Involvement of intrarenal protein-tyrosine phosphatase ζ receptor in progression of renal fibrosis in mice with unilateral ureteral obstruction

Involvement of intrarenal protein-tyrosine phosphatase ζ receptor in progression of renal fibrosis in mice with unilateral ureteral obstruction | 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 Involvement of intrarenal protein-tyrosine phosphatase ζ receptor in progression of renal fibrosis in mice with unilateral ureteral obstruction Tetsuya Abe, Yukihiro Wada, Emiko Takeuchi, Masashi Satoh, Takayuki Imanishi, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9386889/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Protein-tyrosine phosphatase ζ receptor (PTP-ζ), a secondary receptor for interleukin (IL)-34 involved in macrophage (Mø) proliferation, is expressed in damaged kidneys. However, its precise localization and role in renal fibrosis remain unclear. Unilateral ureteral obstruction (UUO) was induced in wild-type (WT) and PTP-ζ-knockout (KO) C57BL/6 mice, then kidneys were analyzed on day 14. WT mice developed severe renal fibrosis with marked intrarenal IL-34 and PTP-ζ expressions. PTP-ζ-KO mice exhibited a significantly reduced fibrotic area and lower expressions of fibrogenic genes, despite comparable IL-34 levels. In UUO kidneys, infiltration of CD206+ Møs and accumulation of myofibroblasts were markedly suppressed in PTP-ζ-KO mice. Flow cytometry revealed a lower ratio of CD11c-CD206+ cells to CD11c+CD206- cells within the intrarenal CD11b+F4/80+ Mø population in KO mice. Mouse bone marrow-derived macrophages (BMDMs) isolated in vitro showed PTP-ζ expression in WT mice, but not in PTP-ζ-KO mice. In WT BMDMs, TGF-β and recombinant IL (rIL)-4 increased arginase-1 and interferon regulatory factor (IRF)-4 expressions, with further amplification upon rIL-34 stimulation. In PTP-ζ-KO BMDMs, additional rIL-34 did not further increase arginase-1 or IRF-4 expressions. IL-34 binding to PTP-ζ appears to promote M2-like Mø infiltration and fibrosis in UUO, suggesting PTP-ζ as a potential target for treating renal fibrosis. Health sciences/Diseases Biological sciences/Immunology Health sciences/Nephrology PTP-ζ IL-34 macrophage renal fibrosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 INTRODUCTION Chronic kidney disease (CKD) affects more than 10% of the global population and is significantly associated with risk of mortality 1-3 . In aging societies such as Japan, CKD has become a major public health concern and imposes considerable burdens on healthcare systems and medical expenditures 4 . Renal fibrosis is an inevitable outcome of all progressive CKD, including chronic glomerulonephritis, hypertensive CKD, and diabetic CKD 5 . In other words, renal fibrosis is a final common pathway for CKD progressing to end-stage renal disease 2,5 . With regard to the mechanisms underlying renal fibrosis, activated myofibroblasts are the principal effectors responsible for the production of extracellular matrix (ECM) proteins in the fibrotic kidney 6-8 . Moreover, the accumulation of intrarenal macrophages (Møs) is another important cause of the development and progression of renal fibrosis 9-11 . Kidney Møs are characterized by wide heterogeneity and marked plasticity and play critical roles in not only maintaining renal homeostasis, but also contributing to renal inflammation and fibrosis 12,13 . Generally, activated Møs are conceptually divided into two major polarization states: classical type 1 (M1); and alternative type 2 (M2) 14,15 . Simplistically, M1Møs are pro-inflammatory or cytotoxic Møs, secreting pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α. In contrast, M2Møs are involved in immune regulation, anti-inflammation, repair or regeneration, and fibrosis 12,14-16 . Regarding the contributions of Møs to renal disease, aberrantly proliferative M1Møs have been recognized to aggravate acute kidney injury (AKI), and the inadequate removal of M2Møs causes renal fibrosis 12,17 . In addition, several previous studies have mentioned that accumulated M2Møs promote renal fibrosis by inducing overproduction of ECM components and secretion of profibrotic factors 8,11,17,18 . In short, M2Møs, rather than M1Møs, are significantly involved in the development of renal fibrosis. This suggests that preventing the intrarenal accumulation of M2Møs could be a promising therapeutic strategy for renal fibrosis. Protein-tyrosine phosphatase ζ receptor (PTP-ζ, PTPRZ1) has been discovered as a secondary receptor of interleukin (IL)-34 that is involved in Mø proliferation 19 . PTP-ζ is mainly expressed in brain cells such as neurons and astrocytes. Consisting of several domains and three isoforms, PTP-ζ is involved in neuronal migration, neuroaxonal elongation, synapse formation, and synaptic regulation 20 . Several studies, including our own and those of our colleagues, have demonstrated that PTP-ζ is expressed in not only brain, but also renal tubular epithelial cells (TECs) 21,22 , Møs 23,24 , and lymphocytes 23,25 . In addition to IL-34, various other ligands for PTP-ζ have been identified, such as tenascin-C, pleiotrophin, and midkine 25-27 . However, among these ligands, intrarenal IL-34 expressions were significant and activation of intrarenal IL-34 through the two representative receptors, colony-stimulating factor 1 (CSF-1) receptor (CSF-1R; also known as cFMS and CD115) and PTP-ζ, was clearly associated with progression of renal disorder in experimental models of renal disease such as ischemia-reperfusion injury (I/R)-induced AKI 21 , cisplatin-induced AKI 22 , advanced lupus nephritis 23 , and AKI to CKD caused by I/R 25 . Of note, alongside the elevation of intrarenal IL-34, both receptors cFMS and PTP-ζ are also highly expressed in damaged renal tissues. In particular, expression of PTP-ζ was dominant in the chronic phases of the aforementioned renal disease models 21,23,25 . We therefore hypothesized that the intrarenal IL-34/PTP-ζ axis, rather than the IL-34/cFMS axis, is deeply associated with the progression of CKD, particularly renal fibrosis. In the present study, we attempted to elucidate the influence of PTP-ζ on renal fibrosis and Mø polarization by focusing on the physiological properties of the IL-34/PTP-ζ axis in vivo in a unilateral ureteral obstruction (UUO) model and in vitro analysis using PTP-ζ-knockout (KO) mice. No such study has been reported to date. Herein, we demonstrated that the genetic ablation of PTP-ζ attenuated renal fibrosis by UUO via suppression of profibrotic Mø accumulation. RESULTS Intrarenal expressions of IL-34 and PTP-ζ in mice with UUO During the experiment period up to day 14 after UUO induction, no mortality was observed in any groups. Body weight changes from baseline to sacrifice were comparable among study groups (Fig 1A). Intrarenal mRNA levels for IL-34, a primary ligand for PTP-ζ, were significantly upregulated in UUO mice, with no significant difference between wild-type (WT) and PTP-ζ-KO mice (Fig 1B). Similarly, protein levels of IL-34 evaluated by enzyme-linked immunosorbent assay (ELISA) in damage kidneys were significantly elevated in UUO mice, but no differences were seen among WT and PTP-ζ-KO mice (Fig 1C). Regarding intrarenal PTP-ζ expressions in UUO mice, western blot (WB) analysis showed clear expressions in the WT group (Fig 1D). Densitometric values of PTP-ζ expression were significantly increased in WT mice with UUO (WT-UUO) when compared to sham-operated WT normal control (NC) mice. In addition, intrarenal PTP-ζ mRNA levels were also upregulated in WT-UUO mice (Fig 1E, F). Meanwhile, neither protein nor mRNA levels for intrarenal PTP-ζ were detected in PTP-ζ-KO mice with UUO (PTP-ζ-KO-UUO) (Fig 1E, F). Effects of PTP-ζ on tubular damage and renal fibrosis in UUO mice Representative images of kidney tissues stained with Masson’s trichrome among the study group mice are shown in Figure 2A–F. Compared to the NC group, the tubulointerstitial (TI) injury score of WT-UUO mice was significantly increased in the cortex, outer medulla, and combined cortical and outer medullary area (Fig 2G–I). Of note, elevated TI injury scores were significantly attenuated in PTP-ζ-KO-UUO mice compared to WT-UUO mice in each area (Fig 2G–I). Regarding renal fibrosis as evaluated by the Sirius red-positive area, representative images of fibrotic kidneys among study groups are shown in Figure 3A–F. The Sirius red-positive renal fibrotic area was significantly increased in WT-UUO mice compared to sham-operated WT mice in the cortex, outer medulla, and combined cortex and outer medulla. The area with markedly elevated fibrosis was significantly suppressed in PTP-ζ-KO-UUO mice compared to WT-UUO mice (Fig 3G–I). In addition, mRNA levels for intrarenal fibrogenetic genes (including TGF-β, α-SMA, collagen type I [Col-1], and fibronectin) were significantly upregulated in UUO mice, whereas significant increases in levels of transcripts for these intrarenal fibrogenetic genes were suppressed in PTP-ζ-KO-UUO mice compared to WT-UUO mice (Fig 3J–M). Effects of PTP-ζ on intrarenal fibroblasts and myofibroblasts in UUO mice Accumulation of fibroblasts in renal tissue was evaluated by immunofluorescence (IF) for platelet-derived growth factor receptor β (PDGFRβ), which is expressed in interstitial fibroblasts and is associated with promoting fibroblast proliferation 28 . Representative images of PDGFRβ staining in kidney tissues among study group mice are shown in Figure 4A–I. WT-UUO mice showed a significant increase in the number of PDGFRβ-positive fibroblasts compared to sham-operated WT mice (Fig 4A, B, and J). PTP-ζ-KO-UUO mice showed a significantly reduced number of PDGFRβ-positive fibroblasts compared to WT-UUO mice (Fig 4B, C, and J). To evaluate accumulation of myofibroblasts in kidney tissues, IF staining of α-SMA 8 among study groups was performed (Fig 4K–S). In WT-UUO mice, the number of α-SMA-positive myofibroblasts was significantly increased when compared to sham-operated WT mice (Fig 4K, L, and T). This increased number of myofibroblasts was significantly attenuated in PTP-ζ-KO-UUO mice compared to WT-UUO mice (Fig 4L, M, and T). Effects of PTP-ζ on intrarenal Mø accumulation and polarization in UUO mice Møs are thought to play a key role in renal fibrosis by skewing from an M1-dominant to an M2-dominant state. We therefore evaluated Mø accumulation and polarization within the renal tissues of mice from each experimental group using real-time reverse transcriptase polymerase chain reaction (RT-PCR) (Fig 5A–E) and IF staining (Fig 5F–S). Expression of cFMS is known to occur predominantly on Mø as a primary receptor for IL-34 to promote Mø proliferation 27 . As shown in Figure 5A, mRNA levels for cFMS were significantly elevated in the kidneys of UUO mice, and expression of this gene was significantly suppressed in PTP-ζ-KO-UUO mice compared to WT-UUO mice. The accumulation of F4/80-positive Mø in the fibrotic UUO kidneys of both WT and PTP-ζ-KO mice was shown by IF images (Fig 5G, K, and O). Quantitative analysis showed that the number of F4/80-positive Mø was significantly higher in UUO kidneys compared to the NC group. However, the accumulated Mø infiltration was significantly decreased in PTP-ζ-KO-UUO mice compared to WT-UUO mice (Fig 5R). To evaluate polarization of Mø, intrarenal transcripts for TNF-α and IL-1β, which are commonly secreted by M1Mø, were measured by RT-PCR. Up-regulation of mRNA for TNF-α and IL-1β were comparable between WT-UUO mice and PTP-ζ-KO-UUO mice (Fig 5B, C). In contrast, elevated intrarenal mRNA levels for Yim-1/Chil-3 and TIMP-1, regarded as markers for profibrotic M2Mø 11,29 , were elevated in UUO mice and clearly higher in WT-UUO mice compared to PTP-ζ-KO-UUO mice (Fig 5D, E). Double staining for F4/80 and CD206, markers (for identifying M2Mø 8 ) revealed marked M2Mø accumulation in the kidneys of UUO mice compared to sham-operated controls (Fig 5I, M, and Q). Notably, the number of F4/80 + CD206 + M2Mø in UUO kidneys was significantly reduced in PTP-ζ-KO-UUO mice compared to WT-UUO mice (Fig 5S). Fluorescence-activated cell sorting (FACS) analysis was performed to evaluate intrarenal Mø polarization across study groups. Representative FACS plots showing the gating strategy for CD11b + F4/80 + CD11c + (M1-like) and CD11b + F4/80 + CD206 + (M2-like) Mø populations in sham-operated NC mice, WT-UUO mice, and PTP-ζ-KO-UUO mice are presented in Figure 6A–C. Based on previous findings 30 , CD11b + F4/80 + CD11c + cells were broadly identified as cyto-destructive, proinflammatory M1-like Mø, while CD11b + F4/80 + CD206 + cells were recognized as cyto-protective, profibrotic M2-like Mø. As shown in Figure 6D, the number of intrarenal Møs identified by FACS analysis tended to be higher in UUO mice compared to the NC WT group. Although the number of M1-like Møs were comparable among the study group (Fig 6E), the number of M2-like Møs tended to be higher in WT-UUO mice than in PTP-ζ-KO UUO mice (Fig 6F), although this difference did not reach statistical significance. Similarly, the elevated M2/M1-like Mø ratio tended to be reduced in PTP-ζ-KO-UUO mice compared to WT-UUO mice, although no significant difference was detected (P=0.0612) (Fig 6G). Expressions of PTP-ζ in bone marrow-derived Møs (BMDMs) These in vivo study results led us to hypothesize that PTP-ζ might be expressed on Møs, and that the IL-34/ PTP-ζ axis may be involved in skewing toward an M2-like phenotype. To test this hypothesis, we performed in vitro experiments using mouse BMDMs isolated from WT B6 mice and PTP-ζ-KO B6 mice (Fig 7). As shown in Figure 7A, BMDMs from the two groups were cultured with medium as demonstrated previously 31 . After washing with phosphate-buffered saline (PBS), BMDMs were stimulated with recombinant IL-34 (rIL-34) (5 ng/mL), TGF-β (5 ng/mL), or both for 24 hours. WB analysis revealed that PTP-ζ was constitutively expressed in WT-BMDMs and its expression remained relatively stable under all stimulation conditions (Fig 7B). In contrast, PTP-ζ expression was upregulated in vivo during UUO-induced inflammation. No detectable PTP-ζ expression was observed in PTP-ζ-KO BMDMs following stimulation with rIL-34 alone, TGF-β alone, or the combination. These findings suggest that the regulatory mechanisms controlling PTP-ζ expression may differ between the in vivo UUO inflammatory environment and the in vitro BMDM culture system. Similarly, the NIH/3T3 murine fibroblast cell line was cultured in specific medium and stimulated with equal volumes of saline, TGF-β, and rIL-34 following an 8-hour starvation period. Unlike BMDMs, transcripts for PTP-ζ were undetectable in NIH/3T3 cells under any stimulation conditions (data not shown). Effects of PTP-ζ on polarization and skewing of BMDMs Since persistent tissue fibrosis typically requires stimulation by TGF-β and IL-4, we performed the following experiments to clarify the influence of the IL-34/PTP-ζ axis on Møs with a fibrotic phenotype. We performed in vitro experiments using BMDMs isolated from WT B6 and PTP-ζ-KO mice. Briefly, BMDMs isolated from WT and PTP-ζ-KO mice were cultured in standard growth medium. After adherence and stabilization until reaching approximately 80% confluence, cells were washed with PBS and subsequently stimulated for 24 hours with a cocktail of rIL-4 (20 ng/mL) and TGF-β (5 ng/mL), with or without rIL-34 (5 ng/mL). To induce definitive skewing toward the M2 phenotype, Møs were stimulated with rIL-4 as previously described 8 . As shown in Figure 8A, mRNA levels for TNF-α, secreted by M1Mø, were not upregulated in either WT-BMDMs or PTP-ζ-KO BMDMs following rIL-34 stimulation, although transcripts of iNOS, another marker of M1Mø, were upregulated in both groups. However, this increase was not further enhanced by additional stimulation with rIL-34, particularly in WT-BMDMs (Fig 8B). In contrast, stimulation with TGF-β and rIL-4 significantly increased arginase-1 transcripts (a marker of M2Mø) in WT-BMDMs. This increase was further enhanced by additional treatment with rIL-34. However, this augmenting effect was lost in PTP-ζ-KO BMDMs due to knockout of the IL-34/PTP-ζ axis (Fig 8C). Moreover, expression of interferon regulatory factor (IRF)-4, a key mediator of Møs polarization toward the M2 phenotype 32 , was also analyzed by WB in the same condition (Fig 8D). Upregulated expressions of IRF-4 were observed in both WT-BMDMs and PTP-ζ-KO BMDMs following stimulation with rIL-4 and TGF-β. Notably, additional stimulation with rIL-34 significantly increased IRF-4 expression levels in WT-BMDMs, whereas no such enhancement after rIL-34 stimulation was observed in PTP-ζ-KO BMDMs. IRF-4 expressions in PTP-ζ-KO BMDMs stimulated with TGF-β, rIL-4, and rIL-34 were significantly suppressed compared to those in WT-BMDMs (Fig 8E). FACS analysis was performed to evaluate the polarization of WT-BMDMs and PTP-ζ-KO BMDMs. Figure 9A-C shows representative FACS plots illustrating the gating strategy used to identify CD11b + F4/80 + CD11c + (M1-like) and CD11b + F4/80 + CD206 + (M2-like) Møs. BMDM counts were normalized to 1,000 CD11b + F4/80 + cells for statistical analysis. Cultured BMDMs without stimulation did not skew toward either CD11c + or CD206 + populations (Fig 9A). In both WT-BMDMs and PTP-ζ-KO BMDMs, stimulation with recombinant cytokines increased both CD11c + M1-like and CD206 + M2-like Mø populations (Fig 9D, E). However, in the presence of IL-34, the proportion of PTP-ζ-KO BMDMs skewing toward CD206 + cells was reduced compared with WT-BMDMs. Consequently, the M2/M1 ratio was significantly decreased in PTP-ζ-KO BMDMs following stimulation with rIL-4, TGF-β, and rIL-34 (Fig 9F). DISCUSSION To our knowledge, the present study provides the first confirmation that genetic deficiency of PTP-ζ prevents the progression of renal fibrosis in mice with UUO. We uncovered the following points: 1) expression levels of PTP-ζ and its primary ligand IL-34, were increased in the UUO kidneys of WT mice; 2) increased expression of profibrogenic genes and accumulation of fibroblasts and myofibroblasts in UUO kidneys were significantly suppressed in PTP-ζ-KO mice; 3) marked Mø accumulation in UUO kidneys, particularly of profibrotic M2-like Møs, was suppressed in PTP-ζ-KO mice; and 4) WT-BMDM stimulated with rIL-4 and TGF-β exhibited enhanced polarization toward the M2 phenotype following additional stimulation with rIL-34, whereas this enhancement was not confirmed in PTP-ζ-KO BMDM. Intrarenal PTP-ζ expression has been demonstrated to be notably elevated during the chronic phase of experimental renal disease models. In lupus-susceptible mice, kidneys exhibiting not only glomerular lesions, but also moderate to severe tubulointerstitial injury and fibrotic change showed a significant increase in both PTP-ζ transcript and protein levels, correlating with progression of lupus nephritis up to 5 months of age 23 . Similarly, in the inflamed kidneys of C57BL/6 mice, PTP-ζ transcript and protein levels were markedly elevated from days 20 to 40 following I/R injury. This increase was associated with fibrosis and tubular damage due to AKI to CKD caused by I/R 21,25 . PTP-ζ expression has not been described in human fibrotic kidney samples with fibrotic change, except for AKI just after renal transplantation 25 . Nevertheless, the above previous findings, together with our current results, indicate the possibility that intrarenal PTP-ζ expression is closely associated with the chronic phase of renal diseases characterized by renal fibrosis. Regarding PTP-ζ ligands in renal fibrosis, clinical studies have shown that elevated serum IL-34 levels are associated with the presence and severity of fibrosis in CKD, as well as with coronary artery disease in patients with heart failure 33 . In the mouse model, both intrarenal IL-34 level and PTP-ζ increased during the CKD phase from day 20 to 40 following I/R injury 21,25 . Moreover, recent analyses have revealed that the other ligands of PTP-ζ (e.g., tenascin-C, pleiotrophin, and midkine) were also upregulated in fibrotic kidneys at days 20 and 40 after I/R 25 . Although we did not assess the intrarenal expression of ligands other than IL-34 in this study, our findings implied that IL-34 was a primary driver of PTP-ζ-mediated signaling in UUO-induced renal fibrosis. Intriguingly, our analysis showed that intrarenal IL-34 expression remained significantly elevated in PTP-ζ-KO UUO kidneys, suggesting that IL-34 expression is not dependent on PTP-ζ itself. Cultured mouse BMDMs consistently showed stable expressions of PTP-ζ with or without additional IL-34 (Fig 7B), contrary to our presumption. Therefore, to better recreate the in vivo microenvironment of UUO-induced renal fibrosis, we performed additional experiments using co-stimulation with rIL-34 together with rIL-4 or TGF-β. Consistent with previous reports 23-25 , expression of PTP-ζ on Mø was detected in the present study. Previous studies, including ours, focusing on the IL-34/PTP-ζ axis have demonstrated that PTP-ζ is expressed on BMDMs 24 . To further investigate the role of the IL-34/PTP-ζ axis in Mø skewing, we performed additional analyses using BMDMs. While tissue-resident Møs under steady-state conditions are generally considered to exist in a neutral M0 state 12,34 , PTP-ζ expression was already detectable in cultured BMDMs under unstimulated conditions in this study, counter to our expectation that expression would be induced upon exposure to polarization stimuli. Our analysis using several phenotypic markers suggested that cultured BMDMs were not fully differentiated M1Møs, but had already initiated polarization toward an M1-like phenotype. The phenotypic changes induced by TGF-β and rIL-4 stimulation in these BMDMs thus may differ from the physiological process in which tissue-resident M0Møs differentiate into M2Møs in vivo . Nevertheless, our results suggest that stimulation with IL-34 was able to further drive a subset of these incompletely polarized M1-like Møs toward an M2-like phenotype (Figs 8, 9). WT-BMDMs stimulated with M2-inducing cytokines and rIL-34 upregulated IRF-4, a factor linked to the renal fibrosis of resident Møs via M2 polarization 32,35 , implying that PTP-ζ signaling in Møs may influence the transition from circulating Møs to resident-like Møs. Although further studies using reliable markers to distinguish resident vs. BM-derived and M1 vs. M2 Møs are needed to clarify the precise role of PTP-ζ in renal fibrosis, we suggest that WT-BMDMs exhibited a stronger shift toward the M2 phenotype than PTP-ζ-deficient BMDMs under above-described experimental conditions. Therefore, IL-34/PTP-ζ axis may accelerate Mø polarization toward the M2 phenotype. Targeting the IL-34/PTP-ζ axis could suppress excessive M2-driven fibrotic responses in CKD, highlighting the potential of this axis as a therapeutic target. Renal fibrosis is characterized by excessive accumulation of ECM, primarily produced by fibroblasts and myofibroblasts 36 . In healthy kidneys, myofibroblasts are basically absent, but it has been considered that myofibroblasts emerge during fibrotic processes and substantially contribute to ECM deposition 36 . Although the precise origin of intrarenal fibroblasts has remained unclear, lineage-tracing experiments using myelin protein zero-Cre mice (which express Cre recombinase in neural crest cells) have revealed that neural crest-derived cells infiltrate the metanephros during embryogenesis and differentiate into renal fibroblasts 36,37 . It is now widely accepted that most myofibroblasts arise from resident renal fibroblasts 38 . In this study, genetic ablation of PTP-ζ significantly attenuated renal fibrosis in a UUO model. This attenuation was accompanied by reduced expression of profibrogenic genes such as TGF-β, along with a marked decrease in the accumulation of fibroblasts and myofibroblasts in fibrotic kidneys. Similarly, in a recent analysis using a renal I/R model, Sirius red and αSMA staining at day 40 post-injury revealed a significant reduction in fibrotic area and area with the presence of myofibroblast in PTP-ζ-KO mice 25 . These findings initially implied that PTP-ζ may regulate fibroblast activation. However, attempts to confirm PTP-ζ expression in fibroblasts using the NIH/3T3 were inconclusive. Accordingly, attention was directed to another important feature of the UUO model: the marked suppression of intrarenal Mø infiltration, particularly M2-like Møs, in PTP-ζ-KO mice. In conclusion, upregulated intrarenal expression of PTP-ζ, along with Mø accumulation, contributes to the progression of renal fibrosis in the UUO mouse model. IL-34 binding to PTP-ζ might promote M2Mø proliferation in UUO kidneys, enhancing profibrotic responses. These findings suggest that PTP-ζ on Mø could represent a potential therapeutic target for renal fibrosis. METHODS Mice The C57BL/6J-Ptprz1 em1Cya (PTP-ζ-KO) mice were purchased from Cyagen (Suite E Santa Clara, CA, USA). Two male PTP-ζ-KO mice and two female PTP-ζ-KO mice were purchased and bred, and the male KO mice obtained from their offspring were used for all experiments in the present study. Briefly, the PTP-ζ-KO mouse model (C57BL/6J background) was generated via CRISPR/Cas-mediated genome engineering. Detailed information regarding the Mouse Ptprz1 Knockout Project is provided in the Supplementary Document. Six-week-old male C57BL/6 (B6) mice were purchased from Oriental Yeast Co. (Tokyo, Japan). All mice were maintained under specific pathogen-free conditions with ad libitum access to food and water in the animal facility of the Analysis Center of Integral Genomic Functions at Kitasato University. Experimental protocol The experimental protocol was approved by the Animal Experimentation and Ethics Committee of Kitasato University (permit nos. M2025-007 and M2025-008). Male, 10-week-old WT B6 and PTP-ζ-KO mice underwent UUO. The left kidney was exposed through a flank incision, then UUO was induced by ligating the left ureter with nylon thread. Mice were sacrificed at 14 days after UUO to collect kidneys. Age-matched male B6 mice received sham operation as NC mice. All surgeries were performed under anesthesia using a mixture of medetomidine hydrochloride (0.75 mg/kg), butorphanol tartrate (5.0 mg/kg) and midazolam (4.0 mg/kg) on a 37°C warming pad. Light microscopy Two experienced kidney pathologists who were blinded to each experimental group evaluated and scored the kidney pathology in Masson’s trichrome-stained paraffin sections, as previously detailed 39 . To evaluate the TI injury, 10 high-power fields (HPFs) (5 from the cortex and 5 from the outer-medulla) from each section were evaluated under 200´ magnification. The extent of TI injury was scored by the percentage of areas with round cell infiltration, tubular dilatation, tubular casts, and tubular epithelial cell necrosis per field. Scores from 0 to 5 were used (0, none; 1, 75% of area), and the results were averaged. Collagen detection To assess the renal fibrotic area, we stained paraffin sections after rehydration in picrosirius red solution (srs250, ScyTek Laboratories, Logan, UT, USA) for 1 hour and rinsed with acidified water. Ten HPFs (5 from the cortex and 5 from the outer-medulla) from each section were selected for assessment, and two renal pathologists blindly evaluated the picrosirius red-positive area in UUO kidneys of the study groups under 400´ magnification. Consequently, the Sirius red-positive area/HPF (as a percentage) was calculated using Image J software (IJ 1.46r; NIH, Bethesda, MD, USA), then results were averaged. Immunofluorescence staining The antibodies (Abs) used for staining are summarized in Supplementary Table S1. The detailed procedure has been described previously 21,22 . Briefly, cut frozen sections (5-µm-thick) were fixed and incubated overnight at 4°C with primary Abs, following incubation with secondary Abs for 2 h. Embedded sections with mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) (ab104139; Abcam, Cambridge, UK) were blindly evaluated by two renal pathologists. The quantification of positive cells for each staining was performed in 10 randomly selected HPFs (5 from the cortex, 5 from the outer medulla) under 400´ magnification 21-23,40 . The mean number of positive cells per HPF was calculated. Homogenization of kidney tissues Kidney tissues (cortex) were homogenized with T-PER Mammalian Protein Extraction Reagent (20 mL/g renal tissue; #78501, Thermo Fisher Scientific, Waltham, MA, USA) containing 1% (v/v) protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) using a TissueLyser (QIAGEN, Hilden, Germany). Harvested lysates were then centrifuged at 3000 g for 10 min at 4°C to remove the cellular debris. Supernatants were collected and stored at -80°C. Protein concentration was measured using the BCA Protein Assay Kit (#23225; Pierce Biotechnology, Rockford, IL, USA) 22,41 . Real-time reverse transcriptase polymerase chain reaction Target gene expression was evaluated using qRT-PCR (TaqMan) assays. The procedure for qRT-PCR was as described previously 22,39,42 . All primers were purchased from Applied Biosystems (Carlsbad, CA, USA), and detailed information is summarized in Supplementary Table S2. Expressions of mRNA were normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control to correct for the differences in the amount of total RNA added to each reaction. Fluorescence-activated cell sorting Briefly, kidneys were digested in collagenase IV (C4-BIOC, Sigma-Aldrich) for 1 h at 37°C, then mashed through a 40-μm sieve, then collected by centrifugation. Single-cell suspensions from kidneys were incubated with 2.4G2 monoclonal Ab (mAb) (anti-FcγRIII/II) to block non-specific binding, then cells were stained with primary mAb as previously described 43 . Abs for FACS are listed in Supplementary Table S3. Western blot analysis Homogenized kidney and harvested lysates from cultured cells were used for WB analysis. For each sample, 20 µg of protein from kidney tissue or 10 µg of protein from cultured cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 4–20% gradient gel (#E-T520L; ATTO CORPORATION, Tokyo, Japan), and the proteins were transferred to a polyvinylidene difluoride membrane. The detailed procedures were as described previously 40,44 . Abs for WB are listed in Supplemental Table S4. Changes in each expression level were normalized by correction to the densitometric intensity of β-actin or GAPDH for each sample using Image J software. Measurement of IL-34 protein levels in kidney tissue homogenate Total IL-34 protein levels were measured in kidney tissue homogenates from each sample using the IL-34 ELISA kit (#M3400; R&D Systems, Abingdon, UK) in accordance with the instructions from the manufacturer 22 . To control for differences between samples, the concentration was corrected based on the amount of total tissue protein 41 . Isolation of bone marrow-derived macrophages To isolate BMDMs from WT B6 mice and PTP-ζ-KO mice (8–12 weeks old), mice were euthanized by cervical dislocation. The abdomen and hind limbs were sterilized with 70% ethanol, and the femurs were dissected out by cutting the bones at both ends. BM was flushed out from the dissected femurs using a 27-gauge needle with RPMI 1640 medium (#11875093; Thermo Fisher Scientific). The detailed procedure has been described previously 31 . To eliminate residual RBCs, RBC lysis buffer (#42030; BioLegend, San Diego, CA, USA) was used. The isolated BMDMs (3.0 ×10 6 cells/well) were initially thawed onto six-well plates, and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), antibiotics, and CSF-1 (20 ng/mL, #416-ML-050; R&D Systems) for 72–96 h until around 80% confluence. We verified the characteristic phenotype of BMDM by analyzing cFMS expression. The cultured BMDMs were washed with sterile PBS to remove residual CSF-1, and the culture medium was changed to RMPI 1640 medium supplemented with 10% FBS and antibiotics during the stimulation or treatment for experiments. In the experiments, BMDMs were stimulated with equal volumes of saline, TGF-β (5 ng/mL, #7666-MB-005; R&D) or rIL-34 (5 ng/mL, #577602; BioLegend) or rIL-4 (20 ng/mL, #214-14-20UG; Thermo Fisher Scientific). All cells were cultured in an atmosphere of 5% CO 2 -95% air at 37°C in a humidified incubator. Cell cultures for mouse fibroblasts NIH/3T3 cells were purchased from ATCC (University Boulevard, Manassas, VA, USA). Quality control and authentication of NIH/3T3 cells are guaranteed and validated by ATCC, as described in the product sheet (CRL-1658™). NIH/3T3 were cultured in ATCC-formulated Dulbecco's modified Eagle's medium (#30-2002; ATCC) supplemented with 10% FCS and antibiotics for 48 h until around 80–90% confluence for analysis. Statistical analysis Data were shown as the mean ± standard error of the mean (SEM), and were analyzed using GraphPad Prism software (version 10.0; GraphPad Software, San Diego, CA, USA). The Mann–Whitney U test was applied for comparisons between groups. Multiple groups were compared using one-way analysis of variance with a post-hoc Tukey’s honestly significant difference test. P value of < 0.05 was considered statistically significant. Declarations Funding This study was supported by the Japan Society for the Promotion of Science KAKENHI (grant no. 22K08319). Acknowledgements The authors greatly appreciate the excellent technical assistance provided by Ms. Masako Takamatsu, Ms. Yui Onoda, and Ms. Miki Hashimura. Author contributions TA, YW, ET, and YT wrote the manuscript. TA, YW, ET, MS, TI, MO, TS, and YT collected data and reviewed the manuscript. All authors contributed to the article and approved the submitted version. Data availability statement All relevant data are within the manuscript and associated supporting information files. Conflicts of Interest None of the authors have any conflicts of interest to declare. References Carney, E. F. The impact of chronic kidney disease on global health. Nat Rev Nephrol 16, 251, doi:10.1038/s41581-020-0268-7 (2020). Chen, T. K., Knicely, D. H. & Grams, M. E. Chronic Kidney Disease Diagnosis and Management: A Review. JAMA 322, 1294-1304, doi:10.1001/jama.2019.14745 (2019). Neuen, B. L., Chadban, S. J., Demaio, A. R., Johnson, D. W. & Perkovic, V. 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Iyoda, M., Shibata, T., Hirai, Y., Kuno, Y. & Akizawa, T. Nilotinib attenuates renal injury and prolongs survival in chronic kidney disease. J Am Soc Nephrol 22, 1486-1496, doi:10.1681/asn.2010111158 (2011). Wada, Y. et al. Epidermal growth factor receptor inhibition with erlotinib partially prevents cisplatin-induced nephrotoxicity in rats. PLoS One 9, e111728, doi:10.1371/journal.pone.0111728 (2014). Yamamoto, Y. et al. Erlotinib attenuates the progression of chronic kidney disease in rats with remnant kidney. Nephrol Dial Transplant 33, 598-606, doi:10.1093/ndt/gfx264 (2018). Tachibana, S. et al. Recombinant human soluble thrombomodulin attenuates anti-glomerular basement membrane glomerulonephritis in Wistar-Kyoto rats through anti-inflammatory effects. Nephrol Dial Transplant 34, 774-782, doi:10.1093/ndt/gfy201 (2019). Satoh, M. et al. Adipose invariant NKT cells interact with CD1d-expressing macrophages to regulate obesity-related inflammation. Immunology 165, 414-427, doi:10.1111/imm.13447 (2022). Imanishi, T. et al. RIPK1 blocks T cell senescence mediated by RIPK3 and caspase-8. Sci Adv 9, eadd6097, doi:10.1126/sciadv.add6097 (2023). Additional Declarations No competing interests reported. Supplementary Files SupplementaryTableS1.docx SupplementaryTableS2.rtf SupplementaryTableS3.docx SupplementaryTableS4.docx SupplementaryfileforPTPzKOmice.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 17 Apr, 2026 Editor assigned by journal 14 Apr, 2026 Submission checks completed at journal 14 Apr, 2026 First submitted to journal 11 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9386889","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":624808137,"identity":"491597bf-d049-41a7-8d63-0a8c924a5ebc","order_by":0,"name":"Tetsuya Abe","email":"","orcid":"","institution":"Kitasato University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Tetsuya","middleName":"","lastName":"Abe","suffix":""},{"id":624808139,"identity":"60837760-3b77-4451-9407-7300c3406b45","order_by":1,"name":"Yukihiro Wada","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYHACAyC2MWBDFeQhqCXNgA2sJ4F4LYcNGFC14FN//PDGD4xt54355Jsff/j5w4bB4ADzww8MMndwazmTVizB2HbbjI2NzUyyJyENqIXNWIKB5xlOLWYHcgyk/7bdtgH6xYyBJ+Fw/YYDQAYDz2HcWs6/Mf7B2HYOqIX988c/CYeBtrB/w6/lRo4Z0GEHgA7jMZDmAWvhwW+L/Y1nZRYM55KN2dhyyqRl0tIYJA/zFEsk4PGLZH/y5hsMZXaG85uPb/74xsaGge94+8YPH3twhxgYMKJEPTMQJ/YcwK+F4Q+GyA9CWkbBKBgFo2AEAQAgzk4mRw49nAAAAABJRU5ErkJggg==","orcid":"","institution":"Kitasato University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Yukihiro","middleName":"","lastName":"Wada","suffix":""},{"id":624808149,"identity":"ee3a366d-e457-4426-ac5a-206491f09528","order_by":2,"name":"Emiko Takeuchi","email":"","orcid":"","institution":"Kitasato University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Emiko","middleName":"","lastName":"Takeuchi","suffix":""},{"id":624808150,"identity":"07e0db54-39a4-4e4d-b4ab-7e9a8b0fa32b","order_by":3,"name":"Masashi Satoh","email":"","orcid":"","institution":"Kitasato University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Masashi","middleName":"","lastName":"Satoh","suffix":""},{"id":624808152,"identity":"f0ca46e6-0bdb-449a-9fff-916c66848958","order_by":4,"name":"Takayuki Imanishi","email":"","orcid":"","institution":"Kitasato University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Takayuki","middleName":"","lastName":"Imanishi","suffix":""},{"id":624808155,"identity":"ec0c6f98-d531-4d9e-b302-b3f950e59e75","order_by":5,"name":"Makoto Otsu","email":"","orcid":"","institution":"Kitasato University School of Allied Health Sciences","correspondingAuthor":false,"prefix":"","firstName":"Makoto","middleName":"","lastName":"Otsu","suffix":""},{"id":624808157,"identity":"f6902e21-bb74-47d5-bca4-442347d9823c","order_by":6,"name":"Tadahiro Suenaga","email":"","orcid":"","institution":"Kitasato University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Tadahiro","middleName":"","lastName":"Suenaga","suffix":""},{"id":624808158,"identity":"aaa23d9b-50b8-4018-a65b-8017f4d00cfb","order_by":7,"name":"Yasuo Takeuchi","email":"","orcid":"","institution":"Kitasato University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yasuo","middleName":"","lastName":"Takeuchi","suffix":""}],"badges":[],"createdAt":"2026-04-11 10:10:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9386889/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9386889/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107485807,"identity":"ba6161a0-9f9f-4d4b-a27f-da56b44d5745","added_by":"auto","created_at":"2026-04-22 02:36:22","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":75787,"visible":true,"origin":"","legend":"\u003cp\u003eIntrarenal expressions of IL-34 and PTP-ζ\u003c/p\u003e\n\u003cp\u003eA) Body weight (BW) change from baseline to day 14 (at time of sacrifice) after sham operation or induction of UUO among sham-operated WT mice, WT mice with UUO, and PTP-ζ-KO mice with UUO. B) Intrarenal IL-34 expressions analyzed by real-time RT-PCR among the study groups. Values are normalized to levels of GAPDH transcript and expressed as the relative ratio. C) IL-34 protein levels in homogenate kidney tissues (50 μg) analyzed by ELISA among the study groups. D) Representative WB analysis for PTP-ζ and β-actin among the study groups. E) Densitometric analysis of WB for PTP-ζ (fold-change of PTP-ζ/β-actin ratio from control) among the study groups. F) Intrarenal PTP-ζ expressions analyzed by real-time RT-PCR among the study groups. Values are normalized to levels of GAPDH transcript and expressed as the relative ratio. All data are expressed as mean ± SEM. The Mann–Whitney U test was used for statistical analyses.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9386889/v1/13b040737749e0f9d755452e.jpg"},{"id":107296336,"identity":"5ace7e82-2f25-4168-915f-b4e0a6df8d68","added_by":"auto","created_at":"2026-04-20 06:45:59","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":140796,"visible":true,"origin":"","legend":"\u003cp\u003eRenal histological findings in mice with UUO\u003c/p\u003e\n\u003cp\u003eA–F) Representative photos of kidney tissues stained with Masson’s trichrome in a sham-operated WT mouse (Aand D), WT mouse with UUO (B and E), and PTP-ζ-KO mouse with UUO (C, F). A–C) Original magnification, ×100. D–F)Original magnification, ×200. G–I) Quantification of the tubulointerstitial damage score per field in the cortex (G), outer medulla (H), and cortex and outer medulla (I) among the study groups. Data are expressed as mean ± SEM. The Mann–Whitney U test was used for statistical analysis.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9386889/v1/a69cfb52b924179b53ab061c.jpg"},{"id":107296337,"identity":"ab12cd00-7b38-42a8-a7c0-d26b6a4a0e48","added_by":"auto","created_at":"2026-04-20 06:45:59","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":160790,"visible":true,"origin":"","legend":"\u003cp\u003eRenal fibrosis in mice with UUO\u003c/p\u003e\n\u003cp\u003eA–F) Representative images of kidney tissues stained with Sirius red in a sham-operated WT mouse (Aand D), WT mouse with UUO (B and E), and PTP-ζ-KO mouse with UUO (C and F). Upper panels (A–C) show the renal cortex; lower panels (D–F) show the outer medulla. Original magnification, 200×. G–I) Percentages of Sirius red-positive area in the cortex (G), outer medulla (H), and combined cortex and outer medulla (I). J–M) Real-time RT-PCR for genes encoding TGF-β (J), α-SMA (K), collagen type I (L), and fibronectin (M). Values are normalized to the GAPDH transcript and given as the relative ratio. Data are expressed as mean ± SEM. The Mann–Whitney U test was used for statistical analysis.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9386889/v1/0ca6b03287ce512af5acabfe.jpg"},{"id":107296341,"identity":"5a9b6bdc-2bb9-477c-8354-5ae80364a639","added_by":"auto","created_at":"2026-04-20 06:46:00","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":135287,"visible":true,"origin":"","legend":"\u003cp\u003eIntrarenal fibroblasts and myoblasts in mice with UUO\u003c/p\u003e\n\u003cp\u003eA–I) Representative images of fibroblasts by immunofluorescence analysis (PDGFRβ staining, ×400; DAPI staining, ×400; and merged PDGFRβ and DAPI staining, ×400) in kidney tissues among sham-operated WT (A, D, and G), WT with UUO (B, E, and H), and PTP-ζ with UUO (C, F, and I) mice. Scale bar, 50 μm. J) Quantitative evaluation of PDGFRβ-positive cells per HPF by immunofluorescence among study groups. K–S) Representative images of myofibroblasts by immunofluorescence analysis (α-SMA staining, ×400; DAPI staining, ×400; and merged α-SMA and DAPI staining, ×400) in kidney tissues in a sham-operated WT mouse (K, N, and Q), WT mouse with UUO (L, O, and R), and PTP-ζ-KO mouse with UUO (M, P, and S). Scale bar, 50 μm. T) Quantitative evaluation of PDGFRβ-positive cells per HPF by immunofluorescence among study groups. Data are expressed as mean ± SEM. The Mann–Whitney U test was used for statistical analyses.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9386889/v1/bf29446c5dd55fd7fda138e7.jpg"},{"id":107296344,"identity":"c49397b0-5ff5-4551-a19f-50f511c01afd","added_by":"auto","created_at":"2026-04-20 06:46:00","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":128493,"visible":true,"origin":"","legend":"\u003cp\u003eIntrarenal Mø infiltration and polarization in mice with UUO\u003c/p\u003e\n\u003cp\u003eA–E) Real-time RT-PCR for genes encoding Mø mediators such as cFMS (A), TNF-α (B), IL-1β (C), Yim-1/Chil 3 (D), and Timp-1 (E). Values are normalized to the GAPDH transcript, and expressed as the relative ratio. F–Q) Representative images of F4/80-positive Mø infiltration and CD206-positive M2-like Mø polarization by immunofluorescence analysis (DAPI staining, ×400; F4/80 staining, ×400; CD206 staining, ×400; and merged F4/80 and CD206 staining, ×400) in fibrotic UUO kidneys tissue among sham-operated WT (F–I), WT with UUO (J–M), and PTP-ζ-KO with UUO (N–Q) mice. Scale bar, 50 μm. Intrarenal F4/80\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e cell infiltration is shown by white triangles (M, Q) Quantitative evaluation of F4/80-positive cells per HPF (R) and cells positive for both F4/80 and CD206 per HPF (S) on immunofluorescence among study groups. Data are expressed as mean ± SEM. The Mann–Whitney U test was used for statistical analyses.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9386889/v1/20ab3d5a5281b5ea4c053ac7.jpg"},{"id":107484240,"identity":"cd128879-4592-4101-b833-201252f5e507","added_by":"auto","created_at":"2026-04-22 02:31:12","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":115772,"visible":true,"origin":"","legend":"\u003cp\u003eIntrarenal Mø polarization in mice with UUO in FACS analysis\u003c/p\u003e\n\u003cp\u003eA–C) Stained single-cell suspensions from kidneys of sham-operated WT mice, WT mice with UUO, and PTP-ζ-KO mice with UUO. The viable CD45\u003csup\u003e+\u003c/sup\u003e peripheral blood cell population was gated first, then the CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e or CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e cell population was gated. Representative FACS plots in kidneys of WT mouse CL (A), WT mouse with UUO (B), and PTP-ζ-KO mouse with UUO (C). D–G) Graphs of the number of intrarenal CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e Møs (D), intrarenal CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e M1-like Møs (E) and CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e M2-like Møs (F), and the ratio of M1-like Møs to M2-like Møs (G) in the kidneys of mouse groups. Data are expressed as mean ± SEM. The Mann–Whitney U test was used for statistical analyses.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9386889/v1/f2c4f6bf83d6e1c0f5c33433.jpg"},{"id":107485624,"identity":"18cb64f8-b663-4e29-808f-b829bfdc1d7d","added_by":"auto","created_at":"2026-04-22 02:35:39","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":44100,"visible":true,"origin":"","legend":"\u003cp\u003ePTP-ζ expressions in bone marrow-derived macrophages (BMDMs)\u003c/p\u003e\n\u003cp\u003eA) Scheme for the analysis. Mouse BMDMs were isolated from WT and PTP-ζ KO B6 mice. Isolated BMDMs, as demonstrated in photo, were cultured with medium and stimulated with equal volumes of saline, TGF-β (5 ng/mL), and recombinant IL-34 (rIL-34, 5 ng/mL), followed by harvest of cells for analysis. B) Representative WB analysis for PTP-ζ and β-actin in BMDMs from WT or PTP-ζ-KO mice following indicated stimulations.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9386889/v1/618af80cb2325370f79e88cd.jpg"},{"id":107868667,"identity":"2f2c5bdc-4a08-4382-bee9-b9f6a79dcdb2","added_by":"auto","created_at":"2026-04-27 07:31:00","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":121845,"visible":true,"origin":"","legend":"\u003cp\u003ePolarization and skewing of BMDMs after stimulations\u003c/p\u003e\n\u003cp\u003eMouse BMDMs were isolated from WT and PTP-ζ-KO B6 mice. The isolated BMDMs were cultured with medium and stimulated with equal volume of saline, recombinant IL (rIL)-4 (20 ng/mL), TGF-β (5 ng/mL), and rIL-34 (5 ng/mL), followed by harvest of cells for analysis. A–C) Real-time RT-PCR for genes encoding mediators for Mø polarization such as TNF-α (M1 Mø marker) (A), iNOS (M1Mø marker) (B), and Arg-1 (M2Mø marker) (C). Values are normalized to the GAPDH transcript, and are expressed as the relative ratio. D) Representative WB analysis for interferon regulatory factor 4 (IRF-4) and β-actin on BMDMs from WT or PTP-ζ-KO mice following indicated stimulations. E) Values represent protein expressions after normalization to β-actin expression, and are depicted as the relative ratio of IRF-4 to β-actin (fold-change of IRF-4/β-actin ratio from control) among study groups. Data are expressed as mean ± SEM. The Mann–Whitney U test was used for statistical analyses.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9386889/v1/9c8107741c0443532ab17452.jpg"},{"id":107296345,"identity":"1b768c6a-b600-47f8-a58f-c7629bc2d80d","added_by":"auto","created_at":"2026-04-20 06:46:00","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":178713,"visible":true,"origin":"","legend":"\u003cp\u003eBMDM polarization in FACS analysis\u003c/p\u003e\n\u003cp\u003eMouse BMDMs were isolated from WT and PTP-ζ-KO B6 mice. Isolated BMDMs were cultured with medium and stimulated with recombinant IL-4 (rIL-4 20 ng/mL), TGF-β (5 ng/mL), and recombinant IL-34 (rIL-34, 5 ng/mL), followed by harvest of cells for analysis. The F4/80\u003csup\u003e+\u003c/sup\u003e cell population was gated first, then the CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e (M1-like) or CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e (M2-like) cell population was gated. A–C) Representative FACS plots illustrate the gating strategy to identify M1-like and M2-like Mø populations in cultured WT BMDMs (A), WT BMDMs stimulated with rIL-4, TGF-β, and rIL-34 (B), and PTP-ζ-KO BMDMs stimulated with rIL-4, TGF-β, and rIL-34 (C). D–F) Graphs of the number of CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e Møs, CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e M1-like Møs (D), and CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e M2-like Møs (E), and the ratio of M2-like Møs to M1-like Møs (F). Data are expressed as mean ± SEM. BMDM counts are normalized to 1,000 cells for statistical analysis. The Mann–Whitney U test was used for statistical analyses.\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9386889/v1/8ba8b679825f4cab2cd26eac.jpg"},{"id":108006891,"identity":"eb55cc85-a0ed-48cf-96fb-96d6c17097f1","added_by":"auto","created_at":"2026-04-28 12:57:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1434827,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9386889/v1/03e0ab54-ebbb-4ea1-9c2f-ce542d9f1010.pdf"},{"id":107296334,"identity":"26516bd8-fe5f-43cf-bd06-6675c8e69aa5","added_by":"auto","created_at":"2026-04-20 06:45:59","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":20012,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9386889/v1/5ff05b2ff9da06693d5b93d9.docx"},{"id":107486751,"identity":"6cc3d419-c480-43dc-b1dc-94b6b4d570f4","added_by":"auto","created_at":"2026-04-22 02:38:52","extension":"rtf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":61269,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS2.rtf","url":"https://assets-eu.researchsquare.com/files/rs-9386889/v1/28f8dba1a824363eabb9b36f.rtf"},{"id":107296339,"identity":"5b70ad5e-640d-4d71-aad0-fabf8630ad98","added_by":"auto","created_at":"2026-04-20 06:45:59","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":22824,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-9386889/v1/93ebbe7c7e9e02b11106c8ca.docx"},{"id":107296338,"identity":"56e222bb-7993-4f57-9148-498f6c591142","added_by":"auto","created_at":"2026-04-20 06:45:59","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":24546,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS4.docx","url":"https://assets-eu.researchsquare.com/files/rs-9386889/v1/caf003a9c865d9cb06d901c3.docx"},{"id":107296340,"identity":"bcf802a4-d812-4f51-b1a7-e3e8a45454ce","added_by":"auto","created_at":"2026-04-20 06:46:00","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":40438,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryfileforPTPzKOmice.docx","url":"https://assets-eu.researchsquare.com/files/rs-9386889/v1/9a601f3804fc763839d63f97.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Involvement of intrarenal protein-tyrosine phosphatase ζ receptor in progression of renal fibrosis in mice with unilateral ureteral obstruction","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eChronic kidney disease (CKD) affects more than 10% of the global population and is significantly associated with risk of mortality\u003csup\u003e1-3\u003c/sup\u003e. In aging societies such as Japan, CKD has become a major public health concern and imposes considerable burdens on healthcare systems and medical expenditures\u003csup\u003e4\u003c/sup\u003e. Renal fibrosis is an inevitable outcome of all progressive CKD, including chronic glomerulonephritis, hypertensive CKD, and diabetic CKD\u003csup\u003e5\u003c/sup\u003e. In other words, renal fibrosis is a final common pathway for CKD progressing to end-stage renal disease\u003csup\u003e2,5\u003c/sup\u003e. With regard to the mechanisms underlying renal fibrosis, activated myofibroblasts are the principal effectors responsible for the production of extracellular matrix (ECM) proteins in the fibrotic kidney\u003csup\u003e6-8\u003c/sup\u003e. Moreover, the accumulation of intrarenal macrophages (M\u0026oslash;s) is another important cause of the development and progression of renal fibrosis\u003csup\u003e9-11\u003c/sup\u003e. Kidney M\u0026oslash;s are characterized by wide heterogeneity and marked plasticity and play critical roles in not only maintaining renal homeostasis, but also contributing to renal inflammation and fibrosis\u003csup\u003e12,13\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eGenerally, activated M\u0026oslash;s are conceptually divided into two major polarization states: classical type 1 (M1); and alternative type 2 (M2)\u003csup\u003e14,15\u003c/sup\u003e. Simplistically, M1M\u0026oslash;s are pro-inflammatory or cytotoxic M\u0026oslash;s, secreting pro-inflammatory cytokines such as tumor necrosis factor (TNF)-\u0026alpha;. In contrast, M2M\u0026oslash;s are involved in immune regulation, anti-inflammation, repair or regeneration, and fibrosis\u003csup\u003e12,14-16\u003c/sup\u003e. Regarding the contributions of M\u0026oslash;s to renal disease, aberrantly proliferative M1M\u0026oslash;s have been recognized to aggravate acute kidney injury (AKI), and the inadequate removal of M2M\u0026oslash;s causes renal fibrosis\u003csup\u003e12,17\u003c/sup\u003e. In addition, several previous studies have mentioned that accumulated M2M\u0026oslash;s promote renal fibrosis by inducing overproduction of ECM components and secretion of profibrotic factors\u003csup\u003e8,11,17,18\u003c/sup\u003e. In short, M2M\u0026oslash;s, rather than M1M\u0026oslash;s, are significantly involved in the development of renal fibrosis. This suggests that preventing the intrarenal accumulation of M2M\u0026oslash;s could be a promising therapeutic strategy for renal fibrosis.\u003c/p\u003e\n\u003cp\u003eProtein-tyrosine phosphatase \u0026zeta; receptor (PTP-\u0026zeta;, PTPRZ1) has been discovered as a secondary receptor of interleukin (IL)-34 that is involved in M\u0026oslash; proliferation\u003csup\u003e19\u003c/sup\u003e. PTP-\u0026zeta; is mainly expressed in brain cells such as neurons and astrocytes. Consisting of several domains and three isoforms, PTP-\u0026zeta; is involved in neuronal migration, neuroaxonal elongation, synapse formation, and synaptic regulation\u003csup\u003e20\u003c/sup\u003e.\u0026nbsp;Several studies, including our own and those of our colleagues, have demonstrated that PTP-\u0026zeta; is expressed in not only brain, but also renal tubular epithelial cells (TECs)\u003csup\u003e21,22\u003c/sup\u003e, M\u0026oslash;s\u003csup\u003e23,24\u003c/sup\u003e, and lymphocytes\u003csup\u003e23,25\u003c/sup\u003e. In addition to IL-34, various other ligands for\u0026nbsp;PTP-\u0026zeta;\u0026nbsp;have been identified, such as tenascin-C, pleiotrophin, and midkine\u003csup\u003e25-27\u003c/sup\u003e. However, among these ligands, intrarenal IL-34 expressions were significant and activation of intrarenal IL-34 through the two representative receptors, colony-stimulating factor 1 (CSF-1) receptor (CSF-1R; also known as cFMS and CD115) and PTP-\u0026zeta;, was clearly associated with progression of renal disorder in experimental models of renal disease such as ischemia-reperfusion injury (I/R)-induced AKI\u003csup\u003e21\u003c/sup\u003e, cisplatin-induced AKI\u003csup\u003e22\u003c/sup\u003e, advanced lupus nephritis\u003csup\u003e23\u003c/sup\u003e, and AKI to CKD caused by I/R\u003csup\u003e25\u003c/sup\u003e. Of note, alongside the elevation of intrarenal IL-34, both receptors cFMS and PTP-\u0026zeta; are also highly expressed in damaged renal tissues. In particular, expression of PTP-\u0026zeta; was dominant in the chronic phases of the aforementioned renal disease models\u003csup\u003e21,23,25\u003c/sup\u003e. We therefore hypothesized that the intrarenal IL-34/PTP-\u0026zeta; axis, rather than the IL-34/cFMS axis, is deeply associated with the progression of CKD, particularly renal fibrosis.\u003c/p\u003e\n\u003cp\u003eIn the present study, we attempted to elucidate the influence of PTP-\u0026zeta; on renal fibrosis and M\u0026oslash; polarization by focusing on the physiological properties of the IL-34/PTP-\u0026zeta; axis \u003cem\u003ein vivo\u003c/em\u003e in a unilateral ureteral obstruction (UUO) model and \u003cem\u003ein vitro\u003c/em\u003e analysis using PTP-\u0026zeta;-knockout (KO) mice. No such study has been reported to date. Herein, we demonstrated that the genetic ablation of PTP-\u0026zeta; attenuated renal fibrosis by UUO via suppression of profibrotic M\u0026oslash; accumulation.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eIntrarenal expressions of IL-34 and PTP-ζ in mice with UUO\u003c/p\u003e\n\u003cp\u003eDuring the experiment period up to day 14 after UUO induction, no mortality was observed in any groups. Body weight changes from baseline to sacrifice were comparable among study groups (Fig 1A). Intrarenal mRNA levels for IL-34, a primary ligand for PTP-ζ, were significantly upregulated in UUO mice, with no significant difference between wild-type (WT) and PTP-ζ-KO mice (Fig 1B). Similarly, protein levels of IL-34 evaluated by enzyme-linked immunosorbent assay (ELISA) in damage kidneys were significantly elevated in UUO mice, but no differences were seen among WT and PTP-ζ-KO mice (Fig 1C). Regarding intrarenal PTP-ζ expressions in UUO mice, western blot (WB) analysis showed clear expressions in the WT group (Fig 1D). Densitometric values of PTP-ζ expression were significantly increased in WT mice with UUO (WT-UUO) when compared to sham-operated WT normal control (NC) mice. In addition, intrarenal PTP-ζ mRNA levels were also upregulated in WT-UUO mice (Fig 1E, F). Meanwhile, neither protein nor mRNA levels for intrarenal PTP-ζ were detected in PTP-ζ-KO mice with UUO (PTP-ζ-KO-UUO) (Fig 1E, F).\u003c/p\u003e\n\u003cp\u003eEffects of PTP-ζ on tubular damage and renal fibrosis in UUO mice\u003c/p\u003e\n\u003cp\u003eRepresentative images of kidney tissues stained with Masson’s trichrome among the study group mice are shown in Figure 2A–F. Compared to the NC group, the tubulointerstitial (TI) injury score of WT-UUO mice was significantly increased in the cortex, outer medulla, and combined cortical and outer medullary area (Fig 2G–I). Of note, elevated TI injury scores were significantly attenuated in PTP-ζ-KO-UUO mice compared to WT-UUO mice in each area (Fig 2G–I).\u003c/p\u003e\n\u003cp\u003eRegarding renal fibrosis as evaluated by the Sirius red-positive area, representative images of fibrotic kidneys among study groups are shown in Figure 3A–F. The Sirius red-positive renal fibrotic area was significantly increased in WT-UUO mice compared to sham-operated WT mice in the cortex, outer medulla, and combined cortex and outer medulla. The area with markedly elevated fibrosis was significantly suppressed in PTP-ζ-KO-UUO mice compared to WT-UUO mice (Fig 3G–I). In addition, mRNA levels for intrarenal fibrogenetic genes (including TGF-β, α-SMA, collagen type I [Col-1], and fibronectin) were significantly upregulated in UUO mice, whereas significant increases in levels of transcripts for these intrarenal fibrogenetic genes were suppressed in PTP-ζ-KO-UUO mice compared to WT-UUO mice (Fig 3J–M).\u003c/p\u003e\n\u003cp\u003eEffects of PTP-ζ on intrarenal fibroblasts and myofibroblasts in UUO mice\u003c/p\u003e\n\u003cp\u003eAccumulation of fibroblasts in renal tissue was evaluated by immunofluorescence (IF) for platelet-derived growth factor receptor β (PDGFRβ), which is expressed in interstitial fibroblasts and is associated with promoting fibroblast proliferation\u003csup\u003e28\u003c/sup\u003e. Representative images of PDGFRβ staining in kidney tissues among study group mice are shown in Figure 4A–I. WT-UUO mice showed a significant increase in the number of PDGFRβ-positive\u0026nbsp;fibroblasts compared to sham-operated WT mice (Fig 4A, B, and J). PTP-ζ-KO-UUO mice showed a significantly reduced number of PDGFRβ-positive fibroblasts compared to WT-UUO mice (Fig 4B, C, and J). To evaluate accumulation of myofibroblasts in kidney tissues, IF staining of α-SMA\u003csup\u003e8\u003c/sup\u003e among study groups was performed (Fig 4K–S). In WT-UUO mice, the number of α-SMA-positive myofibroblasts was significantly increased when compared to sham-operated WT mice (Fig 4K, L, and T). This increased number of myofibroblasts was significantly attenuated in PTP-ζ-KO-UUO mice compared to WT-UUO mice (Fig 4L, M, and T).\u003c/p\u003e\n\u003cp\u003eEffects of PTP-ζ on intrarenal Mø accumulation and polarization in UUO mice\u003c/p\u003e\n\u003cp\u003eMøs are thought to play a key role in renal fibrosis by skewing from an M1-dominant to an M2-dominant state. We therefore evaluated Mø accumulation and polarization within the renal tissues of mice from each experimental group using real-time reverse transcriptase polymerase chain reaction (RT-PCR) (Fig 5A–E) and IF staining (Fig 5F–S). Expression of cFMS is known to occur predominantly on Mø as a primary receptor for IL-34 to promote Mø proliferation\u003csup\u003e27\u003c/sup\u003e. As shown in Figure 5A, mRNA levels for cFMS were significantly elevated in the kidneys of UUO mice, and expression of this gene was significantly suppressed in PTP-ζ-KO-UUO mice compared to WT-UUO mice. The accumulation of F4/80-positive Mø in the fibrotic UUO kidneys of both WT and PTP-ζ-KO mice was shown by IF images (Fig 5G, K, and O). Quantitative analysis showed that the number of F4/80-positive Mø was significantly higher in UUO kidneys compared to the NC group. However, the accumulated Mø infiltration was significantly decreased in PTP-ζ-KO-UUO mice compared to WT-UUO mice (Fig 5R). To evaluate polarization of Mø, intrarenal transcripts for TNF-α and IL-1β, which are commonly secreted by M1Mø, were measured by RT-PCR. Up-regulation of mRNA for TNF-α and IL-1β were comparable between WT-UUO mice and PTP-ζ-KO-UUO mice (Fig 5B, C). In contrast, elevated intrarenal mRNA levels for Yim-1/Chil-3 and TIMP-1, regarded as markers for profibrotic M2Mø\u003csup\u003e11,29\u003c/sup\u003e, were elevated in UUO mice and clearly higher in WT-UUO mice compared to PTP-ζ-KO-UUO mice (Fig 5D, E). Double staining for F4/80 and CD206, markers (for identifying M2Mø\u003csup\u003e8\u003c/sup\u003e) revealed marked M2Mø accumulation in the kidneys of UUO mice compared to sham-operated controls (Fig 5I, M, and Q). Notably, the number of F4/80\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e M2Mø in UUO kidneys was significantly reduced in PTP-ζ-KO-UUO mice compared to WT-UUO mice (Fig 5S).\u003c/p\u003e\n\u003cp\u003eFluorescence-activated cell sorting (FACS) analysis was performed to evaluate intrarenal Mø polarization across study groups. Representative FACS plots showing the gating strategy for CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e (M1-like) and CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e (M2-like) Mø populations in sham-operated NC mice, WT-UUO mice, and PTP-ζ-KO-UUO mice are presented in Figure 6A–C. Based on previous findings\u003csup\u003e30\u003c/sup\u003e, CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e cells were broadly identified as cyto-destructive, proinflammatory M1-like Mø, while CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e cells were recognized as cyto-protective, profibrotic M2-like Mø. As shown in Figure 6D, the number of intrarenal Møs identified by FACS analysis tended to be higher in UUO mice compared to the NC WT group. Although the number of M1-like Møs were comparable among the study group (Fig 6E), the number of M2-like Møs tended to be higher in WT-UUO mice than in PTP-ζ-KO UUO mice (Fig 6F), although this difference did not reach statistical significance. Similarly, the elevated M2/M1-like Mø ratio tended to be reduced in PTP-ζ-KO-UUO mice compared to WT-UUO mice, although no significant difference was detected (P=0.0612) (Fig 6G).\u003c/p\u003e\n\u003cp\u003eExpressions of PTP-ζ in bone marrow-derived Møs (BMDMs)\u003c/p\u003e\n\u003cp\u003eThese\u003cem\u003e\u0026nbsp;in vivo\u0026nbsp;\u003c/em\u003estudy results led us to hypothesize that PTP-ζ might be expressed on Møs, and that the IL-34/ PTP-ζ axis may be involved in skewing toward an M2-like phenotype. To test this hypothesis, we performed \u003cem\u003ein vitro\u003c/em\u003e experiments using mouse BMDMs isolated from WT B6 mice and PTP-ζ-KO B6 mice (Fig 7).\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 7A, BMDMs from the two groups were cultured with medium as demonstrated previously\u003csup\u003e31\u003c/sup\u003e. After washing with phosphate-buffered saline (PBS), BMDMs were stimulated with recombinant IL-34 (rIL-34) (5 ng/mL), TGF-β (5 ng/mL), or both for 24 hours. WB analysis revealed that PTP-ζ was constitutively expressed in WT-BMDMs and its expression remained relatively stable under all stimulation conditions (Fig 7B). In contrast, PTP-ζ expression was upregulated \u003cem\u003ein vivo\u003c/em\u003e during UUO-induced inflammation. No detectable PTP-ζ expression was observed in PTP-ζ-KO BMDMs following stimulation with rIL-34 alone, TGF-β alone, or the combination. These findings suggest that the regulatory mechanisms controlling PTP-ζ expression may differ between the \u003cem\u003ein vivo\u003c/em\u003e UUO inflammatory environment and the \u003cem\u003ein vitro\u003c/em\u003e BMDM culture system.\u003c/p\u003e\n\u003cp\u003eSimilarly, the NIH/3T3 murine fibroblast cell line was cultured in specific medium and stimulated with equal volumes of saline, TGF-β, and rIL-34 following an 8-hour starvation period.\u0026nbsp;Unlike BMDMs, transcripts for PTP-ζ were undetectable in NIH/3T3 cells under any stimulation conditions (data not shown).\u003c/p\u003e\n\u003cp\u003eEffects of PTP-ζ on polarization and skewing of BMDMs\u003c/p\u003e\n\u003cp\u003eSince persistent tissue fibrosis typically requires stimulation by TGF-β and IL-4, we performed the following experiments to clarify the influence of the IL-34/PTP-ζ axis on Møs with a fibrotic phenotype. We performed \u003cem\u003ein vitro\u003c/em\u003e experiments using BMDMs isolated from WT B6 and PTP-ζ-KO mice. Briefly, BMDMs isolated from WT and PTP-ζ-KO mice were cultured in standard growth medium. After adherence and stabilization until reaching approximately 80% confluence, cells were washed with PBS and subsequently stimulated for 24 hours with a cocktail of rIL-4 (20 ng/mL) and TGF-β (5 ng/mL), with or without rIL-34 (5 ng/mL). To induce definitive skewing toward the M2 phenotype, Møs were stimulated with rIL-4 as previously described\u003csup\u003e8\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 8A, mRNA levels for TNF-α, secreted by M1Mø, were not upregulated in either WT-BMDMs or PTP-ζ-KO BMDMs following rIL-34 stimulation, although transcripts of iNOS, another marker of M1Mø, were upregulated in both groups. However, this increase was not further enhanced by additional stimulation with rIL-34, particularly in WT-BMDMs (Fig 8B). In contrast, stimulation with TGF-β and rIL-4 significantly increased arginase-1 transcripts (a marker of M2Mø) in WT-BMDMs. This increase was further enhanced by additional treatment with rIL-34. However, this augmenting effect was lost in PTP-ζ-KO BMDMs due to knockout of the IL-34/PTP-ζ axis (Fig 8C). Moreover, expression of interferon regulatory factor (IRF)-4, a key mediator of Møs polarization toward the M2 phenotype\u003csup\u003e32\u003c/sup\u003e, was also analyzed by WB in the same condition (Fig 8D). Upregulated expressions of IRF-4 were observed in both WT-BMDMs and PTP-ζ-KO BMDMs following stimulation with rIL-4 and TGF-β. Notably, additional stimulation with rIL-34 significantly increased IRF-4 expression levels in WT-BMDMs, whereas no such enhancement after rIL-34 stimulation was observed in PTP-ζ-KO BMDMs. IRF-4 expressions in PTP-ζ-KO BMDMs stimulated with TGF-β, rIL-4, and rIL-34 were significantly suppressed compared to those in WT-BMDMs (Fig 8E).\u003c/p\u003e\n\u003cp\u003eFACS analysis was performed to evaluate the polarization of WT-BMDMs and PTP-ζ-KO BMDMs. Figure 9A-C shows representative FACS plots illustrating the gating strategy used to identify CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e (M1-like) and CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e (M2-like) Møs. BMDM counts were normalized to 1,000 CD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e cells for statistical analysis. Cultured BMDMs without stimulation did not skew toward either CD11c\u003csup\u003e+\u003c/sup\u003e or CD206\u003csup\u003e+\u003c/sup\u003e populations (Fig 9A). In both WT-BMDMs and PTP-ζ-KO BMDMs, stimulation with recombinant cytokines increased both CD11c\u003csup\u003e+\u003c/sup\u003e M1-like and CD206\u003csup\u003e+\u003c/sup\u003e M2-like Mø populations (Fig 9D, E). However, in the presence of IL-34, the proportion of PTP-ζ-KO BMDMs skewing toward CD206\u003csup\u003e+\u003c/sup\u003e cells was reduced compared with WT-BMDMs. Consequently, the M2/M1 ratio was significantly decreased in PTP-ζ-KO BMDMs following stimulation with rIL-4, TGF-β, and rIL-34 (Fig 9F).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eTo our knowledge, the present study provides the first confirmation that genetic deficiency of PTP-\u0026zeta; prevents the progression of renal fibrosis in mice with UUO. We uncovered the following points: 1) expression levels of PTP-\u0026zeta; and its primary ligand IL-34, were increased in the UUO kidneys of WT mice; 2) increased expression of profibrogenic genes and accumulation of fibroblasts and myofibroblasts in UUO kidneys were significantly suppressed in PTP-\u0026zeta;-KO mice; 3) marked M\u0026oslash; accumulation in UUO kidneys, particularly of profibrotic M2-like M\u0026oslash;s, was suppressed in PTP-\u0026zeta;-KO mice; and 4) WT-BMDM stimulated with rIL-4 and TGF-\u0026beta; exhibited enhanced polarization toward the M2 phenotype following additional stimulation with rIL-34, whereas this enhancement was not confirmed in PTP-\u0026zeta;-KO BMDM.\u003c/p\u003e\n\u003cp\u003eIntrarenal PTP-\u0026zeta; expression has been demonstrated to be notably elevated during the chronic phase of experimental renal disease models. In lupus-susceptible mice, kidneys exhibiting not only glomerular lesions, but also moderate to severe tubulointerstitial injury and fibrotic change showed a significant increase in both PTP-\u0026zeta; transcript and protein levels, correlating with progression of lupus nephritis up to 5 months of age\u003csup\u003e23\u003c/sup\u003e. Similarly, in the inflamed kidneys of C57BL/6 mice, PTP-\u0026zeta; transcript and protein levels were markedly elevated from days 20 to 40 following I/R injury. This increase was associated with fibrosis and tubular damage due to AKI to CKD caused by I/R\u003csup\u003e21,25\u003c/sup\u003e. PTP-\u0026zeta; expression has not been described in human fibrotic kidney samples with fibrotic change, except for AKI just after renal transplantation\u003csup\u003e25\u003c/sup\u003e. Nevertheless, the above previous findings, together with our current results, indicate the possibility that intrarenal PTP-\u0026zeta; expression is closely associated with the chronic phase of renal diseases characterized by renal fibrosis.\u003c/p\u003e\n\u003cp\u003eRegarding PTP-\u0026zeta; ligands in renal fibrosis, clinical studies have shown that elevated serum IL-34 levels are associated with the presence and severity of fibrosis in CKD, as well as with coronary artery disease in patients with heart failure\u003csup\u003e33\u003c/sup\u003e. In the mouse model, both intrarenal IL-34 level and PTP-\u0026zeta; increased during the CKD phase from day 20 to 40 following I/R injury\u003csup\u003e21,25\u003c/sup\u003e. Moreover, recent analyses have revealed that the other ligands of PTP-\u0026zeta; (e.g., tenascin-C, pleiotrophin, and midkine) were also upregulated in fibrotic kidneys at days 20 and 40 after I/R\u003csup\u003e25\u003c/sup\u003e. Although we did not assess the intrarenal expression of ligands other than IL-34 in this study, our findings implied that IL-34 was a primary driver of PTP-\u0026zeta;-mediated signaling in UUO-induced renal fibrosis. Intriguingly, our analysis showed that intrarenal IL-34 expression remained significantly elevated in PTP-\u0026zeta;-KO UUO kidneys, suggesting that IL-34 expression is not dependent on PTP-\u0026zeta; itself. Cultured mouse BMDMs consistently showed stable expressions of PTP-\u0026zeta; with or without additional IL-34 (Fig 7B), contrary to our presumption. Therefore, to better recreate the \u003cem\u003ein vivo\u003c/em\u003e microenvironment of UUO-induced renal fibrosis, we performed additional experiments using co-stimulation with rIL-34 together with rIL-4 or TGF-\u0026beta;.\u003c/p\u003e\n\u003cp\u003eConsistent with previous reports\u003csup\u003e23-25\u003c/sup\u003e, expression of PTP-\u0026zeta; on M\u0026oslash; was detected in the present study. Previous studies, including ours, focusing on the IL-34/PTP-\u0026zeta; axis have demonstrated that PTP-\u0026zeta; is expressed on BMDMs\u003csup\u003e24\u003c/sup\u003e. To further investigate the role of the IL-34/PTP-\u0026zeta; axis in M\u0026oslash; skewing, we performed additional analyses using BMDMs. While tissue-resident M\u0026oslash;s under steady-state conditions are generally considered to exist in a neutral M0 state\u003csup\u003e12,34\u003c/sup\u003e, PTP-\u0026zeta; expression was already detectable in cultured BMDMs under unstimulated conditions in this study, counter to our expectation that expression would be induced upon exposure to polarization stimuli. Our analysis using several phenotypic markers suggested that cultured BMDMs were not fully differentiated M1M\u0026oslash;s, but had already initiated polarization toward an M1-like phenotype. The phenotypic changes induced by TGF-\u0026beta; and rIL-4 stimulation in these BMDMs thus may differ from the physiological process in which tissue-resident M0M\u0026oslash;s differentiate into M2M\u0026oslash;s \u003cem\u003ein vivo\u003c/em\u003e. Nevertheless, our results suggest that stimulation with IL-34 was able to further drive a subset of these incompletely polarized M1-like M\u0026oslash;s toward an M2-like phenotype (Figs 8, 9). WT-BMDMs stimulated with M2-inducing cytokines and rIL-34 upregulated IRF-4, a factor linked to the renal fibrosis of resident M\u0026oslash;s via M2 polarization\u003csup\u003e32,35\u003c/sup\u003e, implying that PTP-\u0026zeta; signaling in M\u0026oslash;s may influence the transition from circulating M\u0026oslash;s to resident-like M\u0026oslash;s. Although further studies using reliable markers to distinguish resident vs. BM-derived and M1 vs. M2 M\u0026oslash;s are needed to clarify the precise role of PTP-\u0026zeta; in renal fibrosis, we suggest that WT-BMDMs exhibited a stronger shift toward the M2 phenotype than PTP-\u0026zeta;-deficient BMDMs under above-described experimental conditions. Therefore, IL-34/PTP-\u0026zeta; axis may accelerate M\u0026oslash; polarization toward the M2 phenotype. Targeting the IL-34/PTP-\u0026zeta; axis could suppress excessive M2-driven fibrotic responses in CKD, highlighting the potential of this axis as a therapeutic target.\u003c/p\u003e\n\u003cp\u003eRenal fibrosis is characterized by excessive accumulation of ECM, primarily produced by fibroblasts and myofibroblasts\u003csup\u003e36\u003c/sup\u003e.\u0026nbsp;In healthy kidneys, myofibroblasts are basically absent, but it has been considered that myofibroblasts emerge during fibrotic processes and substantially contribute to ECM deposition\u003csup\u003e36\u003c/sup\u003e. Although the precise origin of intrarenal fibroblasts has remained unclear, lineage-tracing experiments using myelin protein zero-Cre mice (which express Cre recombinase in neural crest cells) have revealed that neural crest-derived cells infiltrate the metanephros during embryogenesis and differentiate into renal fibroblasts\u003csup\u003e36,37\u003c/sup\u003e. It is now widely accepted that most myofibroblasts arise from resident renal fibroblasts\u003csup\u003e38\u003c/sup\u003e. In this study, genetic ablation of PTP-\u0026zeta; significantly attenuated renal fibrosis in a UUO model. This attenuation was accompanied by reduced expression of profibrogenic genes such as TGF-\u0026beta;, along with a marked decrease in the accumulation of fibroblasts and myofibroblasts in fibrotic kidneys. Similarly, in a recent analysis using a renal I/R model, Sirius red and \u0026alpha;SMA staining at day 40 post-injury revealed a significant reduction in fibrotic area and area with the presence of myofibroblast in PTP-\u0026zeta;-KO mice\u003csup\u003e25\u003c/sup\u003e. These findings initially implied that PTP-\u0026zeta; may regulate fibroblast activation. However, attempts to confirm PTP-\u0026zeta; expression in fibroblasts using the NIH/3T3 were inconclusive. Accordingly, attention was directed to another important feature of the UUO model: the marked suppression of intrarenal M\u0026oslash; infiltration, particularly M2-like M\u0026oslash;s, in PTP-\u0026zeta;-KO mice.\u003c/p\u003e\n\u003cp\u003eIn conclusion, upregulated intrarenal expression of PTP-\u0026zeta;, along with M\u0026oslash; accumulation, contributes to the progression of renal fibrosis in the UUO mouse model. IL-34 binding to PTP-\u0026zeta; might promote M2M\u0026oslash; proliferation in UUO kidneys, enhancing profibrotic responses. These findings suggest that PTP-\u0026zeta; on M\u0026oslash; could represent a potential therapeutic target for renal fibrosis.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003eMice\u003c/p\u003e\n\u003cp\u003eThe C57BL/6J-Ptprz1\u003csup\u003eem1Cya\u003c/sup\u003e (PTP-ζ-KO) mice were purchased from Cyagen (Suite E Santa Clara, CA, USA). Two male PTP-ζ-KO mice and two female PTP-ζ-KO mice were purchased and bred, and the male KO mice obtained from their offspring were used for all experiments in the present study. Briefly, the PTP-ζ-KO mouse model (C57BL/6J background) was generated via CRISPR/Cas-mediated genome engineering. Detailed information regarding the Mouse Ptprz1 Knockout Project is provided in the Supplementary Document. Six-week-old male C57BL/6 (B6) mice were purchased from Oriental Yeast Co. (Tokyo, Japan). All mice were maintained under specific pathogen-free conditions with ad libitum access to food and water in the animal facility of the Analysis Center of Integral Genomic Functions at Kitasato University.\u003c/p\u003e\n\u003cp\u003eExperimental protocol\u003c/p\u003e\n\u003cp\u003eThe experimental protocol was approved by the Animal Experimentation and Ethics Committee of Kitasato University (permit nos. M2025-007 and M2025-008). Male, 10-week-old WT B6 and PTP-ζ-KO mice underwent UUO. The left kidney was exposed through a flank incision, then UUO was induced by ligating the left ureter with nylon thread. Mice were sacrificed at 14 days after UUO to collect kidneys. Age-matched male B6 mice received sham operation as NC mice. All surgeries were performed under anesthesia using a mixture of medetomidine hydrochloride (0.75 mg/kg), butorphanol tartrate (5.0 mg/kg) and midazolam (4.0 mg/kg) on a 37°C warming pad.\u003c/p\u003e\n\u003cp\u003eLight microscopy\u003c/p\u003e\n\u003cp\u003eTwo experienced kidney pathologists who were blinded to each experimental group evaluated and scored the kidney pathology in Masson’s trichrome-stained paraffin sections, as previously detailed\u003csup\u003e39\u003c/sup\u003e. To evaluate the TI injury, 10 high-power fields (HPFs) (5 from the cortex and 5 from the outer-medulla) from each section were evaluated under 200´\u0026nbsp;magnification. The extent of TI injury was scored by the percentage of areas with round cell infiltration, tubular dilatation, tubular casts, and tubular epithelial cell necrosis per field. Scores from 0 to 5 were used (0, none; 1, \u0026lt;10%; 2, 10–25%; 3, 26–50%; 4, 51–75%; 5, \u0026gt;75% of area), and the results were averaged.\u003c/p\u003e\n\u003cp\u003eCollagen detection\u003c/p\u003e\n\u003cp\u003eTo assess the renal fibrotic area, we stained paraffin sections after rehydration in picrosirius red solution (srs250, ScyTek Laboratories, Logan, UT, USA) for 1 hour and rinsed with acidified water. Ten HPFs (5 from the cortex and 5 from the outer-medulla) from each section were selected for assessment, and two renal pathologists blindly evaluated the picrosirius red-positive area in UUO kidneys of the study groups under 400´\u0026nbsp;magnification. Consequently, the Sirius red-positive area/HPF (as a percentage) was calculated using Image J software (IJ 1.46r; NIH, Bethesda, MD, USA), then results were averaged.\u003c/p\u003e\n\u003cp\u003eImmunofluorescence staining\u003c/p\u003e\n\u003cp\u003eThe antibodies (Abs) used for staining are summarized in Supplementary Table S1. The detailed procedure has been described previously\u003csup\u003e21,22\u003c/sup\u003e. Briefly, cut frozen sections (5-µm-thick) were fixed and incubated overnight at 4°C with primary Abs, following incubation with secondary Abs for 2 h. Embedded sections with mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) (ab104139; Abcam, Cambridge, UK) were blindly evaluated by two renal pathologists. The quantification of positive cells for each staining was performed in 10 randomly selected HPFs (5 from the cortex, 5 from the outer medulla) under 400´\u0026nbsp;magnification\u003csup\u003e21-23,40\u003c/sup\u003e. The mean number of positive cells per HPF was calculated.\u003c/p\u003e\n\u003cp\u003eHomogenization of kidney tissues\u003c/p\u003e\n\u003cp\u003eKidney tissues (cortex) were homogenized with T-PER Mammalian Protein Extraction Reagent (20 mL/g renal tissue; #78501, Thermo Fisher Scientific, Waltham, MA, USA) containing 1% (v/v) protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) using a TissueLyser (QIAGEN, Hilden, Germany). Harvested lysates were then centrifuged at 3000\u003cem\u003eg\u003c/em\u003e for 10 min at 4°C to remove the cellular debris. Supernatants were collected and stored at -80°C. Protein concentration was measured using the BCA Protein Assay Kit (#23225; Pierce Biotechnology, Rockford, IL, USA)\u003csup\u003e22,41\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eReal-time reverse transcriptase polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eTarget gene expression was evaluated using qRT-PCR (TaqMan) assays. The procedure for qRT-PCR was as described previously\u003csup\u003e22,39,42\u003c/sup\u003e. All primers were purchased from Applied Biosystems (Carlsbad, CA, USA), and detailed information is summarized in Supplementary Table S2. Expressions of mRNA were normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control to correct for the differences in the amount of total RNA added to each reaction.\u003c/p\u003e\n\u003cp\u003eFluorescence-activated cell sorting\u003c/p\u003e\n\u003cp\u003eBriefly, kidneys were digested in collagenase IV (C4-BIOC, Sigma-Aldrich) for 1 h at 37°C, then mashed through a 40-μm sieve, then collected by centrifugation. Single-cell suspensions from kidneys were incubated with 2.4G2 monoclonal Ab (mAb) (anti-FcγRIII/II) to block non-specific binding, then cells were stained with primary mAb as previously described\u003csup\u003e43\u003c/sup\u003e. Abs for FACS are listed in Supplementary Table S3.\u003c/p\u003e\n\u003cp\u003eWestern blot analysis\u003c/p\u003e\n\u003cp\u003eHomogenized kidney and harvested lysates from cultured cells were used for WB analysis. For each sample, 20 µg of protein from kidney tissue or 10 µg of protein from cultured cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 4–20% gradient gel (#E-T520L; ATTO CORPORATION, Tokyo, Japan), and the proteins were transferred to a polyvinylidene difluoride membrane. The detailed procedures were as described previously\u003csup\u003e40,44\u003c/sup\u003e. Abs for WB are listed in Supplemental Table S4. Changes in each expression level were normalized by correction to the densitometric intensity of β-actin or GAPDH for each sample using Image J software.\u003c/p\u003e\n\u003cp\u003eMeasurement of IL-34 protein levels in kidney tissue homogenate\u003c/p\u003e\n\u003cp\u003eTotal IL-34 protein levels were measured in kidney tissue homogenates from each sample using the IL-34 ELISA kit (#M3400; R\u0026amp;D Systems, Abingdon, UK) in accordance with the instructions from the manufacturer\u003csup\u003e22\u003c/sup\u003e. To control for differences between samples, the concentration was corrected based on the amount of total tissue protein\u003csup\u003e41\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIsolation of bone marrow-derived macrophages\u003c/p\u003e\n\u003cp\u003eTo isolate BMDMs from WT B6 mice and PTP-ζ-KO mice (8–12 weeks old), mice were euthanized by cervical dislocation. The abdomen and hind limbs were sterilized with 70% ethanol, and the femurs were dissected out by cutting the bones at both ends. BM was flushed out from the dissected femurs using a 27-gauge needle with RPMI 1640 medium (#11875093; Thermo Fisher Scientific). The detailed procedure has been described previously\u003csup\u003e31\u003c/sup\u003e. To eliminate residual RBCs, RBC lysis buffer (#42030; BioLegend, San Diego, CA, USA) was used. The isolated BMDMs (3.0 ×10\u003csup\u003e6\u003c/sup\u003e cells/well) were initially thawed onto six-well plates, and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), antibiotics, and CSF-1 (20 ng/mL, #416-ML-050; R\u0026amp;D Systems) for 72–96 h until around 80% confluence. We verified the characteristic phenotype of BMDM by analyzing cFMS expression. The cultured BMDMs were washed with sterile PBS to remove residual CSF-1, and the culture medium was changed to RMPI 1640 medium supplemented with 10% FBS and antibiotics during the stimulation or treatment for experiments. In the experiments, BMDMs were stimulated with equal volumes of saline, TGF-β (5 ng/mL, #7666-MB-005; R\u0026amp;D) or rIL-34 (5 ng/mL, #577602; BioLegend) or rIL-4 (20 ng/mL, #214-14-20UG; Thermo Fisher Scientific). All cells were cultured in an atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e-95% air at 37°C in a humidified incubator.\u003c/p\u003e\n\u003cp\u003eCell cultures for mouse fibroblasts\u003c/p\u003e\n\u003cp\u003eNIH/3T3 cells were purchased from ATCC (University Boulevard, Manassas, VA, USA). Quality control and authentication of NIH/3T3 cells are guaranteed and validated by ATCC, as described in the product sheet (CRL-1658™). NIH/3T3 were cultured in ATCC-formulated Dulbecco's modified Eagle's medium (#30-2002; ATCC) supplemented with 10% FCS and antibiotics for 48 h until around 80–90% confluence for analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStatistical analysis\u003c/p\u003e\n\u003cp\u003eData were shown as the mean ± standard error of the mean (SEM), and were analyzed using GraphPad Prism software (version 10.0; GraphPad Software, San Diego, CA, USA). The Mann–Whitney U test was applied for comparisons between groups. Multiple groups were compared using one-way analysis of variance with a post-hoc Tukey’s honestly significant difference test. P value of \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Japan Society for the Promotion of Science KAKENHI (grant no. 22K08319).\u003c/p\u003e\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThe authors greatly appreciate the excellent technical assistance provided by Ms. Masako Takamatsu, Ms. Yui Onoda, and Ms. Miki Hashimura.\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eTA, YW, ET, and YT wrote the manuscript. TA, YW, ET, MS, TI, MO, TS, and YT collected data and reviewed the manuscript. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003eData availability statement\u003c/p\u003e\n\u003cp\u003eAll relevant data are within the manuscript and associated supporting information files.\u003c/p\u003e\n\u003cp\u003eConflicts of Interest\u003c/p\u003e\n\u003cp\u003eNone of the authors have any conflicts of interest to declare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCarney, E. F. The impact of chronic kidney disease on global health. \u003cem\u003eNat Rev Nephrol\u003c/em\u003e 16, 251, doi:10.1038/s41581-020-0268-7 (2020).\u003c/li\u003e\n\u003cli\u003eChen, T. K., Knicely, D. H. \u0026amp; Grams, M. E. Chronic Kidney Disease Diagnosis and Management: A Review. \u003cem\u003eJAMA\u003c/em\u003e 322, 1294-1304, doi:10.1001/jama.2019.14745 (2019).\u003c/li\u003e\n\u003cli\u003eNeuen, B. L., Chadban, S. J., Demaio, A. R., Johnson, D. W. \u0026amp; Perkovic, V. 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However, its precise localization and role in renal fibrosis remain unclear. Unilateral ureteral obstruction (UUO) was induced in wild-type (WT) and PTP-ζ-knockout (KO) C57BL/6 mice, then kidneys were analyzed on day 14. WT mice developed severe renal fibrosis with marked intrarenal IL-34 and PTP-ζ expressions. PTP-ζ-KO mice exhibited a significantly reduced fibrotic area and lower expressions of fibrogenic genes, despite comparable IL-34 levels. In UUO kidneys, infiltration of CD206+ Møs and accumulation of myofibroblasts were markedly suppressed in PTP-ζ-KO mice. Flow cytometry revealed a lower ratio of CD11c-CD206+ cells to CD11c+CD206- cells within the intrarenal CD11b+F4/80+ Mø population in KO mice. Mouse bone marrow-derived macrophages (BMDMs) isolated in vitro showed PTP-ζ expression in WT mice, but not in PTP-ζ-KO mice. In WT BMDMs, TGF-β and recombinant IL (rIL)-4 increased arginase-1 and interferon regulatory factor (IRF)-4 expressions, with further amplification upon rIL-34 stimulation. In PTP-ζ-KO BMDMs, additional rIL-34 did not further increase arginase-1 or IRF-4 expressions. IL-34 binding to PTP-ζ appears to promote M2-like Mø infiltration and fibrosis in UUO, suggesting PTP-ζ as a potential target for treating renal fibrosis.","manuscriptTitle":"Involvement of intrarenal protein-tyrosine phosphatase ζ receptor in progression of renal fibrosis in mice with unilateral ureteral obstruction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-20 06:45:53","doi":"10.21203/rs.3.rs-9386889/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-17T08:24:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-15T01:57:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-15T01:56:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-04-11T10:02:30+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":"86ea2ddf-c22e-4652-b2ea-a09fcf8193a1","owner":[],"postedDate":"April 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":66505140,"name":"Health sciences/Diseases"},{"id":66505141,"name":"Biological sciences/Immunology"},{"id":66505142,"name":"Health sciences/Nephrology"}],"tags":[],"updatedAt":"2026-05-05T23:38:06+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-20 06:45:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9386889","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9386889","identity":"rs-9386889","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}