One Year of Enzyme Replacement Therapy Reduces Globotriaosylceramide Inclusions in Podocytes in Male Adult Patients with Fabry Disease

Abstract

Fabry nephropathy is associated with progressive accumulation of globotriaosylceramide (GL3) in podocytes. Reducing this GL3 burden may reduce podocyte injury. Sensitive methods to quantify podocyte GL3 content may determine whether a given strategy can benefit podocytes in Fabry disease. We developed an unbiased electron microscopic stereological method to estimate the average volume of podocytes and their GL3 inclusions in 6 paired pre- and post-enzyme replacement therapy (ERT) biopsies from 5 men with Fabry disease. Podocyte GL3 content was regularly reduced (average 73%) after 11-12 months of ERT. This was not detectable using a semi-quantitative approach. Parallel to GL3 reduction, podocytes became remarkably smaller (average 63%). These reductions in podocyte GL3 content or size were not significantly correlated with changes in foot process width (FPW). However, FPW after ERT was significantly correlated with the magnitude of the decrease in podocyte GL3 content from baseline to 11-12 months of ERT. Also podocytes exocytosed GL3 inclusions, a phenomenon correlated with their reduction in their GL3 content. Demonstrable after11-12 months, reduction in podocyte GL3 content allows for early assessment of treatment efficacy and shorter clinical trials in Fabry disease.

Conflict of interest statement

Competing Interests: BN is a recipient of investigator initiated Genzyme research grants, a consultant to Genzyme, and received speaker's honoraria and travel support from Genzyme. He is also a member of the Medical Advisory Board of Amicus and performs kidney biopsy studies for Amicus. These interests have been reviewed and managed by the University of Washington in according to its conflict of interest policies. CT has no conflict of interest. ES received speaker fees and travel support from Shire and Genzyme. AS and KS have no conflict of interest. MM is a member and Chair of the Genzyme sponsored North American Fabry Registry Advisory Board*, a recipient of investigator initiated Genzyme research grants, a consultant to Genzyme for clinical trial design*, and a speaker at Genzyme educational meetings*. He is also a consultant to and performs kidney biopsy studies for Amicus and a grant reviewer for Shire. *This interest has been reviewed and managed by the University of Minnesota in according to its conflict of interest policies. This did not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

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 l0,03=AB, l0,i−3=PC, and l0,i+3=PD.

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.