Nephrin dissociates from actin, and its expression is reduced in early experimental membranous nephropathy.
These studies examined the expression of the podocyte slit diaphragm protein nephrin and its association with actin at the onset of proteinuria in passive Heymann nephritis (PHN), a rat model of human membranous nephropathy. Four days after immunization, 58% of PHN rats had mild proteinuria. At that time, most slit diaphragms were still visible on electron microscopy; however, in those locations where the deposits encroached on the filtration slits, the slit diaphragms were either displaced or absent. On day 7, the PHN rats were severely proteinuric, and most slit diaphragms were either absent, displaced, or replaced by occluding-type junctions. Immunofluorescence microscopy with antibodies to the external and cytoplasmic domains of nephrin showed a progressive loss of staining and a change in the distribution of nephrin from an interrupted linear pattern in normal controls to a more dispersed and clustered pattern in PHN. In contrast, the intensity of staining for ZO-1 and CD2-associated protein (CD2AP), two other proteins that are located on the cytoplasmic face of the slit diaphragm, was undiminished. Immunogold electron microscopy confirmed the progressive disappearance of nephrin from podocyte foot processes and retention of CD2AP. Glomeruli and glomerular cell membranes were extracted sequentially with Triton X-100, followed by DNase I or potassium iodide to depolymerize actin. Western blot analysis of the extracts showed a progressive decline of total nephrin on days 4 and 7 of PHN as well as a reduction in the actin-associated fraction. These findings show that nephrin partly dissociates from actin at the onset of podocyte injury in PHN. This is accompanied by a progressive loss of nephrin from the podocyte foot processes and prominent changes in the morphology of the slit diaphragms. These events may underlie the loss of podocyte barrier function in membranous nephropathy.
Yuan
Huaiping
H
*Renal Section and Department of Medicine, Evans Biomedical Research Center, Boston University Medical Center, and Renal Unit and Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.
Takeuchi
Emiko
E
*Renal Section and Department of Medicine, Evans Biomedical Research Center, Boston University Medical Center, and Renal Unit and Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.
Taylor
Gregory A
GA
*Renal Section and Department of Medicine, Evans Biomedical Research Center, Boston University Medical Center, and Renal Unit and Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.
McLaughlin
Margaret
M
*Renal Section and Department of Medicine, Evans Biomedical Research Center, Boston University Medical Center, and Renal Unit and Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.
Brown
Dennis
D
*Renal Section and Department of Medicine, Evans Biomedical Research Center, Boston University Medical Center, and Renal Unit and Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.
Salant
David J
DJ
*Renal Section and Department of Medicine, Evans Biomedical Research Center, Boston University Medical Center, and Renal Unit and Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.
D000199:Actins / Q000378:metabolism*; D000818:Animals; D005455:Fluorescent Antibody Technique; D005921:Glomerulonephritis / Q000378:metabolism* / Q000473:pathology / Q000652:urine; D007678:Kidney Glomerulus / Q000378:metabolism / Q000473:pathology; D008297:Male; D008565:Membrane Proteins; D008854:Microscopy, Electron; D016253:Microscopy, Immunoelectron; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011507:Proteinuria / Q000209:etiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012995:Solubility
D000199:Actins; D008565:Membrane Proteins; D011506:Proteins; C113267:nephrin