JPET Fast Forward. Published on December 12, 2014 as DOI: 10.1124/jpet.114.221085 This article has not been copyedited and formatted. The final version may differ from this version. JPET #221085 Suppression of Mitochondrial Biogenesis through TLR4-dependent MEK/ERK Signaling in Endotoxin-Induced Acute Kidney Injury Joshua A. Smith, L. Jay Stallons, Justin B. Collier, Kenneth D. Chavin, and Rick G. Schnellmann Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina (J.A.S., L.J.S., J.B.C., and R.G.S.); Division of Transplant Surgery, Department of Surgery, Medical University of South Carolina, Charleston, South Carolina (K.D.C.); and Ralph H. Johnson Veterans Administration Medical Center, Charleston, South D o w n Carolina (R.G.S.) lo a d e d fro m jp e t.a s p e tjo u rn a ls .o rg a t A S P E T J o u rn a ls o n M a rc h 2 , 2 0 2 3 JPET Fast Forward. Published on December 12, 2014 as DOI: 10.1124/jpet.114.221085 This article has not been copyedited and formatted. The final version may differ from this version. JPET #221085 1 Running Title: Mitochondrial Biogenesis in Endotoxic AKI Corresponding Author: Rick G. Schnellmann, PhD Department of Drug Discovery and Biomedical Sciences Medical University of South Carolina 280 Calhoun St., MSC140 Charleston, SC 29425 USA Phone: 843-792-3754 Fax: 843-792-2620 E-mail: [email protected] No. of Text Pages: 33 D o w n No. of Tables: 1 lo a d e d No. of Figures: 11 fro m jp e No. of References: 59 t.a s p e tjo Abstract: 238 words u rn a ls Introduction: 687 words .org a t A Discussion: 1,500 words S P E T J Nonstandard Abbreviations: MB, mitochondrial biogenesis; AKI, acute kidney injury; ETC, o u electron transport chain; LPS, lipopolysaccharide; PGC-1α, peroxisome proliferator-activated rna γ ls receptor coactivator-1α; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; MEK, on M mitogen activated protein kinase kinase; ERK, extracellular signal-regulated kinase; TLR4, toll- arc h like receptor 4; RBF, renal blood flow; IL-6, interleukin 6; CLP, cecal ligation and puncture; 2 , 2 BUN, blood urea nitrogen; NRF-1, nuclear respiratory factor-1; TFAM, mitochondrial 02 3 transcription factor a; NDUFS1, NADH dehydrogenase (ubiquinone) Fe-S protein 1; NDUFB8, NADH dehydrogenase (ubiquinone) beta subcomplex 8; ATPSβ, ATP synthase β; COX1, cytochrome c oxidase subunit 1; ND1, NADH dehydrogenase subunit 1; PBS, phosphate buffered saline; GFR, glomerular filtration rate; KIM-1, kidney injury molecule-1; NGAL, γ neutrophil gelatinase-associated lipocalin; PGC-1β, peroxisome proliferator-activated receptor coactivator-1β; PRC, peroxisome proliferator-activated receptor gamma coactivator-related protein 1; MAPK, mitogen activated protein kinase; JNK, c-Jun N-terminal kinase; TPL-2, tumor progression locus 2; NFκB, nuclear factor κ B; RPTC, renal proximal tubule cell Recommended Section: Gastrointestinal, Hepatic, Pulmonary, and Renal JPET Fast Forward. Published on December 12, 2014 as DOI: 10.1124/jpet.114.221085 This article has not been copyedited and formatted. The final version may differ from this version. JPET #221085 2 Abstract: Although disruption of mitochondrial homeostasis and biogenesis (MB) is a widely accepted pathophysiological feature of sepsis-induced AKI, the molecular mechanisms responsible for this phenomenon are unknown. In this study, we examined the signaling pathways responsible for the suppression of MB in a mouse model of lipopolysaccharide (LPS)- induced AKI. Down-regulation of PGC-1α, a master regulator of MB, was noted at the mRNA level at 3 h and protein level at 18 h in the renal cortex, and was associated with loss of renal D o function following LPS treatment. LPS-mediated suppression of PGC-1α led to reduced w n lo a d e expression of downstream regulators of MB and electron transport chain (ETC) proteins along d fro m with a reduction in renal cortical mitochondrial DNA content. Mechanistically, TLR4 knockout jp e t.a s p mice were protected from renal injury and disruption of MB after LPS. Immunoblot analysis e tjo u rn revealed activation of TPL-2/MEK/ERK signaling in the renal cortex by LPS. Pharmacological als .o rg inhibition of MEK/ERK signaling attenuated renal dysfunction and loss of PGC-1α, and was at A S P E associated with a reduction in pro-inflammatory cytokine (e.g. TNF-α, IL-1β) expression at 3 h T J o u rn post-LPS. Neutralization of TNF-α also blocked PGC-1α suppression, but not renal als o n M dysfunction, following LPS-induced AKI. Finally, systemic administration of recombinant arc h 2 TNF-α alone was sufficient to produce AKI and disrupt mitochondrial homeostasis. These , 20 2 3 findings indicate an important role for the TLR4/MEK/ERK pathway in both LPS-induced renal dysfunction and suppression of MB. TLR4/MEK/ERK/TNF-α signaling may represent a novel therapeutic target to prevent mitochondrial dysfunction and AKI produced by sepsis. JPET Fast Forward. Published on December 12, 2014 as DOI: 10.1124/jpet.114.221085 This article has not been copyedited and formatted. The final version may differ from this version. JPET #221085 3 Introduction: Acute kidney injury (AKI) is characterized by a rapid decrease in renal function over the course of hours to days and is associated with significant morbidity and mortality (~40%) (Uchino et al., 2005). Despite recent efforts to better understand AKI, mortality associated with this clinical disorder remains unchanged over the last five decades (Thadhani et al., 1996; Waikar et al., 2008). Sepsis is the most common contributing factor to the development AKI, mortality resulting from AKI is almost doubled in septic patients (~70%) and treatment is limited D o to dialysis and supportive care (Silvester et al., 2001; Schrier and Wang, 2004; Uchino et al., w n lo a d 2005; Bagshaw et al., 2007; Waikar et al., 2008). Taken together, these data reveal a significant ed fro m need for further study of the pathophysiological mechanisms underlying renal injury with an jp e t.a s p emphasis on identifying therapeutic targets to improve clinical outcomes in septic AKI. e tjo u rn Much of the difficulty in developing effective therapies for sepsis-induced AKI stems als .o rg from the multi-factorial nature of the disease. Septic AKI is thought to arise as a result of at A S P E complex interactions involving alterations in renal hemodynamics, microvascular/endothelial T J o u rn cell dysfunction and direct effects of inflammatory cells and their products a ls o n (cytokines/chemokines) on the kidney (Wan et al., 2008). The degree to which changes in global M a rc h 2 renal blood flow (RBF) contribute to renal injury remains a topic of intense debate. However, it , 2 0 2 3 is generally accepted that microvascular dysfunction leads to sluggish capillary flow and subsequent development of local regions of hypoperfusion and hypoxia in the septic kidney (Wu et al., 2007a; Wu et al., 2007b; Gomez et al., 2014). Reduced microvascular flow also amplifies injury by prolonging exposure of the renal parenchyma to inflammatory cells and various inflammatory molecules including pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6, which are primary mediators of cellular injury in JPET Fast Forward. Published on December 12, 2014 as DOI: 10.1124/jpet.114.221085 This article has not been copyedited and formatted. The final version may differ from this version. JPET #221085 4 sepsis-induced AKI (Cunningham et al., 2002; Chawla et al., 2007; Wang et al., 2011; Xu et al., 2014). Data from post-mortem studies and experimental models indicate that tubular cell apoptosis and necrosis are relatively limited in the septic kidney when compared to other forms of AKI (Guo et al., 2004; Langenberg et al., 2008). However, histological findings including tubular cell vacuolization, tubular dilatation, and the presence of swollen mitochondria provide strong evidence that sub-lethal injury to the proximal tubule may play an important role in the development of sepsis-induced AKI (Langenberg et al., 2008; Tran et al., 2011; Takasu et al., D o 2013). w n lo a d The overt structural changes noted in tubular mitochondria following sepsis-induced AKI ed fro m is of particular note given that the proximal tubule relies heavily on mitochondrial generation of jp e t.a s p ATP to drive active transport of electrolytes and fluids. Thus, mitochondrial and/or proximal e tjo u rn tubule dysfunction may contribute to loss of renal function and disease progression in AKI als .o rg (Soltoff, 1986; Thadhani et al., 1996). Recent studies demonstrated suppression of peroxisome at A S P proliferator-activated receptor γ coactivator-1α (PGC-1α) and consequently MB in experimental ET J o u rn models of sepsis-induced AKI including systemic endotoxin exposure and cecal ligation and a ls o n puncture (CLP) (Tran et al., 2011). PGC-1α, a “master regulator of MB,” promotes Ma rc h 2 transcriptional activity in both the nucleus and mitochondria to facilitate generation of new, , 2 0 2 3 functional mitochondria in response to a variety of physiological stimuli (Finck and Kelly, 2006; Sanchis-Gomar et al., 2014). Loss of PGC-1α following sepsis-induced AKI was closely associated with renal and mitochondrial dysfunction and reduced expression of electron transport chain proteins. In addition, proximal tubule-specific PGC-1α knockout delayed recovery of renal function following saline resuscitation in mice treated with LPS (Tran et al., 2011). These JPET Fast Forward. Published on December 12, 2014 as DOI: 10.1124/jpet.114.221085 This article has not been copyedited and formatted. The final version may differ from this version. JPET #221085 5 findings indicate that suppression of PGC-1α and MB may play an important role in disease progression and recovery in the setting of septic AKI. The aim of the current study was to determine the signaling mechanisms responsible for suppression of MB in the renal cortex following endotoxic AKI. We report that LPS exposure leads to down-regulation of PGC-1α and mitochondrial markers in the renal cortex. LPS-induced renal dysfunction and disruption of MB was dependent on TLR4/MEK/ERK and production of the pro-inflammatory cytokine TNF-α. Inhibition of the TLR4/MEK/ERK/TNF-α signaling may D o w offer a novel therapeutic approach to reverse suppression of MB and loss of renal function in n lo a d e d septic AKI. fro m jp e t.a s p e tjo u rn a ls .o rg a t A S P E T J o u rn a ls o n M a rc h 2 , 2 0 2 3 JPET Fast Forward. Published on December 12, 2014 as DOI: 10.1124/jpet.114.221085 This article has not been copyedited and formatted. The final version may differ from this version. JPET #221085 6 Materials and Methods: LPS Model of Sepsis-Induced AKI Six to eight week old male C57BL/6 mice were acquired from the National Institutes of Health National Cancer Institute / Charles River Laboratories (Frederick, MD). Mice were given an intraperitoneal (i.p.) injection of 0.5, 2 or 10 mg/kg lipopolysaccharide (LPS) derived from Escherichia coli serotype O111:B4 (Sigma Aldrich, St. Louis, MO). Control mice received an i.p. injection of an equal volume of 0.9% normal saline. Mice were euthanized by isoflurane D asphyxiation and cervical dislocation at 1, 3, and 18 h after LPS administration, and kidneys and ow n lo a serum were collected for molecular analysis. For experiments utilizing TLR4-deficient animals, de d fro TLR4KO mice were generated by crossing C57BL/10ScN mice with the tlr4LPS-d mutation onto jpm e t.a s the C57BL/6 background for at least five generations (Ellett et al., 2009). All studies were p e tjo u conducted in accordance with the recommendations in the Guide for the Care and Use of rna ls .o rg Laboratory Animals of the National Institutes of Health. Animal use was approved by the a t A S P Institutional Animal Care and Use Committee at the Medical University of South Carolina. E T J o u rn a To determine the role of MEK/ERK signaling in LPS-induced AKI, the MEK inhibitor ls o n M GSK1120212 (trametinib, chemical structure provided in (Gilmartin et al., 2011)) was obtained arc h 2 from Selleckchem Chemicals (Houston, TX). GSK1120212 is a potent and specific inhibitor of , 20 2 3 MEK1/2 which has been previously used in mouse models (Yamaguchi et al., 2011; Yamaguchi et al., 2012). Mice received an i.p. injection of GSK1120212 (1 mg/kg) or vehicle control (DMSO) 1 h prior to administration of LPS. To assess the effects of TNF-α on regulation of MB in this model, rat anti-TNF-α neutralizing antibody (clone MP6-XT22) and the appropriate rat IgG1 κ isotype control antibody (clone RTK2071) were purchased from BioLegend (San Diego, CA). Mice were randomly JPET Fast Forward. Published on December 12, 2014 as DOI: 10.1124/jpet.114.221085 This article has not been copyedited and formatted. The final version may differ from this version. JPET #221085 7 assigned to one of three groups: 1) control, 2) LPS + isotype control antibody (25 mg/kg), and 3) LPS + anti-TNF-α neutralizing antibody (25 mg/kg). Isotype control antibody and anti-TNF-α neutralizing antibody were administered intravenously (i.v.) 1 h prior to LPS via tail vein injection. Control mice received an i.v. injection of vehicle control (PBS). Recombinant mouse TNF-α was obtained from BioLegend to evaluate whether TNF-α alone reproduced LPS-mediated changes in renal function and/or MB. Wild-type C57BL/6 male μ mice (6 to 8 weeks in age) were treated i.v. with either vehicle control (diluent), 20 g/kg D o w μ n recombinant murine TNF-α, or 50 g/kg TNF-α via tail vein injection. Animals were loa d e d euthanized at 18 h after TNF-α administration and kidneys and serum were collected for fro m jp e analysis. t.a s p e tjo u rn Blood Urea Nitrogen Measurement a ls .o rg a Blood urea nitrogen (BUN) was determined using the QuantiChrom Urea Assay kit t A S P E T (BioAssay Systems, Hayward, CA) based on the manufacturer’s directions. All values are Jo u rn a expressed as blood urea nitrogen concentration in milligrams per deciliter. ls o n M a rc h Quantitative Real-Time PCR Analysis of mRNA Expression 2 , 2 0 2 3 Total RNA was isolated from renal cortical tissue with TRIzol reagent (Life Technologies, Grand Island, NY). The iScript Advanced cDNA Synthesis Kit for RT-qPCR μ (Bio-Rad, Hercules, CA) was used to produce a cDNA library from 1 g total RNA according to the manufacturer’s protocol. Quantitative real-time PCR was performed with the generated cDNA using the SsoAdvanced Universal SYBR Green Supermix reagent (Bio-Rad). Relative mRNA expression of all genes was determined by the 2-ΔΔCt method and the 18S ribosomal RNA JPET Fast Forward. Published on December 12, 2014 as DOI: 10.1124/jpet.114.221085 This article has not been copyedited and formatted. The final version may differ from this version. JPET #221085 8 (18S rRNA) was used as a reference gene for normalization as previously described (Wills et al., 2012). Primer pairs used for PCR were as described in Table 1. Analysis of Mitochondrial DNA Content Mitochondrial DNA content was determined by quantitative real-time PCR analysis. Total DNA was isolated from the renal cortex using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA) as described in the manufacturer’s protocol. Extracted DNA was quantified and 5 D ng was used for PCR. Relative mitochondrial DNA content was assessed by the mitochondrial- o w n lo encoded NADH Dehydrogenase 1 (ND1) and was normalized to nuclear-encoded β-Actin. ad e d fro Primer sequences for ND1 and β-Actin were: ND1 sense: 5’-TAGAACGCAAAATCTTAGGG- m jp e t.a 3’; ND1 antisense: 5’-TGCTAGTGTGAGTGATAGGG-3’; β-Actin sense: 5’- sp e tjo u GGGATGTTTGCTCCAACCAA-3’; and β-Actin antisense: 5’- rna ls .o rg GCGCTTTTGACTCAGGATTTAA-3’. at A S P E T Immunoblot Analysis Jo u rn a ls o Protein was extracted from renal cortex using RIPA buffer (50 mM Tris-HCl, 150 mM n M a rc h NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, pH 7.4) with protease inhibitor 2 , 2 0 2 cocktail (1:100), 1 mM sodium fluoride, and 1 mM sodium orthovanadate (Sigma Aldrich). 3 Total protein amount was determined by BCA protein assay. Equal protein quantities (50 – 100 μ g) were loaded onto 4 – 15% SDS-PAGE gels (Bio-Rad). Proteins were resolved by gel electrophoresis and transferred onto nitrocellulose membranes (Life Technologies). Membranes were blocked in 2.5% BSA and incubated overnight with primary antibody at 4°C. Primary antibodies used in these studies included NGAL/Lipocalin-2 (1:1000), phospho-TPL2 (1:500), total TPL2 (1:1000, all from Abcam, Cambridge, MA), phospho-ERK1/2 (1:1000), total ERK1/2 JPET Fast Forward. Published on December 12, 2014 as DOI: 10.1124/jpet.114.221085 This article has not been copyedited and formatted. The final version may differ from this version. JPET #221085 9 (1:1000, both from Cell Signaling Technology, Danvers, MA), KIM1 (1:1000, from R&D systems, Minneapolis, MN), PGC-1 (1:100, Cayman Chemical, Ann Arbor, MI), and β-Actin (1:1000, Santa Cruz Biotechnology, Dallas, TX). Membranes were incubated with the appropriate horseradish peroxidase(HRP)-conjugated secondary antibody before visualization using enhanced chemiluminescence (Thermo Scientific, Waltham, MA) and the GE ImageQuant LAS4000 (GE Life Sciences, Pittsburgh, PA). Optical density was determined using NIH ImageJ software (version 1.46). D o w n Statistical Analysis lo a d e d fro All data are shown as mean ± S.E.M. When comparing two experimental groups, an m jp e t.a unpaired, two-tailed t-test was used to determine statistical differences. A one-way analysis of sp e tjo u variance (ANOVA) followed by Tukey’s post-hoc test was performed for comparisons of rn a ls .o rg multiple groups. A p-value < 0.05 was considered statistically significant. All statistical tests a t A S P were performed using GraphPad Prism software. E T J o u rn a ls o n M a rc h 2 , 2 0 2 3