How cell membrane repair proteins regulate transcription factor signal transduction to control renal fibrosis
Mar 15, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
Haichang Li
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 the 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 intactMG53 in the obstructed kidney compared to the contralateral unobstructed kidney or the kidneys of sham-operated mice. The ureteral obstructed kidneys from MG53defificient 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-kB, 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-kB activation and inflammation and attenuated kidney fibrosis. Thus, MG53may have a therapeutic role in treating chronic kidney inflammation and thereby provide protection against fibrosis that leads to the chronic kidney disease phenotype.
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Translational Statement Chronic inflammation leads to fibrotic remodeling that may also underlie the transition from acute kidney injury to chronic kidney disease. Activation of the protein-inflammatory transcription factor nuclear factor-kB (NF-kB) 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-kB 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-kB 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.1,2 AKI and CKD are more common in older individuals, mainly due to increased susceptibility to injury plus a diminished ability to repair the aging kidney. Clinical studies have shown that CKD is a major risk factor for AKI, and, conversely, episodes of AKI can accelerate the progression of CKD toward end-stage kidney disease. Currently, the treatment of AKI is mainly supportive, and the treatment of established CKD is focused on slowing progression through inhibition of the renin-angiotensin-aldosterone system and now possibly through sodium-glucose cotransporter-2 inhibition.3,4
Depending on the severity of the injury, proximal tubular cells, the major target of acute injury, may undergo changes in cell cycle progression,5,6 metabolism,7 secretion of protein- inflammatory and profibrotic cytokines, or partial epithelial-mesenchymal transition,8 which will determine whether remodeling/repair will be successful or maladaptive and result in kidney fibrosis, a hallmark of CKD.9,10 This remodeling generally occurs on a background of kidney inflammation,
diminished vascular supply, and the production of extracellular matrix (ECM) proteins and includes loss of epithelial cells and accumulation of collagens, a-smooth muscle actin, and fibronectin; it also has a high correlation with deterioration of kidney function.
Chronic inflammation leads to fibrotic remodeling that may also underlie the transition from AKI to CKD.11–13 Cumulative research suggests the involvement of the nuclear factor-kB (NF-kB) transcription factor in the pathogenesis of kidney inflammation caused by infection, injury, or transplantation.11,12,14 Activation of the canonical NF-kB pathway starts with activation of the inhibitor of NF-kB (IkB) kinase, which leads to phosphorylation and degradation of IkBa and the nuclear translocation of NF-kB heterodimers.15 Activation of NF-kB 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.16 Molecular profiling studies reveal nfkb1 as a major driver of kidney fibrosis.17 In addition to kidney tubular cells, innate immune cells such as macrophages and dendritic cells also contribute to kidney injury, inflammation, and fibrotic remodeling.13,18–20
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Growing evidence suggests that muscle-derived secretory factors (i.e., myokines) modulate systemic physiology via tissue crosstalk to influence the progression of kidney diseases.21 MG53 (also named TRIM72) is a muscle-enriched tripartite motif (TRIM) protein with a critical function in cell membrane repair.22,23 TRIM family proteins have diverse functions ranging from the regulation of immune signaling to tissue repair.24 MG53-mediated membrane repair is involved in mitigating acute injury to skeletal muscle,25 kidneys,26 heart,27 lungs,28 brains,29,30 and skin.31 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.26 We also recently demonstrated that sustained elevation of MG53 in the circulation enhances tissue injury repair and regeneration.32 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-kB signaling, has evolved. For example, MG53 attenuates LPS-induced neurotoxicity and neuro-inflammation by inhibiting the TLR4/NF-kB pathway,33 and cardiac MG53 regulates KChIP2 expression to control electrophysiological remodeling during cardio hypertrophy.34 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-b production, via suppression of IRF3/NF-kB activation and inhibition of aberrant intracellular Ca2þ signaling in macrophages.35 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-b, interleukin-6, and interleukin-1b) through negative regulation of the NLRP3 inflammasome and prevention of pyroptosis of the lung.36 Moreover, high-dose chronic treatment of exogenous MG53 was linked to suppression of NF-kB– mediated inflammation in the aged heart.37 These studies suggest MG53 can modulate aberrant NF-kB 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-kB 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.
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METHODS
Animal studies
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 used22 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.38 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.39 RAW 264.7 macrophages were infected with doxycycline (DOX)-dependent AdtPA-MG53-cherry viral particles for 24 hours. The infected cells (at a concentration of 2 -106 /100 ml 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.
Statistical analyses Data are expressed as means -SD. A comparison within groups was performed using the Student’s t-test when comparing 2 experimental basic research H Li et al.: MG53 modulates NF-kB in kidney fibrosis 2 Kidney International (2021) -, -–-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.
Supplementary methods Full methods including histologic evaluation, serum creatinine measurements, plasmid constructs, cell culture and primary tubular epithelial cell isolation, adenovirus preparation and cell infection, NFkB p65 nuclear translocation assay, immunohistochemistry and confocal images, NF-kB 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.
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RESULTS
Mice with ablation of MG53 develop age-dependent kidney fibrosis
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 a-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).
UUO induces aggravated kidney inflammation and fibrosis in mg53-/- mice 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).
MG53 modulates NF-kB activation and p65 nuclear localization
The activity of NF-kB (p65) was assessed in the UUO model. The number of total p65 (t-p65) was significantly increased in UUO kidneys from WT and mg53 / mice, but NF-kB 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 b1 (TGF-b1), plasminogen activator inhibitor-1 (PAI-1), and collagen type I alpha 1 (Col1a1) in the obstructed kidney.40 We detected low levels of gene expression in sham animals and similar induction (TGF-b1, w10 fold; PAI-1, w40 fold; Col1a1, w40 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 the cytosol and the sarcolemmal membrane (sodium-potassium adenosine triphosphatase as a marker of membrane fraction)22,23,25 but also detected enriched nuclear MG53 from skeletal muscle.
MG53 interacts with p65 to control tumor necrosis factor-a– induced transcriptional activity 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 mg) for 8 hours, pro-inflammatory cytokine secretion was significantly greater in mg53 / cells than WT cells (Figure 4b).
Because these data suggested MG53 may interact with p65 to regulate inflflammatory 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-kB (p65) transcriptional activity after TNF-stimulation by measuring the luciferase activity from a stable NF-kB/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-kB transcriptional response elements (kB site) upstream. In the absence of TNF-a, there was very low intrinsic luciferase activity detected in the reporter cells expressing MG53 proteins. MG53 suppressed TNF-a–elicited NF-kB (p65) transcriptional activity by about 40% (Figure 4e).
Overexpression of MG53 prevented NF-kB p65 nuclear translocation upon TNF-a treatment NF-kB p65 translocated to the nucleus upon TNF-a treatment in the kidney proximal tubule epithelium.41 To explore whether treatment with MG53 could attenuate NF-kB p65 nuclear translocation upon TNF-a 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 an 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/mg cellular lysate. In contrast, HKC-8 took up and expressed a much lesser amount of rhMG53 (Figure 5a). The uptake of FL fluorescent-labeled rhMG53 by HKC-8 was confirmed by confocal microscopy (Supplementary Figure S3). After TNF-a treatment, the time to peak p65 activation was 15 to 30 minutes (Figure 5b). Under basal conditions, NF-kB p65 resided mainly in the cytosol, and overexpression of MG53 did not change this (Figure 5c and d). However, after TNF-a 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).
Adoptive transfer of engineered RAW cells into the UUOmouse 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 receivedDOX (2 mg/ml) in drinking water. UUO mice received RAWevery another day for a total of 4 injections. Kidneys were harvested 7 days after surgery.
Mice infused with engineered RAW followed by DOX induction displayed significantly reduced (by 30%) kidney fifibrosis compared with those who received no cells or those infused with engineered RAW cells but without Dox induction (66% fibrotic area; Figure 6a and b).
Only a small RAW/Ad-tPA-MG53 population survived the surveillance of the reticuloendothelial system by the end of the experiments. Immunohistochemistry staining confirmed that DOX treatment-induced RAW-mediated MG53 expression in the kidney (Figure 6c). Interestingly, UUO kidneys from RAW-infused mice and DOX-induced MG53 exhibited a lower p-65/t-p65 ratio (by about 40%) than those who either did not receive RAW or those given RAW without DOX (Figure 6d and e). The reduced p-p65 level was confirmed with p-p65 (S536) immunohistochemistry staining of UUO kidneys (Figure 6f). We think circulating MG53 delivered by Raw/Ad-tPA-MG53/þDOX and uptake by kidney proximal tubule cells could participate in reducing p-p65 (S536) and decrease UUO kidney inflammation. Compared with UUO-only kidneys, TGF-b1, PAI- 1, and Col1a1 expression was increased significantly in UUO kidneys from mice infused with RAW without DOX (no MG53 induction). Elevation of these profibrotic genes could be attributed to the induction of a strong allograft reaction from infused cells into immunocompetent mice. However, UUO kidneys from mice infused with RAW and given DOX (MG53 induction) expressed significantly lower (60%–72% reduction) PAI-1 and relatively lower levels of TGF-b1 and Col1a1 than mice without DOX treatment (Figure 6g).
Systemic administration of rhMG53 mitigates NF-kB activation and fibrosis development in mice subjected to UUO
We tested whether rhMG53 displayed a similar antifibrotic effect as the engineered RAW cell therapy described earlier. Our prior pharmacokinetic data indicated the short, circulating half-life (about 90 minutes in rodents) of rhMG53.42 Mice tolerated chronic administration of 2 to 6 mg/kg
rhMG5326,37 well, so the administration strategy shown in Figure 7a was used. UUO mice were randomly given either vehicle (saline) or rhMG53 (2 mg/kg) daily for the first 7 days post-surgery and then every other day until they were killed on day 12. Sham and contralateral kidneys exhibited roughly equivalent t-p65 and fibronectin expression levels, with the p-p65 levels slightly increased in contralateral kidneys compared with sham kidneys. After UUO, kidney t-p65 increased. Compared with saline-treated animals, the p-65/t-p65 ratio in the UUO kidneys of rhMG53-treated animals was reduced by about 37%, whereas IkBa increased by about 27% (Figure 7b and c). Additionally, fibronectin decreased by about 33% (Figure 7b and c). Immunohistochemistry confirmed rhMG53 was present in UUO kidneys after treatment (Figure 7d). Total fibrosis was reduced in rhMG53-treated UUO kidneys (Figure 7e). However, RNA expression of TGF-b1, PAI-1, and Col1a1 was similar in mice treated with either saline or rhMG53 (Figure 7f).
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DISCUSSION
MG53 is a muscle-enriched TRIM protein initially identified as a prominent component of plasma membrane repair machinery.22,26,42,43 However, the actions of MG53 are not confined to skeletal muscle, and MG53 can be released into the circulation as a myokine.32 We have previously shown MG53 can control inflammation during tissue damage in remote organs.27,29,33,35–37 The present investigation extends the scope of MG53-mediated organ protection to the kidney. We conclude that MG53 can attenuate intrarenal inflammation and kidney fibrosis by blocking cytokine-induced activation of the proinflammatory and profibrotic transcription factor NF-kB. This conclusion is based on the following observations: (i) genetic deletion of MG53 in normal mice.
resulted in a decline in kidney function as the mice aged that was accompanied by an increase in kidney fibrosis and infiltration of the kidney by leukocytes and macrophages; (ii) in a murine UUO kidney injury model, the obstructed kidneys ofMG53-deficient mice exhibited more fibrosis and inflammation than MG53-replete mice; (iii) TNF-a and interleukin-6production by LPS-treated proximal tubular epithelial cells were attenuated in the presence of MG53; (iv) treatment with or overexpression of MG53 in a proximal tubular epithelial cell line diminished TNFa-induced NF-kB activation by blocking the nuclear translocation of the p65 component of-kB (MG53 was found to bind directly to p65, albeit weakly); and (v) phosphorylation of p65, a necessary step in the activation of NF-kB, was decreased in the obstructed kidneys of UUO mice treated with recombinant MG53 induced to overexpress MG53 in macrophages. Furthermore, the inhibitor of NF-kB activation, IkBa, was increased in the obstructed kidneys of UUO mice injected with recombinantMG53.
Genetic deletion of MG53 in normal mice was permissive to the adverse effects of aging on the kidneys. This suggests that the presence of endogenous MG53 provides a baseline level of protection during aging. Similarly, the loss of endogenous MG53 made AKI due to obstruction worse. The UUO kidney accumulated MG53, possibly reflecting remote myokine activity from skeletal muscle. It remains unclear how muscle MG53 may be recruited to the kidney during injury; however, a damaged kidney often develops hypoxia and undergoes oxidative stress,44,45 signals that could serve to recruit MG53. If so, this “muscle-kidney-immune cell” axis could be leveraged to attenuate kidney damage.46 Alternatively, kidney cells do express low levels of MG53 at baseline,26 and, during acute or chronic injury, MG53 production could be upregulated.
NF-kB is a prominent transcriptional driver of inflammation and fifibrosis in kidney injury and aging.14,17,47,48 We were prompted to investigate whether the effects of MG53 could be mediated via NF-kB because the presence of MG53 in the nuclear compartment of kidney homogenates suggested the potential for transcriptional interference. Nuclear localization of MG53 is not without precedent. In a transgenic model overexpressing myocardial MG53, nuclear MG53 regulated the expression of peroxisome proliferator-activated receptor-alpha at the transcriptional level and affected cardiac lipid metabolism.49 Moreover, we recently demonstrated rhMG53 suppressed NF-kB activation to mitigate age-related heart failure through reducing pro-inflammatory cytokines, apoptosis, and oxidative stress in aged cardiomyocytes.37
The molecular mechanism of how MG53 modulates p65 nuclear/cytoplasmic mobilization remains unclear. MG53 contains no nuclear localization signal, but it contains 3 putative N-terminal leucine-rich nuclear export signals, a motif recognized by nuclear export protein exportin.50 The direct interaction between MG53 and p65 was rather weak as the only or main disruptive effect of MG53 on NF-kB activation. Although MG53 appears to affect the dynamic nucleocytoplasmic shuttling of NF-kB critical components, the exact mechanisms remain to be determined.
The critical outcome of MG53-mediated renoprotection appears to be to decrease fibrotic remodeling through the attenuation of NF-kB–mediated inflammation. Kidney fifibrosis is a complex process involving the pathologic deposition of various ECM proteins.51 MG53 seemed to have clear effects on the ECM protein fibronectin. Fibronectin increased in UUO kidneys deficient in MG53 and decreased in UUO mice treated with MG53. The expression of other ECM proteins in the presence and absence of MG53 is more difficult to understand. We previously demonstrated that rhMG53 negatively regulates TGF-b1/Smad signaling and suppresses the synthesis of ECM proteins in an in vitro study.31 In vivo, TGFb1, PAI-1, and Col1a1 transcripts did not increase in UUO kidneys from mg53 / deficient animals. These transcripts did decrease in UUO animals induced to overexpress MG53 through the administration of virally induced mouse
macrophages, but when recombinant MG53 was given, no effect was seen. This may simply be a case of sufficient dosing because the intrarenal MG53 levels achieved were far greater in animals given the macrophage vector as opposed to recombinant MG53. Additionally, protein levels were not measured, and post-transcriptional events could modify the ECM in UUO. Further research is needed to elucidate the emerging role of MG53 in modulating specific ECM proteins. In conclusion, this study has demonstrated that a myokine may help regulate baseline inflammation in a remote organ like the kidney and, in the setting of kidney injury, may help to attenuate and control inflammation. Figure 8 presents a working model of the role of MG53 in renoprotection. During damage-associated molecular patterns (DAMP, such as TNF-a) and pathogen-associated molecular patterns (PAMP, such as LPS) stimulation and ischemic stress, the NF-kB signalosome is activated in the kidney. MG53 protects the kidney by inhibiting the phosphorylation and activation of NF-kB signaling, stabilizing IkBa, and reducing p65 nuclear mobilization, blocking the activation of pro-inflammatory programs. Although endogenous MG53 may mediate these effects, we suggest more robust protection may be afforded using MG53 therapeutically, which is feasible based on the ability of exogenous MG53 to reduce injury in UUO. Further investigation is warranted to understand how this myokine may be clinically exploited.
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DISCLOSURE
JM has an equity interest in TRIM-edicine, Inc., which developsrhMG53 for the treatment of human diseases. Patents on the use of MG53are held by Rutgers University and The Ohio State University. All the other authors declared no competing interests.
ACKNOWLEDGMENTS
This work was supported by the National Institutes of Health (NIH) R01-DK106394 (JM, BHR, and P-HL) and NIH R01-AG062896 (P-HL andBHR). This project was also partly supported by an intramuralLockwood fund from The Ohio State University (P-HL) and an award from the National Center for Advancing Translational Sciences(NCATS; P-HL, award number UL1TR002733). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NCATS or the NIH.
AUTHOR CONTRIBUTIONSHL,
PD, P-HL, JM, and BHR conceived and/or designed the studies; HL, PD, P-HL, ZL, and XZ conducted the experiments, acquired data, and performed data analysis; PD, P-HL, and BHR interpreted the results; and P-HL and BHR drafted, revised, and approved the manuscript. All authors read and approved the final version.
SUPPLEMENTARY MATERIAL
Supplementary File (PDF) Supplementary Methods.
Table S1. Primer sequences. Figure S1. mg53-/- mice display enhanced ECM protein deposition. Age-matched (10 months old) wild-type (WT) and mg53- /- kidney tissues were stained with 40 6-diamidino-2-phenylindole (DAPI; blue, left panels), a- smooth muscle actin (SMA; green, second panels), fibronectin (red, third panels), and merged (right panels). Bar ¼ 25 mm. Quantification of renal fibrosis area (n ¼ 3 per group). Data are presented as means -SD. Student’s t-test. ***P < 0.001.
Figure S2. Distribution of skeletal muscle MG53 between nucleus and cytoplasm. Skeletal muscle-derived from wild-type (WT) ormg53-/- mice were fractionated and immunoblotted with a custom-made anti-MG53 antibody (3 mg lysates) and cell compartment markers (20 mg lysates). Sodium-potassium adenosine triphosphatases are used as a cell membrane marker, histone H3 as a nuclear marker, and tubulin as a cytosol marker.
Figure S3. HKC-8 kidney proximal tubule cells specifically uptake AlexaFluor 647–labeled rhMG53. rhMG53 (Trimedicine) and bovine serum albumin (BSA; ThermoFisher Scientific) were labeled with Alexa Fluor(AF) Antibody Labeling kits (Life Technologies) according to the manufacturer's protocol. HKC-8 cells were cultured on Delta TPG glass-bottomed dish (Bioptechs Inc.) in Dulbecco’s modified Eagle’s medium(DMEM) with 10% fetal bovine serum (FBS) in the presence of labeled proteins, BSA-AF647 (left) and rhMG53-AF647 (right), for 24 hours. Z-stack images were taken from an LSM780 confocal microscope (Zeiss). Images show cells at the bottom of the dishes. Bar ¼ 25 mm.
Figure S4. Engineering RAW cells to mediate inducible MG53secretion. (A) RAW cells were infected with Ad-tPA-MG53 for 24 hours and treated without ( doxycycline [Dox]) or with Dox (þDox, 1 mg/ml)for an additional 24 hours to induce tPA-MG53 expression. Confocal images showed the induction of MG53 expression (custom-made anti-MG53 antibody, red, lower panels) from macrophage-like RAW cells(F4/80 staining, green, top panels). Bars ¼ 25 mm. (B, C) Western blot analysis of cell lysates (in the amount of 0.6, 3, and 6 mg) for induced expression of tPA-MG53 and MG53 from infected RAW cells (B) and the corresponding culture media concentrates (in volumes of 2, 5, and 10ml) (C). Various quantities of rhMG53 (in the amount of 0.2, 1, 0.05, and0.025 ng) were loaded as standards. Mw, molecular weight markers.
