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Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions
Published on: July 2, 2020
Differentiation-induced cultured podocytes express endocytically active megalin, a heymann nephritis antigen
Hajime Yamazaki1, Akihiko Saito, Hidemi Ooi
1Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
Background/Aims:
Megalin is a multiligand endocytic receptor expressed in a number of epithelia. In the Lewis rat kidney, podocytes, as well as proximal tubule cells, express megalin that acts as a pathogenic antigen for Heymann nephritis (HN), an experimental model of membranous nephropathy. To obtain a tool to investigate the molecular mechanisms of megalin-mediated endocytosis and the pathogenesis of HN, we examined whether a differentiation-inducible mouse podocyte cell line expressed endocytically active megalin.
Methods:
Immunofluorescence and immunoprecipitation analyses with an anti-rat megalin antibody were carried out to investigate whether megalin was expressed in the differentiated and undifferentiated podocytes. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed to elucidate whether the cells synthesize megalin mRNA. 125I-labeled receptor-associated protein (RAP), an endocytic ligand for megalin, was used for cellular internalization and degradation assays.
Results:
Immunofluorescence, immunoprecipitation and RT-PCR analyses revealed that megalin was synthesized in both differentiated and undifferentiated cells and localized to the cell surfaces. Effective endocytosis of RAP via megalin was shown under the differentiated condition.
Conclusion:
Endocytically active megalin is expressed in differentiation-induced cultured podocytes. This cell line could be a useful tool for studies on megalin-mediated endocytosis and the pathogenesis of HN.
Insights
A mouse podocyte cell line expresses endocytically active megalin (a receptor involved in Heymann nephritis) upon differentiation. This provides a valuable tool for studying megalin-mediated endocytosis and kidney disease pathogenesis.
Area of Science:
- Nephrology
- Cell Biology
- Molecular Medicine
Background:
- Megalin is a key endocytic receptor found in kidney epithelia, including podocytes.
- Megalin functions as a pathogenic antigen in Heymann nephritis (HN), an experimental model of membranous nephropathy.
- Understanding megalin's role in podocytes is crucial for investigating HN pathogenesis.
Purpose of the Study:
- To determine if a differentiation-inducible mouse podocyte cell line expresses functional megalin.
- To establish a cellular model for studying megalin-mediated endocytosis.
- To facilitate research into the molecular mechanisms of Heymann nephritis.
Main Methods:
- Immunofluorescence and immunoprecipitation assays to detect megalin expression.
- Reverse transcriptase-polymerase chain reaction (RT-PCR) to confirm megalin mRNA synthesis.
- Cellular uptake and degradation assays using radiolabeled receptor-associated protein (RAP) to assess endocytic activity.
Main Results:
- Megalin was synthesized and localized to the cell surface in both differentiated and undifferentiated podocytes.
- Effective endocytosis of RAP via megalin was observed in differentiated podocytes.
- The results confirm the presence of endocytically active megalin in this cell model.
Conclusions:
- A differentiation-inducible mouse podocyte cell line expresses endocytically active megalin.
- This cell line serves as a valuable tool for investigating megalin-mediated endocytosis.
- The model is suitable for further studies on the pathogenesis of Heymann nephritis.
