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      Imaging Glomeruli in Renal Biopsy Specimens

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          Glomerular capillary loops are complex vascular filters composed of interdigitating podocytes and fenestrated endothelial cells with an intervening proteoglycan-rich extracellular matrix. This arrangement is crucial to maintaining the filtration barrier but renders the glomerulus difficult to analyze by conventional two-dimensional histochemical techniques. When pathologic lesions distort glomerular architecture, its complex morphology is even more challenging to interpret. Fortunately, recent advances in microscopes and computer software now enable glomerular enthusiasts to dissect this complex structure with finer detail. In this review we explore the application of new methodologies such as two-photon microscopy that optimize three-dimensional, multicolor imaging and single-cell segmentation of glomerular components.

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          Most cited references 14

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          Identification and characterization of podocalyxin--the major sialoprotein of the renal glomerular epithelial cell

          The glomerular epithelial polyanion is a specialized cell surface component found on renal glomerular epithelial cells (podocytes) that is rich in sialoprotein(s), as detected by staining with cationic dyes (colloidal iron, alcian blue) and wheat germ agglutinin (WGA). We have isolated rat glomeruli and analyzed their protein composition by SDS PAGE in 5-10% gradient gels. When the gels were stained with alcian blue or "Stains All," a single band with an apparent Mr of 140,000 was detected that also stained very prominently with silver, but not with Coomassie Blue. This band predominated in fluorograms of gels of isolated glomeruli that had been labeled in their sialic acid residues by periodate-[3H]borohydride. In lectin overlays, the 140-kilodalton (kd) band was virtually the only one that bound [125I]wheat germ agglutinin, and this binding could be prevented by predigestion with neuraminidase. [125I]Peanut lectin bound exclusively to the 140-kd band after neuraminidase treatment. An antibody was prepared that specifically recognizes only the 140-kd band by immunoprecipitation and immuneoverlay. By immunoperoxidase and immunogold techniques, it was localized to the surface coat of the glomerular epithelium and, less extensively, to that of endothelial cells. When analyzed (after electroelution from preparative SDS gels), the 140-kd band was found to contain approximately 20% hexose and approximately 4.5% sialic acid. These findings indicate that the 140-kd protein is the major sialoprotein of the glomerulus, and it is the only component of glomerular lysates with an affinity for cationic dyes and lectins identical to that defined histochemically for the epithelial polyanion in situ. Since this molecule is a major component of the cell coat or glycocalyx of the podocytes, we have called it "podocalyxin."
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            Germline mutations in the Wilms' tumor suppressor gene are associated with abnormal urogenital development in Denys-Drash syndrome.

            Denys-Drash syndrome is a rare human condition in which severe urogenital aberrations result in renal failure, pseudohermaphroditism, and Wilms' tumor (nephroblastoma). To investigate its possible role, we have analyzed the coding exons of the Wilms' tumor suppressor gene (WT1) for germline mutations. In ten independent cases of Denys-Drash syndrome, point mutations in the zinc finger domains of one WT1 gene copy were found. Nine of these mutations are found within exon 9 (zinc finger III); the remaining mutation is in exon 8 (zinc finger II). These mutations directly affect DNA sequence recognition. In two families analyzed, the mutations were shown to arise de novo. Wilms' tumors from three individuals and one juvenile granulosa cell tumor demonstrate reduction to homozygosity for the mutated WT1 allele. Our results provide evidence of a direct role for WT1 in Denys-Drash syndrome and thus urogenital system development.
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              WT1 is a key regulator of podocyte function: reduced expression levels cause crescentic glomerulonephritis and mesangial sclerosis.

              Glomerular disease is one of the most common causes of end-stage renal failure. Increasing evidence suggests that these glomerulopathies are frequently caused by primary lesions in the renal podocytes. One of the major consequences of podocyte lesions is the accumulation of mesangial matrix in the glomerular basement membrane, a process called glomerulosclerosis. Mesangial sclerosis is one of the most consistent findings in Denys-Drash patients and can be caused by dominant mutations in the Wilms' tumor 1 gene (WT1). The underlying mechanism, however, is poorly understood. WT1 is expressed in the podocytes throughout life, but its function in this cell type is unknown. Combining Wt1-knockout and inducible yeast artificial chromosome transgenic mouse models, we demonstrate that reduced expression levels of WT1 result in either crescentic glomerulonephritis or mesangial sclerosis depending on the gene dosage. Strikingly, the two podocyte-specific genes nphs1 and podocalyxin are dramatically downregulated in mice with decreased levels of Wt1, suggesting that these two genes act downstream of Wt1. Taken together, our data provide genetic evidence that reduced levels of Wt1 are responsible for the pathogenesis of two distinct renal diseases and offer a molecular explanation for the increased occurrence of glomerulosclerosis in patients with WAGR syndrome.

                Author and article information

                Nephron Physiol
                Nephron Physiology
                S. Karger AG
                March 2006
                11 April 2006
                : 103
                : 2
                : p75-p81
                aDepartment of Medicine, Division of Nephrology and Indiana Center for Biological Microscopy, and Departments of bPathology and cAnatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, Ind., USA
                90623 Nephron Physiol 2006;103:p75–p81
                © 2006 S. Karger AG, Basel

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                Page count
                Figures: 2, Tables: 1, References: 31, Pages: 1
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                Microscopic Imaging


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