAMP for AKI, at least in part by activating macrophage-inducible C-type lectin (Mincle) signaling; neutralization of serum Prdx1 substantially prevents mice from developing AKI. This research reveals therapeutic potential of managing AKI via reduction of circulating Prdx1. Clinical protocols were approved by the Ethics Committee of Xiangya Hospital, Central South University, Changsha, China (approval number 201612700). Consent for this study was obtained from the participating healthy subjects and the AKI patients. Blood samples and kidney biopsy tissues from healthy subjects and AKI patients were collected from Xiangya Hospital, Central South University, Changsha, China. The diagnosis of AKI was performed according to the Kidney Disease: Improving Global Outcomes (KDIGO) guidelines.24Kellum J.A. Lameire N. Diagnosis, evaluation, and management of acute kidney injury: a KDIGO summary (Part 1).Crit Care. 2013; 17: 204Crossref PubMed Scopus (1384) Google Scholar Prdx1 knockout (Prdx1–/–) C57BL/6J mice were generated using the CRISPR/Cas9 technique as previously described.23He Y. Li S. Tang D. et al.Circulating peroxiredoxin-1 is a novel damage-associated molecular pattern and aggravates acute liver injury via promoting inflammation.Free Radic Biol Med. 2019; 137: 24-36Crossref PubMed Scopus (40) Google Scholar Male Prdx1–/– and wild-type (WT) mice, aged 8 to 10 weeks, were used. All animal experimental protocols and procedures were approved by the Ethics Review Committee for Animal Experimentation of Central South University, Changsha, China (approval number 201803491). A lipopolysaccharide (LPS)-induced AKI mouse model was established through i.p. injection of LPS (#L2630, Sigma‒Aldrich) at a dose of 15 mg/kg; mice were sacrificed 12 or 24 hours later. A renal IRI mouse model for AKI was established as previously described.25Vinas J.L. Porter C.J. Douvris A. et al.Sex diversity in proximal tubule and endothelial gene expression in mice with ischemic acute kidney injury.Clin Sci (Lond). 2020; 134: 1887-1909Crossref PubMed Scopus (13) Google Scholar Briefly, bilateral renal pedicles were clamped for 30 minutes using nontraumatic microvascular clips, and then the clips were removed to start reperfusion. Body temperature was maintained at 36.5 °C–37 °C throughout the procedures by using a temperature-controlled heating device. Mice were sacrificed 1, 3, or 7 days after reperfusion. For a macrophage depletion model, clodronate liposomes (CLs; #40337ES08, Yeasen) and control liposomes (#40338ES05, Yeasen) were i.p. injected into WT mice (200 μl per mouse). The control liposomes were used to evaluate and eliminate the effect of the liposomes. At day 2 after CLs injection, AKI was induced by IRI as described previously, and primary peritoneal macrophages (PPMs; 6 × 106 cells per mouse) with or without Mincle knockdown were adoptively transferred into the mouse immediately after reperfusion. The mice were sacrificed 24 hours after macrophage transfer. The efficiency of macrophage depletion was confirmed by examining the number of kidney-resident macrophages by flow cytometry and immunohistochemical staining. All data are expressed as mean ± SD. Statistical analysis was performed with SPSS 22.0 software. The Student's t test was used for comparisons between 2 groups, and multiple groups were compared using 1-way analysis of variance followed by a post hoc Bonferroni test. Linear correlations were analyzed using the Pearson correlation coefficient. Values of P < 0.05 were considered statistically significant. Additional details for all methods are provided in the Supplementary Methods. To address a potential functional consequence of circulating Prdx1 in AKI, we induced AKI in mice using LPS. Sepsis is the main cause of AKI, which is commonly mimicked or modeled using LPS infusion in mice.26Xu C. Chang A. Hack B.K. et al.TNF-mediated damage to glomerular endothelium is an important determinant of acute kidney injury in sepsis.Kidney Int. 2014; 85: 72-81Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar,27Kim D.H. Jung Y.J. Lee A.S. et al.COMP-angiopoietin-1 decreases lipopolysaccharide-induced acute kidney injury.Kidney Int. 2009; 76: 1180-1191Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar I.p. injection of LPS resulted in kidney injury starting at 12 hours post-injection, with conditions worsened at 24 hours post-injection, evident by increases in regions with tubular epithelial cell death, loss of brush border, tubular dilation, and intratubular cast formation (Figure 1a). Higher tubular injury scores were graded at 24 hours post-injection than at 12 hours post-injection (Figure 1b). Disease progression was associated with decreases in kidney function (Figure 1c and d). An important observation was that a significant upregulation of serum Prdx1 occurred in mice with AKI at 12 hours following LPS injection, which was further increased at 24 hours post-injection (Figure 1e). To further support the upregulation of serum Prdx1 in mice with AKI, we directly induced AKI using renal IRI, another classic AKI model. Structural alterations in kidneys with IRI were demonstrated (Figure 1f and g), which are similar to the alterations observed in LPS-induced kidney injury (Figure 1a). Kidneys with AKI were associated with kidney function decline (Figure 1h and i). As observed in mice with AKI induced by LPS, a significant increase in serum Prdx1 occurred in IRI-induced AKI mice (Figure 1j). Investigations of AKI in the IRI model commonly focus on the acute phase of kidney injury within 24 hours.28Skrypnyk N.I. Gist K.M. Okamura K. et al.IL-6-mediated hepatocyte production is the primary source of plasma and urine neutrophil gelatinase-associated lipocalin during acute kidney injury.Kidney Int. 2020; 97: 966-979Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 29Lau A. Rahn J.J. Chappellaz M. et al.Dipeptidase-1 governs renal inflammation during ischemia reperfusion injury.Sci Adv. 2022; 8eabm0142Crossref Scopus (11) Google Scholar, 30Yang D. Tang M. Zhang M. et al.Downregulation of G protein-coupled receptor kinase 4 protects against kidney ischemia-reperfusion injury.Kidney Int. 2023; 103: 719-734Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar To confirm our IRI model for capturing the acute phase, we observed a significant improvement in kidney injury from day 3 forward (Supplementary Figure S1). Collectively, the increase in serum Prdx1 in 2 different AKI mouse models strongly suggests its involvement in AKI. The detection of serum Prdx1 in AKI caused by different mechanisms (Figure 1e and j) implies that circulating Prdx1 is physiologically relevant to AKI. To investigate the pathologic contributions of circulating Prdx1 to AKI, we examined the impact of Prdx1 absence on AKI using Prdx1 knockout (Prdx1–/–) mice (Supplementary Figure S2).23He Y. Li S. Tang D. et al.Circulating peroxiredoxin-1 is a novel damage-associated molecular pattern and aggravates acute liver injury via promoting inflammation.Free Radic Biol Med. 2019; 137: 24-36Crossref PubMed Scopus (40) Google Scholar Prdx1 deficiency attenuated tubular injury with significant preservation of kidney function in mice treated with LPS (Figure 2a–f). Significant protection of AKI with preservation of kidney function also occurred in Prdx1–/– mice treated with IRI (Figure 2g–l). To further examine this concept, we induced AKI in Prdx1–/– mice along with i.v. reintroduction of recombinant Prdx1 (rPrdx1). An i.v. injection of 200 ng rPrdx1/mouse made Prdx1–/– mice vulnerable to kidney injury coupled with kidney function decline in response to LPS treatment (Figure 3a–e). The dose of rPrdx1 injected was based on the serum Prdx1 level observed in LPS-induced AKI mice. An average serum Prdx1 level of 140 ng/ml was detected at 24 hours following LPS injection in AKI mice (Figure 1e). With an average blood volume of 1.5 ml for male mice at age 8–10 weeks, i.v. injection of rPrdx1 at 200 ng/mouse is within the pathologic range of serum Prdx1 produced in LPS-induced AKI mice. Similarly, rPrdx1 increased the susceptibility of Prdx1–/– mice to kidney injury caused by IRI (Figure 3f–j). To obtain additional evidence for a pathologic role of circulating Prdx1 in AKI, we investigated the impact of a Prdx1-neutralizing antibody on AKI. Compared with control IgG, the Prdx1-neutralizing antibody offered protection from LPS-induced kidney damage (Figure 4a and b ) and preservation of kidney functions (Figure 4c–e). Similarly, the neutralizing antibody displayed remarkable protections of kidney structure integrity (Figure 4f and g) and kidney function decline caused by IRI in WT mice (Figure 4h–j). Taken together, we provide a comprehensive demonstration that circulating Prdx1 facilitates AKI. We subsequently analyzed the origin of serum Prdx1 in AKI mice. Given the typical feature of tubular epithelial cell death in AKI,31Bonventre J.V. Yang L. Cellular pathophysiology of ischemic acute kidney injury.J Clin Invest. 2011; 121: 4210-4221Crossref PubMed Scopus (1368) Google Scholar,32Sancho-Martinez S.M. Lopez-Novoa J.M. Lopez-Hernandez F.J. Pathophysiological role of different tubular epithelial cell death modes in acute kidney injury.Clin Kidney J. 2015; 8: 548-559Crossref PubMed Scopus (71) Google Scholar we were able to show that LPS induced the release of Prdx1 into the extracellular space from mouse primary renal tubular epithelial cells (RTECs) in a dose- and time-dependent manner (Figure 5a and b ). In human renal tubular epithelial cells (HK2) cells, LPS treatment also resulted in dose-dependent increases of extracellular Prdx1 (Figure 5c). Heat stress, another stronger cell-death inducer, resulted in the death of human renal tubular epithelial cells and the release of abundant extracellular Prdx1 (Figure 5d; Supplementary Figure S3A). In control mice, renal Prdx1 was expressed mainly in proximal tubular epithelial cells, evident by the colocalization of Prdx1 and lotus tetragonolobus lectin (LTL), a marker of proximal tubules (Figure 5e). In mice with LPS-induced AKI, reductions of Prdx1 in renal proximal tubular epithelial cells occurred, and the level of reduction followed the development of AKI (Figure 5e and f). Additionally, in the IRI model in which the insult was applied directly toward the kidney, a significant decrease of Prdx1 in renal proximal tubular epithelial cells was also demonstrated (Figure 5g and h). Further, reductions in renal Prdx1 protein expression in AKI induced by LPS and IRI were not due to decreases in gene expression, as Prdx1 mRNA in the kidneys increased following the pathologic insults (Figure 5i). Despite the release of Prdx1 from mesangial cells after LPS treatment (Supplementary Figure S3B), tubular epithelial cells represent the most abundant resident cells in the kidney, compared with other cell types, including mesangial cells, and are the main cells impaired in AKI.31Bonventre J.V. Yang L. Cellular pathophysiology of ischemic acute kidney injury.J Clin Invest. 2011; 121: 4210-4221Crossref PubMed Scopus (1368) Google Scholar,32Sancho-Martinez S.M. Lopez-Novoa J.M. Lopez-Hernandez F.J. Pathophysiological role of different tubular epithelial cell death modes in acute kidney injury.Clin Kidney J. 2015; 8: 548-559Crossref PubMed Scopus (71) Google Scholar Collectively, the above evidence indicates that the renal proximal tubular epithelial cells are an origin of circulating Prdx1. This concept is consistent with the brain cells and hepatocyte origin of Prdx1 in brain IRI and acute liver injury.21Shichita T. Hasegawa E. Kimura A. et al.Peroxiredoxin family proteins are key initiators of post-ischemic inflammation in the brain.Nat Med. 2012; 18: 911-917Crossref PubMed Scopus (333) Google Scholar,23He Y. Li S. Tang D. et al.Circulating peroxiredoxin-1 is a novel damage-associated molecular pattern and aggravates acute liver injury via promoting inflammation.Free Radic Biol Med. 2019; 137: 24-36Crossref PubMed Scopus (40) Google Scholar However, our observed kidney origin of serum Prdx1 in AKI does not exclude the contributions of other origins to serum Prdx1 (see Discussion sections for details). In view of the demonstrated DAMP property of circulating Prdx1 in brain IRI and liver injury,21Shichita T. Hasegawa E. Kimura A. et al.Peroxiredoxin family proteins are key initiators of post-ischemic inflammation in the brain.Nat Med. 2012; 18: 911-917Crossref PubMed Scopus (333) Google Scholar,23He Y. Li S. Tang D. et al.Circulating peroxiredoxin-1 is a novel damage-associated molecular pattern and aggravates acute liver injury via promoting inflammation.Free Radic Biol Med. 2019; 137: 24-36Crossref PubMed Scopus (40) Google Scholar as well as the central role of sterile inflammation in AKI,7Rabb H. Griffin M.D. McKay D.B. et al.Inflammation in AKI: current understanding, key questions, and knowledge gaps.J Am Soc Nephrol. 2016; 27: 371-379Crossref PubMed Scopus (341) Google Scholar, 8Zhang B. Ramesh G. Uematsu S. et al.TLR4 signaling mediates inflammation and tissue injury in nephrotoxicity.J Am Soc Nephrol. 2008; 19: 923-932Crossref PubMed Scopus (243) Google Scholar, 9Anders H.J. Schaefer L. Beyond tissue injury-damage-associated molecular patterns, toll-like receptors, and inflammasomes also drive regeneration and fibrosis.J Am Soc Nephrol. 2014; 25: 1387-1400Crossref PubMed Scopus (203) Google Scholar we suspected that serum Prdx1 plays a role in facilitating inflammation and—important to note—kidney inflammation in AKI. To examine this possibility, we showed significant increases in the serum levels of the proinflammatory cytokines IL-1β and IL-6 in mice treated with LPS (Figure 6a and b ). Important to note is that the elevations were significantly reduced in LPS-treated Prdx1–/– mice (Figure 6a and b). Similar findings were observed in kidney tissues also (Figure 6c and d). The induction of serum and renal IL-1β and IL-6 is attributed to circulating Prdx1, as evidenced by the significant increases in serum (Figure 6e) and renal IL-1β and IL-6 (Figure 6f) in Prdx1–/– mice reconstituted via i.v. injection of rPrdx1 in response to LPS treatment. Both IL-1β and IL-6 play key roles in facilitating inflammation and can be derived from macrophages.33Crayne C.B. Albeituni S. Nichols K.E. et al.The immunology of macrophage activation syndrome.Front Immunol. 2019; 10: 119Crossref PubMed Scopus (344) Google Scholar In line with this knowledge, LPS induced the infiltration of F4/80+ macrophages in the kidneys of WT mice, the level of which was significantly reduced in Prdx1–/– mice (Figure 7a and c ). These alterations were demonstrated by flow cytometry analysis also (Supplementary Figure S4A). Macrophage infiltration also occurred in AKI induced by IRI in WT mice, and the infiltration was reduced in Prdx1–/– mice (Figure 7b and d). The macrophage infiltration was attributed at least partially to circulating Prdx1, as i.v. injection of rPrdx1 into Prdx1–/– mice significantly increased renal macrophage infiltration in AKI induced by LPS and IRI (Figure 7e and f). We also explored the relationship of Prdx1 with the infiltration of other inflammatory cells in the kidney. Prdx1 deficiency slightly decreased the renal content of cluster of differentiation (CD)11C+ major histocompatibility complex II+ cells, compared to that in WT mice treated with LPS (Supplementary Figure S4B). CD11C and major histocompatibility complex II are biomarkers of dendritic cells.34Helft J. Bottcher J. Chakravarty P. et al.GM-CSF mouse bone marrow cultures comprise a heterogeneous population of CD11c(+)MHCII(+) macrophages and dendritic cells.Immunity. 2015; 42: 1197-1211Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar No differences were present in neutrophils or monocytes in the injured kidneys of WT versus Prdx1–/– mice (Supplementary Figure S4C and D). A well-established finding is that M1-polarized macrophages favor inflammation during the early stage of AKI, whereas M2-polarized anti-inflammatory macrophages create renoprotection during the repair process.35Cao Q. Wang Y. Harris D.C. Pathogenic and protective role of macrophages in kidney disease.Am J Physiol Renal Physiol. 2013; 305: F3-F11Crossref PubMed Scopus (54) Google Scholar LPS is a classic M1 polarization inducer.36Lawrence T. Natoli G. Transcriptional regulation of macrophage polarization: enabling diversity with identity.Nat Rev Immunol. 2011; 11: 750-761Crossref PubMed Scopus (1493) Google Scholar Consistently, we observed an enrichment of CD80+ M1 macrophages in the renal F4/80+CD11b+ macrophage population in WT mice, compared to those in Prdx1–/– mice following LPS challenge (Figure 7g and i). In comparison, a significant enrichment of CD206+ M2 macrophages occurred in Prdx1–/– mice treated with LPS (Figure 7h and i). In vitro, LPS preferentially induced M1 polarization of F4/80+CD11b+ PPMs isolated from WT mice, compared to PPMs derived from Prdx1–/– mice (Figure 7j). Considering that LPS caused elevations in serum Prdx1 in mice (Figure 1e), we suspected that extracellular Prdx1 plays a role in the M1 polarization of macrophages. Indeed, rPrdx1 dose-dependently induced the polarization of PPMs derived from WT mice to CD80+ M1 macrophages (Figure 7k; Supplementary Figure S4E) but not CD206+ M2 macrophages (Figure 7l; Supplementary Figure S4F), suggesting that Prdx1 plays an intriguing role in switching macrophage polarization. Additionally, transforming growth factor beta 1 (TGF-β1) and IL-4 enhanced M2 polarization of PPMs derived from Prdx1–/– mice, compared to PPMs isolated from WT mice (Figure 7m). Taken together, these observations support a role for serum Prdx1 in enhancing renal inflammation by facilitating macrophage infiltration and M1 polarization. Considering the typical role of DAMPs-PRRs in initiating inflammation, we investigated 7 common PRRs that might mediate Prdx1-contributed renal inflammatory actions. rPrdx1 treatment upregulated both Mincle and nucleotide oligomerization domain 2 (Nod2) early (at 6 hours) and at all 3 time points in PPMs (Figure 8a; Supplementary Figure S5A). In comparison, upregulations of TLR9, Ticam-1, TLR5, AGER, and MD2 either did not occur (Supplementary Figure S5D–F) or happened late (Supplementary Figure S5B and C) and to a substantially lower level (comparing Supplementary Figure S5A to S5B and S5C). The level of upregulation of Mincle was substantially higher than that of Nod2 (comparing Figure 8a to Supplementary Figure S5A). We focused on Mincle as a candida