In vivo characterization of a podocyte-expressed short podocin isoform
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In vivo characterization of a podocyte-expressed short podocin isoform. / Butt, Linus; Unnersjö-Jess, David; Reilly, Dervla; Hahnfeldt, Robert; Rinschen, Markus M; Bozek, Katarzyna; Schermer, Bernhard; Benzing, Thomas; Höhne, Martin.
in: BMC NEPHROL, Jahrgang 24, Nr. 1, 378, 19.12.2023.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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T1 - In vivo characterization of a podocyte-expressed short podocin isoform
AU - Butt, Linus
AU - Unnersjö-Jess, David
AU - Reilly, Dervla
AU - Hahnfeldt, Robert
AU - Rinschen, Markus M
AU - Bozek, Katarzyna
AU - Schermer, Bernhard
AU - Benzing, Thomas
AU - Höhne, Martin
N1 - © 2023. The Author(s).
PY - 2023/12/19
Y1 - 2023/12/19
N2 - The most common genetic causes of steroid-resistant nephrotic syndrome (SRNS) are mutations in the NPHS2 gene, which encodes the cholesterol-binding, lipid-raft associated protein podocin. Mass spectrometry and cDNA sequencing revealed the existence of a second shorter isoform in the human kidney in addition to the well-studied canonical full-length protein. Distinct subcellular localization of the shorter isoform that lacks part of the conserved PHB domain suggested a physiological role. Here, we analyzed whether this protein can substitute for the canonical full-length protein. The short isoform of podocin is not found in other organisms except humans. We therefore analysed a mouse line expressing the equivalent podocin isoform (podocinΔexon5) by CRISPR/Cas-mediated genome editing. We characterized the phenotype of these mice expressing podocinΔexon5 and used targeted mass spectrometry and qPCR to compare protein and mRNA levels of podocinwildtype and podocinΔexon5. After immunolabeling slit diaphragm components, STED microscopy was applied to visualize alterations of the podocytes' foot process morphology.Mice homozygous for podocinΔexon5 were born heavily albuminuric and did not survive past the first 24 h after birth. Targeted mass spectrometry revealed massively decreased protein levels of podocinΔexon5, whereas mRNA abundance was not different from the canonical form of podocin. STED microscopy revealed the complete absence of podocin at the podocytes' slit diaphragm and severe morphological alterations of podocyte foot processes. Mice heterozygous for podocinΔexon5 were phenotypically and morphologically unaffected despite decreased podocin and nephrin protein levels.The murine equivalent to the human short isoform of podocin cannot stabilize the lipid-protein complex at the podocyte slit diaphragm. Reduction of podocin levels at the site of the slit diaphragm complex has a detrimental effect on podocyte function and morphology. It is associated with decreased protein abundance of nephrin, the central component of the filtration-slit forming slit diaphragm protein complex.
AB - The most common genetic causes of steroid-resistant nephrotic syndrome (SRNS) are mutations in the NPHS2 gene, which encodes the cholesterol-binding, lipid-raft associated protein podocin. Mass spectrometry and cDNA sequencing revealed the existence of a second shorter isoform in the human kidney in addition to the well-studied canonical full-length protein. Distinct subcellular localization of the shorter isoform that lacks part of the conserved PHB domain suggested a physiological role. Here, we analyzed whether this protein can substitute for the canonical full-length protein. The short isoform of podocin is not found in other organisms except humans. We therefore analysed a mouse line expressing the equivalent podocin isoform (podocinΔexon5) by CRISPR/Cas-mediated genome editing. We characterized the phenotype of these mice expressing podocinΔexon5 and used targeted mass spectrometry and qPCR to compare protein and mRNA levels of podocinwildtype and podocinΔexon5. After immunolabeling slit diaphragm components, STED microscopy was applied to visualize alterations of the podocytes' foot process morphology.Mice homozygous for podocinΔexon5 were born heavily albuminuric and did not survive past the first 24 h after birth. Targeted mass spectrometry revealed massively decreased protein levels of podocinΔexon5, whereas mRNA abundance was not different from the canonical form of podocin. STED microscopy revealed the complete absence of podocin at the podocytes' slit diaphragm and severe morphological alterations of podocyte foot processes. Mice heterozygous for podocinΔexon5 were phenotypically and morphologically unaffected despite decreased podocin and nephrin protein levels.The murine equivalent to the human short isoform of podocin cannot stabilize the lipid-protein complex at the podocyte slit diaphragm. Reduction of podocin levels at the site of the slit diaphragm complex has a detrimental effect on podocyte function and morphology. It is associated with decreased protein abundance of nephrin, the central component of the filtration-slit forming slit diaphragm protein complex.
KW - Humans
KW - Animals
KW - Mice
KW - Podocytes/metabolism
KW - Intracellular Signaling Peptides and Proteins/genetics
KW - Protein Isoforms/genetics
KW - Nephrotic Syndrome/genetics
KW - RNA, Messenger/metabolism
U2 - 10.1186/s12882-023-03420-x
DO - 10.1186/s12882-023-03420-x
M3 - SCORING: Journal article
C2 - 38114895
VL - 24
JO - BMC NEPHROL
JF - BMC NEPHROL
SN - 1471-2369
IS - 1
M1 - 378
ER -