A biocompatible nanoparticle-based approach to inhibiting renal ischemia reperfusion injury in mice by blocking thrombospondin-1 activity

Thrombospondin-1 (TSP-1) is a key mediator of renal ischemia-reperfusion injury (IRI), a major cause of kidney dysfunction under various disease conditions and a risk factor of renal allograft rejection. In this study, we developed a nanotechnology-based therapy targeting TSP-1 to prevent renal IRI. A biocompatible nanoparticle (NP) capable of specific binding to TSP-1 was prepared by conjugating NPs with TSP-1-binding (LSKL) peptides. LSKL/NPs not only effectively adsorbed recombinant TSP-1 proteins in vitro, but also efficiently neutralized TSP-1 in mice undergoing renal IRI. IRI-induced elevation of TSP-1 in the kidney was significantly inhibited by post-IR treatment with LSKL/NPs, but not free LSKL or NPs. Furthermore, TSP-1 proteins adsorbed on LSKL/NPs were functionally inactive and unable to induce apoptosis in renal tubular epithelial cells. Importantly, LSKL/NPs induced strong protection against renal IRI, as shown by markedly diminished serum creatinine levels and improved histological lesions of the kidney. Thus, LSKL/NPs provide a useful means of depleting and inactivating TSP-1 and a potential therapy for renal IRI. Thrombospondin-1 (TSP-1) is a key mediator of renal ischemia-reperfusion injury (IRI), a major cause of kidney dysfunction under various disease conditions and a risk factor of renal allograft rejection. In this study, we developed a nanotechnology-based therapy targeting TSP-1 to prevent renal IRI. A biocompatible nanoparticle (NP) capable of specific binding to TSP-1 was prepared by conjugating NPs with TSP-1-binding (LSKL) peptides. LSKL/NPs not only effectively adsorbed recombinant TSP-1 proteins in vitro, but also efficiently neutralized TSP-1 in mice undergoing renal IRI. IRI-induced elevation of TSP-1 in the kidney was significantly inhibited by post-IR treatment with LSKL/NPs, but not free LSKL or NPs. Furthermore, TSP-1 proteins adsorbed on LSKL/NPs were functionally inactive and unable to induce apoptosis in renal tubular epithelial cells. Importantly, LSKL/NPs induced strong protection against renal IRI, as shown by markedly diminished serum creatinine levels and improved histological lesions of the kidney. Thus, LSKL/NPs provide a useful means of depleting and inactivating TSP-1 and a potential therapy for renal IRI. Ischemia-reperfusion (IR), characterized by an initial restriction of blood to an organ followed by subsequent restoration of blood supply and concomitant reoxygenation, is involved in many pathological conditions as an important cause of tissue injury.1Eltzschig HK Eckle T Ischemia and reperfusion–from mechanism to translation.Nat Med. 2011; 17: 1391-1401Crossref PubMed Scopus (2085) Google Scholar,2Patschan D Patschan S Müller GA Inflammation and microvasculopathy in renal ischemia reperfusion injury.J Transplant. 2012; 2012: 764154Crossref PubMed Google Scholar Renal IR injury (IRI) is also unavoidable and is a major factor impairing graft function and augmenting acute rejection following kidney transplantation.3Nieuwenhuijs-Moeke GJ Pischke SE Berger SP et al.Ischemia and reperfusion injury in kidney transplantation: relevant mechanisms in injury and repair.J Clin Med. 2020; 9: 253Crossref PubMed Scopus (89) Google Scholar, 4Zhao H Alam A Soo AP George AJT Ma D Ischemia-reperfusion injury reduces long term renal graft survival: mechanism and beyond.EBioMedicine. 2018; 28: 31-42Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 5Qiu L Lai X Wang J-J et al.Kidney-intrinsic factors determine the severity of ischemia/reperfusion injury in a mouse model of delayed graft function.Kidney Int. 2020; 98: 1489-1501Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 6Batal I Mohan S De Serres SA et al.Analysis of dendritic cells and ischemia-reperfusion changes in postimplantation renal allograft biopsies may serve as predictors of subsequent rejection episodes.Kidney Int. 2018; 93: 1227-1239Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar There has therefore been a long-standing effort to improve kidney transplants by preventing or ameliorating renal graft IRI. Multiple mechanisms contribute to IR-induced tissue injury, including hypoxia resulted from restrained blood supply and post-reperfusion proinflammatory responses.1Eltzschig HK Eckle T Ischemia and reperfusion–from mechanism to translation.Nat Med. 2011; 17: 1391-1401Crossref PubMed Scopus (2085) Google Scholar,2Patschan D Patschan S Müller GA Inflammation and microvasculopathy in renal ischemia reperfusion injury.J Transplant. 2012; 2012: 764154Crossref PubMed Google Scholar Thrombospondin-1 (TSP1) is a 450 kDa multifunctional homotrimeric glycoprotein.7Chen H Herndon ME Lawler J The cell biology of thrombospondin-1.Matrix Biol. 2000; 19: 597-614Crossref PubMed Scopus (0) Google Scholar TSP-1 was found to be upregulated rapidly following renal IRI and a key mediator of acute kidney injury (AKI).8Thakar CV Zahedi K Revelo MP et al.Identification of thrombospondin 1 (TSP-1) as a novel mediator of cell injury in kidney ischemia.J Clin Invest. 2005; 115: 3451-3459Crossref PubMed Scopus (100) Google Scholar Blockade of TSP-1 receptor CD47 was reported to ameliorate renal IRI,9Rogers NM Thomson AW Isenberg JS Activation of parenchymal CD47 promotes renal ischemia-reperfusion injury.J Am Soc Nephrol. 2012; 23: 1538-1550Crossref PubMed Scopus (48) Google Scholar, 10Isenberg JS Roberts DD The role of CD47 in pathogenesis and treatment of renal ischemia reperfusion injury.Pediatr Nephrol. 2019; 34: 2479-2494Crossref PubMed Scopus (7) Google Scholar, 11Xu M Wang X Banan B et al.Anti-CD47 monoclonal antibody therapy reduces ischemia-reperfusion injury of renal allografts in a porcine model of donation after cardiac death.Am J Transplant. 2018; 18: 855-867Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar indicating that the CD47 signaling plays an important role in mediating IRI induced by TSP-1. In support of these findings, it was shown that upon ligation with TSP-1, CD47 signaling inhibits cell cycle progression and induces senescence and apoptosis in vascular endothelial cells.12Gao Q Chen K Gao L Zheng Y Yang YG Thrombospondin-1 signaling through CD47 inhibits cell cycle progression and induces senescence in endothelial cells.Cell Death Dis. 2016; 7: e2368Crossref PubMed Scopus (52) Google Scholar,13Meijles DN Sahoo S Al Ghouleh I et al.The matricellular protein TSP1 promotes human and mouse endothelial cell senescence through CD47 and Nox1.Sci Signal. 2017; 10: eaaj1784Crossref PubMed Scopus (35) Google Scholar In addition to CD47 signaling, TSP-1 also activates transforming growth factor beta (TGF-β), another factor contributing to IRI.14Murphy-Ullrich JE Suto MJ Thrombospondin-1 regulation of latent TGF-beta activation: a therapeutic target for fibrotic disease.Matrix Biol. 2018; 68–69: 28-43Crossref PubMed Scopus (136) Google Scholar, 15Yang Q Ren GL Wei B et al.Conditional knockout of TGF-betaRII/Smad2 signals protects against acute renal injury by alleviating cell necroptosis, apoptosis and inflammation.Theranostics. 2019; 9: 8277-8293Crossref PubMed Scopus (62) Google Scholar, 16Geng H Lan R Wang G et al.Inhibition of autoregulated TGFbeta signaling simultaneously enhances proliferation and differentiation of kidney epithelium and promotes repair following renal ischemia.Am J Pathol. 2009; 174: 1291-1308Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 17Camarata TD Weaver GC Vasilyev A Arnaout MA Negative regulation of TGFbeta signaling by stem cell antigen-1 protects against ischemic acute kidney injury.PLoS One. 2015; 10: e0129561Crossref PubMed Scopus (10) Google Scholar Thus, TSP-1 is considered a potential therapeutic target for IRI. Nanoparticles (NPs) are promising vehicles for delivery of small molecule drugs and peptides, which suffer from poor pharmacokinetics, rapid clearance, enzymatic instability, and low efficacy reaching the target tissues.18Spicer CD Jumeaux C Gupta B Stevens MM Peptide and protein nanoparticle conjugates: versatile platforms for biomedical applications.Chem Soc Rev. 2018; 47: 3574-3620Crossref PubMed Google Scholar There has been a long-standing interest in the use of NPs for targeted drug delivery to tumors and other diseased tissues.19Goldberg MS Improving cancer immunotherapy through nanotechnology.Nat Rev Cancer. 2019; 19: 587-602Crossref PubMed Scopus (304) Google Scholar,20Han SJ Williams RM D’Agati V Jaimes EA Heller DA Lee HT Selective nanoparticle-mediated targeting of renal tubular Toll-like receptor 9 attenuates ischemic acute kidney injury.Kidney Int. 2020; 98: 76-87Abstract Full Text Full Text PDF PubMed Google Scholar In this study, we attempted to develop a nanotechnology-based strategy to neutralize or deplete TSP-1, hence preventing renal IRI. To enable specific binding to TSP-1, we conjugated LSKL peptides that contain the TSP-1 binding sequence of the latent TGF-β,21Ribeiro SM Poczatek M Schultz-Cherry S Villain M Murphy-Ullrich JE The activation sequence of thrombospondin-1 interacts with the latency-associated peptide to regulate activation of latent transforming growth factor-beta.J Biol Chem. 1999; 274: 13586-13593Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar to biocompatible NPs made of poly(D,L-lactide-co-glycolide) polymer and DSPE-PEG-NHS. We found that LSKL-conjugated NPs (LSKL/NPs) were highly effective in adsorbing TSP-1 proteins in vitro and in kidney tissues following IRI. Importantly, treatment with LSKL/NPs, but not free LSKL peptides or NPs, induced strong protection against renal IRI, as shown by improved renal function and histology. Our results support the use of LSKL/NPs to prevent renal IRI. Materials and methods are detailed in Supporting Information. Protocols involved in the use of animals were reviewed and approved by the Institutional Animal Care and Use Committee of the First Hospital of Jilin University. LSKL/NPs were prepared by mixing LSKL peptides and biocompatible NHS ester functionalized PLGA NPs overnight as detailed in the Methods. LSKL conjugation did not significantly alter the shape (Figure 1A), hydrodynamic diameter (Figure 1B), or negative zeta potentials (Figure 1C) of the NPs. The efficacy of LSKL/NPs to adsorb TSP-1 proteins was determined using AF647-labeled recombinant TSP-1 (AF647-TSP-1). Neither PBS nor free LSKL controls had detectable fluorescence (Figure 2A and 2B). Although fluorescence signals were detectable in both NP and LSKL/NP pellets, the latter was significantly stronger than the former (Figure 2A and 2B). These results indicate that there was limited non-specific binding of TSP-1 to NPs, and that highly efficient adsorption of TSP-1 by LSKL/NPs was predominantly due to specific binding of TSP-1 to LSKL peptides.FIGURE 2LSKL/NPs are capable of adsorbing TSP-1 and inhibiting apoptosis induced by TSP-1 in vitro. (A, B) AF647-conjugated TSP1 (AF647-TSP1) was mixed with NPs or LSKL/NPs, and incubated at 4°C overnight. After removing the supernatants by centrifugation, percentages of AF647-TSP1 adsorbed by NPs were determined by measuring fluorescence intensity of the pellets using the Xenogen IVIS Lumina system (as detailed in Methods section). Tubes containing xa mixture of AF647-TSP1 with PBS or free LSKL were used as controls. (A) Representative fluorescent images; (B) Percentages (means ± SEM; n = 3 per group) of AF647-TSP1 adsorbed onto NPs or LSKL/NPs. ****, p < .0001 (one-way ANOVA with Tukey’s multiple comparison test). (C) HK-2 cells were incubated with PBS, free TSP-1, free LSKL + TSP-1, NPs + TSP-1, or TSP-1 adsorbed on LSKL/NPs for 72 h, and analyzed for apoptosis by flow cytometry. Shown are representative staining profiles (top) and percentages (means ± SEMs; n = 5 per group; bottom) of apoptotic of HK-2 cells (early apoptosis, Q3; late apoptosis, Q2; and total apoptosis, Q2 + Q3). ns, not significant; **, p < .005; ***, p < .0005, ****, p < .0001 (one-way ANOVA with Tukey’s multiple comparison test)View Large Image Figure ViewerDownload Hi-res image Download (PPT) We then determined bioactivity of TSP-1 adsorbed onto LSKL/NPs by measuring their ability to induce apoptosis of human renal tubular epithelial HK-2 cells, which were found to be susceptible to apoptosis induced by soluble TSP-1 (Fig. S1). Briefly, HK-2 cells were cultured for 72 h in medium containing free TSP-1, free LSKL +TSP-1, NPs +TSP-1, or LSKL/NP-adsorbed TSP-1 (at a final TSP-1 concentration of 0.8 µg/mL), or same volume of PBS, and cell death was determined by flow cytometry. A significant increase in apoptosis was detected in HK-2 cells incubated with free TSP-1, free LSKL +TSP-1, and NPs +TSP-1, but not LSKL/NP-adsorbed TSP-1, compared to those incubated with PBS (Figure 2C), indicating a loss of bioactivity for LSKL/NP-adsorbed TSP-1. A mouse model of renal IRI was created by clipping the left renal pedicle for 28 min (Fig. S2A),22Hesketh EE Czopek A Clay M et al.Renal ischaemia reperfusion injury: a mouse model of injury and regeneration.J vis Exp. 2014; 88Google Scholar in which renal IRI was detected 24 h after reperfusion presented by a significant increase in serum creatine (Fig. S2B), progressive renal injury shown as vacuolar degeneration, dilation of renal tubules, disappearance of brush border, and necrosis of epithelial cells (Fig. S2C), and extensive renal infiltration by CD45+ lymphohematopoietic cells (Fig. S2D). Similar to previous reports that tissue injury enhances NP infiltration,23Yu H Lin T Chen W et al.Size and temporal-dependent efficacy of oltipraz-loaded PLGA nanoparticles for treatment of acute kidney injury and fibrosis.Biomaterials. 2019; 219: 119368Crossref PubMed Scopus (46) Google Scholar renal IRI resulted in an increase in LSKL/NP accumulation in the kidney (Figure 3A). There was no significant difference in renal accumulation between LSKL/NPs and NPs (Fig. S3), indicating that LSKL conjugation did not affect NP infiltration into IRI kidneys. To determine the efficacy of LSKL/NPs to neutralize TSP-1 in vivo, we compared the levels of TSP-1 proteins in kidneys of renal IRI mice that were treated 3h after IRI with PBS, free LSKL, NPs, or LSKL/NPs, in which sham-operated (SO) mice were used as controls. Histological analysis of heart, lung, liver, and spleen tissues revealed no acute toxicity associated with administration (i.v.) of LSKL/NPs (Fig. S4). Immunofluorescence staining of renal sections at 24 h post-IR revealed a marked increase in TSP-1 production in the kidney from PBS-treated mice compared to SO controls, which was significantly diminished by treatment with LSKL/NPs, but not free LSKL or NPs (Figure 3B). Consistent with the immunohistology results, western blot analysis also showed a significantly less TSP-1 proteins in the kidney of LSKL/NP-treated compared to free LSKL- or NP-injected mice (Figure 3C). Because TSP-1 adsorbed on LSKL/NPs can be recognized and bound by the anti-TSP-1 antibody (Fig. S5), it is likely that TSP-1 proteins adsorbed on LSKL/NPs were rapidly taken up by cells then degraded, and/or removed out of the kidney via blood circulation and urination. To evaluate the therapeutic potential of LSKL/NPs against IR-induced AKI, mice were treated 3h after renal IR with PBS, free LSKL, NPs, or LSKL/NPs and followed for kidney function. PBS-injected IRI mice showed a significant increase in serum creatinine levels 24 h after IR compared to the pre-IR levels, indicating that IR induced rapid progression of renal dysfunction (Figure 4A). However, treatment with LSKL/NPs nearly completely inhibited IR-induced elevation of serum creatinine, while no significant difference was observed between mice treated with fee LSKL or NPs and PBS controls (Figure 4A). Furthermore, compared to SO mice, renal histologic lesions, characterized by tubular dilation, tubular epithelial cell vacuolar degeneration and necrosis, and loss of the brush border in cortex and medulla, were present in mice at 24h following IR, but significantly less severe in mice treated with LSKL/NPs than in those receiving PBS, free LSKL or NPs (Figure 4B). The renal pathological scores in LSKL/NP-treated mice were significantly lower than those injected with PBS, free LSKL, or NPs (Figure 4C). In line with these observations, TUNEL staining of renal sections revealed that IR-induced renal cell apoptosis was also substantially diminished by treatment with LSKL/NPs (Figure 4D). The cytokine response after renal IRI is dominated by the local induction of IL-6 in the kidney.24Nechemia-Arbely Y Barkan D Pizov G et al.IL-6/IL-6R axis plays a critical role in acute kidney injury.J Am Soc Nephrol. 2008; 19: 1106-1115Crossref PubMed Scopus (259) Google Scholar, 25de Vries DK Lindeman JHN Tsikas D et al.Early renal ischemia-reperfusion injury in humans is dominated by IL-6 release from the allograft.Am J Transplant. 2009; 9: 1574-1584Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 26Lemay S Rabb H Postler G Singh AK Prominent and sustained up-regulation of gp130-signaling cytokines and the chemokine MIP-2 in murine renal ischemia-reperfusion injury.Transplantation. 2000; 69: 959-963Crossref PubMed Google Scholar Although IL-6 signaling has been reported to mediate protection against AKI, IL-6 production correlates with the severity of injury and may exacerbate AKI by mediating proinflammatory responses.24Nechemia-Arbely Y Barkan D Pizov G et al.IL-6/IL-6R axis plays a critical role in acute kidney injury.J Am Soc Nephrol. 2008; 19: 1106-1115Crossref PubMed Scopus (259) Google Scholar,25de Vries DK Lindeman JHN Tsikas D et al.Early renal ischemia-reperfusion injury in humans is dominated by IL-6 release from the allograft.Am J Transplant. 2009; 9: 1574-1584Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar Consistent with these reports, significantly increased IL-6 production was detected in mice following IRI compared to SO controls (Figure 4E). Furthermore, IR-induced elevation of IL-6 was significantly inhibited by treatment with LSKL/NPs, but not fee LSKL or NPs (Figure 4E), correlating with the severity of renal injury in these mice. Together, our results revealed that post-IR treatment with LSK/NPs induced a strong protection against renal IRI. In this study, we show that conjugation with TSP-1-binding LSKL peptides enables NPs to adsorb TSP-1 proteins specifically and effectively both in vitro and in mice undergoing renal IRI. Furthermore, TSP-1 adsorbed on LSKL/NPs, unlike free TSP-1, failed to induce cell death, indicating a loss of bioactivity. Importantly, LSKL/NPs induced strong protection against renal IRI. Mice treated with LSKL/NPs 3 h after IR showed markedly reduced serum creatinine and improved histology compared to those injected with free LSKL peptides or NPs. Thus, LSKL/NPs provide a potential therapy for preventing renal IRI. Using fluorescence-conjugated NPs, we observed a significantly increased accumulation of NPs in the kidney following IRI, which is consistent with previous studies showing increased permeability of the glomerular filtration barrier by renal IRI.23Yu H Lin T Chen W et al.Size and temporal-dependent efficacy of oltipraz-loaded PLGA nanoparticles for treatment of acute kidney injury and fibrosis.Biomaterials. 2019; 219: 119368Crossref PubMed Scopus (46) Google Scholar We also found that there was no significant difference in renal accumulation between LSKL/NPs and NPs, indicating that LSKL conjugation does not affect the efficacy of NPs to enter IR kidneys. These observations explain the high efficacy of LSKL/NPs in diminishing TSP-1 proteins in IRI kidneys and support the use of NPs for targeted drug delivery to treat renal IRI. Previous studies have shown that blocking TSP-1/CD47 signaling by anti-CD47 antibodies could also attenuate IRI.9Rogers NM Thomson AW Isenberg JS Activation of parenchymal CD47 promotes renal ischemia-reperfusion injury.J Am Soc Nephrol. 2012; 23: 1538-1550Crossref PubMed Scopus (48) Google Scholar, 10Isenberg JS Roberts DD The role of CD47 in pathogenesis and treatment of renal ischemia reperfusion injury.Pediatr Nephrol. 2019; 34: 2479-2494Crossref PubMed Scopus (7) Google Scholar, 11Xu M Wang X Banan B et al.Anti-CD47 monoclonal antibody therapy reduces ischemia-reperfusion injury of renal allografts in a porcine model of donation after cardiac death.Am J Transplant. 2018; 18: 855-867Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar However, because CD47 is widely expressed and functions as a ‘don’t eat me’ signal preventing phagocytosis, treatment with anti-CD47 antibodies has been found to cause adverse events such as anemia and thrombocytopenia.27Advani R Flinn I Popplewell L et al.CD47 blockade by Hu5F9-G4 and rituximab in non-hodgkin’s lymphoma.N Engl J Med. 2018; 379: 1711-1721Crossref PubMed Scopus (0) Google Scholar, 28Zeidan AM DeAngelo DJ Palmer J et al.Phase 1 study of anti-CD47 monoclonal antibody CC-90002 in patients with relapsed/refractory acute myeloid leukemia and high-risk myelodysplastic syndromes.Ann Hematol. 2022; 101: 557-569Crossref PubMed Scopus (13) Google Scholar, 29Kojima Y Volkmer J-P McKenna K et al.CD47-blocking antibodies restore phagocytosis and prevent atherosclerosis.Nature. 2016; 536: 86-90Crossref PubMed Scopus (305) Google Scholar These adverse effects could be further escalated in allotransplant recipients in which macrophages are often activated. Furthermore, signaling through CD47 may not be the only mechanism by which TSP-1 induces IRI, and the CD47-independent activities of TSP-1 cannot be prevented by anti-CD47 antibodies. Thus, LSKL/NP is considered to be superior to anti-CD47 antibody in terms of safety and efficacy. Rapid upregulation of TSP-1, peaking around 3 h and returning to baseline levels by 48 h after reperfusion, is an important mechanism for renal IRI.8Thakar CV Zahedi K Revelo MP et al.Identification of thrombospondin 1 (TSP-1) as a novel mediator of cell injury in kidney ischemia.J Clin Invest. 2005; 115: 3451-3459Crossref PubMed Scopus (100) Google Scholar Given that TSP-1 colocalizes with activated caspase-3 in kidney nephrons and induces proapoptotic changes in proximal tubule cells, TSP-1 is believed to be a direct mediator of AKI following IR.8Thakar CV Zahedi K Revelo MP et al.Identification of thrombospondin 1 (TSP-1) as a novel mediator of cell injury in kidney ischemia.J Clin Invest. 2005; 115: 3451-3459Crossref PubMed Scopus (100) Google Scholar TSP-1 is a matricellular protein that signals through interaction with multiple cell surface receptors, including TGF-β1, CD47 and CD36.30Bornstein P Thrombospondins as matricellular modulators of cell function.J Clin Invest. 2001; 107: 929-934Crossref PubMed Google Scholar LSKL contains the TSP-1 binding sequence of the latent TGF-β,21Ribeiro SM Poczatek M Schultz-Cherry S Villain M Murphy-Ullrich JE The activation sequence of thrombospondin-1 interacts with the latency-associated peptide to regulate activation of latent transforming growth factor-beta.J Biol Chem. 1999; 274: 13586-13593Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar TSP-1 adsorbed on LSKL/NPs should therefore lose the ability to activate TGF-β, providing one of the possible mechanisms for LSKL/NP-induced protection against renal IRI.14Murphy-Ullrich JE Suto MJ Thrombospondin-1 regulation of latent TGF-beta activation: a therapeutic target for fibrotic disease.Matrix Biol. 2018; 68–69: 28-43Crossref PubMed Scopus (136) Google Scholar, 15Yang Q Ren GL Wei B et al.Conditional knockout of TGF-betaRII/Smad2 signals protects against acute renal injury by alleviating cell necroptosis, apoptosis and inflammation.Theranostics. 2019; 9: 8277-8293Crossref PubMed Scopus (62) Google Scholar, 16Geng H Lan R Wang G et al.Inhibition of autoregulated TGFbeta signaling simultaneously enhances proliferation and differentiation of kidney epithelium and promotes repair following renal ischemia.Am J Pathol. 2009; 174: 1291-1308Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 17Camarata TD Weaver GC Vasilyev A Arnaout MA Negative regulation of TGFbeta signaling by stem cell antigen-1 protects against ischemic acute kidney injury.PLoS One. 2015; 10: e0129561Crossref PubMed Scopus (10) Google Scholar However, LSKL should not affect the interaction of TSP-1 with CD47,31Lawler J The functions of thrombospondin-1 and-2.Curr Opin Cell Biol. 2000; 12: 634-640Crossref PubMed Scopus (373) Google Scholar which has been shown to play an important role in renal IRI9Rogers NM Thomson AW Isenberg JS Activation of parenchymal CD47 promotes renal ischemia-reperfusion injury.J Am Soc Nephrol. 2012; 23: 1538-1550Crossref PubMed Scopus (48) Google Scholar, 10Isenberg JS Roberts DD The role of CD47 in pathogenesis and treatment of renal ischemia reperfusion injury.Pediatr Nephrol. 2019; 34: 2479-2494Crossref PubMed Scopus (7) Google Scholar, 11Xu M Wang X Banan B et al.Anti-CD47 monoclonal antibody therapy reduces ischemia-reperfusion injury of renal allografts in a porcine model of donation after cardiac death.Am J Transplant. 2018; 18: 855-867Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar and TSP-1-induced endothelial apoptosis.12Gao Q Chen K Gao L Zheng Y Yang YG Thrombospondin-1 signaling through CD47 inhibits cell cycle progression and induces senescence in endothelial cells.Cell Death Dis. 2016; 7: e2368Crossref PubMed Scopus (52) Google Scholar,13Meijles DN Sahoo S Al Ghouleh I et al.The matricellular protein TSP1 promotes human and mouse endothelial cell senescence through CD47 and Nox1.Sci Signal. 2017; 10: eaaj1784Crossref PubMed Scopus (35) Google Scholar Our results showed that, unlike free TSP-1, TSP-1 adsorbed on LSKL/NPs failed to induce tubular epithelial cell death, indicating that TSP-1 adsorbed on LSKL/NPs was also incapable of signaling through CD47. Coating large amounts of proteins on the surface of small NPs has been shown to diminish bioactivity or function of the coated proteins,32Saptarshi SR Duschl A Lopata AL Interaction of nanoparticles with proteins: relation to bio-reactivity of the nanoparticle.J Nanobiotechnology. 2013; 11: 26Crossref PubMed Scopus (711) Google Scholar,33Dhar S Sood V Lohiya G Deivendran H Katti DS Role of physicochemical properties of protein in modulating the nanoparticle-bio interface.J Biomed Nanotechnol. 2020; 16: 1276-1295Crossref PubMed Scopus (3) Google Scholar which is likely responsible for the observed function loss of TSP-1 adsorbed on LSKL/NPs. In addition, LSKL/NP recirculation is considered an important mechanism for removing TSP-1 proteins out of the tissue, i.e., reducing TSP-1 concentration in IR kidneys. In summary, our data demonstrate that LSKL/NPs provide an efficient means of adsorbing/neutralizing TSP-1, hence inhibiting renal IRI. While other mechanisms may also be involved, protection against IRI by LSKL/NPs is assumed to be largely conferred by its ability to block TSP1-induced CD47 and TGF-β activation. We thank the pathologists, Drs. Shan Wu and Jinyu Yu at Department of Nephrology, The First Hospital of Jilin University, for helping evaluate renal pathology. This work was supported by grants from National Key Research and Development Program of China (2021YFA1100700; 2017YFA0208100), NSFC (81871478, 91642208, 81422026), Jilin Scientific and Technological Development Program (20190201094JC, 20200301007RQ), Jilin Provincial Department of Education (JJKH20201050KJ), Interdisciplinary Innovation Project of the First Hospital of Jilin University (JDYYJCHX001), and the Fundamental Research Funds for the Central Universities, JLU. The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation. The data that support the findings of this study are available from the corresponding author upon reasonable request. Additional supporting information may be found in the online version of the article at the publisher’s website. Download .pdf (.95 MB) Help with pdf files Supplementary Material