1. Volume 95, Issue 1, Pages 149-159 (January 2019)
α-galactosidase A deficiency promotes von Willebrand factor secretion in models of Fabry disease 
Justin J. Kang, Nayiri M. Kaissarian, Karl C. Desch, Robert J. Kelly, Liming Shu, Peter F. Bodary, James A. Shayman 
Kidney International 
Volume 95, Issue 1, Pages (January 2019)
DOI: /j.kint
Copyright © 2018 International Society of Nephrology Terms and Conditions

2. Figure 1
Age-dependent endothelial activation in mice with Fabry disease. Blood was drawn via the retro-orbital plexus from male wild-type (WT) and Gla-deficient mice at the indicated ages. (a) Levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) were measured by enzyme-linked immunosorbent assay (n = 5–10 per group). *P < 0.05 compared wiht the age-matched WT mice, and †P < compared with the same genotype at 2 months. (b) Circulating plasma von Willebrand factor (VWF) levels were measured by AlphaLISA (PerkinElmer, Waltham, MA) method as described in the Methods section (n = 4–8 in each age group). A dilution series of pooled platelet-poor plasma (PPP) from C56BL/6 mice (n = 10) was used as a reference (100%). *P < 0.05 compared with the age-matched WT. †P < 0.02 compared with the same genotype at 2 months.
Kidney International  , DOI: ( /j.kint )
Copyright © 2018 International Society of Nephrology Terms and Conditions

3. Figure 2
Elevated von Willebrand factor (VWF) secretion in EA.hy926 cells following α-galactosidase A (GLA) knockdown. (a) Representative immunoblot showing the knockdown efficiency of siRNA against GLA in EA.hy926 cells. (b) Cells were transfected with serum media under control (SCR) or GLA siRNA (siGLA). After the second transfection, the transfection media were replaced with 2% serum media and incubated for 3 days. Levels of VWF accumulation in the media were determined by enzyme-linked immunosorbent assay using a dilution series of pooled normal plasma with known VWF antigen levels as standards and expressed as fold changes with respect to SCR siRNA (siSCR) (n = 6–8 per group). *P < To optimize viewing of this image, please see the online version of this article
Kidney International  , DOI: ( /j.kint )
Copyright © 2018 International Society of Nephrology Terms and Conditions

4. Figure 3
Decreased nitrous oxide (NO) production and suppression of von Willebrand factor (VWF) secretion by an exogenous NO donor, DETA-NONOate. (a) The cells were collected after siRNA transfection. Endothelial NO synthase activity was measured by monitoring the conversion from radiolabeled L-arginine to the formation of L-citrulline. The activity was expressed as L-citrulline count per minute (cpm), and normalized to each sample’s total protein level (n = 6 per group) *P < (b) Following transfection, cells were incubated with vehicle (10 mM HEPES) or DETA-NONOate (50 μM) for 3 days. VWF levels in the cell culture supernatants were determined by enzyme-linked immunosorbent assay and expressed as fold changes with respect to serum media under control siRNA (siSCR) vehicle (n = 4 per group). *P < 0.01 compared with siSCR vehicle, and †P = 0.02 compared with α-galactosidase A siRNA (siGLA) vehicle.
Kidney International  , DOI: ( /j.kint )
Copyright © 2018 International Society of Nephrology Terms and Conditions

5. Figure 4
Time course of basal von Willebrand factor (VWF) secretion from CRISPR wild-type (CR-WT) and α-galactosidase A (GLA) deficient cells. (a) Equal amounts of protein (3 μg) from cell lysates were used. GLA activity in lysates from CRISPR-WT (CR-WT) and CRISPR-GLA (CR-GLA) was expressed as mmol/mg protein per hour para-nitrophenyl released (n = 6 per group). N.D., not detected. (b) Normal growth media were replaced with 2% serum media after confluency. The cell culture supernatants were collected at the various time points thereafter (n = 4 per group). VWF levels were expressed as fold changes with respect to CR-WT at 12 hours. (c) VWF secretion after 2 to 3 days (n = 11 per group). *P < (d) After confluency, cells were incubated with vehicle (10 mM HEPES) or DETA-NONOate (100 μM) for 3 days. VWF levels in the cell culture supernatants were determined by enzyme-linked immunosorbent assay and expressed as fold changes with respect to CR-WT vehicle (n = 8 per group). †P < compared with vehicle in the same group, and *P < compared with wild-type vehicle.
Kidney International  , DOI: ( /j.kint )
Copyright © 2018 International Society of Nephrology Terms and Conditions

6. Figure 5
Endothelial nitric oxide synthase (eNOS) dysregulation and sepiapterin treatment in CRISPR (CR) wild-type (WT) and α-galactosidase A (GLA) cells. (a) Equal amounts of protein (30 μg) from CR-WT and CR-GLA cells were separated on 4% to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (n = 9 per group). *P < (b) Cells were incubated with vehicle (dimethyl sulfoxide, DMSO) or various concentrations of sepiapterin for 24 hours. Trypsinized cells were pelleted and subjected to eNOS activity assay. eNOS activity was determined as described in Figure 3. The data are an average of triplicate assays from 2 separate sets of samples. (c) After confluency, cells were incubated with vehicle (DMSO) or sepiapterin (300 μM) for 3 days. von Willebrand factor (VWF) levels in the cell culture supernatants were determined by enzyme-linked immunosorbent assay and expressed as fold changes with respect to CR-WT vehicle (n = 4 per group). P = 0.01 between CR-WT vehicle and CR-GLA with sepiapterin, †P < compared with vehicle in the same group, *P < compared with CR-WT vehicle, and ‡P < compared with CR-WT with sepiapterin. (d) After confluency, cells were incubated with vehicle (DMSO) or sepiapterin (300 μM) for 3 days. These cells were trypsinized and subjected to eNOS activity assay as described above (n = 4 per group). †P < compared with vehicle in the same group, *P < compared with CR-WT vehicle, and ‡P < compared with CR-WT with sepiapterin. To optimize viewing of this image, please see the online version of this article
Kidney International  , DOI: ( /j.kint )
Copyright © 2018 International Society of Nephrology Terms and Conditions

7. Figure 6
Increased N-ethylmaleimide sensitive factor (NSF) S-nitrosylation and decreased TRX-1 in α-galactosidase A (GLA)–deficient cells. (a) EA.hy926 cells treated with siSCR or siGLA were subjected to the biotin switch assay as described in the Methods section. Isolated biotin-labeled proteins were separated on 4% to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The level of S-nitroyslation of NSF was normalized to its total protein level (n = 6 per group). *P < (b) An equal amount of protein from siRNA-treated cells was loaded and separated on SDS PAGE (n = 6 per group). * P < (c) Confluent CRISPR wild-type (CR-WT) and CRISPR-GLA (CR-GLA) cells were subjected to the biotin switch assay. Isolated biotin-labeled proteins were separated on 4% to 12% SDS PAGE. The level of S-nitroyslation of NSF was normalized to its total protein level (n = 6 per group). *P < (d) An equal amount of protein from CR-WT and CR-GLA cells was loaded and separated on SDS PAGE (n = 6 per group). *P < To optimize viewing of this image, please see the online version of this article
Kidney International  , DOI: ( /j.kint )
Copyright © 2018 International Society of Nephrology Terms and Conditions

8. Figure 7
Decrease in von Willebrand factor (VWF) secretion with antioxidant treatments. (a) After confluency, cells were incubated with vehicle (dimethyl sulfoxide 0.2%) or ebselen (10 μM) for 3 days. VWF levels in the cell culture supernatants were determined by enzyme-linked immunosorbent assay and expressed as fold changes with respect to CRISPR wild-type (CR-WT) vehicle (n = 6 per group). P = between CR-WT vehicle and CRISPR α-galactosidase A (CR-GLA) with ebselen, †P < compared with vehicle in the same group, *P < compared with CR-WT vehicle, and ‡P < 0.05 compared with CR-WT with ebselen. (b) After confluency, cells were incubated with vehicle or Tempol (1 mM) for 3 days. VWF levels in the cell culture supernatants were determined by enzyme-linked immunosorbent assay and expressed as fold changes with respect to CR-WT vehicle (n = 6 per group). †P < compared with vehicle in the same group, *P < compared with CR-WT vehicle, and ‡P < compared with CR-WT with Tempol.
Kidney International  , DOI: ( /j.kint )
Copyright © 2018 International Society of Nephrology Terms and Conditions

9. Figure 8
Treatment of CRISPR wild-type (CR-WT) and CRISPR α-galactosidase A (CR-GLA) cells with recombinant human GLA and eliglustat. (a) CR-WT and CR-GLA cells were seeded in a 24-well plate. The next day, vehicle (25 mM Tris and 150 mM NaCl, pH 7.5) or recombinant human GLA (10 μg/ml, α-Gal A) was added to each well. After 2 days of the treatment, the medium was replaced with 2% serum medium containing vehicle or GLA and incubated for 3 days. von Willebrand factor (VWF) levels were determined by enzyme-linked immunosorbent assay and expressed as fold changes with respect to CR-WT vehicle (n = 6–10 per group). P = 0.13 between vehicle- and GLA-treated CR-GLA; *P < compared with CR-WT vehicle. (b) CR-WT and CR-GLA cells were seeded in a 24-well plate. The following day, vehicle (Dulbecco’s modified Eagle’s medium media) or eliglustat (200 nM) was added to each well. After 2 days of the treatment, the medium was replaced with 2% serum medium containing vehicle or eliglustat and incubated for an additional 3 days. VWF levels were determined by enzyme-linked immunosorbent assay and expressed as fold changes with respect to CR-WT vehicle (n = 6 per group). *P < compared with CR-WT. †P < 0.02 compared with each vehicle condition.
Kidney International  , DOI: ( /j.kint )
Copyright © 2018 International Society of Nephrology Terms and Conditions