A WT1 co-regulator controls podocyte phenotype by shuttling between adhesion structures and nucleus.

Glomerular podocyte differentiation state is critical for filtration barrier function and is regulated by WT1, a zinc finger transcription factor. A yeast two-hybrid assay identified a novel, WT1-interacting protein (WTIP) that maps to human chromosome 19q13.1, a region with genes linked to familial focal segmental glomerulosclerosis. The domain structure of WTIP is similar to the zyxin subfamily of cytosolic LIM domain-containing proteins, which contain three carboxyl-terminal LIM protein-protein interaction domains and a proline-rich, pre-LIM region with a nuclear export signal. Other LIM domain-containing proteins (zyxin and mouse muscle LIM protein) did not interact with WT1 in two-hybrid assays, and WTIP did not interact with an unrelated transcription factor, LMX1B. WTIP mRNA was detected in cultured podocytes and was developmentally regulated, with expression peaking in mouse kidney at embryonic day 15-16 (E15-E16) in kidney but persisting into adulthood. In situ hybridization demonstrated WTIP expression in developing E15 glomeruli and in cultured podocytes. The partial WTIP clone, which interacted with WTIP in the two-hybrid assay, co-localized with WT1 in nuclei, co-precipitated with WT1, and inhibited WT1-dependent transcriptional activation of the amphiregulin promoter. In contrast, full-length WTIP was excluded from cell nuclei, but after the addition of leptomycin B, an inhibitor of Crm1-mediated nuclear export, it accumulated in the nucleus and co-precipitated with WT1 in whole cell lysates. Epitope-tagged WTIP co-localized with the adaptor protein CD2AP (CMS) in podocyte actin spots and with Mena at cell-cell junctions. We propose that WTIP monitors slit diaphragm protein assembly as part of a multiple protein complex, linking this specialized adhesion junction to the actin cytoskeleton, and shuttles into the nucleus after podocyte injury, providing a mechanism whereby changes in slit diaphragm structure modulate gene expression.

Congenital diaphragmatic hernia, kidney agenesis and cardiac defects associated with Slit3-deficiency in mice.

Slit3 along with Slit1 and Slit2 comprise the Slit family of proteins. The latter two proteins are known to be involved in axon guidance and cell migration during animal development. However, little is know about the functions of Slit3. We created a Slit3-deficient mouse model from an OmniBank ES cell line with a Slit3 allele trapped by insertional mutagenesis to analyze the in vivo functions of this protein. In this model, congenital diaphragmatic hernia is the most obvious phenotype. Herniation was found to be caused by a defective central tendon (CT) of the diaphragm that remained fused with the liver. Electron microscopic analyses of the defective CT revealed disorganized collagen fibrils that failed to form tight collagen bundles. The hearts of Slit3-deficient mice have an enlarged right ventricle. In addition, 20% of homozygous mice also showed a range of kidney defects that include unilateral or bilateral agenesis of the kidney and ureter, or varying degrees of renal hypoplasia. Thus, we concluded that Slit3 is involved in the development of multiple organ systems that include the diaphragm and the kidney. Slit3-deficient mice represent a genetic animal model for physiological and pathological studies of congenital diaphragmatic hernia.

DNA expression analysis: serial analysis of gene expression, microarrays and kidney disease.

PURPOSE OF REVIEW: Expression profiling using serial analysis of gene expression and microarray technologies allows global description of expressed genes present in biological systems. Although relatively new technologies, each having been developed in the mid-1990s, both have become established and widely used tools for identification of gene networks and gene function. RECENT FINDINGS: This review highlights DNA expression analyses published in 2002, emphasizing primarily serial analysis of gene expression and microarray technologies. We focus on the applicability of DNA expression analysis to renal disease, especially as some investigators have developed custom serial analysis of gene expression kidney libraries and kidney disease-specific 'designer chip' microarrays. Data analysis techniques and statistics are also discussed, since the challenge is generation of accurate messenger RNA profiles and interpretation of data in a manner that is both coherent and reproducible. SUMMARY: Because kidney disease pathophysiology is complex, expression analysis can identify candidate nephropathy pathogenesis genes and gene networks, which eventually could become targets for therapeutic intervention.

Linkage analysis of candidate loci for end-stage renal disease due to diabetic nephropathy.

Diabetic nephropathy (DN), a major cause of ESRD, is undoubtedly multifactorial and is caused by environmental and genetic factors. To identify a genetic basis for DN susceptibility, we are collecting multiplex DN families in the Caucasian (CA) and African-American (AA) populations for whole genome scanning and candidate gene analysis. A candidate gene search of diabetic sibs discordantly affected, concordantly affected and concordantly unaffected for DN was performed with microsatellite markers in genomic regions suspected to harbor nephropathy susceptibility loci. Regions examined were at human chromosome 10p,10q (orthologous to the rat renal susceptibility Rf-1 locus), and at NPHS1 (nephrin), CD2AP, Wilms tumor (WT1), and NPHS2 (podocin) loci. Linkage analyses were conducted using model-free methods (SIBPAL, S.A.G.E.) for AA, CA, and the combined sample. Allele frequencies and the identity by descent sharing were estimated separately for AA and CA, and race was included as a covariate in the final linkage analysis. To date, we have collected 212 sib pairs from 46 CA and 50 AA families. The average age of diabetes onset was 46.8 yr versus 36.2 yr for CA and 39.5 yr versus 40.2 yr for AA, in males versus females respectively. Genotyping data were available for 106 sib pairs (43 CA, 63 AA) from 27 CA (44% male probands) and 38 AA families (43% male probands). Average AA and CA sibship size was 2.73. Singlepoint and multipoint linkage analyses indicate that marker D10S1654 on chromosome 10p is potentially linked to DN (CA only multipoint P = 4 x 10(-3)). Interestingly, the majority of the linkage evidence derives from the CA sib pairs. We are now adding sib pairs and increasing marker density on chromosome 10. We have excluded linkage with candidate regions for nephrin, CD2AP, WT1, and podocin in this sample. In conjunction with previous reports, our data support evidence for a DN susceptibility locus on chromosome 10.

Kidney disease, genotype and the pathogenesis of vasculopathy.

PURPOSE OF REVIEW: The two leading causes of end-stage renal disease in the United States are diabetes mellitus and hypertensive nephrosclerosis, accounting for over two-thirds of all cases. In many patients both diseases are associated with small- and large-vessel disease, commonly attributed to hypertension or accelerated atherosclerosis. Recent investigations, however, have suggested that renal large-vessel and microvascular disease may be independent contributors to progressive kidney failure. RECENT FINDINGS: Although genes have not been definitely linked to renal vascular disease, population- and family-based epidemiology of kidney disease, segregation analysis of Pima and Caucasian families in which diabetic nephropathy is clustered, and the positional cloning of genes responsible for rare, familial glomerulosclerosis syndromes support the hypothesis that genes regulate the pathogenesis of renal disease. This review highlights developments in our current understanding of vasculopathy and its role in renal disease, and it summarizes evidence suggesting that genetic determinants for the vascular phenotype are associated with common causes of chronic renal failure. SUMMARY: With the application of genomics and proteomics methodologies to drug discovery, the identification of renal susceptibility genes should identify new mechanisms of progressive renal disease pathogenesis and generate novel target molecules for the treatment of kidney disease.