Accumulation of Globotriaosylceramide in Podocytes in Fabry Nephropathy Is Associated with Progressive Podocyte Loss

Abstract

Background: In males with classic Fabry disease, the processes leading to the frequent outcome of ESKD are poorly understood. Defects in the gene encoding α-galactosidase A lead to accumulation of globotriaosylceramide (GL3) in various cell types. In the glomerular podocytes, accumulation of GL3 progresses with age. Of concern, podocytes are relatively resistant to enzyme replacement therapy and are poorly replicating, with little ability to compensate for cell loss.

Methods: In this study of 55 males (mean age 27 years) with classic Fabry disease genotype and/or phenotype, we performed unbiased quantitative morphometric electron microscopic studies of biopsied kidney samples from patients and seven living transplant donors (to serve as controls). We extracted clinical information from medical records and clinical trial databases.

Results: Podocyte GL3 volume fraction (proportion of podocyte cytoplasm occupied by GL3) increased with age up to about age 27, suggesting that increasing podocyte GL3 volume fraction beyond a threshold may compromise survival of these cells. GL3 accumulation was associated with podocyte injury and loss, as evidenced by increased foot process width (a generally accepted structural marker of podocyte stress and injury) and with decreased podocyte number density per glomerular volume. Worsening podocyte structural parameters (increasing podocyte GL3 volume fraction and foot process width) was also associated with increasing urinary protein excretion-a strong prognosticator of adverse renal outcomes in Fabry disease-as well as with decreasing GFR.

Conclusions: Given the known association between podocyte loss and irreversible FSGS and global glomerulosclerosis, this study points to an important role for podocyte injury and loss in the progression of Fabry nephropathy and indicates a need for therapeutic intervention before critical podocyte loss occurs.

Keywords: Fabry disease; chronic kidney disease; pathology; podocyte.

Graphical abstract

Figure 1.

Illustration of morphometric methods. (A) Systematic uniform random sampling of a glomerular profile for stereologic measurements from a patient with Fabry disease. Red boxes represent locations where higher-magnification images are obtained by transmission electron microscopy. Red arrows show the path of sampling. Yellow asterisk marks the box that is magnified in (B) (montage low magnification, about 8000×). (B) Magnified view of a portion of glomerular tuft with a superimposed point grid used for fractional volume estimation. (C) Higher magnification (approximately 30,000×) image with an unbiased counting frame superimposed for estimation of FPW based on the number of line intercepts with the glomerular basement membrane (yellow arrowheads) and number of slits (green arrows).

Figure 2.

Relationships between podocyte structural parameters and age. (A) Relationship between the podocyte inclusion volume fraction (Vv[Inc/PC]) and age. Regression line shows an exponential model with plateau. (B) Relationship between podocyte profile area and Vv(Inc/PC). (C) Inverse relationship between the fractional volume of podocytes per glomerulus (Vv[PC/glom]) and age (r=−0.54, P=0.0001). (D) Relationship between number density of podocytes per glomerulus (Nv[PC/glom]) and age. Solid lines represent regression lines. Dashed lines represent 95% confidence interval.

Figure 3.

Relationships between podocyte structural parameters and urinary protein excretion, and podocyte loss (A) Relationship between UPER (logarithmic scale, in mg/g if urine protein-creatinine ratio or g/d if 24-hour collection) and podocyte inclusion volume fraction (Vv[Inc/PC]; R=0.44, P=0.003). (B) Relationship between UPER (logarithmic scale) and podocyte FPW (R=0.41, P=0.007). (C) Relationship between number density of podocytes per glomerular volume (Nv[PC/glom]) and podocyte total inclusion volume. Regression line shows an exponential model with two-phase decay. Solid line represents regression lines. Dashed lines represent 95% confidence interval.

Figure 4.

Visual depiction of proposed relationships between age and podocyte parameters in Fabry patients and normal controls. Relationships between aging and podocyte GL3 volume (dotted line), podocyte GL3 volume fraction (dashed line), and podocyte loss in Fabry disease (black bold line). The gray line represents physiologic podocyte loss with aging. Initially, the rate of GL3 accumulation is greater than the rate of podocyte enlargement, this leading to increasing podocyte GL3 volume fraction with increasing age up to 25–30 years of age. Thereafter, podocyte GL3 volume fraction plateaus while GL3 accumulation continues in parallel with podocyte enlargement, and this is associated with podocyte loss from aging aggravated by additional podocyte loss from Fabry disease.