Focal segmental glomerulosclerosis (FSGS) is a disease showing severe proteinuria, and the disease progresses to end-stage kidney failure in many cases. However, the pathogenic mechanism of FSGS is not well understood. The slit diaphragm (SD), which bridges the neighboring foot processes of glomerular epithelial cells, is understood to function as a barrier of the glomerular capillary wall. To investigate the role of SD dysfunction in the development of FSGS, we analyzed the expression of SD-associated molecules in rat adriamycin-induced nephropathy, a mimic of FSGS. The staining of the SD molecules nephrin, podocin, and NEPH1 had already shifted to a discontinuous dotlike pattern at the initiation phase of the disease, when neither proteinuria nor any morphological alterations were detected yet. The alteration of NEPH1 expression was the most evident among the molecules examined, and NEPH1 was dissociated from nephrin at the initiation phase. On day 28, when severe proteinuria was detected and sclerotic changes were already observed, alteration of the expressions of nephrin, podocin, and NEPH1 worsened, but no alteration in the expression of other SD-associated molecules or other podocyte molecules was detected. It is postulated that the dissociation of NEPH1 from nephrin initiates proteinuria and that the SD alteration restricted in these molecules plays a critical role in the development of sclerotic changes in FSGS. Copyright © 2008 the American Physiological Society.