TNFRSF12A Human

What is the molecular structure of human TNFRSF12A and how does it function as a receptor?

TNFRSF12A (Fn14) is a 129 amino acid transmembrane receptor that binds to TNFSF12/TWEAK (Tumor necrosis factor ligand superfamily member 12). Structurally, it functions as a weak inducer of apoptosis in certain cell types while promoting angiogenesis and endothelial cell proliferation . The receptor contains an extracellular domain (Glu28-Trp79) that serves as the binding site for TWEAK .

For experimental investigation of receptor function, researchers should employ multiple complementary approaches:

• Recombinant protein studies using TWEAK R/TNFRSF12 Fc Chimera

• Cell proliferation assays with HUVEC cells which demonstrate TWEAK-dependent proliferation

• Western blot analysis, which typically reveals two main bands representing the full-length protein and its extracellular domain

Methodologically, neutralization assays can be performed using anti-human TWEAK R/TNFRSF12 antibodies, with an ND50 typically ranging from 0.3-1.2 μg/mL in the presence of 30 ng/mL recombinant human TWEAK R/TNFRSF12 Fc Chimera and 20 ng/mL recombinant human TWEAK/TNFSF12 .

How does TNFRSF12A interact with downstream signaling partners in the inflammatory cascade?

TNFRSF12A engages with multiple downstream signaling partners, primarily through TNF receptor-associated factors (TRAFs). Upon TWEAK binding, TNFRSF12A recruits TRAF2 and TRAF3, which regulate activation of NF-kappa-B and JNK pathways .

This signaling mechanism can be experimentally studied through:

• Protein interaction assays to examine TRAF recruitment

• Phosphorylation studies of downstream targets

• NF-κB activation assays using Southwestern blotting techniques

• qRT-PCR to measure induction of target genes such as IRF4 and NFKB1

TRAF2 promotes 'Lys-63'-linked ubiquitination of target proteins (BIRC3, RIPK1, TICAM1), while TRAF3 regulates pathways leading to cytokine and interferon production . Researchers should note that TNFRSF12A signaling increases mRNA levels of both IRF4 and NFKB1, amplifying the inflammatory response through transcriptional regulation .

What are the optimal methods for detecting TNFRSF12A expression in human tissue samples?

For comprehensive analysis of TNFRSF12A expression in human tissues, researchers should implement multiple complementary techniques:

• Immunohistochemistry (IHC): Optimal for tissue localization using specific antibodies like Human TWEAK R/TNFRSF12 Antibody (AF1199) . This method has successfully visualized TNFRSF12A expression in renal biopsies from IgAN patients, revealing expression in glomerular crescents with differential staining compared to controls .

• Western blot analysis: For protein quantification, noting that two distinct bands typically appear representing the full-length protein and extracellular domain of this glycosylated protein .

• Real-time RT-PCR: For mRNA expression analysis with demonstrated sensitivity in detecting TNFRSF12A upregulation in disease states .

• ELISA: For quantitative measurement in serum samples, which has been validated in clinical studies comparing 93 septic AKI patients with 42 healthy controls .

• Single-cell transcriptomics: For cell-specific expression analysis, which has identified TNFRSF12A as one of the highest expressed TNFRSFs in podocytes in vivo .

Each method should include appropriate controls and standardization procedures for reliable quantification.

How does TNFRSF12A expression vary across different cell types within the kidney?

TNFRSF12A demonstrates significant cell type-specific expression patterns within the kidney, which can be methodically analyzed using single-cell transcriptomics and immunohistochemistry:

• Podocytes: Single-cell transcriptomic data reveals Fn14 (TNFRSF12A) as one of the highest expressed TNFRSF members in podocytes in vivo . This high expression makes podocytes particularly responsive to TWEAK stimulation.

• Glomerular cells: In membranous nephropathy (MN), glomerular TNFRSF12A gene expression is significantly upregulated (>2-fold increase) compared to healthy controls . This can be detected through glomerular microarray analysis.

• Tubular cells: Display intense TNFRSF12A staining even in minimal change disease specimens . Tubular Fn14 upregulation occurs during abdominal sepsis and contributes to septic AKI pathogenesis .

• Cell-specific responses: When conducting expression analysis, researchers should distinguish between:

  • Glomerular expression patterns (predominant in MN)

  • Tubular expression (pronounced in various kidney diseases)

  • Expression in glomerular crescents (characteristic of IgAN)

These differential expression patterns suggest cell type-specific roles for TNFRSF12A in kidney pathophysiology and should inform experimental design when targeting specific nephron compartments.

How does TNFRSF12A contribute to membranous nephropathy development and progression?

TNFRSF12A plays a critical role in membranous nephropathy (MN) through several mechanisms that can be experimentally investigated:

• Glomerular upregulation: Transcriptomic analysis from Nephroseq demonstrates that glomerular TNFRSF12A gene expression is significantly upregulated (>2-fold) in MN patients (n=21) compared to healthy living donors (n=21) . This finding was independently validated in a second dataset comparing MN (n=9) with minimal change nephrotic syndrome (n=7) .

• PLA2R regulation: Mechanistically, TWEAK increases PLA2R (phospholipase A2 receptor) expression in podocytes both in vivo and in cultured human podocytes . This is particularly significant as PLA2R is the major autoantigen in primary MN.

• Experimental methodology:

  • In cultured human podocytes, TWEAK (100 ng/mL) treatment for 6 hours significantly increases PLA2R mRNA and protein expression

  • This effect can be prevented by pretreatment with tacrolimus (25 ng/mL)

  • Western blot analysis reveals two main bands representing different glycosylated forms of PLA2R

• Transcriptional effects: TWEAK stimulation also increases mRNA levels of IRF4 and NFKB1, amplifying inflammatory responses through transcriptional regulation .

Researchers investigating this pathway should consider the therapeutic potential of interrupting TWEAK-Fn14 signaling, as evidenced by the inhibitory effect of tacrolimus on TWEAK-induced PLA2R expression.

What is the relationship between TNFRSF12A and neutrophil extracellular traps (NETs) in septic acute kidney injury?

The interaction between TNFRSF12A and neutrophil extracellular traps (NETs) in septic acute kidney injury (AKI) represents a critical disease mechanism that can be experimentally investigated through the following approaches:

• Co-occurrence analysis: NETs formation concurs with Fn14 upregulation in multiple experimental models:

  • Murine models of abdominal, endotoxemic, and multidrug-resistant sepsis

  • Serum samples from patients with septic AKI

  • This correlation can be visualized using principal components analysis (PCA)

• Functional relationship: PAD4 (essential for NETs formation) functionally coexpresses with the Fn14 signaling network as demonstrated by bioinformatic analyses . Statistically significant correlations exist between PAD4 and multiple components of Fn14 signaling:

  • TAB2 (P = 7.16e-006)

  • RIPK1 (P = 1.98e-005)

  • MAP3K7 (P = 5.66e-006)

  • TNFRSF1A (P = 0.002)

  • TNF (P = 0.010)

• Combined intervention approach: Experimental evidence shows that:

  • Pharmacological blockade of NETs formation (using sivelestat or Cl-Amidine) synergizes with ITEM-2 (anti-Fn14 mAb) to prolong survival and protect against AKI in sepsis models

  • Similar benefits were observed in genetic models (CMV-Cre; PAD4fl/fl mice)

  • This synergistic effect is macrophage-dependent, as macrophage depletion abrogates the protection

These findings suggest a mechanistic framework where combined targeting of both NETs formation and Fn14 signaling represents a promising therapeutic strategy for septic AKI.

What are the most appropriate experimental models for investigating TNFRSF12A function in kidney disease?

Researchers investigating TNFRSF12A function in kidney disease should consider these validated experimental models:

• In vitro cellular models:

  • Cultured human podocytes: Respond to TWEAK (100 ng/mL) with increased PLA2R expression after 6-hour treatment

  • HUVEC human umbilical vein endothelial cells: Exhibit TWEAK-dependent proliferation that can be inhibited by recombinant TWEAK R/TNFRSF12 Fc Chimera

  • These models allow investigation of cell-specific responses and signaling mechanisms

• In vivo disease models:

  • Cecal Ligation and Puncture (CLP): A model of abdominal sepsis that upregulates tubular Fn14 expression

  • LPS-induced endotoxemia (LIE): Alternative sepsis model for studying Fn14 regulation

  • Multidrug-resistant sepsis models: For testing therapeutic approaches in clinically relevant contexts

• Genetic models:

  • PAD4-deficient mice (CMV-Cre; PAD4fl/fl): For studying NET formation in relation to Fn14 signaling

  • Fn14 knockout mice: For loss-of-function studies

  • TWEAK-deficient mice: Protect against cyclosporin A-induced nephrotoxicity

• Pharmacological intervention models:

  • Anti-Fn14 antibody (ITEM-2) treatment: Provides protection against septic AKI

  • NETs inhibition: Using neutrophil elastase inhibitor (sivelestat) or PAD4 inhibitor (Cl-Amidine)

  • Tacrolimus: Prevents TWEAK-induced PLA2R expression in podocytes

Each model offers distinct advantages for investigating specific aspects of TNFRSF12A biology, from molecular mechanisms to therapeutic applications.

What methodological approaches can be used to study TNFRSF12A-TWEAK interaction inhibition?

Multiple methodological approaches can be employed to study TNFRSF12A-TWEAK interaction inhibition, each providing complementary information:

• Recombinant protein competition assays:

  • Recombinant Human TWEAK R/TNFRSF12 Fc Chimera inhibits TWEAK/TNFSF12-induced proliferation in HUVEC cells in a dose-dependent manner

  • This inhibition can be neutralized by increasing concentrations of anti-human TWEAK R/TNFRSF12 antibody

  • Quantification is typically assessed by determining the ND50 (neutralizing dose), which ranges from 0.3-1.2 μg/mL

• Cell-based functional assays:

  • Proliferation assays using HUVEC cells treated with TWEAK (20 ng/mL)

  • Assessment of downstream signaling (NF-κB activation, IRF4/NFKB1 mRNA expression)

  • PLA2R expression in human podocytes (mRNA via RT-PCR and protein via Western blot)

• Antibody-based inhibition:

  • ITEM-2 (anti-Fn14 monoclonal antibody) for in vivo and in vitro studies

  • Goat Anti-Human TWEAK R/TNFRSF12 Antigen Affinity-purified Polyclonal Antibody for neutralization assays

• Small molecule inhibitors:

  • Tacrolimus (25 ng/mL) prevents TWEAK-induced PLA2R expression in podocytes

  • This suggests a potential mechanism for repurposing existing drugs

• Combination approaches:

  • Combined inhibition of NETs formation and Fn14 signaling shows synergistic effects

  • This can be achieved using ITEM-2 together with either sivelestat or Cl-Amidine

These methodologies provide a comprehensive toolkit for researchers investigating potential therapeutic targeting of the TNFRSF12A-TWEAK interaction.

How do TNFRSF12A expression levels correlate with clinical outcomes in kidney diseases?

TNFRSF12A expression demonstrates significant correlations with clinical outcomes in several kidney diseases, providing important translational insights:

• Membranous Nephropathy (MN):

  • Glomerular TNFRSF12A gene expression is >2-fold higher in MN patients compared to healthy controls

  • This upregulation is specific to MN when compared with minimal change disease

  • The correlation with PLA2R (major autoantigen) suggests a mechanistic link to disease pathogenesis

• Septic Acute Kidney Injury (AKI):

  • Serum levels of Fn14 are significantly elevated in patients with septic AKI (n=93) compared to healthy controls (n=42)

  • Principal component analysis (PCA) reveals distinct expression patterns between patients and controls

  • NETs and Fn14 levels show concurrent elevation, suggesting a coordinated inflammatory response

• IgA Nephropathy (IgAN):

  • Immunohistochemistry shows TWEAK and Fn14 detection in glomerular crescents of IgAN patients

  • This localization contrasts with minimal change disease, where expression is predominantly tubular

• Biomarker potential:

  • The distinct expression patterns in different kidney diseases suggest TNFRSF12A could serve as a diagnostic or prognostic biomarker

  • Combined assessment of NETs and Fn14 might improve disease classification

• Therapeutic implications:

  • Correlation with disease severity suggests TNFRSF12A as a viable therapeutic target

  • Preclinical models show that Fn14 blockade (alone or in combination with NETs inhibition) improves outcomes

These correlations highlight the potential of TNFRSF12A as both a biomarker and therapeutic target across multiple kidney diseases.

What therapeutic approaches targeting TNFRSF12A are being developed for kidney diseases?

Several therapeutic approaches targeting TNFRSF12A are under development for kidney diseases, with varying mechanisms and stages of investigation:

• Monoclonal antibody therapies:

  • ITEM-2: An anti-Fn14 monoclonal antibody that has demonstrated efficacy in preclinical models of septic AKI

  • Mechanism: Pharmacological deactivation of tubular cell-intrinsic Fn14

  • Preclinical evidence: Prolongs survival and protects against septic AKI in murine models

• Combination therapies:

  • Combined NETs and Fn14 blockade: Shows synergistic protective effects in septic AKI

  • Implementation methods:

    • NETs inhibition via neutrophil elastase inhibitor sivelestat (SIVE)

    • NETs inhibition via peptidylarginine deiminase 4 (PAD4) inhibitor Cl-Amidine

    • Combined with ITEM-2 anti-Fn14 mAb

  • Mechanism: Enhances infiltration and survival of efferocytic GAS6+ macrophages

• Recombinant protein approaches:

  • Recombinant Human TWEAK R/TNFRSF12 Fc Chimera: Functions as a decoy receptor

  • Mechanism: Inhibits TWEAK/TNFSF12-induced proliferation in HUVEC cells in a dose-dependent manner

  • Experimental evidence: ND50 typically 0.3-1.2 μg/mL in in vitro neutralization assays

• Small molecule inhibitors:

  • Tacrolimus: Prevents TWEAK-induced PLA2R expression in podocytes

  • Mechanism: Likely interferes with TWEAK-Fn14 signaling cascades

  • Dosage: Effective at 25 ng/mL in vitro

• RNA-based therapeutics:

  • microRNA-19a mimics: Downregulate tubular cell-intrinsic Fn14 expression

  • Mechanism: Post-transcriptional regulation of Fn14 expression

These approaches represent a diverse therapeutic pipeline targeting TNFRSF12A in kidney diseases, with most advanced evidence for monoclonal antibodies and combination therapies in preclinical models.

How does the heterogeneity of TNFRSF12A expression across kidney cell types influence therapeutic targeting strategies?

The heterogeneous expression of TNFRSF12A across kidney cell types presents both challenges and opportunities for therapeutic targeting that should inform research design:

• Cell-specific expression patterns:

  • Podocytes: Single-cell transcriptomics identifies TNFRSF12A as one of the highest expressed TNFRSFs in podocytes in vivo

  • Tubular cells: Show intense TNFRSF12A staining even in minimal change disease

  • Glomerular cells: Significant upregulation in membranous nephropathy (>2-fold)

  • Glomerular crescents: Present in IgA nephropathy but not minimal change disease

• Implications for therapeutic design:

  • Cell-specific targeting approaches may be required to maximize efficacy while minimizing off-target effects

  • The predominant expression in different compartments varies by disease state, suggesting disease-specific targeting strategies

• Methodological considerations:

  • Single-cell RNA sequencing should be employed to map expression across all kidney cell types

  • Cell-specific conditional knockout models can determine compartment-specific contributions to disease

  • Biodistribution studies of therapeutic candidates are essential to ensure appropriate targeting

• Combination approaches:

  • The synergistic effect of combined NETs and Fn14 blockade suggests that targeting multiple pathways may overcome the limitations of heterogeneous expression

  • Strategies that address both the direct effects of TNFRSF12A signaling and secondary inflammatory processes may be most effective

• Research priorities:

  • Development of cell-specific delivery systems for TNFRSF12A-targeting drugs

  • Investigation of differential downstream signaling in different cell types

  • Assessment of cell-specific biomarkers to predict response to TNFRSF12A-targeted therapies

Understanding this heterogeneity is essential for developing precision medicine approaches to TNFRSF12A-targeted therapy in kidney diseases.

What mechanistic interactions exist between TNFRSF12A signaling and epigenetic regulation in kidney disease models?

Emerging evidence suggests complex interactions between TNFRSF12A signaling and epigenetic regulation in kidney disease models that warrant sophisticated research approaches:

• HOXA5-mediated transcriptional regulation:

  • Interrupting NETs formation enhances transcription of tubular cell-intrinsic Fn14 through dismantling the proteasomes-mediated turnover of homeobox protein Hox-A5 (HOXA5)

  • This occurs in a DNA methyltransferase 3a (DNMT3a)-independent manner

  • Methodological approach: Requires chromatin immunoprecipitation (ChIP) assays to detect HOXA5 binding to TNFRSF12A promoter regions

• Proteasome-dependent regulation:

  • The proteasome pathway regulates HOXA5 turnover, which subsequently affects Fn14 transcription

  • Experimental approach: Cycloheximide (CHX) pulse-chase assays can determine protein stability and degradation rates

• DNA methylation independence:

  • The DNMT3a-independence of this regulation suggests alternative epigenetic mechanisms

  • Investigation methods: CRISPR technology can be used to manipulate specific epigenetic modifiers

• Transcriptional effects of TWEAK stimulation:

  • TWEAK increases mRNA levels of IRF4 and NFKB1

  • These transcription factors may further influence epigenetic landscapes

  • Analytical approach: Luciferase reporter assays can measure transcriptional activity

• Research directions:

  • Genome-wide analysis of histone modifications in TWEAK-stimulated kidney cells

  • Investigation of chromatin accessibility changes using ATAC-seq

  • Exploration of non-coding RNA involvement in TNFRSF12A regulation

These complex interactions between signaling and epigenetic regulation represent an advanced frontier in TNFRSF12A research, with potential implications for developing epigenetic-targeted therapeutic approaches for kidney diseases.