PKHD1 Antibody

What is PKHD1 and why is it important in kidney research?

PKHD1 is a large membrane-associated protein that plays a crucial role in tubule formation and maintenance in kidneys and other organs. In humans, the canonical PKHD1 protein has 4,074 amino acids with a mass of approximately 447 kDa . It contains a single transmembrane domain near its C-terminus and multiple immunoglobulin-like plexin-transcription-factor domains in its extracellular portion .

PKHD1 is critical in kidney research because mutations in the PKHD1 gene cause autosomal recessive polycystic kidney disease (ARPKD), a severe form of polycystic kidney disease affecting infants and children . The protein promotes ciliogenesis in renal epithelial cells and maintains the architecture of kidney tubule lumens . It also influences cellular symmetry by regulating centrosome duplication, mitotic spindle assembly, and oriented cell division during tubular elongation through the planar cell polarity pathway .

Understanding PKHD1's structure, function, and interactions provides insights into the mechanisms of cyst formation in kidney diseases and potential therapeutic targets.

What are the key structural features of PKHD1 protein relevant when selecting antibodies?

When selecting antibodies for PKHD1 research, several critical structural features should be considered:

• Size and Processing:

  • PKHD1 is an extremely large protein (447 kDa) that undergoes post-translational modifications, including glycosylation

  • Standard Western analysis often fails to detect the full-length protein without specialized techniques

• Domains and Epitopes:

  • N-terminal extracellular region with immunoglobulin-like plexin-transcription-factor domains and parallel beta-helix 1 repeats

  • Single transmembrane domain near the C-terminus

  • Short cytoplasmic C-terminal tail (approximately 192 amino acids)

• Isoforms:

  • Multiple alternatively spliced variants exist

  • Some isoforms lack the transmembrane domain and may be secreted

  • A specific isoform of approximately 135 kDa associated with the C-terminus has been identified

• Species Differences:

  • The intracellular C-termini show only 43% identity between humans and mice

  • Antibodies generated against human PKHD1 may have variable cross-reactivity with rodent orthologs

• PKHD1-Like Proteins:

  • PKHD1L1 shares approximately 25% identity with PKHD1

  • In humans, PKHD1L1 is 4,243 amino acids with a mass of 465.7 kDa

When selecting antibodies, targeting different regions allows detection of different aspects of PKHD1 biology. For instance, C-terminal antibodies may detect both full-length protein and truncated isoforms, while N-terminal antibodies might be more specific for the full-length protein .

How does PKHD1 expression vary across tissues and developmental stages?

PKHD1 exhibits distinct spatiotemporal expression patterns:

Developmental Expression:

• First detected in epithelial cells of the neural tube at embryonic day (E) 9.5 in mice

• By E10.5, expression appears in the main bronchi and primordial gut

• At E11.5, expression occurs in the epithelia of the early ureteric bud and mesonephric tubules, primarily at apical surfaces

• By E15.5, expression continues in branching ureteric buds as they differentiate into collecting ducts

• Also expressed in developing adrenal cortex and immature hepatocytes

Adult Tissue Expression:

• Highly expressed in both fetal and adult kidney

• In adult kidneys, PKHD1 is widely expressed in the epithelia of:

  • Collecting ducts

  • Proximal convoluted tubules

  • Thick ascending limbs of Henle's loop

• Expression is predominantly at the apical domains of polarized epithelial cells

• Also expressed in liver, particularly in biliary cells

Subcellular Localization:

• Predominantly localized to basal bodies/primary cilia in renal epithelial cells

• Also found at the apical membrane domain of polarized epithelial cells

• Some expression detected in ciliary shafts, microvilli, and plasma membrane in cultured cells

This expression pattern reflects PKHD1's role in tubulogenesis and maintenance of ductal structures across multiple organs, explaining why mutations affect both kidneys and liver in ARPKD .

What are the recommended applications for different types of PKHD1 antibodies?

PKHD1 antibodies can be utilized across various applications, with specific considerations for each technique:

When selecting antibodies:

• For detection of full-length protein: C-terminal antibodies often perform better

• For subcellular localization: Antibodies successfully used in IF/ICC (like those targeting basal body epitopes)

• For detecting multiple isoforms: Consider using antibodies against different regions

Many commercial PKHD1 antibodies are available with varying applications:

• Polyclonal antibodies: Often offer high sensitivity but may have more background

• Monoclonal antibodies: Provide consistent results across experiments

• Antibodies conjugated to various tags (FITC, HRP, PerCP) for specialized applications

Various host species including rabbit, rat, and mouse are available, allowing flexibility in experimental design when performing double-labeling studies .

How can I validate the specificity of my PKHD1 antibody?

Validating antibody specificity is crucial for reliable research results. Several complementary approaches should be used:

• Multiple Antibody Comparison:

  • Use antibodies targeting different regions of PKHD1 (N-terminus, C-terminus)

  • Similar staining patterns with different antibodies increase confidence in specificity

  • In published research, hAR-Np, hAR-C2p, and hAR-Cm3G6 antibodies showed concordant staining patterns

• Peptide Competition Assays:

  • Pre-incubate the antibody with the immunizing peptide

  • If staining is blocked or significantly reduced, this supports antibody specificity

  • "Immunoreactive staining was efficiently competed by preincubation with the hAR-N antigen"

• Recombinant Protein Expression:

  • Express a tagged version of the protein region recognized by the antibody

  • Test if the antibody detects the recombinant protein at the expected size

  • "Transient transfection of HEK293 cells with the Flag-tag construct yielded immunoreactive bands at the expected size using the hAR-Np antiserum, the hAR-Nm3G12 mAb, and the Flag-M2 antibody"

• Correlation with mRNA Expression:

  • Compare antibody staining patterns with mRNA expression by in situ hybridization

  • "The in situ cRNA hybridization and the hAR-Np antiserum gave similar staining patterns in mouse kidneys"

• Testing in PKHD1-Deficient Models:

  • Use tissue from PKHD1 mutant models (like pck rats) to assess signal reduction

  • "In the kidneys of the pck rats, the level of PKHD1 was significantly reduced but not completely absent"

• Western Blot Analysis:

  • Confirm the antibody detects a protein of the expected size

  • For PKHD1, immunoprecipitation followed by Western blot is often necessary

  • Look for the expected ~400 kDa band for full-length PKHD1

• Subcellular Localization Assessment:

  • Verify that the antibody shows the expected subcellular localization

  • For PKHD1, this would be primary cilia/basal bodies and apical membranes

  • "PKHD1 immunoreactivity was restricted to one or two spots per cell when viewed en face"

• Cross-Reactivity Testing:

  • Test antibody against related proteins like PKHD1L1

  • Particularly important since PKHD1L1 shares 25% identity with PKHD1

Documentation of these validation steps should be maintained, as antibody performance can vary between lots and applications.

What controls should I include when performing immunostaining with PKHD1 antibodies?

When performing immunostaining with PKHD1 antibodies, including appropriate controls is essential for valid interpretation:

Primary Controls:

• Positive Tissue Controls:

  • Known PKHD1-expressing tissues:

    • Kidney (collecting ducts, proximal tubules, thick ascending limbs)

    • Developing neural tube

    • Fetal lung bronchi

    • Liver (especially biliary cells)

• Negative Tissue Controls:

  • Tissues with minimal PKHD1 expression

  • Within kidney sections, glomeruli should show minimal staining compared to tubules

• Peptide Competition:

  • Pre-incubate antibody with the antigenic peptide

  • This should eliminate or significantly reduce specific staining

  • "Immunoreactive staining was efficiently competed by preincubation with the hAR-N antigen"

• Multiple Antibody Verification:

  • Use different antibodies targeting distinct regions of PKHD1

  • Similar patterns increase confidence in specificity

  • Published research used antibodies against N-terminal (hAR-Np, hAR-Nm3G12), C-terminal (hAR-C2p, hAR-C2m3C10), and middle regions (hAR-Cm3G6)

Technical Controls:

• Primary Antibody Omission:

  • Omit primary antibody but include all other steps

  • Controls for non-specific binding of secondary antibody

• Isotype Controls (for monoclonal antibodies):

  • Include isotype-matched irrelevant antibody

  • Controls for non-specific binding due to antibody class

• Endogenous Peroxidase Blocking (for IHC with HRP detection):

  • Verify adequate blocking of endogenous peroxidase activity

Disease Model Controls:

• PKHD1 Mutant Models:

  • Include tissue from PKHD1 mutant models (like pck rats)

  • Should show reduced or altered staining pattern

  • "The pck rats exhibited significantly less immunoreactivity of PKHD1 than did WT rats"

Co-Localization Controls:

• Double-Staining Controls:

  • For co-localization studies, include single-stained controls

  • Use established markers for specific kidney structures:

    • DBA (Dolichos biflorus agglutinin) for cortical collecting ducts

    • NHE3 (Na+/H+ exchanger 3) for proximal tubules

    • AQP2 (Aquaporin-2) for collecting ducts

    • Tamm-Horsfall protein for thick ascending limbs

  • For ciliary localization, include polycystin-2 as a marker

These controls should be processed alongside experimental samples under identical conditions to ensure valid comparisons and interpretations.

Why might I see multiple bands in Western blots when using PKHD1 antibodies?

Multiple bands in PKHD1 Western blots are common and can be attributed to several factors:

• Alternative Splicing:

  • PKHD1 undergoes extensive alternative splicing

  • "Diverse alternatively spliced variants of PKHD1 could putatively generate isoforms of the PKHD1 product"

  • A specific ~135 kDa band detected with C-terminal antibodies may represent a splice variant

• Protein Processing:

  • PKHD1 undergoes post-translational modifications including glycosylation

  • Proteolytic cleavage may generate fragments of different sizes

  • "Several transcripts are predicted to encode truncated products which lack the TM and may be secreted"

• Technical Limitations:

  • Standard Western analysis using some antibodies failed to detect the expected ~400-kDa bands

  • "Immunoprecipitation (IP) followed by Western blot was required to obtain the expected bands"

  • The large size makes it technically challenging to transfer and detect by standard Western blotting

• Protein Degradation:

  • Large proteins like PKHD1 (447 kDa) are particularly susceptible to degradation

  • Sample preparation and handling can affect integrity

• Related Proteins:

  • PKHD1L1 shares 25% identity and 41% similarity with PKHD1

  • "In humans, the canonical [PKHD1L1] protein has a reported length of 4243 amino acid residues and a mass of 465.7 kDa"

To address multiple bands:

• Use peptide competition to determine which bands are specific

• Compare results using antibodies against different regions

• Optimize protocols for large proteins (lower % gels, longer transfer times)

• Consider IP-Western for detecting full-length protein

• Document all bands observed and their reproducibility across experiments

How do I differentiate between specific and non-specific staining when using PKHD1 antibodies in immunohistochemistry?

Differentiating between specific and non-specific staining is crucial for accurate data interpretation:

Characteristics of Specific PKHD1 Staining:

• Anatomical Distribution:

  • Primarily in kidney tubules but not glomeruli

  • Biliary epithelium in liver

  • Epithelial cells of developing neural tube, bronchi, and gut during embryogenesis

• Subcellular Localization:

  • In polarized epithelial cells: primarily at apical domains

  • In cultured cells: localized to basal bodies/primary cilia

  • "PKHD1 immunoreactivity was restricted to one or two spots per cell when viewed en face"

  • "In the lateral view, the stained bodies were localized to the supranuclear region of the cells"

• Competition Sensitivity:

  • Specific staining should be reduced when the antibody is pre-incubated with the immunizing peptide

  • "Immunoreactive staining was efficiently competed by preincubation with the hAR-N antigen"

• Consistency with Multiple Antibodies:

  • Similar staining patterns with antibodies targeting different regions of PKHD1

Characteristics of Non-Specific Staining:

• Diffuse Pattern:

  • Uniform staining across all tissue types

  • Lack of subcellular specificity

• Inconsistent Localization:

  • Staining that doesn't match known PKHD1 localization

  • Random distribution rather than in specific structures

• Resistance to Competition:

  • Staining that remains after pre-incubation with immunizing peptide

• Non-Cellular Structures:

  • Staining of structures not expected to express PKHD1

  • Edge artifacts or staining of necrotic areas

Methods to Minimize Non-Specific Staining:

• Antibody Optimization:

  • Titrate antibody concentration (typically 1:200-1:500 for PKHD1 antibodies)

  • Too high concentration increases non-specific binding

• Blocking Optimization:

  • Use appropriate serum (typically 5-10%)

  • Consider protein blockers like BSA

• Antigen Retrieval Adjustment:

  • Optimize retrieval conditions to maintain epitope integrity while minimizing artifacts

• Use Validated Antibodies:

  • Select antibodies with demonstrated specificity

  • Commercial PKHD1 antibodies have varying performance across applications

• Multiple Staining Approaches:

  • Confirm key findings with independent antibodies and techniques

  • "The in situ cRNA hybridization and the hAR-Np antiserum gave similar staining patterns"

By systematically applying these criteria and controls, researchers can confidently distinguish between specific and non-specific staining patterns in their PKHD1 immunohistochemistry experiments.

How can PKHD1 antibodies be used to study cilia-related functions in kidney cells?

PKHD1 antibodies offer sophisticated tools for investigating cilia-related functions, particularly given PKHD1's localization to primary cilia in kidney cells:

• Co-localization Studies with Ciliary Markers:

  • Double immunofluorescence with PKHD1 and ciliary proteins

  • "PKHD1 was colocalized with polycystin-2 at the basal bodies/primary cilia"

  • Examine spatial relationships between PKHD1 and ciliary components

• Subciliary Localization:

  • High-resolution microscopy to determine precise localization

  • "Immunoelectron microscopy analysis confirmed that PKHD1 is highly agglomerated at the basal body. Some positive immunoreactivity was also seen in the ciliary shaft, microvilli and plasma membrane"

  • Understand PKHD1's distribution within the ciliary compartment

• Dynamic Studies of Ciliogenesis:

  • Track PKHD1 localization during ciliary assembly and disassembly

  • Correlate with cell cycle phases

  • PKHD1 "promotes ciliogenesis in renal epithelial cells"

• Disease Model Analysis:

  • Compare PKHD1 ciliary localization in normal vs. diseased states

  • "The pck rats exhibited significantly less immunoreactivity of PKHD1 than did WT rats"

  • Assess ciliary abnormalities in ARPKD models

• Functional Correlations:

  • Combine PKHD1 staining with functional ciliary assays:

    • Calcium influx measurements

    • Flow sensing experiments

    • Ciliary length and motility assessments

• Combined Genetic and Imaging Approaches:

  • Correlate PKHD1 mutations with ciliary defects

  • Study ciliary PKHD1 in CRISPR-edited cell lines

  • Rescue experiments with wild-type PKHD1

• Developmental Timing Studies:

  • Track PKHD1 ciliary expression during kidney development

  • Correlate with tubule formation and maturation

  • "During embryogenesis, PKHD1 is widely expressed in epithelial derivatives"

• Interdisciplinary Techniques:

  • Combine antibody-based imaging with proteomics of isolated cilia

  • Use proximity labeling to identify PKHD1 interactors specifically within cilia

  • Correlate with functional genomics data

These approaches leverage PKHD1 antibodies to understand fundamental questions about ciliary biology and pathogenesis of ciliopathies like ARPKD.

What approaches can be used to study the interaction between PKHD1 and other polycystic kidney disease proteins?

The co-localization of PKHD1 with polycystin-2 (PC2) at primary cilia suggests functional interactions between polycystic kidney disease proteins. Multiple complementary approaches can investigate these interactions:

• Biochemical Interaction Analysis:

  • Co-immunoprecipitation using PKHD1 antibodies followed by blotting for potential partners

  • Reciprocal co-IP (pull down PC2, blot for PKHD1)

  • Cross-linking prior to IP to capture transient interactions

  • "PKHD1 was colocalized with polycystin-2 at the basal bodies/primary cilia"

• Advanced Microscopy Techniques:

  • Super-resolution microscopy (STORM, PALM) for precise co-localization

  • Förster Resonance Energy Transfer (FRET) to detect direct interactions

  • Proximity Ligation Assay (PLA) for in situ detection of protein proximity

  • Live-cell imaging to track dynamic interactions

• Protein Domain Analysis:

  • Map interaction domains using truncated constructs

  • Test specific domains for binding to potential partners

  • "PKHD1 predominantly localizes to the basal bodies of the primary cilia in cultured renal cells"

• Pathogenic Mutation Impact:

  • Assess how disease-causing mutations affect protein interactions

  • Study interaction patterns in patient-derived cells

  • "Its coexpression with polycystin-2 suggests that PKHD1 acts in concert with polycystins and may be directly or indirectly involved in a common cystogenic pathway"

• Functional Consequence Studies:

  • Determine how disrupting interactions affects:

    • Ciliary localization of either protein

    • Calcium signaling pathways

    • Cell polarity and oriented cell division

  • "PKHD1 has an impact on cellular symmetry by ensuring correct bipolar cell division"

• Genetic Interaction Analysis:

  • Study phenotypic severity in models with combined mutations

  • Look for genetic modifiers that affect both PKHD1 and PC2

  • "The dramatic reduction of PKHD1 levels as compared to age-matched WT is consistent with a haploinsufficiency mechanism contributing to cystogenesis in ARPKD"

• Proteomic Approaches:

  • Tandem affinity purification of PKHD1 complexes

  • Mass spectrometry identification of interacting proteins

  • BioID or APEX proximity labeling to identify neighboring proteins

• Temporal Dynamics:

  • Study interaction patterns during development

  • Examine whether interactions change during ciliogenesis

  • "PKHD1 is expressed in epithelial cells at critical stages of kidney, lung, liver, and CNS morphogenesis"

These multidisciplinary approaches can reveal how PKHD1 functions within protein networks and how disruptions contribute to disease pathogenesis.

How can I use PKHD1 antibodies to investigate the functional consequences of PKHD1 mutations?

PKHD1 antibodies provide powerful tools for investigating how mutations affect protein function and contribute to disease pathogenesis:

• Expression Level Analysis:

  • Quantify PKHD1 protein levels in normal vs. mutant samples

  • "The pck rats exhibited significantly less immunoreactivity of PKHD1 than did WT rats"

  • Assess whether mutations cause protein instability or reduced expression

• Subcellular Localization Studies:

  • Determine if mutations alter PKHD1 trafficking or localization

  • Focus on ciliary/basal body localization and apical membrane targeting

  • Use co-staining with organelle markers (ER, Golgi) to identify potential retention sites

• Domain-Specific Detection:

  • Use antibodies targeting different PKHD1 domains

  • Detect truncated proteins resulting from nonsense or frameshift mutations

  • "Only ≈5% of the pck renal cysts retained PKHD1 labeling, and most were at the early stage of cystogenesis"

• Protein-Protein Interaction Assessment:

  • Analyze how mutations affect interactions with partners

  • Compare co-immunoprecipitation profiles between wild-type and mutant PKHD1

  • Focus on interactions with polycystin-2 and other ciliary proteins

• Functional Correlations:

  • Correlate PKHD1 mutation status with:

    • Ciliary structure and function

    • Cell polarization defects

    • Oriented cell division abnormalities

    • Signaling pathway disruptions

  • "PKHD1 promotes ciliogenesis in renal epithelial cells"

• Patient-Derived Models:

  • Analyze PKHD1 in cells from ARPKD patients

  • Compare with results from animal models (pck rats)

  • Establish genotype-phenotype correlations

• Alternative Isoform Analysis:

  • Determine how mutations affect different PKHD1 isoforms

  • "At least one previously undescribed isoform at ≈135 kDa associated with the COOH terminus of PKHD1"

  • Assess changes in isoform ratios in disease states

• Developmental and Tissue-Specific Effects:

  • Analyze timing of PKHD1 dysfunction during development

  • Compare effects across kidney, liver, and other affected tissues

  • "We did not observe any PKHD1 labeling in large cysts of the pck rat kidneys"

• Therapeutic Response Monitoring:

  • Use PKHD1 antibodies to assess responses to potential therapies

  • Monitor changes in protein expression, localization, or interactions

  • Establish PKHD1 status as a biomarker for treatment efficacy

• Haploinsufficiency Assessment:

  • Quantitative analysis of PKHD1 thresholds required for normal function

  • "The dramatic reduction of PKHD1 levels is consistent with a haploinsufficiency mechanism contributing to cystogenesis in ARPKD"

These approaches provide a comprehensive framework for understanding how PKHD1 mutations lead to disease and for developing potential therapeutic strategies for ARPKD.