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Page 4 of 11 Feriozzi et al. Rare Dis Orphan Drugs J 2024;3:11 https://dx.doi.org/10.20517/rdodj.2023.37

Further studies using other cells or animal models showed different potential effects of excess misfolded
GLA proteins in the ER. A GLA-knock-out HEK293 cell line model induced by transient transfection and
[24]
expression of mutated GLA proteins caused ER stress and UPR activation . Different responses to ER
stress and UPR may be a cell-type dependent mechanism, implying that not every cell line or tissue
responds equally to an excess of misfolded proteins , meaning that pathogenic mechanisms associated
[25]
with UPR could be tissue-dependent.

In a fly model, expression of GLA variants resulted in ER retention, ER-associated degradation, and UPR
activation, which was improved by the pharmacological chaperone migalastat . Interestingly, further
[26]
research in the flies showed that UPR activation resulted in the death of dopaminergic cells and a shorter
life span of the flies.

Nikolaenko et al. recently highlighted another point of view of ER stress, which is not directly associated
with misfolded GLA due to changes in their primary sequence but with exposure to high Lyso-Gb3 levels.
The evidence from a proteomic study revealed that exposure to Lyso-Gb3 affected protein folding and
ubiquitination pathways. High Lyso-Gb3 levels may cause direct disruption of the chaperone system and the
[27]
cytosol in the ER, increasing ubiquitination .

The Role of ER stress and UPR may suggest that FD is not only a storage disease but also a gain of function
component due to ER retention of mutant protein and ER stress due to an excess of accumulated
glycolipids.

THE GLOMERULAR AND VASCULAR COMPARTMENT
Upon routine histochemical observation of glomeruli from Fabry kidney biopsies, Gb3 deposits are
displayed as empty vacuoles in the cytoplasm of all cells, prominently in podocytes. Usually, mesangial areas
are enlarged with an increase in the extracellular matrix and a proliferation of the mesangial cells. Moreover,
focal segmental glomerulosclerosis can typically be present due to podocyte damage. Over time, these
lesions result in diffuse global glomerular sclerosis, and clinically, the changes translate into proteinuria and
progressive impairment of renal function with an overt chronic renal disease .
[28]

The podocyte has been extensively investigated in FN. Sanchez-Nino demonstrated that upon exposure of
human podocytes in vitro to increasing concentrations of Lyso-Gb3, these cells release inflammatory
mediators of glomerular damage, such as Transforming Growth Factor beta [TGFβ]. TGFβ is a powerful
activator of collagen and fibronectin synthesis and determines an increase in extracellular matrix
production and tissue fibrosis . Later, she demonstrated that Gb3 increases the NOTCH-1 signaling, a
[29]
podocyte injury mediator, with the NFκB activation. NFκB is the trigger to stimulate the release of pro-
inflammatory and profibrotic cytokines . The deposition of Gb3 into the podocytes causes a progressive
[30]
injury and the detachment of the cell into urine: so-called podocyturia . The podocyte has limited or no
[31]
turnover. The reduction of podocytes is associated with segments of the denuded glomerular basement
membrane, changes in the slit diaphragm, and subsequent areas of segmental glomerular sclerosis.

The mesangial cells proliferate, and an increase in mesangial extracellular matrix coexists. Indeed, these
[32]
cells, under Gb3 deposition, release pro-inflammatory and profibrotic cytokines . Mesangial cells express
the plasma membrane TLR4 that, following the recognition of Gb3, start the innate immune response
[33]
with the release of cytokines . Moreover, the mesangium is colonized by inflammatory circulating cells
[9]
such as myofibroblasts, macrophages, and monocytes, stimulating local inflammation by releasing IL1β and
TNFα . In the urine of Fabry patients, proteomic studies have found many signals of tissue inflammation.
[33]