Bestrophin-3 is differently expressed in normal and injured mouse glomerular podocytes.

AIM: Bestrophins are putative calcium-activated chloride channels. Recently, cell-protective functions for Bestrophin-3 (Best3) were proposed. Best3 exists in different splice variants. We have here examined expression, alternative splicing and localization of Best3 in mouse podocytes under normal conditions and during endoplasmic reticulum (ER) stress. METHODS: Best3 expression was determined on the mRNA level using quantitative PCR and on the protein level by immunohistochemistry and Western blotting. RESULTS: Staining for Best3 was pronounced in glomeruli and was detected in cultured mouse podocytes. Best3 did not co-localize with markers for endothelial cells (CD31), podocyte foot processes (synaptopodin) or microtubules (actin). However, immunogold-based electron microscopy and co-localization with nestin showed Best3 presence in podocyte primary processes and cell bodies. Only two splice variants of Best3 mRNA (both lacking exons 2 and 3, and one also lacking exon 6), but no full-length variant, were detected. ER stress induced by lipopolysaccharides in vivo transiently elevated mRNA levels of total Best3 and its two splice variants with different time courses. In cultured podocytes under ER stress induced by thapsigargin, the expression of total Best3, its splice variants and nestin transiently increased with similar time courses. The ER stress marker C/EBP homologous protein (CHOP) and nestin mRNA increased during ER stress in vivo and in vitro. CONCLUSIONS: Best3 is localized intracellularly in cell bodies and primary processes of mouse podocytes and is co-localized with nestin. Two splice variants of Best3 are expressed in glomeruli and in cultured podocytes, and their expression is differentially regulated in ER stress.

Podocyte proteoglycan synthesis is involved in the development of nephrotic syndrome.

Proteoglycans (PGs) are important for the glomerular barrier, for cell signaling, and for the anchorage of cells to the glomerular basement membrane. They are, however, complex macromolecules, and their production has not yet been thoroughly investigated in podocytes. In the present study, we studied the biosynthesis of PGs by highly differentiated human podocytes and in rats. The cells were treated with puromycin aminonucleoside (PAN; a nephrosis-inducing agent), steroids (used as primary treatment for nephrotic syndrome), or both. Analysis was made by TaqMan real-time PCR, Western blotting, and by metabolic labeling with (35)S and (3)H. We found that podocytes produce versican, syndecan-1, decorin, and biglycan together with the previously known PG syndecan-4, glypican, and perlecan. PAN treatment downregulated the mRNA and the protein expression of both versican (by 24 +/- 6%, P < 0.01, for mRNA and by 50% for protein) and perlecan (by 14 +/- 5%, P < 0.05, for mRNA and by 50% for protein). The decreased expression was confirmed by studying the glomerular gene expression in rats treated with PAN during a time course study. In addition, puromycin decreased the expression of enzymes involved in the glycosaminoglycan biosynthesis. Steroid treatment decreased perlecan (by 24 +/- 3%, P < 0.01) and syndecan-1 expression (by 30 +/- 4%, P < 0.01) but increased the expression of decorin 2.5-fold. The observed alterations of PG synthesis induced by PAN may lead to decreased glomerular anionic charge and disturbed podocyte morphology, factors that are important for the development of a nephrotic syndrome.