Fig 1.
Schematic representation of how, due to a change in cell size, the "reference trap" may mask detection of intracellular GL3 reduction.
While the podocyte on the left loses 50% of its GL3 content, because of a proportional (50%) shrinkage in podocyte cytoplasmic volume, the fraction of the volume of the podocyte cytoplasm filled with GL3 [Vv(Inc/PC)] remains the same (the cell on the right).
Fig 2.
Intercept measurements for the point-sampled intercept method for a podocyte nucleus with concavity.
Dashed lines represent the sampling grid superimposed on podocyte nuclei. One of the cross-points of this grid falling on the nuclear profile (here "P") is randomly selected(i.e., sampling point). The red line represents the random direction line, passing through the sampling point "P" along which the intercepts are measured. The intercepts include ,
, and
.
Table 1.
Clinical characteristic at baseline and follow up after 11–12 months of enzyme replacement therapy.
Fig 3.
(A) A representative glomerulus from an 18 year old male (Case #1, see Table 1 for patient's characteristics) with Fabry disease at baseline (ERT-naïve). Podocytes (PC) are remarkably enlarged with abundant GL3 inclusions (Inc); (B) A representative glomerulus from the same patient after 12 months of ERT (1 mg/kg/EOH agalsidase-beta) shows smaller podocytes. The majority of podocytes still showed many GL3 inclusions (asterisks). However, occasional podocytes showed no GL3 inclusions (Ø); (C)Total volume of GL3 inclusions per podocytes V(Inc/PC) was reduced after 11–12 months of ERT (p = 0.02); (D) Podocyte GL3 score (semiquantitative [14]) did not reduce significantly after 11–12 months (follow-up) of ERT (p = 0.18); (E) Podocyte volume significantly decreased after 11–12 months of ERT compared to baseline (p = 0.02). The dashed area shows the range of podocyte volume in biopsies from 5 healthy kidney donor normal controls. The difference between podocyte volume in Fabry patients after 11–12 months of ERT and these healthy controls was not statistically significant; (F) Podocyte GL3 inclusion volume fraction [Vv(Inc/PC)] did not change significantly after 11–12 months of ERT (p = 0.42). The numbers written by each line in C-F represents case numbers according to Table 1. Panels C-F show the average values of presented parameters in each biopsy.
Fig 4.
Various stages of GL3 inclusion exocytosis in podocytes.
(A) Fusion of GL3 inclusions or their surrounding membranes with the cell membrane (arrows); (B) The space surrounding an intracellular GL3 inclusion is connected to the urinary space through an orifice (arrow); (C) A portion of a GL3 inclusion is squeezed into the urinary space through an orifice (arrow). An adjacent GL3 inclusion is partially extruded from the podocyte while preserving its round shape (arrowhead): (D) A GL3 inclusions extruding from a podocyte shows partial unfolding of its multilamellar structure (arrow); (E) Unfolding of the multilamellar structure of a GL3 inclusion while being squeezed out of a podocyte through an orifice (arrow); (F) Extruded GL3 inclusions in the urinary space (arrow); (G) A large round GL3 inclusion almost completed its extrusion from the podocyte (arrow); (H) An empty round space in a podocyte with partial protrusion into the urinary space, reflecting recent exocytosis (asterisk) and a nearby extracellular GL3 inclusion (arrow); (I) A large and partially unfolded GL3 inclusion in the urinary space (arrow).
Fig 5.
(A) Foot process effacement(black arrows) in a biopsy from a Fabry patient at baseline (ERT-naïve). Asterisk marks a GL-3 inclusion in a podocyte (PC). White arrow shows GL-3 inclusions in an endothelial cell (En). (B) A biopsy after 11–12 months of ERT from the same patient still shows areas of glomerular basement membranes with foot process effacement (black arrows). Note that podocytes contain GL-3 inclusions (asterisk), while the endothelial cells are cleared from inclusions. (C) Intact foot processes (black arrows) from a normal control biopsy. (D) Foot process width (FPW) changes estimated by unbiased morphometry. Although FPW was numerically reduced from baseline to follow up in 4/6 cases, the difference was not statistically significant (p = 0.29). The dashed area shows the range of FPW in biopsies from 9 healthy kidney donor normal controls. FPW in baseline and follow up biopsies from Fabry patients were significantly greater than normal controls (p = 0.002 and p = 0.0006, respectively). (E) Foot process effacement (FPE) semi-quantitative scores did not change significantly from baseline to follow up. FPE scoring was based on Tøndel et al. [16], where a score of “0” = no foot identified process effacement; “(+)” = foot process effacement in short segments; and “+” = foot process effacement in longer segments.
Fig 6.
Correlation between volume fraction of GL-3 inclusions per podocyte [Vv(Inc/PC)] % change from baseline to follow up (11–12 months post ERT) and foot process width (FPW) at follow up.