Integrin α3 negative podocytes: A gene expression study

Abstract

Integrin α3β1 is a cell adhesion receptor widely expressed in epithelial cells. Pathogenic variants in the gene encoding the integrin α3 subunit ITGA3 lead to a syndrome including interstitial lung disease, nephrotic syndrome, and epidermolysis bullosa (ILNEB). Renal involvement mainly consists of glomerular disease caused by loss of adhesion between podocytes and the glomerular basement membrane.The aim of this study was to characterize the impact of loss of integrin α3 on human podocytes.ITGA3 was stably knocked-out in the human podocyte cell line AB8/13, designated as PodoA3−, and in human proximal tubule epithelial cell line HK2 using the targeted genome editing technique CRISPR/Cas9. Cell clones were characterized by Sanger sequencing, quantitative PCR, Western Blot and immunofluorescence staining. RNASeq of integrin α3 negative cells and controls was performed to identify differential gene expression patterns.Differentiated PodoA3− did not substantially change morphology and adhesion under standard culture conditions, but displayed significantly reduced spreading and adhesion when seed on laminin 511 in serum free medium. Gene expression studies demonstrated a distinct dysregulation of the adhesion network with downregulation of most integrin α3 interaction partners. In agreement with this, biological processes such as “extracellular matrix organization” and “cell differentiation” as well as KEGG pathways such as “ECM-receptor interaction”, “focal adhesion” and the “PI3K-Akt signaling pathway” were significantly downregulated in human podocytes lacking the integrin α3 subunit.

Keywords

• ILNEB
• Integrin α3
• Skin blistering
• Nephrotic syndrome
• Kidney
• CRISPR/Cas9