Quantification of Urine Podocytes (Podo) and Their Globotriaosylceramide (GL3) Content Using Imaging Flow Cytometry

Background: Fabry disease (FD) is a lysosomal storage disease caused by a deficiency of α-galactosidase A (α-Gal-A) leading to intracellular GL3 (main α-Gal-A substrate) leading to critical end-organ damage. Podo injury and loss play key roles in FD nephropathy pathogenesis. We aimed to develop a non-invasive assay for quantification of urine Podo and intracellular GL3 content. Methods: A CRISPR/Cas9 GLA knock-out (KO) conditionally immortalized human Podo cell line and its wild type (WT) counterpart were used as laboratory controls. Urothelial and proximal tubular cell lines were used as negative controls. IFC protocols for Podo and GL3 quantification were validated. 37 ml random urine sample from a male patient with FD who had been on enzyme replacement therapy (ERT) for over 20 years was analyzed using IFC. Results: IFC reliably separated Podo from urothelial and proximal tubular cells in our laboratory controls based on GLEPP1 expression. In addition, IFC reliably detected intracellular GL3 with a granular pattern based on CD77 staining intensity. The GLA-KO Podo showed 2.4 fold greater GL3 signal compared to the WT Podo. 41% of GL3 signal in GLA-KO Podo was colocalized with LAMP1, consistent with both intra- and extra-lysosomal GL3 accumulation. IFC of the urine sample from the FD patient showed 124 Podo (~3 Podo/ml urine) as a distinct population with variable intracellular GL3, likely reflecting an ERT effect (Figure). Conclusion: IFC is a powerful tool for the quantification of rare cells in the urine, such as Podo. In FD patients, quantifying urine Podo and their GL3 content may serve as potentially useful non-invasive biomarkers of kidney involvement and evaluation of treatment effect. Funding: NIDDK Support - NIDDK Support, NIDDK Support