1. Volume 83, Issue 2, Pages 251-263 (February 2013)
Growth arrest–specific protein 1 is a novel endogenous inhibitor of glomerular cell activation and proliferation 
Claudia R.C. van Roeyen, Stephanie Zok, Jessica Pruessmeyer, Peter Boor, Yoshikuni Nagayama, Stefan Fleckenstein, Clemens D. Cohen, Frank Eitner, Hermann-Josef Gröne, Tammo Ostendorf, Andreas Ludwig, Jürgen Floege 
Kidney International 
Volume 83, Issue 2, Pages (February 2013)
DOI: /ki
Copyright © 2013 International Society of Nephrology Terms and Conditions

2. Figure 1
Platelet-derived growth factor-BB (PDGF-BB) and -DD regulate growth arrest–specific protein-1 (GAS1) expression in mesangial cells (MCs). PDGF-BB and -DD, but not PDGF-AA and -CC, induce a downregulation of GAS1 mRNA as determined by real-time reverse transcriptase PCR and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Before the experiment, human MCs (HMCs) had been cultured for 72h in MCDB medium to upregulate GAS1 expression. Data are means±s.d. of four independent experiments. *Indicates P<0.05 versus unstimulated cells.
Kidney International  , DOI: ( /ki )
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3. Figure 2
Growth arrest–specific protein-1 (GAS1) is secreted in the supernatant by mesangial cells (MCs). (a) Increased concentrations of released GAS1 protein could be detected in the supernatant of rat MCs (RMCs) transfected with an expression vector encoding for full-length GAS1 cDNA compared with mock-transfected rat MCs. In addition, GAS1 protein concentrations were increased in cell lysates of GAS1-overexpressing cells in comparison with mock-transfected cells. Data are means±s.d. of three independent experiments. (b) GAS1 mRNA, as detected by real-time reverse transcriptase PCR and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, following growth arrest in rat MCs. Data are means±s.d. of six independent experiments. *Indicates P<0.05 versus fetal calf serum (FCS)–containing medium. (c) Increased GAS1 protein concentrations could be detected in lysates of rat MCs incubated in MCDB medium for 24h compared with rat MCs growing in the presence of FCS in a western blot analysis. As a loading control, the membrane was re-incubated with an anti-GAPDH antibody. Data of two independent experiments are shown. Mean values of both experiments are shown in the morphometric analysis. (d) Growth arrest of rat MCs resulted in increased secreted endogenous GAS1 protein concentrations. GAS1 protein concentrations were measured in the supernatant following growth arrest in rat MCs. Data are means±s.d. of three independent experiments. *P<0.05 versus FCS-containing medium. (e) Secreted GAS1 protein could be detected in the supernatant of growth-arrested rat MCs by western blot analysis. The figure shows a representative blot.
Kidney International  , DOI: ( /ki )
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4. Figure 3
The proteases disintegrin and metalloproteinase (ADAM) 10 and 17 are involved in the secretion of growth arrest–specific protein-1 (GAS1). (a) Rat mesangial cells (MCs) were grown up to 80–90% confluence in fully supplemented medium, and the medium was changed to MCDB medium or RPMI plus 10% fetal calf serum (FCS) with GI254023x (10μmol/l; inhibits ADAM10), GW280264x (10μmol/l; inhibits ADAM10+17), or dimethylsulfoxide (DMSO). After 3h, the cells were stimulated with phorbol 12-myristate 13-acetate (PMA; 200ng/ml) or ionomycin (1μmol/l). Conditioned media were harvested and analyzed by enzyme-linked immunosorbent assay. Data are means±s.d. of three independent experiments. N: no inhibitors added; *P<0.05 versus DMSO-treated cells; $P<0.05 versus PMA-treated cells; §P<0.05 versus ionomycin-treated cells. (b) ADAM activity was measured in cell lysates using a fluorogenic peptide–based substrate mimicking the α-cleavage site of amyloid β-protein precursor (Ac-RE(EDANS)-VHHQKLVF-K(DABCYL)-R-OH). Data are means±s.d. of three independent experiments; N: no inhibitors added; *P<0.05 versus DMSO-treated cells; $P<0.05 versus PMA-treated cells; §P<0.05 versus ionomycin-treated cells. (c) ADAM10 and 17 expression is regulated during growth arrest of rat MCs. ADAM10 and 17 mRNA, as detected by real-time reverse transcriptase PCR and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, following growth arrest in MCs. For this, rat MCs were switched from RPMI plus 10% FCS at 0h to MCDB medium. Data are means±s.d. of six independent experiments; *P<0.05 versus time point 0h. GI, GI254023x; GW, GW280264x.
Kidney International  , DOI: ( /ki )
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5. Figure 4
Growth arrest–specific protein-1 (GAS1) inhibits the growth of proliferating rat mesangial cells (RMCs). (a) Proliferating rat MCs can be growth-inhibited by adding recombinant GAS1 protein. Cells growing in the presence of 2% fetal calf serum (FCS) were treated with 50, 25, 10, 5, or 1ng/ml recombinant mouse GAS1 protein. Cell proliferation was measured by 5-bromo-2-deoxyuridine incorporation and enzyme-linked immunosorbent assay. Data are means±s.d. of six independent experiments. *Indicates P<0.05 versus medium without GAS1 protein. (b) Overexpression of soluble GAS1/Fc fusion protein downregulated platelet-derived growth factor-B (PDGF-B) mRNA expression in proliferating rat MCs; *indicates P<0.05 versus mock-transfected cells. (c) In growth-arrested rat MCs, the mRNA expression of Gli 1 was downregulated, whereas Gli 2 and 3 were overexpressed. The soluble GAS1/Fc fusion protein reduced the mRNA expression of Gli in growth-arrested, but not in proliferating, rat MCs. *Indicates P<0.05 versus proliferating mock-transfected rat MCs; $ indicates P<0.05 versus mock-transfected growth-arrested rat MCs. PBS, phosphate-buffered saline; pfuse, control-transfection plasmid; VM, medium with FCS.
Kidney International  , DOI: ( /ki )
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6. Figure 5
Growth arrest–specific protein-1 (GAS1) expression and secretion in kidneys of healthy and nephritic rats. (a) In the healthy rat kidney, as well as in nephritic rats during different time points of the anti-Thy1.1 nephritis, GAS1 is expressed in podocytes. Faint overexpression is also present in the expanded mesangium on day 7 (original magnifications: × 600). (b) GAS1 expression in podocytes (WT-1 positive) and the activated mesangium (α-smooth muscle actin (αSMA) positive) was confirmed on double immunostaining. (c) Glomerular expression of GAS1 in the course of anti-Thy1.1 mesangioproliferative nephritis. Rats were killed at 4h and at days 1, 2, 4, 7, 9, 14, 21, and 28 after disease induction (n=9 each). RNA was isolated from glomeruli, and the expression of GAS1 mRNA was measured by real-time reverse transcriptase PCR. The figure shows the transcript expression (means±s.d.) in relation to the expression in non-nephritic rats. *Indicates P<0.05 versus non-nephritic phosphate-buffered saline (PBS)–treated rats. (d) Secretion of GAS1 protein in the urine was decreased in nephritic rats compared with healthy rats. *Indicates P<0.05 versus healthy, PBS-treated rats. U-Crea, urinary creatinine.
Kidney International  , DOI: ( /ki )
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7. Figure 6
Growth arrest–specific protein-1 (GAS1) shedding by disintegrin and metalloproteinase (ADAM) proteases in podocytes in vitro. (a) GAS1 mRNA expression and soluble GAS1 protein concentration in the supernatant was increased in differentiated podocytes in comparison with undifferentiated podocytes in vitro (n=3). The figure shows transcript expression (means±s.d.) in relation to undifferentiated podocytes. *Indicates P<0.05 versus undifferentiated podocytes. (b) By reverse transcriptase PCR, increased mRNA expression of ADAM10 and 17 was detected in differentiated podocytes in comparison with undifferentiated podocytes (n=3). The figure shows the transcript expression (means±s.d.) in relation to undifferentiated podocytes. *Indicates P<0.05 versus undifferentiated podocytes. (c) Podocytes were treated with GI254023x (10μmol/l; inhibits ADAM10), GW280264x (10μmol/l; inhibits ADAM10+17), TAPI-1 (20μmol/l; inhibits predominantly ADAM17), or dimethylsulfoxide (DMSO). ADAM activity was measured in cell lysates using a fluorogenic peptide–based substrate mimicking the α-cleavage site of amyloid β-protein precursor (Ac-RE(EDANS)-VHHQKLVF-K(DABCYL)-R-OH). Data are means±s.d. of seven independent experiments; *P<0.05 versus DMSO-treated cells. (d) Podocytes were treated with ADAM-specific inhibitors or DMSO. Conditioned media were analyzed by enzyme-linked immunosorbent assay. Data are means±s.d. of 3–7 independent experiments. *P<0.05 versus DMSO-treated cells; §P<0.05 versus 2 × DMSO-treated cells. GI, GI254023x; GW, GW280264x.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

8. Figure 7
Disintegrin and metalloproteinase (ADAM) 10 and 17 expression and activity are regulated during the course of anti-Thy1.1 glomerulonephritis. (a) Glomerular expression of ADAM10 and 17 in the course of anti-Thy1.1 mesangioproliferative nephritis. Rats were killed at 4h and at days 1, 2, 4, 7, 9, 14, 21, and 28 after disease induction (n=9 each). RNA was isolated from the glomeruli, and the expression of ADAM10 and 17 mRNA was measured by real-time reverse transcriptase PCR. The figure shows the transcript expression (means±s.d.) in relation to the expression in non-nephritic rats. *Indicates P<0.05 versus non-nephritic rats. (b) Glomerular ADAM activity in the course of anti-Thy1.1 nephritis in rats. Rats were killed at 4h and at days 1, 2, 4, 7, 9, 21, and 28 after disease induction. Glomeruli were isolated and ADAM17 activity was measured (see legend of Figure 3). The figure shows the α-secretase activity (means±s.d.) in relation to the activity in non-nephritic rats. Specificity of α-secretase activity measurement was shown using ADAM10- and 17-specific inhibitors in non-nephritic and nephritic (day 7) rats. *Indicates P<0.05 versus non-nephritic rats; #indicates P<0.05 versus nephritic rats (day 7). PBS, phosphate-buffered saline; GI, GI254023x; GW, GW280264x.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

9. Figure 8
Growth arrest–specific protein-1/Fc (GAS1/Fc) fusion protein inhibits the proliferation of mesangial cells (MCs). (a) Rat MCs were transfected with an expression plasmid encoding for an extracellular GAS1 protein fragment (without glycosyl-phosphatidyl-inositol anchor) fused with a c-terminal rat Fc fragment (pfuse-GAS1). GAS1 mRNA expression was measured using real-time reverse transcriptase PCR and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, following growth arrest in rat MCs. For this, rat MCs were switched from RPMI plus 10% fetal calf serum (FCS) at 0h to MCDB medium and incubated for 24h. Data are means±s.d. of four independent experiments. The overexpressed GAS1/Fc fusion protein was secreted into the supernatant as shown in anti-GAS1 enzyme-linked immunosorbent assay and western blot analyses. (b) Proliferation of rat MCs was measured by 5-bromo-2-deoxyuridine incorporation. Cells overexpressing GAS1/Fc fusion protein showed a reduced proliferation rate in comparison with mock-transfected rat MCs after incubation in MCDB, as well as in FCS-containing medium, for 24h. Data are means±s.d. of four independent experiments. (c) Incubation of rat MCs with conditioned media from rat MCs overexpressing GAS1/Fc fusion proteins resulted in a decreased proliferation compared with incubation with conditioned media from control-transfected rat MCs.
Kidney International  , DOI: ( /ki )
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10. Figure 9
Systemic overexpression of growth arrest–specific protein-1/Fc (GAS1/Fc) fusion protein during anti-Thy1.1 nephritis ameliorates mesangioproliferative changes in vivo. Nephritic rats were electroporated with an expression vector encoding for the GAS1/Fc fusion protein (n=8) or the control plasmid (n=8) pfuse at day 3 after disease induction. (a) GAS1 protein expression could be detected in muscle tissue of GAS1/Fc-overexpressing rats. Increased GAS1 concentrations could be detected in the serum of pfuse-GAS1 electroporated rats from 3 days after electroporation. At day 9, GAS1 serum concentrations were increased threefold compared with control-transfected rats; *indicates P<0.05 versus serum GAS1 concentration at day 2 before electroporation. (b) The de novo expression of α-smooth muscle actin in glomeruli was reduced after systemic GAS1/Fc overexpression as detected in immunohistochemical staining.
Kidney International  , DOI: ( /ki )
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11. Figure 10
Growth arrest–specific protein-1 (GAS1) expression in healthy and diseased human kidneys. (a) GAS1 mRNA expression was measured using real-time reverse transcriptase PCR in glomeruli isolated from renal biopsies obtained from patients with proliferative lupus nephritis (SLE, n=13), IgA nephropathy (IgAN, n=16), or pretransplant biopsies from living donors (LDs, n=7). In IgA nephropathy, decreased glomerular GAS1 mRNA expression was observed. (b) In the healthy human kidney, as well as in biopsies from patients with different renal diseases, GAS1 was expressed in podocytes and downregulated in IgA nephropathy (original magnifications: × 400). GN, glomerulonephritis.
Kidney International  , DOI: ( /ki )
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