RNLS Human

What is the basic function of RNLS in human physiology?

Renalase (RNLS) functions as an enzyme with monoamine oxidase activity and is implicated in the degradation of catecholamines. Beyond its enzymatic role, RNLS has been identified as having significant antiapoptotic properties that protect cells from death. In normal physiological conditions, RNLS-mediated signaling protects normal cells exposed to toxic stress from apoptotic death, serving as a survival factor . This function appears to be dysregulated in pathological conditions such as cancer, where RNLS overexpression can promote tumor cell survival.

How is RNLS expression measured in tissue samples?

RNLS expression in tissue samples can be quantitatively measured using automated immunofluorescence (IF) microscopy systems such as AQUA (Automated Quantitative Analysis). This technique allows for precise measurement of protein expression in formalin-fixed, paraffin-embedded specimens organized in tissue microarrays (TMAs) . For fluid samples such as serum or plasma, ELISA methods are commonly employed with commercially available kits that use Sandwich-ELISA principles. These kits typically have a detection range of 7.82-500ng/mL with a sensitivity around 4.69 ng/mL .

Which human tissues express RNLS?

RNLS expression has been documented in various human tissues. Research has shown progressive expression patterns from normal skin to benign nevi to primary malignant melanoma to metastatic melanoma, indicating tissue-specific regulation that changes during disease progression . Additionally, RNLS is expressed in pancreatic β cells, where its deletion has been studied in relation to Type 1 Diabetes Mellitus . The enzyme can be detected in serum, plasma, and various tissue homogenates, suggesting widespread expression throughout the body .

How does RNLS expression correlate with melanoma progression and survival outcomes?

Studies examining approximately 600 histospots for RNLS protein expression using quantitative automated immunofluorescence microscopy have revealed that progression from normal skin to benign nevi to primary malignant melanoma to metastatic melanoma is accompanied by a significant increase in RNLS expression (P = 0.009, P = 0.0003, and P < 0.001, respectively) . This elevated expression has significant prognostic implications. Patients with high RNLS expression (AQUA score > median score 75,764.45) showed reduced melanoma-specific survival compared to those with low expression, with 5-year and 10-year disease-specific survival rates of 55% versus 69% and 39.7% versus 58.5%, respectively (P = 0.008) . Multivariate analysis confirmed RNLS levels as independently predictive of survival in melanoma (P = 0.004; HR = 3.130).

What methodological approaches can be used to study RNLS-mediated signaling in cancer cell survival?

To investigate RNLS-mediated signaling in cancer cell survival, researchers have employed several methodological approaches:

• Cell viability assays: Comparing the effects of recombinant RNLS (rRNLS) versus control proteins (e.g., BSA) on serum-starved melanoma cell lines (A375.S2, MeWo, SkMel5, and SkMel28), with viability assessed using WST-1 assays .

• Live/dead cell counting: Determining both total cell number and percentage of live cells to differentiate between proliferative effects and antiapoptotic effects of RNLS treatment .

• Inhibition studies: Using neutralizing monoclonal antibodies (e.g., m28-RNLS) or peptide antagonists (e.g., RP-220A) to block RNLS signaling and assess downstream effects on cell survival .

• In vivo xenograft models: Injecting human melanoma cells (A375.S2) subcutaneously into athymic nude mice to generate tumors, then treating with either control antibodies or RNLS-neutralizing monoclonal antibodies to assess effects on tumor growth .

How can RNLS be targeted therapeutically in cancer?

Research indicates three potential approaches for targeting RNLS therapeutically in cancer:

• Neutralizing antibodies: Monoclonal antibodies such as m28-RNLS have shown efficacy in blocking RNLS signaling. In xenograft models, treatment with m28-RNLS decreased tumor volume significantly (P < 0.05) compared to control IgG-treated animals. IHC staining with Ki67 revealed a significant decrease in cellular proliferation within the tumors treated with anti-RNLS antibody (13.4 ±3.0 positive cells/high power field versus 35.1 ±2.3 in controls; P = 0.0004) .

• Peptide antagonists: Engineered peptides like RP-220A, designed by decreasing the net charge of the RNLS peptide RP-220 (replacing 3 Lysine/arginine residues with alanine), can antagonize RNLS action by binding to PMCA4b without activating signaling. These peptides have demonstrated dose-dependent cytotoxicity in melanoma cells in culture (P < 0.005) .

• Small molecule inhibitors: While not explicitly detailed in the search results, the identification of RNLS as a FAD-dependent enzyme suggests potential for developing small molecule inhibitors targeting its catalytic site.

What are the key RNLS polymorphisms studied in relation to human diseases?

Two single nucleotide polymorphisms (SNPs) in the RNLS gene have been prominently studied in relation to human diseases, particularly hypertension:

• rs2576178 A > G: Located in the RNLS gene, this polymorphism has been investigated for association with blood pressure maintenance and hypertension risk .

• rs2296545 C > G: This polymorphism results in a non-synonymous Asp to Glu substitution that deletes a flavin adenine dinucleotide (FAD) binding site, potentially leading to impaired enzyme functionality. Given that RNLS is a FAD-dependent enzyme, this polymorphism may directly affect its catalytic activity .

These polymorphisms have been studied in population-based cohorts such as the Cardiovascular Cohort of the "Malmö Diet and Cancer" (MDC-CC) with 5696 participants .

How do age and other demographic factors interact with RNLS polymorphisms in disease risk assessment?

Research has revealed significant interactions between age and RNLS polymorphisms, particularly the rs2296545 C > G variant. When analyzing a population stratified by age quartiles:

• In the youngest quartile (age <52.3 years), the rs2296545 C > G polymorphism showed protective effects against elevated blood pressure, with decreased systolic BP (β ± SE: -1.351 ± 0.617, P = 0.029), diastolic BP (β ± SE: -1.014 ± 0.347, P = 0.004), and reduced hypertension risk (OR: 0.835, 95% CI: 0.717-0.972, P = 0.02) in the additive genetic model .

• Conversely, in the oldest quartile (age >62.6 years), the same polymorphism showed a trend toward higher systolic BP (β ± SE: 2.237 ± 1.156, P = 0.053), higher diastolic BP (β ± SE: 1.470 ± 0.551, P = 0.008), and increased hypertension risk (OR: 1.248, 95% CI: 0.956-1.628, P = 0.10) in the autosomal dominant model .

This age-dependent effect suggests that the influence of RNLS polymorphisms on disease risk may vary across different life stages, highlighting the need for age-stratified analyses in genetic association studies.

What methodological considerations are important when designing genetic association studies for RNLS polymorphisms?

When designing genetic association studies for RNLS polymorphisms, researchers should consider:

• Adequate sample size: Studies should include large population cohorts (such as the MDC-CC with 5696 participants) to ensure sufficient statistical power for detecting genetic associations .

• Long-term follow-up: For assessing the impact on clinical outcomes, extended follow-up periods (e.g., 15 years as in the MDC-CC study) are necessary to capture sufficient incident events .

• Multiple genetic models: Analyses should evaluate different genetic models (additive, autosomal dominant, recessive) as effects may vary between models .

• Age stratification: Given the significant interaction between age and RNLS polymorphisms, stratification by age quartiles or other relevant age groupings is crucial for revealing age-dependent effects .

• Comprehensive phenotyping: Studies should include detailed phenotypic data beyond the primary outcome, including multiple related physiological parameters (e.g., both systolic and diastolic blood pressure when studying hypertension) .

• Adjustment for covariates: Statistical analyses should adjust for relevant covariates (sex, age, BMI, and other cardiovascular risk factors) to isolate the specific genetic effect .

How does RNLS deletion affect pancreatic β cell function and survival in diabetes models?

RNLS deletion has several significant effects on pancreatic β cell function and survival in diabetes models:

• Protection from autoimmune reactions: RNLS deletion using CRISPR/Cas9 in mice weakens the response of polyclonal β-cell-reactive CD8+ T cells and disrupts immune recognition of β cells, preventing autoimmune-mediated cell killing .

• Endoplasmic Reticulum (ER) stress mitigation: RNLS deletion prevents ER stress by increasing the threshold for triggering the unfolded protein response, making ER stress less likely to occur in β cells .

• Enhanced oxidative stress resistance: RNLS mutations in β cells increase their survivability when exposed to oxidative stress, a key factor in β cell deterioration in diabetes .

These protective effects suggest that targeted RNLS deletion could represent a novel therapeutic approach for Type 1 Diabetes Mellitus by preserving β cell mass and function.

What are the technical considerations for implementing CRISPR/Cas9-mediated RNLS deletion in diabetes research?

Implementing CRISPR/Cas9-mediated RNLS deletion in diabetes research requires attention to several technical aspects:

• Guide RNA design: Careful design of guide RNAs targeting the RNLS gene is essential to ensure specificity and minimize off-target effects.

• Delivery methods: Researchers must optimize delivery systems for CRISPR/Cas9 components to effectively target pancreatic β cells, which may involve viral vectors, nanoparticles, or other delivery platforms.

• Verification of deletion: Thorough validation of successful RNLS deletion through techniques such as sequencing, Western blotting, or functional assays is crucial.

• Model systems: Initial studies utilizing mouse models provide valuable insights, but careful consideration must be given to differences between murine and human RNLS when translating findings.

• Safety assessment: Comprehensive evaluation of potential side effects and long-term consequences of RNLS deletion is necessary before clinical application can be considered .

These considerations highlight the complexity of applying genome editing approaches in diabetes research and the need for rigorous experimental design and validation.

What quantitative techniques are available for measuring RNLS protein levels in different sample types?

Several quantitative techniques are available for measuring RNLS protein levels across different sample types:

• ELISA (Enzyme-Linked Immunosorbent Assay): Commercial sandwich ELISA kits are available with detection ranges of 7.82-500ng/mL and sensitivity around 4.69 ng/mL. These are suitable for measuring RNLS in serum, plasma, tissue homogenates, and other biological samples .

• Automated Quantitative Analysis (AQUA): This automated immunofluorescence microscopy system allows precise quantification of protein expression in formalin-fixed, paraffin-embedded tissue samples organized in tissue microarrays. AQUA scores provide standardized measurements of RNLS expression that can be used for comparative analyses across different tissue types and disease stages .

• Immunohistochemistry (IHC): Standard IHC techniques can be used in conjunction with specific markers (such as Ki67 for proliferation) to correlate RNLS expression with other cellular characteristics .

• Western blotting: While not explicitly mentioned in the search results, Western blotting represents another standard technique for quantifying RNLS protein levels in cell and tissue lysates.

How can researchers design experiments to study the interaction between RNLS and stress responses in different cell types?

To study the interaction between RNLS and stress responses in different cell types, researchers can design experiments using the following approaches:

• Serum starvation models: Exposing cells to serum starvation induces stress that can be modulated by adding recombinant RNLS or RNLS inhibitors to evaluate survival effects. Cell viability can be assessed using assays such as WST-1, along with counting total and live cell numbers to distinguish between proliferative and antiapoptotic effects .

• Oxidative stress induction: Cells with or without RNLS deletion/inhibition can be exposed to oxidative stress inducers (e.g., hydrogen peroxide, paraquat) to assess the protective role of RNLS against oxidative damage .

• ER stress models: Chemicals that induce ER stress (e.g., tunicamycin, thapsigargin) can be used to evaluate how RNLS modulates the unfolded protein response. Readouts might include expression of ER stress markers (BiP, CHOP, XBP1 splicing) and cell survival .

• Combined genetic and pharmacological approaches: Using CRISPR/Cas9 to delete RNLS in conjunction with pharmacological RNLS inhibitors or recombinant RNLS administration allows for complementary approaches to validate findings .

• In vivo stress models: Animal models with tissue-specific RNLS deletion can be challenged with relevant stressors (e.g., streptozotocin for β cells, UVB exposure for skin cells) to assess the role of RNLS in stress responses at the organismal level .

What are the considerations for developing new RNLS inhibitors or modulators for research applications?

Development of new RNLS inhibitors or modulators for research applications should consider:

• Target specificity: Given RNLS's role as a FAD-dependent enzyme with monoamine oxidase activity, inhibitors should be designed for specificity to avoid off-target effects on related enzymes .

• Binding site selection: Different approaches can target distinct sites:

  • FAD binding site: The rs2296545 C > G polymorphism affects a FAD binding site, suggesting this region's importance for enzyme function

  • PMCA4b interaction: Peptide antagonists like RP-220A that bind to PMCA4b can disrupt RNLS signaling without affecting enzymatic activity

  • Neutralizing epitopes: Monoclonal antibodies like m28-RNLS target specific epitopes that neutralize RNLS activity

• Modality options: Multiple therapeutic modalities show promise:

  • Monoclonal antibodies demonstrated efficacy in tumor xenograft studies

  • Peptide antagonists showed dose-dependent cytotoxicity in cancer cells

  • Small molecule inhibitors could potentially target the enzymatic active site

• Evaluation metrics: Cell-based assays should assess:

  • Direct binding to RNLS using techniques like surface plasmon resonance

  • Effects on RNLS enzymatic activity

  • Functional outcomes in relevant cellular models (cancer cell survival, β cell protection)

  • In vivo efficacy in disease models

These considerations represent critical factors for developing effective RNLS-targeting tools for both research applications and potential therapeutic development.

Table 2: Impact of RNLS rs2296545 C > G Polymorphism on Blood Pressure by Age Group