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Chandramohan et al. J Transl Genet Genom 2024;8:394-404 https://dx.doi.org/10.20517/jtgg.2024.38 Page 396

METHODS
Patients and patient characteristics
Females with FD diagnosis who underwent kidney biopsies at the University of Alabama at Birmingham
between 2000 and 2020 were retrieved.

Patients with elevated serum creatinine, proteinuria > 500 mg/day by UPCR, and those with incomplete
serological data or who did not undergo genetic testing were excluded from the analysis. Patients on
medications known to cause intracellular inclusion bodies, such as amiodarone and chloroquine, were
excluded.

The presence and severity of FD’s symptoms, such as acroparasthesia, hypohidrosis/hyperhydrosis,
gastrointestinal symptoms, cornea verticillate, and angiokeratoma, were collected. Information on the
presence of left ventricular hypertrophy by two-dimensional (2D) echocardiography, history of
hypertension, and anti-hypertensive medication use was also collected. Patients were not on enzyme
replacement therapy (ERT) or any other FD-specific therapy at the time of biopsy.

The study protocol was approved by the Institution Review Board at the University of Alabama in
Birmingham.

Kidney biopsy
Renal core biopsies from each patient, received fresh in a transport media (RPMI with L-glutamine), were
triaged for light microscopy (LM), Immunofluorescence (IF), and electron microscopy (EM). For LM, a
portion of cores were fixed in 10% buffered formalin processed in the usual manner and paraffin-embedded;
2-3 μm sections were stained with hematoxylin-eosin, Periodic acid Schiff hematoxylin (PASH), trichrome,
and Jones silver. For IF, 4-6 mm cores were placed in OCT (Optimal Cutting Temperature compound) and
quickly frozen at -20 °C; 2-3 μm sections were stained using antisera for IgG, IgA, IgM, C3, C1q, and
lambda light chain (MP Biomedicals, LLC. Solon, Ohio) and kappa light chain [Agilent Technologies
Singapore (International) Pte Ltd]. For EM, 1-2 mm pieces were initially fixed in 10% Carson's neutral
buffered formalin, post-fixed in 1% osmium tetroxide, and embedded in epoxy resin. Ultrathin sections
(70-90 nm) were cut, mounted on a copper grid, and post-stained with uranyl acetate and lead citrate.
Sections were examined at a Philips Morgagni or Philips CM10 transmission electron microscope.

Kidney biopsy scoring and interpretation
Standard evaluation that includes light microscopy, immunofluorescence, and electron microscopy was
done for kidney biopsy samples. A validated scoring system for renal pathology in FD developed by Fogo
[13]
and members of the International Study Group of Fabry Nephropathy (ISGFN) was used . Histological
features such as glomeruli sclerosis, arteriolar hyalinosis, and the presence of inclusion bodies in the parietal
epithelial cells, tubular epithelium, peritubular capillaries, and vessels were recorded as present or absent.
Podocyte vacuolization was scored as none, mild (< 25%), moderate (25%-50%), and severe (> 50%).
Podocyte inclusions were graded between 0 and 4+. Podocyte effacement was graded in percentage.

Serologic testing
Serum creatinine (SCr), eGFR, urine protein creatinine ratio, GL-3, and Lyso GL-3 enzyme activity,
reported around the same time patients were biopsied, were collected. Renal function was assessed using
serum creatinine and eGFR. Serum creatinine was reported as mg/dL, eGFR was calculated using the
2
chronic kidney disease epidemiology collaboration (CKD-EPI) equation and expressed as mL/min/1.73m ,
and proteinuria detected by UPCR was defined as the ratio of random spot total protein to creatinine and
expressed in mg/g.