In vivo expression of podocyte slit diaphragm-associated proteins in nephrotic patients with NPHS2 mutation.
BACKGROUND
Mutations in NPHS2, encoding podocin, are a prevalent cause of autosomal-recessive steroid-resistant nephrotic syndrome (SRNS). Podocin is a protein associated with the slit diaphragm that interacts with nephrin and CD2-associated protein (CD2AP) within lipid rafts.


METHODS
Using renal biopsies of six patients, we analyzed the in vivo consequences of different types of NPHS2 mutations on (1) the podocyte expression and distribution of podocin using in situ hybridization and immunohistology and (2) the distribution of related podocyte proteins and glomerular extracellular matrix components.


RESULTS
In two patients with homozygous 855_856delAA or 419delG mutation, absence of podocyte labeling with the antibodies against the C-terminal domain contrasted with the normal expression of the N-terminal domain of the protein along the glomerular basement membrane (GBM). In patients carrying compound heterozygous mutations or variants (R168S/467_468insT, R138Q/V180M, and R291W/R229Q), or single heterozygous 976_977insA, podocin transcription appeared unchanged but the distribution of the protein was modified. Podocin was restricted to the podocyte body in the patient carrying the R168S/467_468insT mutation whereas strong immunolabeling of the podocyte body was associated with discrete labeling along the GBM in the three others. In all cases, podocin defect was associated with changes in the distribution of nephrin, CD2AP, and alpha-actinin: the proteins were mainly detected in the podocyte body, with mild expression along the GBM. There were no detectable changes in the distribution of other podocyte proteins or glomerular extracellular matrix components.


CONCLUSIONS
NPHS2 mutations result in profound alteration of podocin expression and/or distribution. Secondary changes in the distribution of nephrin, CD2AP, and alpha-actinin are additional evidences for the scaffolding role of podocin in the organization of the slit diaphragm.
INSERM U574, Université René Descartes, Hôpital Necker-Enfants Malades, Paris, France.
Shao-Yu
SY
Zhang
Arnaud
A
Marlier
Olivier
O
Gribouval
Thierry
T
Gilbert
Laurence
L
Heidet
Corinne
C
Antignac
Marie Claire
MC
Gubler
D000185:Actinin / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000293:Adolescent; D002648:Child; D002675:Child, Preschool; D003598:Cytoskeletal Proteins / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006720:Homozygote; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007678:Kidney Glomerulus / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D008297:Male; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D009404:Nephrotic Syndrome / Q000235:genetics* / Q000473:pathology / Q000503:physiopathology*; D017354:Point Mutation; D011506:Proteins / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C114545:CD2-associated protein; D003598:Cytoskeletal Proteins; D016326:Extracellular Matrix Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C407223:NPHS2 protein; D011506:Proteins; C113267:nephrin; D000185:Actinin