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Mitogen-Activated Protein Kinase 14 Promotes AKI

† ‡ † † Alberto Ortiz,* Holger Husi, Laura Gonzalez-Lafuente,* Lara Valiño-Rivas,* † ‡ ‡ Manuel Fresno,§ Ana Belen Sanz,* William Mullen, Amaya Albalat, Sergio Mezzano,¶ ‡| † Tonia Vlahou,** Harald Mischak, and Maria Dolores Sanchez-Niño*

*Instituto Investigacion Sanitaria-Fundacion Jimenez Diaz-Universidad Autonoma de Madrid and Fundacion Renal Iñigo Alvarez de Toledo-Instituto Reina Sofia de Investigacion Nefrologica, Madrid, Spain; †Red de Investigacion Rena, Madrid, Spain; ‡Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, United Kingdom; §Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas de la Universidad Autonoma de Madrid, Madrid, Spain; |Mosaiques diagnostics GmbH, Hannover, Germany; ¶Unidad de Nefrología, Instituto de Medicina, Universidad Austral de Chile, Valdivia, Chile; and **Biomedical Research Foundation Academy of Athens, Athens, Greece

ABSTRACT An improved understanding of pathogenic pathways in AKI may identify novel therapeutic approaches. Previously, we conducted unbiased liquid chromatography-tandem mass spectrometry–based protein expres- sion profiling of the renal proteome in mice with acute folate nephropathy. Here, analysis of the dataset iden- tified enrichment of pathways involving NFkB in the kidney cortex, and a targeted data mining approach identified components of the noncanonical NFkB pathway, including the upstream kinase mitogen-activated protein kinase kinase kinase 14 (MAP3K14), the NFkB DNA binding heterodimer RelB/NFkB2, and proteins involved in NFkB2 p100 ubiquitination and proteasomal processing to p52, as upregulated. Immunohistochem- istry localized MAP3K14 expression to tubular cells in acute folate nephropathy and human AKI. In vivo,kidney expression levels of NFkB2 p100 and p52 increased rapidly after folic acid injection, as did DNA binding of RelB and NFkB2, detected in nuclei isolated from the kidneys. Compared with wild-type mice, MAP3K14 activity– deficient aly/aly (MAP3K14aly/aly) mice had less kidney dysfunction, inflammation, and apoptosis in acute folate nephropathy and less kidney dysfunction and a lower mortality rate in cisplatin-induced AKI. The exchange of bone marrow between wild-type and MAP3K14aly/aly mice did not affect the survival rate of either group after folic acid injection. In cultured tubular cells, MAP3K14 small interfering RNA targeting decreased inflammation and cell death. Additionally, cell culture and in vivo studies identified the chemokines MCP-1, RANTES, and CXCL10 as MAP3K14 targets in tubular cells. In conclusion, MAP3K14 promotes kidney injury through pro- motion of inflammation and cell death and is a promising novel therapeutic target.

J Am Soc Nephrol 28: 823–836, 2017. doi: 10.1681/ASN.2015080898

The incidence of AKI is increasing.1 However, there is of an array of inflammatory molecules. The combina- currently no therapy that reliably prevents the progres- tion of unbiased protein expression profiling with sion to AKI or accelerates recovery of renal function.2,3

Thus, reliable biomarkers and novel therapeutic ap- Received August 15, 2015. Accepted July 28, 2016. proaches are needed.4 AKI is characterized by kidney A.O. and M.D.S.-N. contributed equally to this work. inflammation and tubular cell death, dedifferentiation, and subsequent proliferation.5–9 However, given the Published online ahead of print. Publication date available at www.jasn.org. complexity and redundancies of the process, it is un- likely that targeting a single inflammatory molecule Correspondence: Dr. Maria Dolores Sanchez-Niño, Fundación Jiménez Díaz, Avda Reyes, Católicos 2, 28040 Madrid, España, or Dr. Alberto provides the kind of benefit that will be clinically rele- Ortiz, Unidad de Diálisis, Fundación Jiménez Díaz, Avda Reyes Católicos vant. Thus, attention has focused on upstream signaling 2, 28040 Madrid, España. Email: [email protected] or [email protected] pathways that may regulate the coordinated expression Copyright © 2017 by the American Society of Nephrology

J Am Soc Nephrol 28: 823–836, 2017 ISSN : 1046-6673/2803-823 823 BASIC RESEARCH www.jasn.org focused data mining is a powerful tool to expand our knowl- focused data analysis was conducted in 24-hour kidney cortex edge of relevant pathways and key factors in disease. Liquid control and folate nephropathy samples.15 LC-MS/MS identified chromatography-tandem massspectrometry (LC-MS/MS) iden- 41,235 peptides in the kidney cortex corresponding to 6516 tified approximately 2000 proteins in murine renal cortex.10 unique, nonredundant proteins (Supplemental Figure 1).15 However, its applications to the study of AKI has been limited This study represents a new complimentary analysis of this pre- and mainly concentrated in the analysis of biofluids such as urine viously generated dataset. KEGG pathway analysis identified the or in the study of the metabolome rather than the proteome.11–14 enrichment of several pathways on the basis of the upregulation To identify novel pathways and mediator networks active in AKI of key proteins in folate nephropathy samples (Table 1). NFkB in a comprehensive manner, we used tissue LC-MS/MS to assess was at the crossroads of several of these pathways. NFkB activa- changes in the renal proteome of experimental acute folate ne- tion is regulated by MAPK, requires ubiquitination and protea- phropathy. Bioinformatics analysis of 41,235 peptides in cortical somal processing or degradation, and regulates apoptosis and kidney tissue by LC-MS/MS proteomics allowed the identifica- chemokine secretion. Canonically activated NFkB signaling has tion of 6516 unique proteins, of which 1480 were differentially long been implicated in kidney injury.16 However, there is much expressed in samples from experimental nephrotoxic AKI as less information on noncanonical NFkB activation and its com- compared with controls.15 On this previously reported raw data- ponents. A targeted data mining approach searched for compo- set, we have now performed novel pathway analysis in search of nents of the noncanonical NFkB pathway. A KEGG-generated cell death or inflammatory pathways that are activated in acute NFkB signaling pathway map (Supplemental Figure 2) summa- folate nephropathy. This analysis indicated enrichment of pro- rizes the expression of noncanonical NFkB signaling pathway teins from the noncanonical activation pathway for transcription components and NFkB2 (p100/p52) ubiquitination and protea- factor NFkB. NFkBpromotesinflammation by modulating gene somal activation. Upregulation was observed for MAP3K14, the transcription.16,17 Canonical NFkB activation is rapidly initiated essential upstream kinase activating the noncanonical NFkB through degradation of IkB proteins by the proteasome, thus pathway,21,22 for proteins required for NFkB2 p100 ubiquitina- releasing complexes that translocate to the nucleus to promote tion and proteasomal processing to active NFkB2 p52, such as transcription of proinflammatory genes and downregulate UBE2M/UBC12 (E2) and CULLIN-1 (E3), and for the two main the expression of anti-inflammatory molecules such as components of noncanonical NFkB DNA-binding heterodimers, Klotho.18,19 By contrast, noncanonical NFkB activation is a NFkB2 and RELB (Table 2). delayed response that is engaged by a limited number of stim- uli and involves activation of the mitogen-activated protein Validation of Noncanonical NFkB Pathway Activation kinase kinase kinase 14 (MAP3K14)/NFkB-inducing kinase, in Acute Folate Nephropathy proteasomal processing of NFkB p100 to p52, and nuclear The proteomics findings of increased MAP3K14, RELB, and translocation of p52/RelB complexes.16,20 The role and regu- NFkB2 p100/p52 were validated by Western blot and immu- lation of MAP3K14 in AKI is poorly understood. nohistochemistry and the mechanisms by which the system is The combined proteomic and bioinformatics approach upregulated were explored by assessing mRNA expression. enabled identification of evidencefor MAP3K14 activation and Kidney Map3k14, RelB,andNFkB2 mRNA expression was the upregulation of several proteins of the noncanonical NFkB activation pathway in acute folate nephropathy that were con- firmed by Western blot and immunohistochemistry. Func- Table 1. Signaling pathways modulated in folate tional studies identified chemokine expression and cell death nephropathy–associated AKI samples and identified by and proliferation as novel MAP3K14-regulated processes in pathway analysis using KEGG database searching tubular cells. Furthermore, MAP3K14 was overexpressed KEGG Map Number of Hits Name during human AKI and genetically modified mice confirmed mmu04151 46 PI3K-Akt signaling pathway the key role of MAP3K14 in acute folate nephropathy and mmu04010 29 MAPK signaling pathwaya cisplatin-induced AKI. mmu04910 27 Insulin signaling pathway mmu04020 22 Calcium signaling pathway mmu04120 21 Ubiquitin mediated proteolysisa a RESULTS mmu04062 19 Chemokine signaling pathway mmu04310 17 Wnt signaling pathway Kidney Tissue Proteomics Bioinformatics Analysis mmu04630 16 Jak-STAT signaling pathway mmu04660 15 T cell receptor signaling pathway Identifies Upregulation of MAP3K14 and Noncanonical k – mmu03320 13 PPAR signaling pathway NF B Components in Folate Nephropathy Associated mmu04064 13 NFkB signaling pathwaya AKI mmu04370 11 VEGF signaling pathway Acute folate nephropathy is characterized by increased serum mmu04912 11 GnRH signaling pathway creatinine (0.5360.25 versus 0.1060.02 mg/dl at 24 hours; mmu04210 11 Apoptosisa P,0.05), tubular cell death, and interstitial inflammation.15 mmu04722 10 Neurotrophin signaling pathway As previously described, unbiased proteomics combined with aNFkB activation is at the crossroads of these pathways.

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Table 2. Noncanonical NFkB signaling pathway and ubiquitination and Figure 4), MCP-1 (Figure 3E, Supplemen- proteasomal degradation proteins significantly modulated in acute folate tal Figure 4), RANTES (Figure 3F, Supple- nephropathy mental Figure 4), CXCL10 (Figure 3G, Name Gene Fold Change P Value Supplemental Figure 4), and Ccl21a Noncanonical NFkB pathway mRNA expression (Figure 3H, Supplemen- Nuclear factor NFkB p100 subunit Nfkb2 1.46 0.03 talFigure4)werelowerthanincontrol Mitogen-activated protein kinase Map3k14 1000 0.05 MAP3K14+/+ mice or MAP3K14+/aly kinase kinase 14 (NIK) heterozygote mice with folate nephropathy. Transcription factor RelB Relb 3.33 0.01 Finally, we tested a different model, Ubiquitination system cisplatin-induced AKI. An initial study at 72 Cullin-1 Cul1 1000 0.02 hours resulted in 5/5 (100%) mortality in NEDD8-conjugating enzyme Ubc12 Ube2m 3.01 0.01 wild-type (WT) mice, whereas mortality in Data represent focused data mining after nonbiased analysis of the significant dataset. NIK, NF-k-B- fi inducing kinase. MAP3K14 de cient mice was 0/5 (0%). Next we studied the outcome at 48 hours. Mortality was 1/7 (14%) in WT mice and 0/5 (0%) in MAP3K14 deficient mice. Fur- increased in folate nephropathy, suggesting transcriptional thermore, renal function was preserved in MAP3K14 deficient upregulation (Figures 1A and 2, A and C). Western blot con- mice as compared with WT mice (Figure 4), suggesting that firmed increased kidney MAP3K14, RELB, NFkB2 p100, and mortality was due to severe AKI. NFkB2 p52 in folate nephropathy (Figures 1B and 2, B, D, and Immunohistochemistry confirmed the lack of RELB (Fig- E). Immunohistochemistry localized the increased expression ure 5A) and NFkB2 P52 expression in MAP3K14 deficient of these proteins to tubular cells (Figure 1C). In addition, mice with folate nephropathy (Figure 5B), and disclosed de- NFkB2 p52 and RELB DNA-binding activity was increased creased F4/80 macrophages and CD3 T lymphocytes (Figure 6, in nuclear extracts from folate nephropathy kidneys (Figure A and B) and deoxynucleotidyl-transferase–mediated dUTP 2F). Thus, evidence for increased activation of MAP3K14 in- nick-end labeling (TUNEL) positive tubular cells representing cludes processing of NFkB2 p100 to NFkB2 p52 and nuclear dying cells (Figure 6C) in MAP3K14 deficient mice compared translocation and increased DNA binding activity of the RelB/ to heterozygous controls with folate nephropathy. NFkB2 p52 transcription factor. The expression of the ubiq- We next induced folate nephropathy in bone marrow uitination pathway CULLIN-1 protein was also confirmed to chimeras to test whether MAP3K14 deficiency in kidney cells be increased in folate nephropathy (Supplemental Figure 3). or in bone marrow–derived cells was responsible for nephro- Given the poor understanding of its role in kidney injury protection. Mice on a MAP3K14aly/aly background were protec- and its upstream situation in the pathway, we further explored ted from folate nephropathy–induced death when compared the role of MAP3K14 in folate nephropathy–associated AKI. In with MAP3K14 +/+ mice and this was independent of the this regard, extensive MAP3K14 immunostaining was also bone marrow genotype (Supplemental Figure 5). observed in kidney tubules in human AKI (Figure 1D). MAP3K14 Function in Tubular Cells MAP3K14 Deficient Mice Were Protected from Folate After the findings of MAP3K14 upregulation and of evidence Nephropathy and Cisplatin-Induced AKI for MAP3K14 activation (NFkB2 P100 processing to P52) in To explore the role of MAP3K14 in folate nephropathy, tubular cells in vivo, and a beneficial effect of MAP3K14 de- we used MAP3K14 activity–deficient alymphoplasia (MAP3- ficiency in vivo, the function of MAP3K14 was explored in K14aly/aly) mice, which carry a point mutation causing an cultured murine tubular epithelial cells by small interfering amino acid substitution in the carboxy-terminal interaction RNA (siRNA) targeting (Figure 7, A and B). Because KEGG domain of MAP3K14.23,24 MAP3K14+/aly heterozygote mice pathway analysis had identified chemokine signaling and ap- and MAP3K14+/+ mice were used as controls. optosis as enriched pathways and MAP3K14 deficiency indeed MAP3K14+/+ or MAP3K14+/aly heterozygote mice devel- resulted in lower inflammation and tubular cell death in vivo, oped folate nephropathy characterized by increased serum we explored the potential regulation of chemokine secretion creatinine and urea levels (Figure 3, A and B, Supplemental and cell death by MAP3K14 in tubular cells. For this we took Figure 4) and increased kidney NFkB2 activation (Figure 3, C advantage of TWEAK, the only cytokine characterized to date and D, Supplemental Figure 4), expression of chemokines to activate the noncanonical NFkB pathway in tubular cells.25 (Figure 3, E–G, Supplemental Figure 4), and interstitial mac- In order to assess for further functions of MAP3K14 we ex- rophages. plored canonical NFkB targets, including CXCL10, whose ex- MAP3K14 deficient mice were protected from folate pression was recently related to MAP3K14 polymorphisms nephropathy. Serum creatinine and urea (Figure 3, A and B, in human lymphoblastoid cells, but that had not previously Supplemental Figure 4) and kidney expression of NFkB2 been linked to MAP3K14 by functional studies.26 MAP3K14 p100/52 protein and mRNA (Figure 3, C and D, Supplemental silencing by specific siRNAs prevented TWEAK-induced

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Figure 1. Increased kidney mRNA and protein expression of MAP3K14 in acute folate nephropathy and human AKI. Kidney mRNA levels were assessed by quantitative RT-PCR and protein levels by Western blot. (A) MAP3K14 mRNA, *p,0.01 versus vehicle. (B) MAP3K14 protein, *p,0.005 versus vehicle. (C) MAP3K14 immunohistochemistry. Increased MAP3K14 expression was localized to tubular cells in nephropathy samples from WT mice at 24 hours. Original magnification 340; n=6 animals per group. (D) MAP3K14 expression in human kidney tissue. Immunohistochemistry was performed in human control and AKI tissue. Increased tubular cell immunostaining for MAP3K14 was observed in AKI. Original magnification 320; detail 3100. upregulation of Cxcl10 mRNA (Figure 7C) and protein (Figure apoptosis in tubular cells cultured in the absence of survival 7D) as well as of canonical NFkB targets such as Mcp1 and factors (Figure 8). Rantes27 (Figure 7, E and F, Supplemental Figure 6). Differ- ences in MCP-1 expression, which peaks earlier than RANTES, were more evident at earlier time points (Supplemental Figure DISCUSSION 6). In this regard some genes are targeted by both canonical and noncanonical NFkB, whereas others such as CCL21 are specif- For the first time we have uncovered evidence that MAP3K14 ically targeted by noncanonical NFkB activation in tubular and is a therapeutic target in kidney injury. A nonbiased proteomics extrarenal cells.25,28,29 characterization of acute folate nephropathy kidneys disclosed Deprivation of the survival factors from serum is a classic enrichment for components of the noncanonical NFkBacti- inducer of apoptosis.30 MAP3K14 targeting decreased vation, chemokine, and apoptosis pathways. Further studies

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Figure 2. Increased kidney RelB and NFkB2 expression and evidence for noncanonical NFkB activation in acute folate nephropathy. Kidney mRNA levels (A and C) were assessed by quantitative RT-PCR and protein levels by Western blot (B and D). (A) RelB mRNA, *p,0.01 versus vehicle. (B) RelB protein, *p,0.03 versus vehicle. (C) NFkB2 mRNA, *p,0.01 versus vehicle. (D) NFkB2 p100 and p52 proteins, representative Western blot. (E) NFkB2 p100 and p52 protein quantification, *p,0.03 and **p,0.05 versus vehicle. NFkB2 p100 is processed to NFkB p52 by the proteasome. (F) Increased nuclear DNA-binding activity of NFkB2 p52 and RelB in folate nephropathy. A DNA-binding ELISA was used to quantify DNA-binding activity of NFkB2 p52 and RelB in nuclei obtained from kidneys 24 hours after induction of folate nephropathy or vehicle administration. *p,0.01 versus vehicle; n=6 animals per group. evidenced activation of the apical kinase of the noncanonical NFkB is a family of structurally homologous proteins, in- NFkB pathway, and showed that in kidney tubular cells cluding NFkB1, NFkB2,RELA,RELB,andC-REL,which MAP3K14 regulates the expression of chemokines not previ- form homo- or hetero-dimers that bind to kB enhancers in ously associated with noncanonical NFkB, such as CXCL10, DNA to promote or inhibit gene transcription.16 Two main and cell survival. In vivo MAP3K14 targeting protected from pathways for NFkB activation are known. Canonical NFkB folate nephropathy and cisplatin-induced AKI, improving renal activation is usually a rapid, protein synthesis–independent function and decreasing inflammation and tubular cell death. and transient response to a wide range of stimuli that involves

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Figure 3. MAP3K14 deficient mice were protected from folate nephropathy–associated AKI. (A) Serum creatinine, *p,0.02 versus heterozygous mice. (B) Serum urea, *p,0.001 versus heterozygous mice. (C) NFkB2 p100 and p52 proteins (representative Western blot). (D) NFkB2 mRNA, *p,0.01 versus heterozygous mice with folate nephropathy. (E) Decreased whole kidney MCP-1, (F) RANTES, and (G) CXCL10 mRNA expression in MAP3K14 deficient mice with folate nephropathy compared with heterozygous mice. *p,0.02 versus heterozygous mice with folate nephropathy. (H) CCL21a mRNA expression. Mean6SD of six mice per group at the 72-hour time- point; *p,0.03 versus heterozygous mice with folate nephropathy. proteasomal degradation of cytosolic IkB inhibitory proteins pathway, only a limited number of stimuli are known to acti- leading to the release of RELA/P50 and other dimers that then vate the noncanonical NFkB pathway. These include advanced migrate to the nucleus. NFkB-driven IkBa resynthesis con- glycosylation end products33 and TNF receptor superfamily tributes to a fast turn-off of the response. By contrast, non- members such as lymphotoxin-b receptor, B-cell activating canonical NFkB activation requires MAP3K14 activation and factor, CD40, receptor activator for NFkB, CD27, and the NFkB2 P100 processing to P52 by the proteasome, resulting TWEAK receptor Fn14.20,25,35 None of these receptors or their in a delayed nuclear translocation of RELB/P52 heterodimers ligands was identified in the proteomic analysis of kidney cor- and in prolonged activation of NFkB target genes.31–33 In- tex. However, some of them had been previously shown to creased transcription of NFkB2 and RELB may contribute to contribute to AKI. A literature search revealed a role in AKI persistence of the response.34 In contrast to the canonical for CD27 and TWEAK/Fn14.36–38 CD27 was localized to

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are low in quiescent cells as a result of rapid degradation. Cytokines and oxidative stress may increase MAP3K14 protein stability, leading to MAP3K14 activation.20 In addition to this universal mechanism of MAP3K14 regulation, we found in- creased steady-state Map3k14 mRNA levels to be an additional regulatory mechanism of MAP3K14 expression in tubular ep- ithelium that also takes place in vivo during AKI. MAP3K14 induces IkB kinase-a–mediated phosphorylation of NFkB2 p100, a prerequisite for p100 ubiquitination and subsequent proteasomal processing to active NFkB2 p52.32 Ubiquitination is required for targeting of specific proteins to the proteasome. F-box proteins provide specificity for Figure 4. MAP3K14 deficient mice were protected from exper- substrate recognition in the S-phase kinase associated protein imental cisplatin-induced AKI. (A) Serum creatinine. (B) Serum 1-cullin 1-F-box protein (SCF) family of the Cullin-RING +/+ urea. *p,0.02, **p,0.04 versus MAP3K14 cisplatin-induced ligases E3 ubiquitin ligase superfamily.43 The F-box protein 6 – AKI mice. Mean SD of 5 6 mice per group at the 48-hour time- b-transducin repeat containing (b-TrCP; FBW1A) provides point. substrate specificity for MAP3K14-phosphorylated p100, al- lowing ubiquitination by SCFb-TrCP.44–47 Efficient NFkB2 p100 ubiquitination requires UBA3 (ube1c) and UBE2M sloughed cells in tubular lumens postischemia and CD27- (UBC12), UBE2D3 (UBCH5c), and intact CULLIN-1 in SCF deficient mice were protected from AKI and tubular cell apo- b-TrCP.45 Interestingly, the UBE2M E2 ubiquitin conjugating ptosis.36,37 In contrast to this single report, multiple studies enzyme and CULLIN-1 of the SCFb-TrCP E3 ubiquitin ligase from several institutions have provided evidence for a role of were found to be upregulated in folate nephropathy. TWEAK/FN14 in kidney injury.39 Furthermore, TWEAK tar- Evidence for MAP3K14 activation in vivo in folate ne- geting decreased the expression of the noncanonical NFkB phropathy included increased MAP3K14 levels, NFkB p100 target CCL21 in tubular cells.25 Thus for cell culture studies processing to NFkB p52, increased nuclear localization and we chose TWEAK as a pathophysiologically relevant activator DNA binding activity of P52/RELB, and decreased kidney in- of the noncanonical NFkB pathway. flammation and cell death and preserved renal function in There is evidence for a role of canonical activation of NFkB MAP3K14 activity–deficient mice. Protection from folate ne- in kidney injury.16 However, no therapeutic approach specif- phropathy may depend on systemic MAP3K14 deficiency ically targeting NFkB systemically is undergoing clinical trials, (e.g., leukocyte MAP3K14 deficiency) and/or kidney suggesting a fundamental lack of understanding of the system. MAP3K14 deficiency. Bone marrow transplantation experi- In this regard, the role of MAP3K14 and noncanonical NFkB ments results suggest that MAP3K14 deficiency in marrow- activation in AKI has not been well characterized. There is very derived cells is not required for nephroprotection, and are little and scattered information on activation of this pathway consistent with, but do not confirm, the hypothesis that renal in kidney disease. MAP3K14 was phosphorylated in tubular cell MAP3K14 targeting is important for nephroprotection. cells during kidney ischemia-reperfusion40 and levels were in- Alternatively, MAP3K14 targeting in additional, nonrenal, creased in experimental diabetic nephropathy and human nonmarrow-derived cells may play a role. The fact that non- delayed graft function.40,41 TWEAK-dependent nuclear trans- renal cells also express MAP3K14 may result in undesired location of RELB and p52 was observed in tubular cells in side effects when targeting MAP3K14 with small molecules. nephrotoxic AKI.25 In this report, kidney tissue proteomics MAP3K14 and noncanonical NFkBgenetargetshavebeen identified upregulation of several proteins in the noncanonical characterized in the immune system, but there is little informa- NFkB pathway, upregulation of these proteins was confirmed tion on kidney cells.16 We now provide evidence of a role of and localized to tubular cells, the contribution of transcrip- MAP3K14 in the regulation of the inflammatory and cell tional regulation was identified, and the role of MAP3K14 in death/proliferation responses in cultured tubular cells that to- tubular cell injury was characterized. In this regard, MAP3K14 gether with the upregulation of MAP3K14 in tubular cells in activity–deficient mice were protected from folate nephropa- folate nephropathy suggest at least a partial contribution of thy and cisplatin-induced AKI. Thus, MAP3K14 represents a MAP3K14 targeting in tubular cells to the therapeutic responses. key regulated step promoting AKI that may potentially be sub- The P52/RELB heterodimers characteristic of MAP3K14- ject to therapeutic manipulation, although at present there are initiated noncanonical NFkB activation share a number of no satisfactory MAP3K14 inhibitors.42 gene targets with RELA-containing, classically activated MAP3K14 is the essential upstream serine/threonine kinase NFkB complexes.16,29 Because canonical NFkB activation is of the noncanonical NFkB pathway that binds to TRAF2 and an early transient response peaking at around 1–3hours, participates in NFkB signaling in response to the TNF super- whereas noncanonical NFkB activation is delayed and peaks family and IL-1 receptors.22 MAP3K14 protein concentrations at around 24 hours, noncanonical NFkB activation may

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Figure 5. MAP3K14 deficient mice are protected from tubular noncanonical NFkB pathway activation in acute folate nephropathy. (A) RelB and (B) p100/52 immunohistochemistry. Nuclear p52 is observed in renal tubules from heterozygous mice with folate nephropathy (arrows), whereas no staining was observed in MAP3K14 deficient mice with folate nephropathy. Immunohistochemistry does not discriminate between NFkB2 p100 and NFkB2 p52. However, Western blot shown in Figure 4C shows the presence of the active NFkB2 p52 protein. Images representative of six animals per group at the 72-hour time-point; original magnification 340; detail 3400; n=6 animals per group. contribute to sustained NFkB-dependent gene expression.16,28,48 is regulated by MAP3K14. CXCL10 (IP-10) had long been asso- In this regard, the CC genotype of the MAP3K14 SNP rs7222094 ciated with kidney injury in animals and humans.51,52 MCP-1 and was recently associated with increased mortality and renal dys- RANTES, which are targets of canonical NFkBactivation,53 were function in septic shock patients.26 CXCL10 was the gene with the also found to be MAP3K14-dependent in tubular cells. CCL21a greatest difference in expression between major and minor was previously shown to be MAP3K14-dependent in this cell MAP3K14 genotypes. The rs7222094 genotype strongly associ- system.27 Thus, a wide spectrum of chemokines, commonly con- ated with decreased CXCL10 levels in lymphoblastoid cell lines sidered as both canonical NFkB targets and noncanonical NFkB and in septic shock patients.26 Urinary CXCL10 is increased in targets, is regulated by MAP3K14 in cultured tubular cells and AKI patients49,50 and, as shown here, in folate nephropathy kidney during folate nephropathy. tissue.Wenowprovideforthefirst time direct functional evi- KEGG pathway analysis also disclosed apoptosis pathways dence that persistent CXCL10 expression in response to TWEAK as overrepresented in the folate nephropathy–associated AKI

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Figure 6. MAP3K14 deficient mice were protected from acute folate nephropathy–induced inflammation and cell death. (A) F4/80 macrophage and (B) CD3 immunohistochemistry. Macrophage infiltration is milder in MAP3K14 deficient mice with folate nephropathy than in heterozygous mice with folate nephropathy. * P,0.001; ** P,0.02; original magnification 320. (C) TUNEL for fragmented DNA characteristic of apoptosis was frequently positive in tubular cells in heterozygous mice with folate nephropathy. The rate of apoptosis was lower in MAP3K14 deficient mice with folate nephropathy. * P,0.03; original magnification 320; mean6SD of six mice per group at the 72-hour time-point. proteome. MAP3K14 had been identified as a cell death regulator CONCISE METHODS in cancer cells. Indeed, Map3k14 siRNA targeting reduced serum deprivation–induced death in tubular cells. These results were con- Animal Model sistent with decreased tubular cell apoptosis in vivo in MAP3K14 Studies were conducted in accord with the National Institutes of activity–deficient mice during folate nephropathy. These results are Health Guide for the Care and Use of Laboratory Animals. Acute also consistent with observations targeting another component of folate nephropathy causes reversible increase in serum creatinine the noncanonical NFkBpathway,RELB.Thus,RelB targeting by and urea, tubular cell death, compensatory tubular cell proliferation, siRNA protected mice against lethal kidney ischemia,54 and in cul- activation of an inflammatory response, and eventual progression to tured proximal tubular cells knockdown of RELB abrogated the mild fibrosis.27,56–58 Indeed, folic acid nephropathy has been reported excess apoptosis induced by TNF in combination with cisplatin.55 in humans.59 C57/BL6 female mice (12- to 14-week-old) from the In conclusion,preclinicalfunctional studiesincellculture and Instituto Investigacion Sanitaria-Fundacion Jimenez Diaz animal fa- in vivo identified MAP3K14 as a promising therapeutic target in cilities received a single intraperitoneal (ip) injection of folic acid kidney injury. In this regard MAP3K14 was upregulated during (Sigma-Aldrich, St. Louis, MO), 250 mg/kg in 0.3 mol/L sodium bi- human kidney injury, suggesting that experimental findings may carbonate or vehicle, and were euthanized 24 hours or 72 hours after be applicable to the clinical settings. This information sets injection (n=6 per group). The kidneys were perfused in situ with the stage for the exploration of the potential of MAP3K14 as a cold saline before removal. One half-kidney from each mouse was therapeutic target in humans. fixedinbufferedformalin,embeddedinparaffin,andusedfor

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Figure 7. Functional characterization of MAP3K14 actions on cultured proximal tubular cells: chemokine expression. (A) MAP3K14 siRNA silencing in cultured murine proximal tubular cells suppressed MAP3K14 protein expression. Representative Western blot. (B) MAP3K14 siRNA silencing in cultured murine proximal tubular cells suppressed MAP3K14 mRNA expression. (C) MAP3K14 siRNA silencing prevents CXCL10 mRNA upregulation induced by a 24-hour stimulation by the noncanonical NFkB activator TWEAK (100 ng/ml). qRT-PCR. *p,0.01 versus control; **p,0.01 versus TWEAK alone. (D) MAP3K14 siRNA silencing prevents the increase in culture supernatants of the CXCL10 chemokine induced by exposure for 24 hours to 100 ng/ml TWEAK (ELISA). *p,0.001 versus control; **p,0.01 versus TWEAK alone. (E) MAP3K14 siRNA silencing prevents MCP1 mRNA upregulation induced by the noncanonical NFkBacti- vatorTWEAK.qRT-PCR.*p,0.001 versus scrambled; **p,0.001 versus TWEAK alone. (F) MAP3K14 siRNA silencing prevents RANTES mRNA upregulation induced by TWEAK. qRT-PCR. *p,0.002 versus scrambled; **p,0.003 versus TWEAK alone. Cells were treated with scramble or MAP3K14 siRNA before addition of 100 ng/ml TWEAK for 24 hours; mean6SD of three independent experiments. immunohistochemistry, and the other half was snap-frozen in liquid solution. The cisplatin dose was based on literature analysis and results nitrogen for RNA and protein studies. The cortex from one kidney of preliminary experiments, showing renal function impairment at obtained 24 hours after folic acid or vehicle injection was carefully day three after cisplatin injection. MAP3K14aly/aly mice (n=10) and separated and snap-frozen for proteomics analysis. MAP3K14+/+ littermate control mice (n=12) were used in these ex- Toassess the roleofMAP3K14 in folatenephropathy, MAP3K14aly/aly periments and sacrificed at 48 and 72 hours. mice deficient in MAP3K14 and MAP3K14+/aly heterozygote or +/+ MAP3K14 littermate control mice received a single ip injection of Generation of Bone Marrow Chimera folic acid (Sigma-Aldrich), 250 mg/kg in 0.3 mol/L sodium bicarbonate Recipient MAP3K14+/+ littermate control mice and MAP3K14aly/aly or vehicle, and were euthanized 72 hours after injection (n=5 per mice at age 6 weeks were g-irradiated with two doses of 5 Gy for group). MAP3K14aly/aly mice deficient in MAP3K14 were from the Cen- ablation of endogenous bone marrow cells. For bone marrow trans- tro Biologia Molecular (Madrid, Spain) animal facilities.23 plantation, bone marrow cells were isolated (donor) by flushing the A different model of AKI was induced by the ip injection of a single femurs and tibias using a 25 g needle with DMEM (Invitrogen, dose of 25 mg/kg cisplatin (Sigma-Aldrich) dissolved in 0.9% saline Carlsbad, CA). After resuspension, bone marrow cells were

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(EMD Millipore, Watford, UK). Eight M urea buffer was added to the protein extract, centrifuged at 14,000 3 g for 15 minutes, and then repeated. The protein extract was then mixed gently for 1 minute with 0.05 M iodoacetamide buffer and incubated for a further 20 minutes before centrifugation. Eight M urea buffer was again added and centri-

fuged (twice). Ammonium bicarbonate buffer (50 mM NH4HCO3,pH 8) was added and centrifuged (twice) before incubating overnight with trypsin. The trypsin homogenate was centrifuged and washed with the ammonium bicarbonate buffer before acidification with 10% formic acid. Sample volumes were adjusted to match final concentration of protein before analysis by LC-MS/MS. Tissue extracts were separated on a Dionex Ultimate 3000 RSLS nano flow system (Dionex, Camberly, UK). A 5 ml sample was loaded in 0.1% formic acid and acetonitrile (98:2) onto a Dionex 100 mm 3 2cm,5mm C18 nano trap column at a flow rate of 5 ml/min. Elution was performed on an Acclaim PepMap C18 nano column 75 mm 3 50 cm, 2 mm, 100 Å with a linear gradient of solvent A, 0.1% formic acid and acetonitrile (98:2), against solvent B, 0.1% formic acid and acetonitrile (20:80), start- ing at 1% B for 5 minutes rising to 30% at 400 minutes then to 50% B at 480 minutes. The sample was ionized in positive ion mode using a Prox- eon nano spray ESI source (Thermo Fisher Scientific, Vernon Hills, IL) andanalyzedinanOrbitrapVelosFTMS(ThermoFinnigan,Bremen, Germany). The MS was operated in a data-dependent mode (top 40) to switch between MS and MS/MS acquisition and parent ions were frag- mented by collision-induced dissociation. Data files were searched against Figure 8. Functional characterization of MAP3K14 actions on the IPI mouse nonredundant database using SEQUEST with enzyme fi fi fi cultured proximal tubular cells: cell death. (A) MAP3K14 siRNA speci ed as trypsin. A xed modi cation of carbamidomethylation was silencing decreases spontaneous apoptosis of serum-deprived set and oxidation of methionine and proline as variable modifications tubular cells. Representative flow cytometry diagrams of cell DNA were selected. Mass error windows of 20 ppm and 0.8 Da were allowed content. Hypodiploid cells consistent with apoptosis are indicated for MS and MS/MS, respectively. In SEQUEST, only peptides that showed by a horizontal bar. (B) Quantification of hypodiploid apoptotic mass deviation of less than 10 ppm were passed, and the peptide data were cells. *p,0.05 versus control; **p,0.03 versus TWEAK/TNFa/ extracted using high peptide confidence and top one peptide rank filters. 6 INFg alone; mean SD of three independent experiments. Statistical P value analysis was performed using the Mann–Whitney U test. centrifuged (300 3 g, 5 minutes, 4 °C). After resuspension with ice-cold Bioinformatics Analysis DMEM, bone marrow cells were filtered through a 35 mm filter. Irra- Protein identification and a significantdatasetof1480entrieswith diated recipient MAP3K14+/+ littermate control and MAP3K14aly/aly P values ,0.05andfoldchangesof.2 have been previously de- mice were injected intravenously with 4 3 106 donor bone marrow scribed.15 This dataset was used for metabolic and signaling pathway cells (in 100 ml per recipient) within 4 hours after the last irradiation analysis using the KEGG web-resource (www.genome.jp/kegg-bin/) dose. Eight weeks after bone marrow transplantation, bone marrow or with PathVisio (www.pathvisio.org). Focused data mining was chimeric mice (four groups of five mice: recipient MAP3K14+/+ with then amplified to all molecules with a P value #0.05. donor MAP3K14+/+, recipient MAP3K14+/+ with donor MAP3K14aly/aly, recipient MAP3K14aly/aly with donor MAP3K14+/+, and recipient Cells and Reagents MAP3K14aly/aly with donor MAP3K14aly/aly) were subjected to folic MCT cells are a cultured line of proximal tubular epithelial cells acid nephropathy and euthanized at 72 hours. harvested originally from the renal cortex of SJL mice and have been extensively characterized.61 MCT cells were cultured in RPMI 1640 Sample Preparation and Mass Spectrometry Analysis (Gibco, Carlsbad, CA), 10% decomplemented FBS, 2 mM gluta- Tissue samples were weighed out and extracted using the filter aided mine, 100 U/ml penicillin, and 100 mg/ml streptomycin, in 5% CO2 at sample preparation method,60 as described previously.15 Briefly, tis- 37°C.61 Recombinant human soluble TWEAK (EMD Millipore, sue samples were homogenized in SDS-lysis buffer (1:10 sample to Billerica, MA) was used at 100 ng/ml. buffer ratio) (0.1 M Tris-HCl pH 7.6 supplemented with 4% SDS and 0.1 M DTT) using an Ultra-Turrax T 25 (IKA, Staufen, Germany), Western Blot Analysis incubated at 95°C for 3 minutes, and clarified by centrifugation at Tissue and cell samples were homogenized in lysis buffer62 then sep- 16,000 3 g for 5 minutes at room temperature. An aliquot of the arated by 10% or 12% SDS-PAGE under reducing conditions and supernatant was taken and placed in a Micron YM-30 filter device transferred to PVDF membranes (EMD Millipore, Bedford, MA),

J Am Soc Nephrol 28: 823–836, 2017 MAP3K14 and AKI 833 BASIC RESEARCH www.jasn.org blocked with 5% skimmed milk in PBS/0.5% v/v Tween 20 for 1 hour, by flow cytometry of DNA content. For assessment of the cell cycle and and washed with PBS/Tween. Primary antibodies were rabbit polyclonal apoptosis, adherent cells were pooled with spontaneously detached cells, anti-p100/52 (1:500; Cell Signaling Technology, Danvers, MA), anti-RelB and stained in 100 mg/ml propidium iodide, 0.05% NP-40, 10 mg/mL (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), anti-MAP3K14 RNAse A in PBS at 4°C for .1 hour. This assay permeabilizes the cells. (1:1000; Cell Signaling Technology), anti-Cyclin D1 (1:1000; Cell Signal- Permeabilization allows entry of propidium iodide into all cells, dead or ing Technology) and anti–cullin-1 (1:500; Santa Cruz Biotechnology). alive. Apoptotic cells are characterized by a lower DNA content (hypodip- Antibodies were diluted in 5% milk PBS/Tween. Blots were washed loid cells) because of nuclear fragmentation. Thus, this assay is not based with PBS/Tween and subsequently incubated with appropriate horserad- on the known ability of propidium iodide to enter dead cells. The per- 30 ish peroxidase–conjugated secondary antibody (1:2000; GE Healthcare, centage of apoptotic cells with decreased DNA content (Ao)wascounted. Waukesha, WI). After washing, the blots were developed with the chemiluminescence method. Blots were then reprobed with mono- ELISA clonal anti-mouse a-tubulin antibody (1:2000; Sigma-Aldrich) and Cells were transfected with MAP3K14 siRNA and stimulated with 100 levels of expression were corrected for minor differences in loading. ng/ml TWEAK. Murine CxCL10 in the supernatants was determined by ELISA (BD Pharmingen, San Diego, CA). Quantitative RT-PCR One mg RNA isolated by Trizol (Invitrogen) was reverse transcribed NFkB DNA-Binding Activity with High Capacity cDNA Archive Kit and real-time PCR was per- RelB and NFkB2 p52 subunits in nuclear extracts from kidney tissue formed on an ABI Prism 7500 PCR system (Applied Biosystems, were assessed by their binding to an oligonucleotide containing the Foster City, CA) using the DeltaDelta Ct method.63 Expression levels NFkB consensus site using TransAM NFkB Family Kit (Active Motif, are given as ratios to GAPDH. Predeveloped primer and probe assays Carlsbad, CA). were from Applied Biosystems, Foster City, CA. Statistics Immunohistochemistry Statistical analysis was performed using SPSS 11.0 statistical software Immunohistochemistry was carried out as previously described on (IBM, White Plains, NY). Results are expressed as mean6SD. Signif- fi 62 paraf n-embedded 5 mm thick tissue sections. Primary antibodies icance at the P,0.05 level was assessed by t test for two groups of data were rabbit polyclonal anti-RelB (1:50; Santa Cruz Biotechnology), anti- and ANOVA for three or more groups. NFkB2 p100/p52 (1:20; Santa Cruz Biotechnology), anti-MAP3K14 (1:100; Cell Signaling Technology, Danvers, MA), rat polyclonal anti-F4/ 80 antigen (1:50; Serotec, Oxford, UK), rabbit monoclonal anti-CD3 (1:100; Dako, Denmark), and anti–Cullin-1 (1:80; Santa Cruz Biotechnol- ACKNOWLEDGMENTS ogy). Sections were counterstained with Carazzi hematoxylin. Negative controls included incubation with a nonspecific immunoglobulin of the Thanks to Beatriz Barrocal, Dr. Daniel Carpio, and M. Eugenia Burgos same isotype as the primary antibody. for their technical help. Apoptosis was assayed by TUNEL (In Situ Cell Death Detection Kit; Grant support: Fondo Europeo de Desarrollo Regional funds and Roche, Basel, Switzerland) according to the manufacturer’sinstructions.63 Fondo de Investigacion en Salud Instituto de Salud Carlos III PI13/ For human kidney immunohistochemistry, control kidney tissue 00047, PI15/00298, CP14/00133, CP12/03262, Red Tematica de In- from nephrectomy specimens (n=4) and AKI tissue (n=7) diagnosed as vestigacion Cooperativa en Salud, Red de Investigacion Renal RD12/ “acute tubular necrosis” was studied. Mean age was 36.4618.6 years, 0021, European Uremic Toxin Work Group, Sociedad Española de four patients were females, and serum creatinine ranged from 1.7 to 10.0 Nefrologia, Comunidad de Madrid (CIFRA S2010/BMD-2378), mg/dl (5.763.5 mg/dl). Immunohistochemistry was performed as de- Fondecyt 1160465. Salary support: FIS Miguel Servet to M.D.S.-N., scribed above by using anti-human MAP3K14 from Abcam. Programa Intensificación Actividad Investigadora (ISCIII/Agencia Laín-Entralgo/CM) to A.O. The research presented in this manu- Transfection with Small Interfering RNA script was supported in part by the FP7 programs “Improvement of Cells were grown in six-well plates (Costar, Cambridge, MA) and transfected tools and portability of MS-based clinical proteomics as applied with a mixture of 20 nmol/ml MAP3K14 siRNA (Santa Cruz Biotechnology), to chronic kidney disease” (Protoclin, PEOPLE-2009-IAPP, GA Opti-MEM I Reduced Serum Medium, and Lipofectamine 2000 (Invitro- 251368), “Clinical and system –omics for the identification of the gen).64 After 18 hours, cells were washed and cultured for 6 hours in com- Molecular Determinants of established Chronic Kidney Disease” plete medium, and serum-depleted for 24 hours before addition of stimulus. (iMODE-CKD, PEOPLE-ITN-GA-2013-608332) and “Systems bi- This time point was selected from a time-course of decreasing MAP3K14 ology towards novel chronic kidney disease diagnosis and treatment” protein expression in response to siRNA. A negative control scrambled (SysKID HEALTH–F2–2009–241544). siRNA provided by the manufacturer did not reduce MAP3K14 protein.

Cell Death and Apoptosis DISCLOSURES Cells were cultured to subconfluence in six-well plates and transfected H.M. is the cofounder and co-owner of Mosaiques Diagnostics (Hannover, withMAP3K14siRNA aspreviouslydescribed.65 Apoptosis was assessed Germany).

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