Abstract
Nephrin is a critical component of glomerular filtration barrier, which is important to maintain glomerular structure and avoid proteinuria. Downregulation of nephrin expression is commonly observed at early stage of glomerular disorders, suggesting that methods to increase nephrin expression in podocytes may have therapeutic utility. Here, we generated a knockin mouse line carrying single copy of 5.5 kb nephrin promoter controlling expression of enhanced green fluorescent protein (EGFP) at Rosa26 genomic locus (Nephrin-EGFP mouse). In these mice, EGFP was specifically expressed in podocytes. Next, we isolated and cultivated glomeruli from these mice, and developed a protocol to automatically quantitate EGFP expression in cultured glomeruli. EGFP signal was markedly reduced after 5 days of culture but reduction was inhibited by vitamin D treatment. We confirmed that vitamin D increased mRNA and protein expression of endogenous nephrin in cultivated glomeruli. Thus, we generated a mouse line converting nephrin promoter activity into fluorescence, which can be used to screen compounds having activity to enhance nephrin gene expression.
Highlights
Slit membrane synthesized by podocytes plays an essential role to form glomerular filtration barrier in the kidney [1]
A donor vector carrying Nephrin+enhanced green fluorescent protein (EGFP) expression cassette and a Cre expression vector were co-injected into fertilized eggs isolated from the seed mice to obtain knockin mice carrying a single copy of Nephrin+EGFP cassette at the Rosa26 locus in the opposite direction to Rosa26 transcription[13, 14] (Fig 1)
We generated Nephrin-EGFP mice, which express EGFP in podocytes under the control of nephrin promoter inserted at the Rosa26 locus
Summary
Slit membrane synthesized by podocytes plays an essential role to form glomerular filtration barrier in the kidney [1]. Loss in the function and numbers of podocytes is a key event in various renal disorders, leading to proteinuria, glomerulosclerosis and, eventually, end-stage renal disease [2]. To alleviate podocyte injury is one of major strategies to treat chronic kidney disease, but still efficient treatment options are limited [3]. Nephrin is a critical component of glomerular filtration barrier [4]. Downregulation of nephrin expression is commonly observed at PLOS ONE | DOI:10.1371/journal.pone.0157497. Downregulation of nephrin expression is commonly observed at PLOS ONE | DOI:10.1371/journal.pone.0157497 June 30, 2016
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