Podocyte‐Specific Rescuing and Deletion of Acid Ceramidase Gene in Mouse Glomeruli

Abstract

We have recently developed a podocyte‐restricted acid ceramidase (AC) gene knockout mouse model (Asah1fl/fl/Podocre, Asah1 is a code of mouse AC gene), which was shown to produce podocyte dysfunction and consequent minimal change disease (MCD). The present study attempted to develop somatic gene intervention methods to perform podocyte‐specific gene deletion, rescuing and silencing of this AC gene in Asah1fl/fl/Podocre mice and their control littermates including Asah1fl/fl/WT mice, which could exclude the potential development or compensatory influence given that Asah1 gene was embryonically deleted in podocytes in those animal strains. We first constructed 2 different vectors which can be transfected to change AC gene expression including a podocyte‐specific AC gene (podo‐Asah1) expression vector and a podocyte‐specific Cre gene (podo‐Cre) expression vector. The pEZX vector containing podocin promoter was used to generate Asah1 and Cre full length gene expression plasmids, which were transfected into Asah1fl/fl/Podocre mice to rescue Asah1 gene and into Asah1fl/fl/WT mice to delete floxed Asah1 gene, respectively. Using isolated podocytes from 2 different mouse strains, we confirmed by immunofluorescence staining, real‐time PCR and Western blot analysis that these vectors could be successfully transfected into podocytes and produced corresponding effects to alter Asah1 gene expression, for example, rescuing of this gene in Asah1fl/fl/Podocre podocytes and deletion of this gene in Asah1fl/fl/WT podocytes. In animal experiments, we used microbubble ultrasound transfection techniques to introduce different plasmids into the kidneys. It was found that Asah1 gene was rescued in podocytes of Asah1fl/fl/Podocre mice by podo‐Asah1 plasmids and deleted in Asah1fl/fl/WT mice by podo‐Cre plasmids. Functionally, we demonstrated that increased Asah1 expression in podocytes decreased desmin levels, but enhanced podocin expression, indicating restoration of podocyte function. It also enhanced the interaction of multivesicular bodies (MVBs) and lysosomes as shown by increase in Vps16 and Lamp‐1 colocalization, which resulted in decreased exosome release from podocytes. Taken together, our results suggest that somatic transfection of podocin promoter‐driven Asah1 and Cre gene expression vectors produced podocyte‐restricted gene changes in different gene engineered animal models for AC function studies.Support or Funding Informationsupported by NIH grant DK54927, DK120491 and HL057244This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.