Glomerulus-specific mRNA transcripts and proteins identified through kidney expressed sequence tag database analysis.

The kidney glomerulus plays a crucial role in blood filtration but the molecular composition and physiology of the glomerulus is not well understood. We previously constructed and large-scale sequenced four mouse glomerular expressed sequence tag (EST) libraries from newborn and adult mouse glomeruli. Here, we compared glomerular EST profiles with whole kidney EST profiles, thereby identifying 497 transcripts corresponding to UniGene clusters that were glomerulus-enriched, that is expressed more abundantly in glomeruli than in whole kidney. These include several known protein-coding glomerulus-specific transcripts critical for glomerulus development and function, but also a large number of gene transcripts, which have not previously been shown to be expressed in the glomerulus, or implicated in glomerular functions. We used in situ hybridization to demonstrate glomerulus-specific RNA expression for six novel glomerular genes and the public Human Protein Atlas to verify glomerular protein expression for another two. The higher mRNA abundance for the eight genes in glomeruli compared with whole kidney was also verified by Taqman quantitative polymerase chain reaction. We surmise that the further characterization of these genes and proteins will increase our understanding of glomerular development and physiology.

Genetic kidney diseases disclose the pathogenesis of proteinuria.

The sieving of plasma components occurs in the kidney through the glomerular capillary wall. This filter is composed of three layers: endothelium, glomerular basement membrane (GBM), and podocyte foot processes connected by slit diaphragms. Defects in this barrier lead to proteinuria and nephrotic syndrome. Previously, defective GBM was regarded to be responsible for proteinuria. However, recent work on genetic diseases has indicated that podocytes and the slit diaphragm are crucial in restricting protein leakage. Congenital nephrotic syndrome of the Finnish type (NPHS1) is caused by mutations in a novel NPHS1 gene, which encodes for a cell adhesion protein, nephrin. This protein is synthesized by podocytes, and seems to be a major component of the slit diaphragm. In severe NPHS1, lack of nephrin leads to missing slit diaphragm. The role of nephrin in acquired kidney diseases remains unknown. In addition to nephrin, other podocyte proteins (podocin, alpha-actinin-4, CD2AP, FAT) have recently been identified and associated with the development of proteinuria. It seems that the slit diaphragm and its interplay with the podocyte cytoskeleton is critical for the normal sieving process, and defects in one of these components easily lead to proteinuria.

Role of nephrin in cell junction formation in human nephrogenesis.

Nephrin is a cell adhesion protein located at the slit diaphragm area of glomerular podocytes. Mutations in nephrin-coding gene (NPHS1) cause congenital nephrotic syndrome (NPHS1). We studied the developmental expression of nephrin, ZO-1 and P-cadherin in normal fetal kidneys and in NPHS1 kidneys. We used in situ hybridization and immunohistochemistry at light and electron microscopic levels. Nephrin and zonula occludens-1 (ZO-1) were first expressed in late S-shaped bodies. During capillary loop stage, nephrin and ZO-1 localized at the basal margin and in the cell-cell adhesion sites between developing podocytes, especially in junctions with ladder-like structures. In mature glomeruli, nephrin and ZO-1 concentrated at the slit diaphragm area. P-cadherin was first detected in ureteric buds, tubules, and vesicle stage glomeruli. Later, P-cadherin was seen at the basal margin of developing podocytes. Fetal NPHS1 kidneys with Fin-major/Fin-major genotype did not express nephrin, whereas the expression of ZO-1 and P-cadherin was comparable to that of control kidneys. Although early junctional complexes proved structurally normal, junctions with ladder-like structures and slit diaphragms were completely missing. The results indicate that nephrin is dispensable for early development of podocyte junctional complexes. However, nephrin appears to be essential for formation of junctions with ladder-like structures and slit diaphragms.

Congenital nephrotic syndrome (NPHS1): features resulting from different mutations in Finnish patients.

BACKGROUND: Congenital nephrotic syndrome (NPHS1) is a rare disease inherited as an autosomally recessive trait. The NPHS1 gene mutated in NPHS1 children has recently been identified. The gene codes for nephrin, a cell-surface protein of podocytes. Two mutations, named Fin-major and Fin-minor, have been found in over 90% of the Finnish patients. In this study, we correlated the NPHS1 gene mutations to the clinical features and renal findings in 46 Finnish NPHS1 children. METHODS: Clinical data were collected from patient files, and kidney histology and electron microscopy samples were re-evaluated. The expression of nephrin was studied using immunohistochemistry, Western blotting, and in situ hybridization. RESULTS: Nephrotic syndrome was detected in most patients within days after birth regardless of the genotype detected. No difference could be found in neonatal, renal, cardiac, or neurological features in patients with different mutations. Nephrin was not expressed in kidneys with Fin-major or Fin-minor mutations, while another slit diaphragm-associated protein, ZO-1, stained normally. In electron microscopy, podocyte fusion and podocyte filtration slits of various sizes were detected. The slit diaphragms, however, were missing. In contrast to this, a nephrotic infant with Fin-major/R743C genotype expressed nephrin in kidney had normal slit diaphragms and responded to therapy with an angiotensin-converting enzyme inhibitor and indomethacin. CONCLUSIONS: The most common NPHS1 gene mutations, Fin-major and Fin-minor, both lead to an absence of nephrin and podocyte slit diaphragms, as well as a clinically severe form of NPHS1, the Finnish type of congenital nephrotic syndrome.