AQP2 Antibody

What is the molecular profile of the AQP2 protein recognized by AQP2 antibodies?

AQP2 is a water channel protein with a canonical length of 271 amino acid residues and a molecular mass of 28.8 kDa in humans. It belongs to the MIP/aquaporin (TC 1.A.8) protein family and undergoes post-translational modifications, including N-glycosylation and phosphorylation. AQP2's subcellular localization spans cytoplasmic vesicles, the Golgi apparatus, and the cell membrane. It is primarily expressed in collecting tubules of the kidney medulla. Synonyms for this target include NDI2, WCH-CD, ADH water channel, aquaporin-CD, and AQP-CD. Orthologous forms have been identified in mouse, rat, bovine, chimpanzee, and chicken species .

Which cell types can be identified using AQP2 antibodies?

AQP2 antibodies serve as effective markers for identifying several specialized kidney cell types. These include Cortical Collecting Duct Principal Cells, Inner Medullary Collecting Duct Cells, Connecting Tubule Principal Cells, Collecting Duct Principal Cells, and Outer Medullary Collecting Duct Principal Cells. This cellular specificity makes AQP2 antibodies valuable tools for nephrology research, particularly in studies examining the cellular architecture and functional organization of kidney tissues .

What species reactivity can researchers expect from commercially available AQP2 antibodies?

Most commercially available AQP2 antibodies are designed to recognize epitopes conserved across multiple species. For example, antibodies targeting the C-terminal region (peptide (C)RQSVELHSPQSLPRGSKA, corresponding to amino acid residues 254-271 of rat AQP2) typically demonstrate cross-reactivity with rat, mouse, and human AQP2 proteins. This cross-species reactivity facilitates comparative studies across different animal models and translation to human research applications .

What are the validated applications for AQP2 antibodies in kidney research?

AQP2 antibodies have been extensively validated for:

• Western blot analysis: Detects both glycosylated (35-50 kDa) and non-glycosylated (29 kDa) forms of AQP2 in kidney tissue lysates. Optimal dilutions typically range from 1:200 to 1:1000 depending on the antibody source and tissue preparation method .

• Immunohistochemistry: Effective for both paraffin-embedded sections (typically at 1:100-1:200 dilution) and cryosections. This application reveals the distribution pattern of AQP2 in kidney structures, with intense staining in collecting ducts but not in thin segments of the loop of Henle .

• Immunoelectron microscopy: Enables high-resolution subcellular localization of AQP2, particularly for distinguishing between apical plasma membrane localization versus intracellular vesicular pools .

• Immunoprecipitation: Useful for protein-protein interaction studies involving AQP2 and its regulatory partners .

How should researchers design experiments to study AQP2 trafficking in response to vasopressin?

To effectively study vasopressin-regulated AQP2 trafficking, researchers should consider:

What control measures should be implemented when using phospho-specific AQP2 antibodies?

When using phospho-specific AQP2 antibodies, researchers should implement the following controls:

• Peptide competition assays: Pre-absorption of antibodies with phosphorylated and non-phosphorylated peptides to confirm specificity. The antibody should recognize only the phosphorylated form .

• Comparison with total AQP2 antibodies: Run parallel experiments with antibodies recognizing total AQP2 regardless of phosphorylation status .

• Phosphopeptide standards: Include phosphopeptide standards spanning the relevant region (e.g., amino acids 241-271 of the COOH-terminal tail of AQP2) for quantitative calibration .

• Dephosphorylation controls: Treatment of samples with phosphatases to confirm that signal loss correlates with phosphorylation status .

• Multiple antibody validation: When possible, confirm results using antibodies generated against different epitopes containing the same phosphorylation site .

What are the key phosphorylation sites in AQP2 and their functional significance?

AQP2 contains multiple phosphorylation sites with distinct functional roles:

• Ser256: Located in a PKA consensus site, this is the most well-characterized phosphorylation site. It appears critically involved in vasopressin-induced trafficking of AQP2. Interestingly, baseline levels of Ser256 phosphorylation are constitutively high in rat kidney, and there may not be a significant increase following acute dDAVP stimulation .

• Ser261: Located in the C-terminal tail, but its functional significance is less well established compared to other sites.

• Ser264: Typically exhibits low levels of phosphorylation (below 5% of total AQP2), even after vasopressin stimulation .

• Ser269: Shows the most dramatic response to vasopressin stimulation. Studies suggest Ser269 phosphorylation may be a more consistent indicator of vasopressin action and AQP2 membrane abundance than Ser256 phosphorylation .

How can researchers quantitatively analyze AQP2 phosphorylation with high precision?

For quantitative analysis of AQP2 phosphorylation, researchers should follow these methodological steps:

• Sample preparation:

  • SDS-PAGE on 12% polyacrylamide gels

  • Include phosphopeptide standards covering a range of quantities (the peptide should span amino acids 241-271 of AQP2's C-terminal tail)

  • Optimize standard range to ensure sample signals fall within the standard curve

• Immunoblotting protocol:

  • Prepare identical duplicate immunoblots on the same day

  • Probe one blot with phospho-specific antibody (against Ser256, Ser261, Ser264, or Ser269)

  • Probe the second blot with total AQP2 antibody

  • Do not strip and reprobe membranes as this can affect quantification accuracy

• Quantification approach:

  • Calculate band densities for both glycosylated and non-glycosylated bands

  • Create standard curves using phosphopeptide standards

  • Calculate the percentage of total AQP2 phosphorylated at each specific site

  • Present data as the ratio of phosphorylated to total AQP2

What experimental considerations are important when comparing different phosphorylation sites of AQP2?

When comparing different phosphorylation sites of AQP2, researchers should consider:

• Baseline phosphorylation levels: Ser256 exhibits constitutively high phosphorylation levels, while Ser264 phosphorylation remains below 5% of total AQP2 even after stimulation. Understanding these baselines is crucial for proper experimental design and interpretation .

• Temporal dynamics: Different sites may exhibit different phosphorylation/dephosphorylation kinetics in response to stimuli. Time-course experiments are essential to capture these differences .

• Model selection: Results may vary between in vivo and in vitro models. For instance, patterns of phosphorylation response observed in rat kidney are similar but not identical to those seen in cultured mpkCCD cells .

• Antibody specificity validation: When comparing multiple phosphorylation sites, rigorous validation of antibody specificity for each site is critical to avoid cross-reactivity issues .

• Functional correlation: Correlate phosphorylation data with functional outcomes such as water permeability or membrane localization to establish physiological relevance .

How does AQP2 phosphorylation regulate its subcellular trafficking?

AQP2 trafficking regulation through phosphorylation involves several key mechanisms:

What methodological approaches can distinguish between trafficking defects and expression level changes in AQP2 mutants?

To differentiate between trafficking defects and expression level changes in AQP2 mutants, researchers should implement:

• Combined lysate and plasma membrane fraction analysis: Prepare both whole cell lysates and isolated plasma membrane fractions from expression systems (such as Xenopus oocytes). This allows determination of whether mutant proteins are synthesized but fail to reach the membrane .

• Immunoblot analysis with site-specific antibodies: Use antibodies that can specifically detect wild-type or mutant AQP2. For C-terminal mutations, specialized antibodies may be needed to recognize the altered sequence .

• Functional water permeability assays: Measure osmotic water permeability (Pf) to correlate protein expression with functional capacity. This helps distinguish between properly trafficked but dysfunctional protein versus trafficking-deficient protein .

• Co-expression studies: Compare the effects of expressing wild-type AQP2 alone, mutant AQP2 alone, and co-expression of both to identify potential dominant-negative effects that might suggest trafficking interference rather than simple expression defects .

• Immunofluorescence microscopy: Visualize the subcellular distribution pattern of wild-type versus mutant AQP2 to directly observe trafficking differences .

How can researchers investigate the dominant-negative effects of AQP2 mutants?

To investigate the dominant-negative effects of AQP2 mutants, researchers should consider these methodological approaches:

• Co-injection expression studies: In systems like Xenopus oocytes, researchers can:

  • Inject wild-type AQP2 cRNA alone

  • Inject mutant AQP2 cRNA alone

  • Co-inject both wild-type and mutant cRNAs

  • Measure osmotic water permeability (Pf) in each condition

  A significant reduction in Pf (>50%) upon co-injection, compared to wild-type alone, suggests a dominant-negative effect rather than simple haploinsufficiency .

• Protein interaction analysis:

  • Perform co-immunoprecipitation experiments using antibodies against wild-type or mutant AQP2

  • Analyze samples by SDS-PAGE and immunoblotting

  • Detect the presence of heteromeric complexes between wild-type and mutant proteins

• Structural analysis of tetramers:

  • AQP2 functions as a tetramer in the membrane

  • Analyze the formation and stability of heterotetramers containing both wild-type and mutant subunits

  • Investigate whether mutant subunits disrupt the trafficking of the entire tetramer

How can phospho-specific AQP2 antibodies be used to study drug effects on renal water handling?

Phospho-specific AQP2 antibodies offer powerful tools for studying pharmacological effects on renal water handling through several approaches:

• Drug-induced nephrotoxicity studies:

  • Example: Gentamicin treatment effects on AQP2 expression and phosphorylation can be monitored in different kidney regions (cortex, outer medulla, inner medulla)

  • Immunohistochemical staining with anti-phospho-AQP2 antibodies enables visualization of region-specific changes after drug administration

  • Temporal monitoring allows distinction between early effects (2 days) versus prolonged exposure (8 days)

• V₂-receptor antagonist evaluation:

  • Administration of V₂-receptor antagonists for 30 minutes produces near-complete disappearance of phosphorylated AQP2 from the apical plasma membrane

  • This rapid response can be quantified through immunoelectron microscopy and immunoblotting

  • The approach provides a sensitive readout for assessing drug potency and kinetics of action

• Therapeutic intervention assessment:

  • Comparative analysis of phosphorylation at different sites (Ser256, Ser261, Ser264, Ser269) provides mechanistic insights into how drugs may differentially affect specific phosphorylation pathways

  • Quantitative analysis using standard curves allows precise determination of drug effects on the percentage of AQP2 phosphorylated at each site

What methodological approaches can resolve contradictory findings in AQP2 phosphorylation studies?

To address contradictions in AQP2 phosphorylation research, the following methodological approaches are recommended:

How can researchers distinguish between AQP2 isoforms and potential degradation products in immunoblot analysis?

When analyzing AQP2 by immunoblotting, researchers face challenges in distinguishing between isoforms, glycosylation states, and degradation products. The following methodological approach is recommended:

• Recognition of characteristic band patterns:

  • Non-glycosylated AQP2: Appears as a 29-kDa band

  • Glycosylated AQP2: Appears as 35-50 kDa bands

  • Combined analysis of both forms is important for accurate quantification

• Identification of potential artifacts:

  • A protein band below the main AQP2 bands has been identified as a histone that is recognized by some C-terminal AQP2 antibodies

  • Researchers should be aware of this potential cross-reactivity to avoid misinterpretation

• Mutant protein detection:

  • C-terminal mutations may alter antibody recognition

  • Special antibodies may be required for mutant proteins (e.g., for C-terminal mutations like 721delG, 763-772del, or 812-818del)

  • Mutant AQP2 proteins may show altered molecular weight (e.g., 34 kDa instead of 29 kDa)

• Sample preparation considerations:

  • Heating samples at 70°C for 10 minutes before SDS-PAGE helps maintain AQP2 stability

  • For membrane fractions, samples from 20 oocytes per lane provide optimal detection

  • For lysates, one-fifth of an oocyte equivalent per lane is typically sufficient