Nephrin TRAP Mice Lack Slit Diaphragms and Show Fibrotic Glomeruli and Cystic Tubular Lesions MAIJA RANTANEN,* TUULA PALME ´ N,* ANU PA ¨ TA ¨ RI,* HEIKKI AHOLA,* SANNA LEHTONEN † , EVA ÅSTRO ¨ M,* THOMAS FLOSS, ‡ FRANZ VAUTI, ‡ WOLFGANG WURST, ‡ PATRIZIA RUIZ, § DONTSCHO KERJASCHKI, ¶ and HARRY HOLTHO ¨ FER* *Biomedicum, Molecular Medicine, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland; † Haartman Institute, Department of Pathology, University of Helsinki, Helsinki, Finland; ‡ GSF Center for Environment and Health, Institute of Mammalian Genetics, Neuherberg, Germany; § Max-Planck Institute for Molecular Genetics, Berlin, Germany; and ¶ Institute of Clinical Pathology, Division of Ultrastructural Pathology and Cell Biology, University of Vienna, Vienna, Austria. Abstract. The molecular mechanisms maintaining glomerular filtration barrier are under intensive study. This study describes a mutant Nphs1 mouse line generated by gene-trapping. Nephrin, encoded by Nphs1, is a structural protein of inter- podocyte filtration slits crucial for formation of primary urine. Nephrin trap/trap mutants show characteristic features of protein- uric disease and die soon after birth. Morphologically, fibrotic glomeruli with distorted structures and cystic tubular lesions were observed, but no prominent changes in the branching morphogenesis of the developing collecting ducts could be found. Western blotting and immunohistochemical analyses confirmed the absence of nephrin in nephrin trap/trap glomeruli. The immunohistochemical staining showed also that the inter- action partner of nephrin, CD2-associated protein (CD2AP), and the slit-diaphragm-associated protein, ZO-1 - , appeared unchanged, whereas the major anionic apical membrane pro- tein of podocytes, podocalyxin, somewhat punctate as com- pared with the wild-type (wt) and nephrin wt/trap stainings. Elec- tron microscopy revealed that 90% of the podocyte foot processes were fused. The remaining interpodocyte junctions lacked slit diaphragms and, instead, showed tight adhering areas. In the heterozygote glomeruli, approximately one third of the foot processes were fused and real-time RT-PCR showed 60% decrease of nephrin-specific transcripts. These results show an effective nephrin gene elimination, resulting in a phenotype that resembles human congenital nephrotic syn- drome. Although the nephrin trap/trap mice can be used to study the pathophysiology of the disease, the heterozygous mice may provide a useful model to study the gene dose effect of this crucial protein of the glomerular filtration barrier. The molecular mechanisms of diseases leading to proteinuria, a frequent medical symptom, are poorly understood. Increasing evidence suggests a key role for podocytes and particularly for the interpodocyte slit diaphragm in the permeability changes (1). Many pathophysiologically important proteins of the podo- cytes have been recently identified. In addition to their specific functions and cellular locations, they all share direct or indirect connection to the cytoskeleton that maintains normal podocyte shape. The identification of NPHS1 as the disease-causing gene in congenital nephrotic syndrome of the Finnish type (CNF) was a milestone in establishing the molecular composition of the interpodocyte slit diaphragm (2). Nephrin, the protein encoded by NPHS1, has been suggested to form the slit diaphragm by either homo- or heterophilic interactions creating pores that act as the ultimate sieve of the glomerular filter (2– 4). Subsequently, CD2-associated protein (CD2AP), originally found to enhance proper CD2-positioning required for antigen presentation (5), has been shown to bind nephrin and cause nephrotic syndrome in null mutant mice (6). A human homo- logue of CD2AP, Cas ligand with multiple Src-homology domains, has been suggested to regulate cytoskeletal rear- rangements (7). Thus, CD2AP is considered as a strong can- didate for linking nephrin to the cytoskeleton. Recently, cloning of NPHS2, disrupted in autosomal reces- sive steroid-resistant nephrotic syndrome (8), and ACTN4, mutated in focal segmental glomerulosclerosis (9), were re- ported. NPHS2 encodes for the membrane-associated protein, podocin, which is the second interaction partner identified for nephrin and capable of modulating its signaling activity (10). The cytosolic protein product of ACTN4, -actinin-4, is an actin-filament crosslinking protein. Despite knowing the important role of nephrin in the glo- merular filtration barrier (11–14), a detailed understanding of its functions is still missing. Our results have shown alternative splicing of nephrin mRNA (11,12,15) and changes of nephrin mRNA levels during experimental renal diseases closely par- Received September 19, 2001. Accepted February 23, 2002. Correspondence to Dr. Harry Holtho ¨fer, Biomedicum, Molecular Medicine, University of Helsinki, PB 63, FIN-00014 Helsinki, Finland. Phone: +358-9- 191-25500; Fax: +358-9-191 25501; E-mail: Harry.Holthofer@Helsinki.Fi 1046-6673/1306-1586 Journal of the American Society of Nephrology Copyright © 2002 by the American Society of Nephrology DOI: 10.1097/01.ASN.0000016142.29721.22 J Am Soc Nephrol 13: 1586–1594, 2002