Table 1.
Demographic & clinical features of study population.
Fig 1.
Urine osmolality and electrolyte analysis of samples from FSGS pediatric patients and age-matched healthy control subjects.
Spot urine collections from FSGS patients and healthy control subjects were used to measure urine osmolality (A), urinary sodium (B), urinary potassium (C), and urinary chloride (D) concentrations. SigmaPlot software Version 14.0 was used to determine statistical significance between the two groups. ** Represents p<0.001.
Fig 2.
Characterization of EV from FSGS patients and control subjects.
A. Summary scatter plot of nanoparticle tracking analysis showing EV size (N = 5 for FSGS group and N = 7 for control group). B. Summary scatter plot of nanoparticle tracking analysis showing EV concentration (N = 5 for FSGS group and N = 5 for control group). C. Western blot analysis of the EV markers Caveolin-1, CD9, and Syntenin. Each EV preparation from the two groups was probed for the non-EV negative control marker HDAC2 and Uromodulin (Tamm-Horsfall protein). The positive control (+ CTRL) lane included cell lysate from the human kidney proximal tubule epithelial cell line HK2. D. Representative electron micrographs showing EVs from the FSGS and control groups. SigmaPlot software Version 14.0 was used to determine statistical significance between the two groups. ** Represents p<0.001.
Table 2.
EV concentration normalized to Uromodulin (Tamm-Horsfall protein).
FSGS n = 5, Control n = 5; p-value of 0.208.
Fig 3.
Western blot analysis of phospho-STAT-3 protein after challenging human mesangial cells with EVs from pediatric FSGS patients or age-matched healthy control subjects.
A. Representative Western blot of phospho-STAT-3 (pSTAT-3) (top blot) and beta actin (bottom blot) after treating human mesangial cells with EVs from FSGS patients or healthy control subjects. The first three lanes represent cellular lysates from three independent experiments of cells treated with control urinary EVs while the last three lanes represent cellular lysates from three independent experiments of cells treated with urinary EVs from FSGS patients. B. Densitometric analysis of the top and bottom immunoreactive bands of the pSTAT-3 blot in panel A normalized to the actin blot. SigmaPlot software Version 14.0 was used to determine statistical significance between the two groups * represents p<0.05 ** represents a p<0.001.
Fig 4.
Western blot analysis of PCNA after treating human mesangial cells treated with urinary EVs from FSGS patients or control subjects.
A. Western blot of PCNA (top blot) and actin (bottom blot). The first three lanes represent cellular lysates from three independent experiments of cells treated with control urinary EVs while the last three lanes represent cellular lysates from three independent experiments of cells treated with urinary EVs from FSGS patients. B. Densitometric analysis of the immunoreactive blot in panel A. SigmaPlot software Version 14.0 was used to determine statistical significance between the two groups. * represents a p-value <0.05. C. Cell proliferation of human mesangial cells treated with either H2O2, EVs (2X10^7 particles/ml) from control subjects, or EVs (2X10^6, 2X10^7, or 2X10^8 particles/ml) from FSGS patients. Additional controls included treatment of human mesangial cells with or without BrdU. A two-way ANOVA was performed using GraphPad Prism 9 to determine statistical significance between the groups. ** represents a p-value <0.01, *** represents a p-value <0.001, **** represents a p-value<0.0001.
Fig 5.
Immunofluorescence microscopy analysis of normal human mesangial cell proliferation after challenging the cells with urinary EVs from pediatric FSGS patients or age-matched healthy control subjects.
A. Representative images of the proliferative markers ki-67 (green) and PCNA (magenta) in acetone:methanol fixed normal human mesangial cells challenged with urinary EVs from FSGS patients (pool of 5 patients) (top panels) or with urinary EVs from healthy control subjects (pool of 5 subjects) (bottom panels). A 2”x2” box was placed over 5–7 cells and the image was cropped and saved in Tiff format for Image J analysis. B. Densitometric analysis of the ki-67 and PCNA positive staining in panel A. Five cells for each marker were analyzed for each group. SigmaPlot software Version 14.0 was used to determine statistical significance between the two groups ** represents a p<0.001.