Disease-causing missense mutations in NPHS2 gene alter normal nephrin trafficking to the plasma membrane

Kidney Int. 2004 Nov;66(5):1755-65. doi: 10.1111/j.1523-1755.2004.00898.x.


Background: Podocin is a membrane-integrated protein that is located at the glomerular slit diaphragm and directly interacts with nephrin. The gene encoding podocin, NPHS2, is mutated in patients with autosomal-recessive steroid-resistant nephrotic syndrome (SRN). In order to study a potential pathomechanism of massive proteinuria in patients with SRN, we have investigated the trafficking and subcellular localization of five common disease-causing missense mutants of human podocin.

Methods: Site-directed mutagenesis was applied to generate cDNA constructs encoding five different missense mutations of human podocin (P20L, G92C, R138Q, V180M, and R291W). To identify the subcellular localization of each mutant in transfected human embryonic kidney (HEK)293 cells, we have generated and characterized a rabbit polyclonal antibody against the human podocin. Specificity of the antibody was determined by light and immunoelectron microscopy, as well as immunoblot analysis using human glomeruli. Confocal microscopy was applied to determine subcellular localization of the wild-type and the mutated podocin molecules, as well as wild-type nephrin in transfected cells. Immunoprecipitation and pull-down studies were carried out to investigate the molecular interaction of podocin mutants and wild-type nephrin.

Results: Immunofluorescence and confocal microscopy showed that wild-type podocin located to the plasma membrane when expressed in HEK293 cells. Two missense mutations, P20L and G92C, located at the N-terminus part of the molecule, were also present at the plasma membrane, indicating that these mutations did not affect the subcellular localization of the mutated podocin molecules. In contrast, subcellular localization of three other missense mutants located in the proximal C-terminus part of the protein was drastically altered, in which R138Q was retained in the endoplasmic reticulum (ER), V180M formed inclusion bodies in the cytoplasm, and the R291W mutant was trapped both in the ER and in small intracellular vesicles. Interestingly, this abnormal subcellular localization of podocin missense mutants also resulted in alteration in protein trafficking of wild-type nephrin in cotransfected cells through the strong protein binding between both molecules.

Conclusion: In patients with SRN, some missense mutations in the NPHS2 gene not only lead to misfolding and mislocalization of the mutated podocin, but they can also interfere with slit diaphragm structure and function by altering the proper trafficking of nephrin to the plasma membrane.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Cell Membrane / metabolism
  • Drug Resistance / genetics*
  • Genes, Recessive
  • Humans
  • Immunologic Techniques
  • Intracellular Signaling Peptides and Proteins
  • Kidney / metabolism*
  • Membrane Proteins / genetics*
  • Membrane Proteins / metabolism
  • Microscopy, Immunoelectron
  • Mutation, Missense*
  • Nephrotic Syndrome / drug therapy
  • Nephrotic Syndrome / genetics*
  • Protein Transport
  • Proteins / metabolism*
  • Steroids / therapeutic use*
  • Subcellular Fractions / metabolism
  • Tissue Distribution
  • Transfection


  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins
  • NPHS2 protein
  • Proteins
  • Steroids
  • nephrin