CLIC5A, a component of the ezrin-podocalyxin complex in glomeruli, is a determinant of podocyte integrity.

The chloride intracellular channel 5A (CLIC5A) protein, one of two isoforms produced by the CLIC5 gene, was isolated originally as part of a cytoskeletal protein complex containing ezrin from placental microvilli. Whether CLIC5A functions as a bona fide ion channel is controversial. We reported previously that a CLIC5 transcript is enriched approximately 800-fold in human renal glomeruli relative to most other tissues. Therefore, this study sought to explore CLIC5 expression and function in glomeruli. RT-PCR and Western blots show that CLIC5A is the predominant CLIC5 isoform expressed in glomeruli. Confocal immunofluorescence and immunogold electron microscopy reveal high levels of CLIC5A protein in glomerular endothelial cells and podocytes. In podocytes, CLIC5A localizes to the apical plasma membrane of foot processes, similar to the known distribution of podocalyxin and ezrin. Ezrin and podocalyxin colocalize with CLIC5A in glomeruli, and podocalyxin coimmunoprecipitates with CLIC5A from glomerular lysates. In glomeruli of jitterbug (jbg/jbg) mice, which lack the CLIC5A protein, ezrin and phospho-ERM levels in podocytes are markedly lower than in wild-type mice. Transmission electron microscopy reveals patchy broadening and effacement of podocyte foot processes as well as vacuolization of glomerular endothelial cells. These ultrastructural changes are associated with microalbuminuria at baseline and increased susceptibility to adriamycin-induced glomerular injury compared with wild-type mice. Together, the data suggest that CLIC5A is required for the development and/or maintenance of the proper glomerular endothelial cell and podocyte architecture. We postulate that the interaction between podocalyxin and subjacent filamentous actin, which requires ezrin, is compromised in podocytes of CLIC5A-deficient mice, leading to dysfunction under unfavorable genetic or environmental conditions.

A human glomerular SAGE transcriptome database.

BACKGROUND: To facilitate in the identification of gene products important in regulating renal glomerular structure and function, we have produced an annotated transcriptome database for normal human glomeruli using the SAGE approach. DESCRIPTION: The database contains 22,907 unique SAGE tag sequences, with a total tag count of 48,905. For each SAGE tag, the ratio of its frequency in glomeruli relative to that in 115 non-glomerular tissues or cells, a measure of transcript enrichment in glomeruli, was calculated. A total of 133 SAGE tags representing well-characterized transcripts were enriched 10-fold or more in glomeruli compared to other tissues. Comparison of data from this study with a previous human glomerular Sau3A-anchored SAGE library reveals that 47 of the highly enriched transcripts are common to both libraries. Among these are the SAGE tags representing many podocyte-predominant transcripts like WT-1, podocin and synaptopodin. Enrichment of podocyte transcript tags SAGE library indicates that other SAGE tags observed at much higher frequencies in this glomerular compared to non-glomerular SAGE libraries are likely to be glomerulus-predominant. A higher level of mRNA expression for 19 transcripts represented by glomerulus-enriched SAGE tags was verified by RT-PCR comparing glomeruli to lung, liver and spleen. CONCLUSION: The database can be retrieved from, or interrogated online at http://cgap.nci.nih.gov/SAGE. The annotated database is also provided as an additional file with gene identification for 9,022, and matches to the human genome or transcript homologs in other species for 1,433 tags. It should be a useful tool for in silico mining of glomerular gene expression.

A SAGE-based comparison between glomerular and aortic endothelial cells.

Endothelial cells have many characteristics in common, but significant morphological and functional differences exist between endothelial cells from different anatomic sites. The specific glomerular endothelial (GEn) cell transcript repertoire is unknown. We sought to determine whether endothelial cells derived from bovine glomeruli display a distinct transcriptional profile compared with bovine aortic endothelium (BAE) under identical conditions. Serial analysis of gene expression (SAGE), which includes known and unknown transcripts, was used to make the comparison. The GEn and BAE SAGE libraries contain 36,844 and 26,452 total tag sequences, respectively. Among 6,524 unique tag sequences represented at least 2 times in the 2 libraries, 2,094 (32%) were matched to well-characterized bovine cDNA sequences (358 tags) or expressed sequence tags (EST). Identification of the human homolog was achieved for 1,035 of these tags. Forty-two tags were differentially expressed in GEn. For 25 of these, the bovine cDNA or EST, and for 17 the human homolog was identified. Among all transcripts with a known bovine and human tag, seven were expressed at levels more than 10-fold higher in cultured GEn cells compared with all other SAGE libraries. The transcript "DKFZp564B076" was localized by in situ hybridization to glomerular endothelium in vivo and was shown by real-time RT-PCR to be highly abundant in glomeruli compared with aortic intima. This work supports the concept that differences in the transcriptional profile of endothelial cells from distinct origins are observed under otherwise equivalent conditions. Furthermore, we have identified the first known transcript predominant in glomerular endothelium in vivo.

Characterization and prevalence of PITX2 microdeletions and mutations in Axenfeld-Rieger malformations.

PURPOSE: Mutations of the homeodomain protein PITX2 produce Axenfeld-Rieger (AR) malformations of the anterior chamber, an autosomal dominant disorder accompanied by a 50% risk of glaucoma. Twenty-nine mutations of PITX2 have been described, with a mutational prevalence estimated between 10% and 60% in AR. In the current study, the possible role of altered PITX2 gene dosage in the etiology of AR was investigated. Gross gene deletions and duplications should alter PITX2 activity analogously to hypomorphic and hypermorphic mutations, respectively. METHODS: Sixty-four patients with AR, iridogoniodysgenesis (IGD), iris hypoplasia (IH), or anterior segment dysgenesis (ASD) were screened for PITX2 mutations by sequencing. PITX2 gene dosage was concurrently examined in these patients by real-time quantitative PCR. Microsatellite markers were used to map 4q25 microdeletions at a contig scale, as well as for haplotype analysis in an extended AR kindred. An additional 27 patients with other assorted ocular phenotypes were evaluated by similar methods, amounting to a total of 91 cases analyzed. RESULTS: Three novel mutations of PITX2 (4.7%) were identified among 64 patients with AR, IGD, IH, or ASD. Deletions of PITX2 were as frequent as mutations in our sample. Chromosome 4q25 microdeletions were physically mapped relative to several microsatellite markers in each patient. Cosegregation of AR and a PITX2 deletion was demonstrated in an extended kindred. CONCLUSIONS: Point mutations and gross deletions of PITX2 appear to produce an equivalent haploinsufficiency phenotype. Quantitative PCR is an efficient means of detecting causative PITX2 deletions in patients with AR and may increase the detection rate at this locus.