The cell membrane repair protein MG53 modulates transcription factor NF-κB signaling to control kidney fibrosis

Kidney fibrosis is associated with the progression of acute kidney injury to chronic kidney disease. MG53, a cell membrane repair protein, has been shown to protect against injury to kidney epithelial cells and acute kidney injury. Here, we evaluated the role of MG53 in modulation of kidney fibrosis in aging mice and in mice with unilateral ureteral obstruction (UUO) a known model of progressive kidney fibrosis. Mice with ablation of MG53 developed more interstitial fibrosis with age than MG53-intact mice of the same age. Similarly, in the absence of MG53, kidney fibrosis was exaggerated compared to mice with intact MG53 in the obstructed kidney compared to the contralateral unobstructed kidney or the kidneys of sham operated mice. The ureteral obstructed kidneys from MG53 deficient mice also showed significantly more inflammation than ureteral obstructed kidneys from MG53 intact mice. In vitro experiments demonstrated that MG53 could enter the nuclei of proximal tubular epithelial cells and directly interact with the p65 component of transcription factor NF-κB, providing a possible explanation of enhanced inflammation in the absence of MG53. To test this, enhanced MG53 expression through engineered cells or direct recombinant protein delivery was given to mice subject to UUO. This reduced NF-κB activation and inflammation and attenuated kidney fibrosis. Thus, MG53 may have a therapeutic role in treating chronic kidney inflammation and thereby provide protection against fibrosis that leads to the chronic kidney disease phenotype. Kidney fibrosis is associated with the progression of acute kidney injury to chronic kidney disease. MG53, a cell membrane repair protein, has been shown to protect against injury to kidney epithelial cells and acute kidney injury. Here, we evaluated the role of MG53 in modulation of kidney fibrosis in aging mice and in mice with unilateral ureteral obstruction (UUO) a known model of progressive kidney fibrosis. Mice with ablation of MG53 developed more interstitial fibrosis with age than MG53-intact mice of the same age. Similarly, in the absence of MG53, kidney fibrosis was exaggerated compared to mice with intact MG53 in the obstructed kidney compared to the contralateral unobstructed kidney or the kidneys of sham operated mice. The ureteral obstructed kidneys from MG53 deficient mice also showed significantly more inflammation than ureteral obstructed kidneys from MG53 intact mice. In vitro experiments demonstrated that MG53 could enter the nuclei of proximal tubular epithelial cells and directly interact with the p65 component of transcription factor NF-κB, providing a possible explanation of enhanced inflammation in the absence of MG53. To test this, enhanced MG53 expression through engineered cells or direct recombinant protein delivery was given to mice subject to UUO. This reduced NF-κB activation and inflammation and attenuated kidney fibrosis. Thus, MG53 may have a therapeutic role in treating chronic kidney inflammation and thereby provide protection against fibrosis that leads to the chronic kidney disease phenotype. Translational StatementChronic inflammation leads to fibrotic remodeling that may also underlie the transition from acute kidney injury to chronic kidney disease. Activation of the proinflammatory transcription factor nuclear factor κB (NF-κB) is involved in the pathogenesis of kidney inflammation. Here, we provide evidence that MG53, a previously identified cell membrane repair protein, directly interacts with NF-κB and reduces its transcriptional activity. Concomitantly, exogenously administered MG53 can decrease fibrotic remodeling of the inflamed kidney. These findings point to a protective interplay between MG53 and NF-κB to attenuate the development of inflammation-mediated kidney fibrosis. Pharmacologic administration of MG53 might be a promising approach for the treatment of progressive kidney fibrosis. Chronic inflammation leads to fibrotic remodeling that may also underlie the transition from acute kidney injury to chronic kidney disease. Activation of the proinflammatory transcription factor nuclear factor κB (NF-κB) is involved in the pathogenesis of kidney inflammation. Here, we provide evidence that MG53, a previously identified cell membrane repair protein, directly interacts with NF-κB and reduces its transcriptional activity. Concomitantly, exogenously administered MG53 can decrease fibrotic remodeling of the inflamed kidney. These findings point to a protective interplay between MG53 and NF-κB to attenuate the development of inflammation-mediated kidney fibrosis. Pharmacologic administration of MG53 might be a promising approach for the treatment of progressive kidney fibrosis. Kidney dysfunction, which can be classified as acute kidney injury (AKI) or chronic kidney disease (CKD), has grown into an epidemic in older populations. In 2017, the prevalence of CKD reached 14.5% of people aged 65 and over, resulting in Medicare expenditures of over $84 billion for treatment.1Saran R. Robinson B. Abbott K.C. et al.US Renal Data System 2018 annual data report: epidemiology of kidney disease in the United States.Am J Kidney Dis. 2019; 73: A7-A8Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar,2Saran R. Robinson B. 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Watanabe I.K. et al.Inflammation in renal diseases: new and old players.Front Pharmacol. 2019; 10: 1192Crossref PubMed Scopus (84) Google Scholar Cumulative research suggests the involvement of the nuclear factor κB (NF-κB) transcription factor in the pathogenesis of kidney inflammation caused by infection, injury, or transplantation.11Zhang H. Sun S.C. NF-kappaB in inflammation and renal diseases.Cell Biosci. 2015; 5: 63Crossref PubMed Scopus (143) Google Scholar,12Song N. Thaiss F. Guo L. NFκB and Kidney Injury.Front Immunol. 2019; 10: 815Crossref PubMed Scopus (39) Google Scholar,14Reid S. Scholey J. Recent approaches to targeting canonical NFκB signalling in the early inflammatory response to renal IRI.J Am Soc Nephrol. 2021; 32: 2117-2124Crossref PubMed Scopus (3) Google Scholar Activation of the canonical NF-κB pathway starts with activation of the inhibitor of NF-κB (IκB) kinase, which leads to phosphorylation and degradation of IκBα and the nuclear translocation of NF-κB heterodimers.15Hoffmann A. Levchenko A. Scott M.L. et al.The IkappaB-NF-kappaB signaling module: temporal control and selective gene activation.Science. 2002; 298: 1241-1245Crossref PubMed Scopus (1436) Google Scholar Activation of NF-κB signaling in kidney epithelial cells and infiltrating immune cells can be elicited by pathophysiological triggers such as exposure to lipopolysaccharides (LPSs) or ischemia-reperfusion injury.16Marko L. Vigolo E. Hinze C. et al.Tubular epithelial NF-kappaB activity regulates ischemic AKI.J Am Soc Nephrol. 2016; 27: 2658-2669Crossref PubMed Scopus (94) Google Scholar Molecular profiling studies reveal nfkb1 as a major driver of kidney fibrosis.17Wu H. Lai C.F. Chang-Panesso M. et al.Proximal tubule translational profiling during kidney fibrosis reveals proinflammatory and long noncoding RNA expression patterns with sexual dimorphism.J Am Soc Nephrol. 2020; 31: 23-38Crossref PubMed Scopus (25) Google Scholar In addition to kidney tubular cells, innate immune cells such as macrophages and dendritic cells also contribute to kidney injury, inflammation, and fibrotic remodeling.13Andrade-Oliveira V. Foresto-Neto O. Watanabe I.K. et al.Inflammation in renal diseases: new and old players.Front Pharmacol. 2019; 10: 1192Crossref PubMed Scopus (84) Google Scholar,18Singbartl K. Formeck C.L. Kellum J.A. Kidney-immune system crosstalk in AKI.Semin Nephrol. 2019; 39: 96-106Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 19Hato T. Dagher P.C. How the innate immune system senses trouble and causes trouble.Clin J Am Soc Nephrol. 2015; 10: 1459-1469Crossref PubMed Scopus (76) Google Scholar, 20Komada T. Muruve D.A. The role of inflammasomes in kidney disease.Nat Rev Nephrol. 2019; 15: 501-520Crossref PubMed Scopus (90) Google Scholar Growing evidence suggests that muscle-derived secretory factors (i.e., myokines) modulate systemic physiology via tissue crosstalk to influence the progression of kidney diseases.21Peng H. Wang Q. Lou T. et al.Myokine mediated muscle-kidney crosstalk suppresses metabolic reprogramming and fibrosis in damaged kidneys.Nat Commun. 2017; 8: 1493Crossref PubMed Scopus (71) Google Scholar MG53 (also named TRIM72) is a muscle-enriched tripartite motif (TRIM) protein with a critical function in cell membrane repair.22Cai C. Masumiya H. Weisleder N. et al.MG53 nucleates assembly of cell membrane repair machinery.Nat Cell Biol. 2009; 11: 56-64Crossref PubMed Scopus (309) Google Scholar,23Cai C. Masumiya H. Weisleder N. et al.MG53 regulates membrane budding and exocytosis in muscle cells.J Biol Chem. 2009; 284: 3314-3322Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar TRIM family proteins have diverse functions ranging from the regulation of immune signaling to tissue repair.24Hatakeyama S. TRIM family proteins: roles in autophagy, immunity, and carcinogenesis.Trends Biochem Sci. 2017; 42: 297-311Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar MG53-mediated membrane repair is involved in mitigating acute injury to skeletal muscle,25Cai C. Weisleder N. Ko J.K. et al.Membrane repair defects in muscular dystrophy are linked to altered interaction between MG53, caveolin-3, and dysferlin.J Biol Chem. 2009; 284: 15894-15902Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar kidney,26Duann P. Li H. Lin P. et al.MG53-mediated cell membrane repair protects against acute kidney injury.Sci Transl Med. 2015; 7: 279ra236Crossref Scopus (67) Google Scholar heart,27Liu J. Zhu H. Zheng Y. et al.Cardioprotection of recombinant human MG53 protein in a porcine model of ischemia and reperfusion injury.J Mol Cell Cardiol. 2015; 80: 10-19Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar lung,28Jia Y. Chen K. Lin P. et al.Treatment of acute lung injury by targeting MG53-mediated cell membrane repair.Nat Commun. 2014; 5: 4387Crossref PubMed Scopus (71) Google Scholar brain,29Guan F. Huang T. Wang X. et al.The TRIM protein Mitsugumin 53 enhances survival and therapeutic efficacy of stem cells in murine traumatic brain injury.Stem Cell Res Ther. 2019; 10: 352Crossref PubMed Scopus (24) Google Scholar,30Yao Y. Zhang B. Zhu H. et al.MG53 permeates through blood-brain barrier to protect ischemic brain injury.Oncotarget. 2016; 7: 22474-22485Crossref PubMed Scopus (33) Google Scholar and skin.31Li H. Duann P. Lin P.H. et al.Modulation of wound healing and scar formation by MG53 protein-mediated cell membrane repair.J Biol Chem. 2015; 290: 24592-24603Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar Our previous studies identified a low level of MG53 expression in the kidney, which is a key component in AKI protection, and mice deficient in MG53 (mg53−/−) were more susceptible to stress-induced AKI.26Duann P. Li H. Lin P. et al.MG53-mediated cell membrane repair protects against acute kidney injury.Sci Transl Med. 2015; 7: 279ra236Crossref Scopus (67) Google Scholar We also recently demonstrated that sustained elevation of MG53 in the circulation enhances tissue injury repair and regeneration.32Bian Z. Wang Q. Zhou X. et al.Sustained elevation of MG53 in the bloodstream increases tissue regenerative capacity without compromising metabolic function.Nat Commun. 2019; 10: 4659Crossref PubMed Scopus (21) Google Scholar The relative roles of kidney-specific and extrinsic circulating MG53 in modulating kidney fibrosis during progressive CKD are still unknown. Recently, an emerging role of MG53 in modulating tissue protection through the inhibition of inflammation, especially via modulation of NF-κB signaling, has evolved. As examples, MG53 attenuates LPS-induced neurotoxicity and neuroinflammation by inhibiting the TLR4/NF-κB pathway,33Guan F. Zhou X. Li P. et al.MG53 attenuates lipopolysaccharide-induced neurotoxicity and neuroinflammation via inhibiting TLR4/NF-kappaB pathway in vitro and in vivo.Prog Neuropsychopharmacol Biol Psychiatry. 2019; 95: 109684Crossref PubMed Scopus (26) Google Scholar and cardiac MG53 regulates KChIP2 expression to control electrophysiological remodeling during cardiohypertrophy.34Liu W. Wang G. Zhang C. et al.MG53, a novel regulator of KChIP2 and Ito,f, plays a critical role in electrophysiological remodeling in cardiac hypertrophy.Circulation. 2019; 139: 2142-2156Crossref PubMed Scopus (20) Google Scholar In addition, we demonstrated a dual function of MG53 in preventing a maladaptive hyperinflammatory response during a sublethal influenza A virus infection, characterized by excessive interferon-β production, via suppression of IRF3/NF-κB activation and inhibition of aberrant intracellular Ca2+ signaling in macrophages.35Sermersheim M. Kenney A.D. Lin P.H. et al.MG53 suppresses interferon-β and inflammation via regulation of ryanodine receptor-mediated intracellular calcium signaling.Nat Commun. 2020; 11: 3624Crossref PubMed Scopus (12) Google Scholar Furthermore, upon a lethal dose of influenza A virus infection, MG53 was shown to prevent mortality and lung damage, not by directly affecting viral titers per se but by mitigating cytokine storm (interferon-β, interleukin-6, and interleukin-1β) through negative regulation of the NLRP3 inflammasome and prevention of pyroptosis of the lung.36Kenney A.D. Li Z. Bian Z. et al.Recombinant MG53 protein protects mice from lethal influenza virus infection.Am J Respir Crit Care Med. 2021; 203: 254-257Crossref PubMed Scopus (7) Google Scholar Moreover, high-dose chronic treatment of exogenous MG53 was linked to suppression of NF-κB–mediated inflammation in the aged heart.37Wang X. Li X. Ong H. et al.MG53 suppresses NFκB activation to mitigate age-related heart failure.JCI Insight. 2021; 6e148375Crossref PubMed Scopus (2) Google Scholar These studies suggest MG53 can modulate aberrant NF-κB signaling during inflammation but whether this occurs in the context of kidney inflammation has not been studied. We postulated that MG53 regulates kidney inflammation by modulating the transcriptional activity of NF-κB and in so doing can attenuate the kidney fibrosis that occurs as a consequence of chronic inflammation. This study was undertaken to address this hypothesis. All experiments with animals adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and followed protocols approved by the Ohio State University Institutional Animal Care and Use Committee. Age-matched wild type (WT) or mg53−/− nonlittermates of the 129S1/SvImJ strain were used22Cai C. Masumiya H. Weisleder N. et al.MG53 nucleates assembly of cell membrane repair machinery.Nat Cell Biol. 2009; 11: 56-64Crossref PubMed Scopus (309) Google Scholar to establish the role of MG53 in the modulation of age-dependent and unilateral ureteric obstruction (UUO)-induced kidney fibrosis. C57B6/J mice (9–10 weeks) were purchased from Jackson Labs. For kidney injury studies, mice (9–12 weeks) were subjected to UUO to induce kidney fibrosis. Mice were anesthetized via isoflurane (1.5%–2.0%) to ensure a deep plane of anesthesia. The UUO procedure was performed by ligating the left ureter with 5-0 surgical silk twice according to published protocols.38Chevalier R.L. Forbes M.S. Thornhill B.A. Ureteral obstruction as a model of renal interstitial fibrosis and obstructive nephropathy.Kidney Int. 2009; 75: 1145-1152Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar Sham mice only had an abdominal skin incision. To evaluate the effect of rhMG53 on obstructive kidney injury, 1 group of UUO mice received rhMG53 (2 mg/kg) via tail vein injection beginning immediately after UUO surgery (day 0) and then daily for 7 days, with 2 additional doses on days 9 and 11. A control group of UUO mice received saline injections according to the same schedule. Mice were killed on day 12 after UUO surgery, kidneys were perfused in situ with phosphate-buffered saline, and tissue samples from the UUO kidney and the contralateral kidney were collected for histology and Western blot analysis. In separate studies, WT and mg53−/− mice underwent UUO or sham surgery and were killed on day 7 after the operation. MG53 was given to mice using a cell-based therapy approach.39Guiteras R. Sola A. Flaquer M. et al.Macrophage overexpressing NGAL ameliorated kidney fibrosis in the UUO mice model.Cell Physiol Biochem. 2017; 42: 1945-1960Crossref PubMed Scopus (27) Google Scholar RAW 264.7 macrophages were infected with doxycycline (DOX)-dependent Ad-tPA-MG53-mcherry viral particles for 24 hours. The infected cells (at a concentration of 2 × 106/100 μl saline per mouse) were tail vein injected into mice immediately after surgery (day 0 injection). Mice received 4 injections of Ad-tPA-MG53 transduced RAW cells on days 0, 1, 3, and 6. Immediately after the day 0 cell injection, mice were randomized into a group that received and a group that did not receive DOX (2 mg/ml in 5% sucrose solution) in their drinking water for 7 days and then killed. Tissue samples from the UUO kidney and contralateral kidney were collected for histology, RNA, and protein analyses. Data are expressed as means ± SD. A comparison within groups was performed using the Student’s t test when comparing 2 experimental groups and 1-way analysis of variance for more than 2 groups (Graphpad Prism 8.2; GraphPad) followed by either the Bonferroni or Holm-Sidak multiple comparison test ad hoc. A value of P < 0.05 was considered statistically significant. Full methods including histologic evaluation, serum creatinine measurements, plasmid constructs, cell culture and primary tubular epithelial cell isolation, adenovirus preparation and cell infection, NF-κB p65 nuclear translocation assay, immunohistochemistry and confocal images, NF-κB luciferase reporter assay, cytokine enzyme-linked immunosorbent assay, tissue fractionation, immunoblotting, coimmunoprecipitation, quantitative real-time polymerase chain reaction (Supplementary Table S1), and rhMG53 production are in the Supplementary Methods. We compared the effect of aging on kidney function and fibrosis in mg53−/− and WT mice. We confirmed our previous observation that although serum creatinine levels of young mice (2 months) with and without MG53 were not significantly different, older (16–20 months) mg53−/− mice exhibited a significantly higher serum creatinine than age-matched WT controls (Figure 1a). Based on trichrome staining, we found that mg53−/− mice had more kidney fibrosis compared with the WT mice, starting at the age of 5 months and continuing through the age of 20 months (Figure 1b and c). Immunohistochemical staining was used to evaluate the impact of MG53 ablation on the infiltration of leukocytes, monocytes, and macrophages in 2-month and 5-month-old mouse kidneys (Figure 1d and e). Kidneys from young WT and mg53−/− mice had similar levels of intrarenal leukocytes, but significantly more inflammatory cells were found in mg53−/− kidneys than WT kidneys in 5-month-old mice. Moreover, immunohistochemistry for α-smooth muscle actin and fibronectin, 2 extracellular matrix proteins commonly found in areas of kidney fibrosis, showed enhanced (3- to 8-fold) staining in the glomeruli and tubulointerstitium of kidneys from 10-month-old mg53−/− mice compared with 10-month-old WT mice (Supplementary Figure S1). Kidney MG53 levels detected by immunoblotting significantly increased 7 days after UUO (Figure 2a). The mg53−/− UUO kidneys displayed exaggerated fibrosis, with a 30% additional trichrome-stained area, than WT UUO kidneys (Figure 2b). Fibronectin was also significantly increased in UUO kidneys (Figure 2c). Kidney infiltration by leukocytes and macrophages was significantly increased after UUO in WT and mg53−/− mice but was further increased in mg53-deficient animals (Figure 2d and e). The activity of NF-κB (p65) was assessed in the UUO model. The amount of total p65 (t-p65) was significantly increased in UUO kidneys from WT and mg53−/− mice, but NF-κB activation, assessed as phosphorylation of p65, was greater (16-fold) in the mg53−/− UUO kidney than the WT UUO kidney (2-fold) compared with sham-operated animals (Figure 3a). We analyzed the relative RNA levels of transforming growth factor β1 (TGF-β1), plasminogen activator inhibitor-1 (PAI-1), and collagen type I alpha 1 (Col1a1) in the obstructed kidney.40Higgins C.E. Tang J. Higgins S.P. et al.The genomic response to TGF-β1 dictates failed repair and progression of fibrotic disease in the obstructed kidney.Front Cell Dev Biol. 2021; 9: 678524Crossref PubMed Scopus (3) Google Scholar We detected low levels of gene expression in sham animals and similar induction (TGF-β1, ∼10 fold; PAI-1, ∼40 fold; Col1a1, ∼40 fold) in the UUO kidney from WT and mg53−/− (Figure 3b). To examine the spatial distribution of MG53 in the kidney cortex under normal conditions, renal tissue fractions were analyzed. We regularly obtained 10-fold more total cytosol protein than the total extracted nuclear protein. The t-p65 levels were comparable in the cytosolic fraction between WT and mg53−/− mice but were greater in the nuclear fraction of mg53−/− mice (Figure 3c). MG53 was detected in the cytosol fraction (with tubulin as a cytosol marker). Unexpectedly, a significant amount of MG53 was detected at the nuclear fraction (with histone H3 as a nuclear fraction marker). To confirm this finding, we also examined MG53 localization in skeletal muscle fractions (Supplementary Figure S2). We reproduced our previous studies in which MG53 localized to cytosol and the sarcolemmal membrane (sodium-potassium adenosine triphosphatase as a marker of membrane fraction)22Cai C. Masumiya H. Weisleder N. et al.MG53 nucleates assembly of cell membrane repair machinery.Nat Cell Biol. 2009; 11: 56-64Crossref PubMed Scopus (309) Google Scholar,23Cai C. Masumiya H. Weisleder N. et al.MG53 regulates membrane budding and exocytosis in muscle cells.J Biol Chem. 2009; 284: 3314-3322Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar,25Cai C. Weisleder N. Ko J.K. et al.Membrane repair defects in muscular dystrophy are linked to altered interaction between MG53, caveolin-3, and dysferlin.J Biol Chem. 2009; 284: 15894-15902Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar but also detected enriched nuclear MG53 from skeletal muscle. To confirm our tissue fractionation results, we conducted the following immunohistochemical studies. In cultured WT proximal tubular epithelial cells, p65 was found mainly in the cytosol but localized to nuclei in the absence of MG53 (Figure 4a). When these cells were treated with LPS (5 μg) for 8 hours, proinflammatory cytokine secretion was significantly greater in mg53−/− cells than WT cells (Figure 4b). Because these data suggested MG53 may interact with p65 to regulate inflammatory cytokine expression, a direct interaction between p65 and MG53 was assessed by coimmunoprecipitation assays using HEK293 cells cotransfected with Flag-p65 and HA-MG53. We demonstrated a weak interaction (Figure 4c). Cotransfection of GFP-p65 and RFP-MG53 plasmids and confocal analysis confirmed colocalization of p65 and MG53 (Figure 4d). We then quantified whether MG53 affects NF-κB (p65) transcriptional activity after TNF-α stimulation by measuring the luciferase activity from a stable NF-κB/293-GFP-Luc transcriptional reporter cell line that was transiently transfected with either pHM6 vector or HA-MG53. The reporter cells contain a transduced lentivirus expression firefly luciferase reporter driven by a minimal cytomegalovirus promoter in conjunction with 4 copies of consensus NF-κB transcriptional response elements (κB site) upstream. In the absence of TNF-α, there was very low intrinsic luciferase activity detected in the reporter cells expressing MG53 proteins. MG53 suppressed TNF-α–elicited NF-κB (p65) transcriptional activity by about 40% (Figure 4e). NF-κB p65 translocated to the nucleus upon TNF-α treatment in the kidney proximal tubule epithelium.41Gong P. Canaan A. Wang B. et al.The ubiquitin-like protein FAT10 mediates NF-kappaB activation.J Am Soc Nephrol. 2010; 21: 316-326Crossref PubMed Scopus (63) Google Scholar To explore whether treatment with MG53 could attenuate NF-κB p65 nuclear translocation upon TNF-α stimulation, we overexpressed MG53 in HKC-8 cells either via DOX-dependent Ad-tPA-MG53 viral transduction or through treatment with recombinant human MG53 (rhMG53). Using a MG53-specific monoclonal antibody, after DOX-dependent induction MG53 was detected by immunoblotting. MG53 was present at a very high level in Ad-tPA-MG53 transduced HKC-8 cells, equivalent to 1 ng rhMG53/μg cellular lysate. In contrast, HKC-8 took up and expressed a much lesser amount of rhMG53 (Figure 5a). Uptake of fluorescent-labeled rhMG53 by HKC-8 was confirmed by confocal microscopy (Supplementary Figure S3). After TNF-α treatment, the time to peak p65 activation was 15 to 30 minutes (Figure 5b). Under basal conditions, NF-κB p65 resided mainly in the cytosol, and overexpression of MG53 did not change this (Figure 5c and d). However, after TNF-α stimulation, p65 translocated to the nuclei in HKC-8 cells not overexpressing MG53. In comparison, nuclear translocation of p65 fell by 33% (cells given rhMG53) to 50% (transduced cells given DOX) in cells overexpressing MG53 (Figure 5c and d). To understand whether treatment with MG53 could attenuate fibrosis, MG53 was increased in the UUO model via engineered RAW 264.7 macrophages (RAW). We engineered RAW via Dox-dependent induction of Ad-tPA-MG53 expression. DOX-dependent induction of tPA-MG53 expression was confirmed by confocal immunostaining of RAW with a MG53-specific monoclonal antibody (Supplementary Figure S4A), immunoblotting RAW cell lysates after viral transduction (Supplementary Figure S4B), and immunoblotting concentrated culture medium of transduced RAW (Supplementary Figure S4C). Adoptive transfer of engineered RAW cells into the UUO mouse model was performed with 1 × 106 cells administered through tail vein injection immediately after UUO surgery. Mice were randomly divided into a group that received normal drinking water (−DOX) and a group that received DOX (2 mg/ml) in drinking water. UUO mice receive