Chloroquine may induce endothelial injury through lysosomal dysfunction and oxidative stress.

COVID-19 is a pandemic with no end in sight. There is only one approved antiviral agent but global stocks are deemed insufficient. Despite in vitro antiviral activity, clinical trials of chloroquine and hydroxychloroquine were disappointing, and they may even impair outcomes. Chloroquine causes zebroid deposits reminiscent of Fabry disease (α-galactosidase A deficiency) and endothelial cells are key targets of COVID-19. We have explored the effect of chloroquine on cultured endothelial cells and its modulation by recombinant α-galactosidase A (agalsidase). Following dose-response studies, 0.5 µg/mL chloroquine was added to cultured human endothelial cells. Neutral red and Lysotracker were used to assess lysosomes. Cytotoxicity was evaluated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) - MTT assay and cell stress by assessing reactive oxygen species (ROS) and nitric oxide (NO). In endothelial cells, chloroquine induced dose-dependent cytotoxicity at in vitro test concentrations for COVID-19 therapy. At a sublethal concentration, chloroquine significantly induced the accumulation of acid organelles (P < 0.05), increased ROS levels, and decreased NO production (P < 0.05). These adverse effects of chloroquine on endothelial cell biology were decreased by agalsidase-ß (P < 0.05). Chloroquine-induced endothelial cell cytotoxicity and stress is attenuated by agalsidase-ß treatment. This suggests that endothelial cell injury may contribute to the failure of chloroquine as therapy for COVID-19 and may be at least in part related to causing dysfunction of the lysosomal enzyme α-galactosidase A.

Lyso-Gb3 Increases αvß3 Integrin Gene Expression in Cultured Human Podocytes in Fabry Nephropathy.

BACKGROUND: Podocyturia in Fabry nephropathy leads to glomerulosclerosis and kidney disease progression. Integrins are involved in podocyte attachment to the glomerular basement membrane. We hypothesized that in Fabry nephropathy, lyso-Gb3 could modulate αvß3 expression in podocytes. Together with UPAR, the αvß3 integrin is a key mechanism involved in podocyte detachment and podocyturia. METHODS: In cultured human podocytes stimulated with lyso-Gb3, the mRNA expression of the ITGAV and ITGB3 genes encoding integrins αv and ß3, respectively, was analyzed by RT-qPCR. RESULTS: In cultured human podocytes, lyso-Gb3 at concentrations encountered in the serum of Fabry patients increased ITGAV and ITGB3 mRNA levels within 3 to 6 h. This pattern of gene expression is similar to that previously observed for PLAUR (UPAR) gene expression but is in contrast to the delayed (24 h) upregulation of other markers of podocyte stress and mediators of injury, such as CD80, TGFß1, CD74, Notch1, and HES. CONCLUSIONS: Human podocyte stress in response to glycolipid overload in Fabry nephropathy, exemplified by lyso-Gb3, is characterized by an early increase in the expression of components of the αvß3/UPAR system, which contrasts with the delayed rise in the expression of other mediators of podocyte injury. This suggests that the αvß3/UPAR system may be a therapeutic target in Fabry nephropathy.

Expression of uPAR in Urinary Podocytes of Patients with Fabry Disease.

Background. Despite enzyme replacement therapy, Fabry nephropathy still progresses. Podocyturia is an irreversible event that antedates proteinuria and leads to chronic renal failure. We evaluated a potential mechanism of podocyte detachment via the expression of the urokinase-type Plasminogen Activator Receptor (uPAR) in urinary podocytes of Fabry patients. Methods. This is a cross-sectional study that included controls (n = 20) and Fabry patients (n = 44) either untreated (n = 23) or treated with agalsidase-ß (n = 21). Variables. Variables are estimated glomerular filtration rate (eGFR), urinary protein : creatinine ratio, and urinary uPAR+ podocyte : creatinine ratio. uPAR mRNA expression in response to lyso-Gb3, a bioactive glycolipid accumulated in Fabry disease, was studied in cultured human podocytes. Results. Controls and Fabry patients had similar age, gender, and renal function. Urinary uPAR+ podocytes were higher in patients than in controls. Untreated patients were significantly younger; had more females, and presented lower urinary protein : creatinine ratios and significantly higher urinary uPAR+ podocytes than treated subjects. In treated patients, urinary uPAR+ podocytes correlated with urinary protein : creatinine ratio (ρ = 0.5; p = 0.02). Lyso-Gb3 at concentrations found in the circulation of Fabry patients increased uPAR expression in cultured podocytes. Conclusions. Urinary podocytes expressing uPAR are increased in Fabry patients, especially in untreated patients. The potential contribution of uPAR expression to podocyte detachment merits further studies.

Enzyme Replacement Therapy for Fabry Disease

Abstract Fabry disease is a rare X-linked disease caused by the deficiency of α-galactosidase that leads to the accumulation of abnormal glycolipid. Untreated patients develop potentially lethal complications by age 30 to 50 years. Enzyme replacement therapy is the current standard of therapy for Fabry disease. Two formulations of recombinant human α-galactosidase A (agalsidase) are available in most markets: agalsidase-α and agalsidase-β, allowing a choice of therapy. However, the US Food and Drug Administration rejected the application for commercialization of agalsidase-α. The main difference between the 2 enzymes is the dose. The label dose for agalsidase-α is 0.2 mg/kg/2 weeks, while the dose for agalsidase-β is 1.0 mg/kg/2 weeks. Recent evidence suggests a dose-dependent effect of enzyme replacement therapy and agalsidase-β is 1.0 mg/kg/2 weeks, which has been shown to reduce the occurrence of hard end points (severe renal and cardiac events, stroke, and death). In addition, patients with Fabry disease who have developed tissue injury should receive coadjuvant tissue protective therapy, together with enzyme replacement therapy, to limit nonspecific progression of the tissue injury. It is likely that in the near future, additional oral drugs become available to treat Fabry disease, such as chaperones or substrate reduction therapy.