Quantification of Urine Podocytes (Podo) Using Imagining Flow Cytometry (IFC)

Background: Podo injury and loss contribute to the progression of most kidney diseases causing ESRD. Quantification of Podo in the urine is a noninvasive approach for early detection of glomerular injuries and monitoring kidney disease progression. Current approaches for urine Podo quantification are time-consuming and difficult to standardize. We aimed to develop a standardized, faster approach using IFC Methods: Laboratory standards for positive and negative controls were developed using human Podo (markers: podocalyxin, GLEPP1), urothelial (marker: UPK3) and proximal tubular (marker: LRP2) cell lines. Validation process included sensitivity, specificity, linearity and lower limit of quantification. Limited urine samples from patients were examined. IFC was done using Mark-II Imagestrem. Data was analyzed using IDEAS software. Results: IFC reliably separated Podo from urothelial and proximal tubular cells. There was close to perfect correlation between podocalyxin and GLEPP1 signals. Analysis of triplicate samples at 5 dilutions demonstrated linearity down to a lower limit of quantification of 500 Podo/75 uL of analyte. IFC of spot urine samples from 5 random patients with membranous nephropathy, pauci-immune glomerulonephritis, diffuse lupus nephritis, IgA nephropathy and Fabry disease quantified a range of 4-146 Podo/ml urine with a detection sensitivity of 0.07% of total events captured. Conclusion: We have developed laboratory standards for the quantification of urine Podo and have validated IFC protocols. In contrast to conventional flow cytometry, IFC is capable of detecting rare cells and can be a powerful tool to quantify urine Podo loss. Further work is needed to explore podocyturia variability in healthy and disease conditions. Funding: NIDDK Support - NIDDK Support, NIDDK Support