ATP6V0A4 Antibody

What is ATP6V0A4 and what is its biological function?

ATP6V0A4 is a subunit of the V0 complex of vacuolar(H+)-ATPase (V-ATPase), a multisubunit enzyme composed of a peripheral complex (V1) that hydrolyzes ATP and a membrane integral complex (V0) that translocates protons. The V-ATPase enzyme is responsible for acidifying and maintaining the pH of intracellular compartments. In some cell types, it is targeted to the plasma membrane, where it is responsible for acidifying the extracellular environment. ATP6V0A4 specifically plays a crucial role in normal vectorial acid transport into the urine by the kidney .

The protein is also known by several synonyms, including:

• V-type proton ATPase 116 kDa subunit a 4

• V-ATPase 116 kDa isoform a 4

• Vacuolar proton translocating ATPase 116 kDa subunit a isoform 4

• Vacuolar proton translocating ATPase 116 kDa subunit a kidney isoform

ATP6V0A4 is tissue-restricted and replaces generic subunits of V-ATPases, with particularly high expression noted in renal α-intercalated cells and the epididymis .

What is the significance of ATP6V0A4 in kidney function and disease?

ATP6V0A4 has significant importance in kidney physiology and pathology. Mutations in the ATP6V0A4 gene are associated with distal renal tubular acidosis (dRTA), a rare genetic disease affecting the secretion of H+ ions in the intercalated cells of the collecting duct .

In certain populations, such as Tunisia, specific founder mutations in ATP6V0A4 have been identified in patients with dRTA without hearing impairment . The involvement of ATP6V0A4 in acid transport makes it critical for maintaining proper kidney function and acid-base balance in the body.

Furthermore, dysregulation of ATP6V0A4 expression has been observed in kidney cancers, particularly clear cell renal cell carcinoma (ccRCC), where it is significantly downregulated compared to adjacent normal kidney tissues .

How is ATP6V0A4 expression altered in renal cell carcinoma?

In contrast to some other cancers, ATP6V0A4 expression is significantly downregulated in clear cell renal cell carcinoma (ccRCC) compared to adjacent normal kidney tissues. Multiple studies have confirmed this finding:

• Analysis of eight GEO datasets (GSE76351, GES6344, GSE15641, GSE16449, GSE47032, GSE66270, GSE53000 and GSE53757) showed significantly lower expression of ATP6V0A4 in ccRCC tissues compared with adjacent normal kidney tissues (all P<0.0001) .

• RT-qPCR validation in 23 pairs of ccRCC tissues and adjacent normal tissues confirmed that ATP6V0A4 expression levels in ccRCC tissues were significantly lower than those in adjacent normal kidney tissues (P=0.0007) .

• Immunohistochemistry further verified the significant downregulation of ATP6V0A4 protein expression levels in ccRCC tissues compared to adjacent normal kidney tissues (P<0.001) .

• In renal cell carcinoma cell lines, ATP6V0A4 was significantly downregulated in 769-P, ACHN, and Caki-2 cell lines compared to HK-2 cells (normal kidney cells) at the transcription level, and was remarkably decreased in all 5 tested RCC cell lines at the translational level .

This pattern distinguishes ATP6V0A4 in kidney cancer from its expression in other cancer types, such as breast cancer and glioma, where it has been reported to be highly expressed in more invasive phenotypes .

What is the prognostic value of ATP6V0A4 expression in ccRCC?

ATP6V0A4 expression levels correlate with patient prognosis in clear cell renal cell carcinoma (ccRCC). Analysis of data from The Cancer Genome Atlas (TCGA) showed that higher expression of ATP6V0A4 is associated with better survival outcomes in ccRCC patients .

Kaplan-Meier survival analysis demonstrated that patients with ccRCC and high ATP6V0A4 expression had a better prognosis compared to those with low expression . This finding was further confirmed by analyzing data from 534 patients with ccRCC obtained from TCGA, which showed that dysregulated expression of ATP6V0A4 in ccRCC was associated with the 5-year survival rate .

Interestingly, ATP6V0A4 expression was not significantly associated with other clinicopathological variables including age, sex, primary tumor (T stage), lymph node involvement (N stage), distant metastasis (M stage), and AJCC stage, as shown in the following data table:

These findings suggest that ATP6V0A4 may serve as an independent prognostic marker in ccRCC .

How does ATP6V0A4 expression differ across various cancer types?

ATP6V0A4 exhibits differential expression patterns across various cancer types, which is important for researchers to consider when investigating its role in specific malignancies:

• In ccRCC: ATP6V0A4 is significantly downregulated compared to normal kidney tissues, and higher expression is associated with better prognosis .

• In breast cancer: Previous studies have reported that ATP6V0A4 is highly expressed in highly invasive breast cancer. Knockdown of ATP6V0A4 has been shown to significantly inhibit cell invasion in breast cancer by decreasing the targeting of V-ATPases to the plasma membrane of MDA-MB-231 cells .

• In glioma: ATP6V0A4 has been reported to be highly expressed in specific subtypes of human gliomas .

• In other cancers: V-ATPases (of which ATP6V0A4 is a subunit) have been found at the plasma membranes of several invasive tumor cells, including melanoma, Ewing sarcoma, and liver, lung, ovarian, esophageal, prostate, and pancreatic cancer .

These contrasting expression patterns suggest that ATP6V0A4 may play context-dependent roles in different cancer types and tissues, potentially reflecting the tissue-specific functions of V-ATPases in normal physiology.

What are the validated applications for ATP6V0A4 antibodies in research?

Based on the available information from commercial antibody suppliers and research literature, ATP6V0A4 antibodies have been validated for several experimental applications:

• Western Blotting (WB): ATP6V0A4 antibodies have been validated for detecting the protein in western blot assays, allowing researchers to measure protein expression levels in cell and tissue lysates .

• Immunohistochemistry (IHC): ATP6V0A4 antibodies can be used for detecting the protein in fixed tissue sections, with recommended dilutions typically in the range of 1:50-1:200 .

• Immunocytochemistry/Immunofluorescence (ICC/IF): Some ATP6V0A4 antibodies have been validated for detecting the subcellular localization of the protein in cultured cells .

• RT-qPCR: While not directly using the antibody, researchers have successfully used RT-qPCR to measure ATP6V0A4 mRNA expression levels in conjunction with protein-level studies using antibodies .

It's important to note that for each specific application, optimization of antibody concentration, incubation conditions, and detection methods may be required based on the sample type and experimental design.

What methodological considerations should be taken when designing experiments using ATP6V0A4 antibodies?

When designing experiments involving ATP6V0A4 antibodies, researchers should consider several methodological aspects:

• Antibody validation: Ensure the antibody has been validated for your specific application and species of interest. For example, some antibodies may be validated for human samples but not for other species .

• Controls:

  • Positive controls: Include tissues known to express ATP6V0A4 (kidney, epididymis) as positive controls .

  • Negative controls: Include appropriate negative controls such as antibody diluent without primary antibody.

  • Knockdown/knockout controls: When possible, include samples where ATP6V0A4 has been knocked down or knocked out to confirm antibody specificity.

• Sample preparation:

  • For Western blot: Proper protein extraction and denaturation conditions should be optimized.

  • For IHC/ICC: Appropriate fixation methods should be used, considering that some epitopes may be sensitive to certain fixatives.

• Detection methods:

  • For Western blot: Consider using loading controls appropriate for your experimental context.

  • For IHC/ICC: Determine whether chromogenic or fluorescent detection is more appropriate for your research question.

• Quantification approaches:

  • For Western blot: Consider using software for densitometric analysis.

  • For IHC: Use appropriate scoring systems (e.g., H-score, Allred score) for semi-quantitative assessment or digital image analysis for quantitative assessment.

• Statistical analysis: Use appropriate statistical methods as demonstrated in published research, such as:

  • Fisher's exact test for comparing expression between tumor and normal tissues

  • Chi-square tests for analyzing associations with clinicopathological variables

  • Kaplan-Meier method with log-rank test for survival analysis

  • Student's t-test or ANOVA with appropriate post-hoc tests for comparing expression levels between groups

How can researchers effectively troubleshoot non-specific binding or weak signals when using ATP6V0A4 antibodies?

When encountering issues with ATP6V0A4 antibody performance, researchers can implement several troubleshooting strategies:

• For non-specific binding:

  • Optimize blocking conditions by testing different blocking agents (BSA, milk, serum) and concentrations.

  • Increase the number and duration of washing steps.

  • Titrate the primary antibody concentration to find the optimal signal-to-noise ratio.

  • Use more stringent washing buffers (increasing salt concentration or adding detergents).

  • Pre-adsorb the antibody with non-specific proteins.

  • Consider using a different detection system that may offer higher specificity.

• For weak signals:

  • Increase protein loading amounts for Western blots.

  • Increase antibody concentration (though this should be balanced against the risk of increased background).

  • Extend primary antibody incubation time (e.g., overnight at 4°C).

  • Use signal enhancement systems compatible with your detection method.

  • For IHC/ICC, optimize antigen retrieval methods (heat-induced or enzymatic).

  • Ensure samples are properly processed to preserve the epitope of interest.

• For validation of results:

  • Use multiple antibodies targeting different epitopes of ATP6V0A4 if available.

  • Correlate protein detection with mRNA expression data as done in published studies .

  • Consider using recombinant ATP6V0A4 as a positive control in Western blot experiments.

How does the role of ATP6V0A4 in cancer differ from other V-ATPase subunits?

ATP6V0A4 represents a tissue-specific subunit of V-ATPases that may have distinct functions compared to other V-ATPase subunits in cancer:

• Tissue specificity: Unlike some more ubiquitously expressed V-ATPase subunits, ATP6V0A4 is tissue-restricted and replaces generic subunits of V-ATPases, with particularly high expression in renal tissues and epididymis . This tissue specificity may contribute to its unique role in kidney cancers.

• Differential regulation: While many V-ATPase subunits are upregulated in various cancers, ATP6V0A4 shows downregulation in ccRCC, contrary to its upregulation in other cancer types like breast cancer and glioma . This suggests context-dependent regulation and function.

• Prognostic implications: Studies have shown that higher expression of ATP6V0A4 is associated with better prognosis in ccRCC , which differs from some other V-ATPase subunits that are often associated with worse outcomes when overexpressed in cancers.

• Potential for targeted therapy: The tissue-specific nature of ATP6V0A4 might offer opportunities for more targeted therapeutic approaches compared to generic V-ATPase inhibitors. Researchers exploring V-ATPase targeting should consider the differential expression of ATP6V0A4 across cancer types when designing experimental approaches.

Understanding these differences is critical when developing research hypotheses about ATP6V0A4's role in cancer progression and when considering it as a potential therapeutic target.

What mechanisms might explain the contradictory expression patterns of ATP6V0A4 across different cancer types?

The contradictory expression patterns of ATP6V0A4 across different cancer types (downregulated in ccRCC but upregulated in breast cancer and glioma) represent an intriguing research question. Several hypotheses might explain these observations:

• Tissue-specific baseline expression: ATP6V0A4 is highly enriched in normal kidney tissues, particularly in α-intercalated cells. As proposed by researchers, "the decreased expression of ATP6V0A4 resulted in the loss of normal tissue structure in renal cancer" . In tissues with naturally lower ATP6V0A4 expression, upregulation might occur during malignant transformation.

• Cell differentiation status: The downregulation in ccRCC might reflect dedifferentiation of specialized kidney cells that normally express high levels of ATP6V0A4. As noted, "Normal renal cells underwent a neoplastic transformation and lost the ability to differentiate and mature to varying degrees, which led to the loss of normal structure in the kidney and replacement by tumor cells" .

• Tumor microenvironment requirements: Different cancer types may have varying requirements for extracellular acidification. In highly invasive cancers like certain breast cancers, upregulation of ATP6V0A4 might facilitate invasion through increased extracellular acidification and activation of proteases .

• Genetic and epigenetic regulation: Different cancer types have distinct genetic and epigenetic landscapes that could differently affect ATP6V0A4 expression. Research into the promoter regulation and epigenetic modifications of ATP6V0A4 across cancer types could provide insights.

• Functional requirements: The role of V-ATPases may differ between cancer types. In some cancers, they may be primarily involved in invasion and metastasis, while in others they might serve different functions in cellular homeostasis.

These hypotheses offer potential research directions for investigating the context-dependent regulation and function of ATP6V0A4 in cancer.

How might targeting V-ATPases in cancer therapy need to account for the differential expression of ATP6V0A4?

The differential expression of ATP6V0A4 across cancer types has important implications for therapeutic strategies targeting V-ATPases:

• Cancer-specific approaches: Given that ATP6V0A4 is downregulated in ccRCC but upregulated in other cancers, therapeutic strategies might need to be tailored to the specific cancer type. For instance:

  • In cancers with ATP6V0A4 overexpression (e.g., breast cancer, glioma), direct inhibition might be beneficial.

  • In ccRCC where ATP6V0A4 is already downregulated and associated with worse prognosis, restoration of expression might be more appropriate than inhibition.

• Potential for specific targeting: The tissue-restricted nature of ATP6V0A4 might allow for more selective targeting compared to generic V-ATPase inhibitors, potentially reducing off-target effects in normal tissues.

• Biomarker potential: ATP6V0A4 expression levels could serve as biomarkers to predict response to V-ATPase-targeting therapies. Research has shown that specific V-ATPase inhibitors such as concanamycin A and bafilomycin have demonstrated anti-tumor effects in various cancer models .

• Combination approaches: Research indicates that "pharmacological inhibition of V-ATPases results in potent antitumor and anti-metastatic effects in vitro and in vivo" . Combination of V-ATPase inhibitors with other therapies might be more effective in certain contexts, particularly where V-ATPase activity contributes to therapy resistance.

• Consideration of functional consequences: Therapeutic targeting should consider the downstream effects of V-ATPase inhibition in different contexts, including effects on:

  • Extracellular acidification

  • Activation of proteases such as cathepsins and matrix metalloproteinases

  • Cell migration and invasion capabilities

  • Tumor microenvironment

Future research should "evaluate the expression patterns of multiple subunits and determine whether the activity of V-ATPases can be regulated by changes in the expression of ATP6V0A4" across different cancer types .

What are the most effective experimental models for studying ATP6V0A4 function in renal physiology and pathology?

For studying ATP6V0A4 function in renal physiology and pathology, researchers should consider multiple experimental models:

• Cell culture models:

  • Renal tubular epithelial cell lines (e.g., HK-2 cells) represent normal kidney cells with endogenous ATP6V0A4 expression .

  • Renal cancer cell lines (e.g., 769-P, ACHN, Caki-2) allow for comparative studies against normal models .

  • Primary cultures of intercalated cells can provide more physiologically relevant models for acid-base transport studies.

• Genetic manipulation approaches:

  • siRNA knockdown of ATP6V0A4 can help elucidate its function in specific cell types.

  • CRISPR/Cas9-mediated knockout or knock-in models can create stable cell lines for long-term studies.

  • Overexpression models in renal cancer cell lines could test hypotheses about the functional consequences of restoring ATP6V0A4 expression.

• Animal models:

  • Knockout or conditional knockout mouse models of ATP6V0A4 can reveal its systemic and tissue-specific roles.

  • Models of renal tubular acidosis could provide insights into the pathophysiological consequences of ATP6V0A4 dysfunction.

  • Xenograft models using manipulated cancer cell lines can assess the impact of ATP6V0A4 expression on tumor growth and metastasis in vivo.

• Patient-derived samples:

  • Paired tumor and normal kidney tissues from ccRCC patients allow for direct translational studies .

  • Analysis of samples from patients with distal renal tubular acidosis due to ATP6V0A4 mutations provides insights into clinical consequences of dysfunction .

• Functional assays:

  • pH measurement assays (intracellular and extracellular) can directly assess the impact of ATP6V0A4 on proton transport.

  • Cell migration and invasion assays can evaluate the role of ATP6V0A4 in cancer cell behavior.

  • Protease activity assays can investigate downstream effects of ATP6V0A4-mediated acidification.

Combining multiple models and approaches provides the most comprehensive understanding of ATP6V0A4 function in both physiological and pathological contexts.

What statistical approaches are most appropriate for analyzing ATP6V0A4 expression data in clinical samples?

Based on published research methodologies, several statistical approaches are appropriate for analyzing ATP6V0A4 expression data in clinical samples:

• For comparing expression between tumor and normal tissues:

  • Paired analyses like paired t-tests for continuous data from matched samples

  • Fisher's exact test for categorical data as used in published studies

  • Wilcoxon signed-rank test for non-parametric paired data

• For correlation with clinicopathological variables:

  • Chi-square test for categorical variables as demonstrated in the literature

  • Fisher's exact test for small sample sizes

  • Mann-Whitney U test or Kruskal-Wallis test for non-parametric comparisons across multiple groups

• For survival analysis:

  • Kaplan-Meier method with log-rank test to compare survival outcomes between high and low expression groups

  • Cox proportional hazards regression for multivariate analysis to assess independent prognostic value

  • Harrell's C-index for evaluating the predictive accuracy of prognostic models

• For gene expression data from microarrays or RNA-seq:

  • Proper normalization methods appropriate to the platform

  • Multiple testing corrections (e.g., Benjamini-Hochberg procedure) to control false discovery rates

  • ANOVA with appropriate post-hoc tests for multiple group comparisons

• For integrated multi-omics analysis:

  • Correlation analyses between mRNA and protein expression levels

  • Pathway enrichment analysis to understand biological context

  • Machine learning approaches for identifying complex patterns

When reporting results, researchers should clearly describe:

• Data distribution characteristics

• Tests for normality

• Specific statistical tests applied

• P-value thresholds used (typically P<0.05 is considered statistically significant)

• Effect sizes in addition to P-values

These approaches help ensure robust and reproducible analysis of ATP6V0A4 expression data in clinical research.