NPHS2 Antibody

What is NPHS2/podocin and why is it significant in kidney research?

NPHS2 encodes podocin, a 42 kDa membrane protein located on podocyte foot processes that is essential for the glomerular filtration barrier. Podocin belongs to the stomatin family and is crucial for proper kidney function. Mutations in NPHS2 cause recessive steroid-resistant nephrotic syndrome and are the most common genetic cause of this condition in children older than 1 month . The protein exists in two isoforms with molecular weights of 42 kDa and 35 kDa .

Podocin's importance stems from its critical role in the slit diaphragm complex, where it interacts with other proteins like nephrin. Understanding podocin function and trafficking provides insights into podocyte biology and the pathophysiology of kidney diseases, particularly nephrotic syndromes.

What applications can NPHS2 antibodies be used for in kidney research?

NPHS2 antibodies are versatile tools with multiple validated applications:

These applications enable researchers to quantify podocin expression, examine its subcellular localization, study protein-protein interactions, and assess changes in disease models or patient samples .

What species reactivity can be expected with NPHS2 antibodies?

Based on validation data, NPHS2 antibodies show reactivity across multiple species:

When selecting an antibody, it's essential to verify its reactivity with your specific model system. Available data confirms reactivity in rat kidney tissue, mouse kidney tissue, and human cell lines including 293T cells . Some antibodies have been successfully used in zebrafish embryos, expanding their utility to developmental studies .

How can researchers use NPHS2 antibodies to study podocin trafficking defects in disease models?

Research on NPHS2 variants has revealed distinct subcellular trafficking patterns that can be effectively studied using antibodies. A methodological approach includes:

• Generate appropriate disease models with NPHS2 mutations (kidney organoids, cell lines, or animal models)

• Use immunofluorescence with organelle markers to track podocin localization

• Perform subcellular fractionation with Western blotting to quantify podocin in different compartments

• Apply live-cell imaging with tagged constructs to monitor trafficking dynamics

Studies using kidney organoids with pathogenic NPHS2 variants have demonstrated that while wild-type podocin localizes to the plasma membrane, variant proteins display unique mistrafficking patterns . These trafficking defects can be effectively visualized using immunofluorescence with NPHS2 antibodies combined with markers for cellular compartments such as the endoplasmic reticulum, Golgi apparatus, and plasma membrane .

What approaches help differentiate between normal and pathogenic NPHS2 variants in experimental systems?

Recent studies have established several key approaches to distinguish normal function from pathogenic variants:

• Protein expression analysis: All pathogenic variant lines showed reduced podocin protein levels despite normal transcription, suggesting post-translational effects .

• Subcellular localization patterns: Different mutations result in distinct trafficking patterns that can be visualized with immunofluorescence. Use co-localization with organelle markers to precisely define these patterns .

• Interaction studies: NPHS2 mutations can specifically disrupt podocin-nephrin association. Co-immunoprecipitation studies with NPHS2 antibodies can detect these altered interactions .

• Functional assessments: Measure functional consequences like increased apoptosis, which has been observed in podocytes with NPHS2 mutations even without endoplasmic reticulum stress .

Kidney organoids derived from induced pluripotent stem cells (iPSCs) with engineered or patient-derived NPHS2 mutations provide physiologically relevant models for these analyses .

How can researchers assess the functional impact of specific NPHS2 mutations using antibodies?

To evaluate how specific mutations affect podocin function:

• Compare protein levels: Use Western blotting with NPHS2 antibodies to quantify protein expression between wild-type and mutant samples. Research has shown reduced protein levels in variants like p.G92C, p.P118L, p.R138Q, p.R168H, and p.R291W .

• Evaluate protein-protein interactions: Employ co-immunoprecipitation to assess how mutations impact podocin's interactions with key partners like nephrin. Multiple studies have shown that NPHS2 mutations can specifically disrupt these critical interactions .

• Assess subcellular distribution: Use immunofluorescence to determine if mutations alter podocin's localization pattern. Create quantitative profiles of protein distribution across cellular compartments .

• Correlation with clinical phenotypes: Compare laboratory findings with clinical data from patients with the same mutations. For example, patients with the p.R168H variant have been studied both at the clinical and molecular levels .

What are the optimal conditions for Western blotting with NPHS2 antibodies?

For reliable Western blot results with NPHS2 antibodies, follow these optimized conditions:

These conditions have been validated for rat kidney tissue and human 293T cell lysates, with specific bands detected at approximately 45 kDa, though the calculated molecular weight of podocin is 42 kDa .

What antigen retrieval methods are effective for immunohistochemistry with NPHS2 antibodies?

The choice of antigen retrieval method significantly impacts NPHS2 antibody staining quality:

For paraffin-embedded sections:

• Heat-mediated antigen retrieval in citrate buffer (pH 6.0) for 20 minutes has been validated for both mouse and rat kidney tissues

• Alternative: TE buffer (pH 9.0) has also shown effective results

For frozen sections:

• Minimal or no antigen retrieval is typically required

• Blocking with 10% goat serum is recommended to reduce background

Detection can be accomplished using:

• Biotinylated secondary antibodies with Streptavidin-Biotin-Complex and DAB chromogen for bright-field microscopy

• Fluorescently-labeled secondary antibodies for immunofluorescence applications

Optimize antibody dilution within the recommended range (1:200-1:800) for each specific tissue type and fixation method .

How should researchers validate NPHS2 antibody specificity for their experimental system?

Comprehensive validation ensures reliable results:

• Control samples:

  • Positive controls: Use rat or mouse kidney tissue with known podocin expression

  • Negative controls: Include tissues not expressing podocin or NPHS2 knockout models

  • Secondary-only controls: Essential for assessing non-specific binding

• Specificity tests:

  • Western blot: Confirm single band at expected molecular weight (42-45 kDa)

  • Peptide competition: Pre-incubation with immunizing peptide should abolish signal

  • Multiple antibodies: Use antibodies targeting different epitopes to confirm results

• Cross-validation approaches:

  • Correlate protein detection with mRNA expression

  • Compare results across multiple detection methods (WB, IHC, IF)

  • For new model systems, benchmark against established podocin-expressing samples

• Application-specific considerations:

  • For WB: Include molecular weight markers and positive control lysates

  • For IHC/IF: Include tissue sections with known expression patterns

  • For co-localization: Use established podocyte markers like nephrin for reference

How should researchers interpret variations in podocin molecular weight across experimental systems?

When analyzing podocin by Western blot, researchers may observe molecular weight variations that require careful interpretation:

• Expected vs. observed molecular weight:

  • Calculated molecular weight: 42 kDa based on amino acid sequence

  • Commonly observed: 42-45 kDa in Western blot analyses

  • Two reported isoforms: 42 kDa and 35 kDa

• Potential explanations for variations:

  • Post-translational modifications affecting protein migration

  • Species-specific differences in podocin processing

  • Effects of sample preparation on protein conformation

  • Presence of mutations that alter protein size or processing

• Validation approaches:

  • Compare observed bands with recombinant podocin controls

  • Assess consistency across multiple antibodies targeting different epitopes

  • Consider whether observed variations correlate with functional differences

Understanding these variations is important for accurately interpreting experimental results, particularly when studying disease-associated variants .

What considerations are important when comparing podocin expression across different experimental models?

When comparing podocin expression in different models, control for these variables:

• Model-specific differences:

  • Cell lines: May lack the 3D architecture of glomeruli and supporting cell types

  • Organoids: More physiologically relevant but with batch-to-batch variability

  • Animal models: Consider species differences in kidney physiology

  • Patient samples: Account for genetic and treatment heterogeneity

• Developmental considerations:

  • Podocin expression changes during kidney development

  • Match developmental stages when comparing across models

  • For organoid models, standardize differentiation protocols and timepoints

• Quantification strategies:

  • Western blot: Normalize to multiple housekeeping proteins

  • IHC/IF: Standardize image acquisition parameters and quantification methods

  • Consider both protein abundance and localization pattern

• Statistical approaches:

  • Distinguish between biological and technical replicates

  • Account for batch effects when combining data from multiple experiments

  • Use appropriate tests based on data distribution and experimental design

These considerations enable meaningful comparisons across different model systems, contributing to a more comprehensive understanding of podocin biology .

How can NPHS2 antibodies help elucidate mechanisms of steroid-resistant nephrotic syndrome?

NPHS2 antibodies are valuable tools for understanding the molecular basis of steroid-resistant nephrotic syndrome (SRNS):

• Genetic-phenotype correlations:

  • Different NPHS2 mutations (e.g., p.R138Q, p.R168H) show distinct molecular phenotypes that can be characterized using antibodies

  • Antibodies can help categorize variants based on their effects on protein expression, localization, and interactions

• Mechanistic insights:

  • Study how mutations affect podocin's interaction with nephrin and other slit diaphragm proteins

  • Assess whether mutations lead to protein degradation, mistrafficking, or functional defects at the membrane

  • Investigate downstream signaling pathways affected by podocin dysfunction

• Therapeutic implications:

  • Screen compounds that may rescue variant protein mistrafficking or degradation

  • Evaluate potential therapies targeting specific molecular mechanisms

  • Assess podocin as a biomarker for disease progression or treatment response

• Translational applications:

  • Correlate molecular findings with transplant outcomes (recurrence rates differ based on genetic status)

  • Inform genetic counseling based on molecular mechanisms of specific variants

  • Guide personalized treatment approaches based on underlying molecular defects

Recent research has demonstrated that patients with biallelic NPHS2 variants have distinct transplant outcomes compared to those without identified pathogenic variants, highlighting the clinical relevance of these molecular studies .