1. Volume 75, Issue 4, Pages 399-407 (February 2009)
Downregulation of α-galactosidase A upregulates CD77: functional impact for Fabry nephropathy 
Thomas Thomaidis, Manfred Relle, Mitra Golbas, Christoph Brochhausen, Peter R. Galle, Michael Beck, Andreas Schwarting 
Kidney International 
Volume 75, Issue 4, Pages (February 2009)
DOI: /ki
Copyright © 2009 International Society of Nephrology Terms and Conditions

2. Figure 1
Successful silencing of α-Gal expression in primary TECs and HK2 kidney cell lines by siRNA transfection. (a) α-Gal A mRNA (425 bp) is degraded by α-Gal A-specific siRNAs in TEC. Different α-Gal A siRNA sequences (S1, S2, S3, and S4, respectively) show distinct blocking efficiencies, the highest being with sequence 1. Laminin siRNA (siL) served as a positive silencing control (TEC (lam.), 250 bp). M: 50 bp marker. (b) A combination of the three efficient siRNA sequences (S1, S3, and S4) leads to rapid and complete suppression of α-Gal A expression by HK2 cells. This combination of silencer RNAs is even more efficient than using sequence 1 alone (S1). Five days after siRNA transfection, the cells resynthesize α-Gal A mRNA, which can be detected by RT-PCR. The expression of α-Gal A mRNA is not influenced by reagents used to transfect the HK2 cells (negative control). β-Actin served as a control for the integrity of the mRNAs. M: 100 bp marker. (c) Western blot of TECs and HK2 cells, which were tested for α-Gal A expression 2 days after siRNA transfection. The protein appears in untreated cells at about 50 kDa (TEC and HK2). Almost no α-Gal A was detected in transfected cells (siTEC and siHK2). (d) Using RT-PCR, peripheral blood mononuclear cells (PBMCs) from patients with AFD were compared with those of a healthy subject (pos.). α-Gal A mRNA (425 bp) could be detected only in the PBMC of the healthy person, but was absent in Fabry PBMC (1–5). The same PBMC mRNAs were tested for β-actin expression (250 bp), which served as a control for the integrity of the mRNA. M: 50 bp marker. AFD, Anderson–Fabry disease; RT-PCR, reverse transcriptase PCR; TECs, tubular epithelial cells.
Kidney International  , DOI: ( /ki )
Copyright © 2009 International Society of Nephrology Terms and Conditions

3. Figure 2
Stable silencing of α-Gal A in HK2 and primary kidney cells by short hairpin RNAs. (a) A stable degradation of α-Gal A mRNA is achieved with shRNAs. TECs were transfected with three different recombinant vectors expressing α-Gal A-specific shRNA sequences (V1, V2, and V3). Cells transfected with sequence 1 (V1) showed a complete knockout of α-Gal A gene expression. (b) Western blots were performed to demonstrate the efficient reduction of α-Gal A protein in primary kidney cells. Although a stable transfection with shRNA vector 1 (shRNA-V1) strongly reduced the amount of α-Gal A protein, complete inactivation of the enzyme was achieved by a transient transfection with the siRNA 1. Recombinant α-Gal A (Replagal, 100 and 200 ng, respectively) served as a positive control (for the rabbit α-human Gal A antiserum). (c) Western blot analysis demonstrated that transfected TECs and HK2 (shTEC and shHK2) do not express α-Gal A protein (50 kDa), as do the native HK2 cells and TECs. β-Actin (Figure 2a) and tubulin (Figure 2c) served as controls for both the integrity of the RNA and the protein lysates, respectively. M: 100 bp marker. shRNAs, short hairpin RNAs; TECs, tubular epithelial cells.
Kidney International  , DOI: ( /ki )
Copyright © 2009 International Society of Nephrology Terms and Conditions

4. Figure 3
Upregulation of intracellular Gb3 after α-Gal silencing in TEC and HK2. Electron micrograph of a tubular epithelial cell after 2 months of stable α-Gal A silencing with shRNA. The lysosomes contain inclusions (arrows) that are pathognomonic for AFD (a). In a higher magnification, these inclusions revealed the typical ultrastructure of concentric arranged lamellae (arrows, b), which are in accordance with the lamellar deposits in human organs with Fabry disease. Even if cellular inclusions could be found in the control cells also (arrow, c), these missed the typical lamellar ultrastructure in higher magnifications (d). After membrane permeabilization, HK2 and TECs were tested for intracellular Gb3 expression under conditions of α-Gal downregulation (e). An almost 1.5- to 2-fold increase in intracellular Gb3 was observed (from 36.6 to 58.1% in HK2 cells and from to 44.5% in TECs, representative examples). AFD, Anderson–Fabry disease; shRNA, short hairpin RNA; TECs, tubular epithelial cells.
Kidney International  , DOI: ( /ki )
Copyright © 2009 International Society of Nephrology Terms and Conditions

5. Figure 4
Vitality of TEC and HK2 after α-Gal A silencing.α-Gal A silencing reduces the proliferation rate of TECs (a) and the HK2 cell line (b). After 2 months in culture, the vitality of the cells was tested with an MTT proliferation assay before (culture) and after (shRNA) shRNA transfection. The control vector had no influence on the viability of the target cells (values given as mean±s.d.; *P< versus culture, Mann–Whitney U-test). MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; shRNA, short hairpin RNA; TECs, tubular epithelial cells.
Kidney International  , DOI: ( /ki )
Copyright © 2009 International Society of Nephrology Terms and Conditions

6. Figure 5
Effect of α-Gal A silencing on CD77/Gb3 expression in TEC and HK2 cells. Flow cytometric analysis of CD77/Gb3 expression in TECs (a) and HK2 cells (b) 2 days after siRNA silencing. The silencing of α-Gal A by siRNA results in a significant upregulation of CD77, on the membrane of both HK2 cells and TECs. Transfection reagents alone had no effect on CD77 expression (data not shown). (Values given are mean±s.d.; *P<0.05, Mann–Whitney U-test). (c–f) Immunostaining of Gb3 in TECs and HK2 cells. α-Gal A ‘silenced’ TECs (d) and HK2 (f) cells demonstrate higher expression of Gb3 in comparison with non-transfected cells (c and e). The membrane distribution of positive signal reflects the upregulation of membranous CD77/Gb3 (arrows). TECs, tubular epithelial cells.
Kidney International  , DOI: ( /ki )
Copyright © 2009 International Society of Nephrology Terms and Conditions

7. Figure 6
CD77 overexpression on PBMC of patients with AFD. (a) PBMCs from Fabry patients and healthy subjects were tested by flow cytometry for membrane expression of CD77. The number of CD77-positive PBMCs from Fabry patients is upregulated (21.18%) compared with healthy subjects (example of Fabry patient). (b) A study of 18 Fabry patients and 18 healthy subjects demonstrating a significant CD77 overexpression in Fabry PBMC. Age and gender of the patients/control group are listed in Tables 1 and 2, respectively (values given are mean±s.d.; *P<0.0005, Mann–Whitney U-test). AFD, Anderson–Fabry disease; PBMC, peripheral blood mononuclear cell.
Kidney International  , DOI: ( /ki )
Copyright © 2009 International Society of Nephrology Terms and Conditions

8. Figure 7
Modulation of CD77 expression on HK2 cells by ERT. CD77 (Gb3) expression of transfected and untreated (-) HK2 cells was examined by FACS analysis before and after ERT. Stable silencing of α-Gal A for more than 2 months with shRNAs caused a significant CD77 increase on the cell membrane (shRNA). The administration of 10 μg/ml of agalsidase-α reduced the expression of CD77 similar to the level of non-transfected cells (shRNA+ERT) (values given are mean±s.d.; *P<0.05 versus untreated HK2, †P<0.05 versus shRNA HK2, Mann–Whitney U-test). ERT, enzyme replacement therapy; FACS, flow cytometry analysis; shRNA, short hairpin RNA.
Kidney International  , DOI: ( /ki )
Copyright © 2009 International Society of Nephrology Terms and Conditions