1. Volume 79, Issue 2, Pages 199-209 (January 2011)
Cell division autoantigen 1 enhances signaling and the profibrotic effects of transforming growth factor-β in diabetic nephropathy 
Yugang Tu, Tieqiao Wu, Aozhi Dai, Yen Pham, Phyllis Chew, Judy B. de Haan, Yu Wang, Ban-Hock Toh, Hongjian Zhu, Zemin Cao, Mark E. Cooper, Zhonglin Chai 
Kidney International 
Volume 79, Issue 2, Pages (January 2011)
DOI: /ki
Copyright © 2011 International Society of Nephrology Terms and Conditions

2. Figure 1
Diabetes induces renal fibrosis in spontaneous hypertensive rats (SHR). (a and b) Paraffin-embedded kidney sections of SHR with streptozotocin-induced diabetes for 32 weeks (Dia) and age-matched non-diabetic control SHR (Con) were stained by Masson's Trichrome method to show extracellular matrix (ECM) in blue color (a) and by immunohistochemical staining using antibodies (1:1000 dilution) to α-smooth muscle actin (α-SMA) to show α-SMA in brown color (b), with scale bars of 300 μm shown (original magnification, × 200). A negative control with no primary antibody is shown for the immunohistochemical staining of an SHR (Ab-). Positive staining was quantified using the Image-Pro Plus program. Approximately 10 random views were measured per section to give an average arbitrary unit. A graph of quantification is shown for ECM (a) and α-SMA (b) in non-diabetic control (empty bar) and diabetic groups (solid bar). (c) mRNA levels of the following proteins in the kidneys of non-diabetic control (empty bar) and diabetic SHR groups (solid bar) as determined by quantitative real-time reverse transcription-PCR are shown: transforming growth factor (TGF)-β1 (TGFβ), TGF-β type I receptor (TβRI), TGF-β type II receptor (TβRII), connective tissue growth factor (CTGF), α-SMA, collagen I (COL1), III (COL3), IV (COL4), and fibronectin (FN). Values are shown as means±s.e. s.e. (n=7 for control; n=9 for diabetic group) is shown as error bar. *P<0.05 and **P<0.001 vs non-diabetic control group.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

3. Figure 2
Cell division autoantigen 1 (CDA1) expression levels are increased in diabetic spontaneous hypertensive rat (SHR) kidneys. CDA1 protein (a, b) was detected in SHR kidney sections by immunohistochemical staining, and CDA1 mRNA levels (c) were determined by quantitative real-time reverse transcription-PCR. (a) CDA1 was stained in brown color mostly in tubules (Ab), which was removed by preabsorption of the antibody with antigen (Ab+Ag). Scale bar of 300 μm is shown (original magnification, × 200) for these two pictures. CDA1 was also detected in podocytes (red arrow) within glomerulus (glo) with a scale bar of 50 μm (original magnification, × 400). (b) CDA1 was stained in the kidney sections of non-diabetic control (Con) and 32-week diabetic (Dia) SHR and shown in brown color (left panels). Scale bar of 600 μm is shown (original magnification, × 100). CDA1 staining was quantified (right panel) and analyzed as described in the legend of Figure 1, and shown as arbitrary units for the non-diabetic (empty bar) and diabetic groups (solid bar). (c) CDA1 mRNA levels in the kidneys of non-diabetic (empty bar) and diabetic groups (solid bar) are shown. Values are means±s.e. s.e. (n=7 for control; n=9 for diabetic group) is shown as error bar. ‡P<0.001 vs non-diabetic control group.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

4. Figure 3
Cell division autoantigen 1 (CDA1) expression in human kidney and proximal tubule cell line HK-2. (a) Immunohistochemical staining of a human kidney section showing CDA1 detected in both tubular cells and glomerular cells (Ab) using anti-CDA1 antibody (1:1000 dilution), which was removed by preabsorption of the antibody with antigen (Ab+Ag). A scale bar of 300 μm is shown (original magnification, × 200). Staining of CDA1 in podocytes (red arrow) within the glomerulus (glo) is shown with a scale bar of 50 μm (original magnification, × 400). (b) Western blot analysis showing CDA1 in human kidney (K) and lung (L) tissue homogenates, and in the human proximal tubule cell line HK-2 (HK2) transfected with Myc-tagged CDA1 expressing plasmid. Blot was reprobed for α-tubulin. (c) Reverse transcription-PCR detection of CDA1 mRNA in podocytes. Total RNA isolated from a human podocyte cell line (Pod)34 and from HK-2 cells as control (HK2) was reverse transcribed using random primers and the synthesized complementary DNA was used as a template for PCR using a pair of CDA1-specific primers (forward primer: 5′-CCGGAATTCGAGGTGGTGATCATGGAAGAC-3′; reverse primer: 5′-ACGCGTCGACTCATCCTCTAGGTCAGAGTCG-3′, underlined is sequence identical to CDA1). The primers for β-actin are as follows: forward primer 5′-GAGGCCCAGAGCAAGAGAG-3′ and reverse primer 5′-GGCTGGGGTGTTGAAGGT-3′. The PCR-amplified products at the expected size of ∼800 bp for CDA1, 225 bp for β-actin, and DNA standards (M) were shown after a 1.2% agarose gel electrophoresis.
Kidney International  , DOI: ( /ki )
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5. Figure 4
Transforming growth factor (TGF)-β, connective tissue growth factor (CTGF), and extracellular matrix genes are affected by alteration in cellular levels of cell division autoantigen 1 (CDA1) in HK-2 cells. (a) HK-2 cells were infected with no virus (white bars), control adenovirus at low dose (∼100 multiplicity of infection, MOI) (gray bars), and CDA1-expressing adenovirus at low dose (∼100 MOI) (hatched bars) and high dose (∼600 MOI) (black bars) for 24 h. mRNA levels of specified proteins as determined by quantitative real-time reverse transcription-PCR are shown as arbitrary units. Values are means±s.e. s.e. (n=6) is shown as error bar. *P<0.001 vs no virus control and †P<0.001 vs low dose (100 MOI) CDA1-expressing adenovirus group. (b) HK-2 cells were stably transduced with no retrovirus (white bars), vector control retrovirus (gray bars), and CDA1 siRNA m957 retrovirus (black bars). mRNA levels of these cells were shown for proteins as specified. Values are means±s.e. s.e. (n=6) is shown as error bar. *P<0.001 and †P<0.05 vs no virus control. (c) Western blot showing CDA1 and α-tubulin in HK-2 cells stably transduced with no virus (NV), control retrovirus (Vect), and CDA1 small interfering RNA (siRNA) m957 retrovirus (siRNA). FN, fibronectin.
Kidney International  , DOI: ( /ki )
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6. Figure 5
Intracellular levels of cell division autoantigen 1 (CDA1) affect transforming growth factor (TGF-β) activity in stimulating TGF-β-responsive reporter constructs in HK-2 cells. (a) HK-2 cells stably transduced with vector retrovirus (Con) and retrovirus expressing CDA1 small interfering RNA (siRNA) m957 (siR) were co-transfected with pGL3-BASIC, a promoter-less luciferase plasmid (BASIC), p3TP-Lux (3TP), or p(CAGA)12-Luc (CAGA) with a cytomegalovirus (CMV)-β-galactosidase plasmid by electroporation as described previously.10 Approximately 24 h after electroporation, the cells cultured in six-well plates were treated with TGF-β at various concentrations for another 30 h. TGF-β concentrations used are 0.00 (white bars), 0.05 (gray bars), 0.10 (hatched bars), and 0.20 ng/ml (black bars). Luciferase activity was normalized against the β-galactosidase activity of each sample. Values are means±s.e. s.e. (n=6) is shown as error bar. *P<0.001 vs no TGF-β treatment (0.00 ng/ml) within the same group and †P<0.001 vs vector retrovirus transduced cells (Con) treated with the same concentration of TGF-β. (b) HK-2 cells were co-transfected with p3TP-Luc and CMV-galactosidase plasmids. After 24 h culture, the cells were infected with a vector control adenovirus (Ad-Con) and CDA1 expressing adenovirus (Ad-CDA1) at, 0, 250, 500, and 1000 multiplicity of infection (MOI) for 24 h, and then treated without or with TGF-β at 0.2 ng/ml for another 30 h. Luciferase activity was normalized against the β-galactosidase activity of each sample. Values are means±s.e. s.e. (n=6) is shown as error bar. *P<0.001 vs no adenovirus and no TGF-β treatment and †P<0.001 vs the same adenovirus dose with no TGF-β treatment.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

7. Figure 6
Cell division autoantigen 1 (CDA1) overexpression enhances and CDA1 knockdown blocks the activity of transforming growth factor (TGF)-β in stimulating expression of extracellular matrix genes. (a and b) Western blot analysis shows CDA1 and α-tubulin in HK-2 cells infected by a control adenovirus (Ad-Con) and CDA1-expressing adenovirus (Ad-CDA1) at specified multiplicity of infection (MOI) for 24 h followed by incubation in fresh medium for a further 30 h (a), or by adenoviruses expressing CDA1 small interfering RNA (siRNA) m95710,16 or CDA1 siRNA 1562 (target sequence: 5′-CAGCGAGAAUCCUGACCACAA-3′) (b). (c–e) Five groups of HK-2 cells were infected for 24 h with no virus (NV), control adenovirus (Con), CDA1-expressing adenovirus (CDA1), and adenoviruses expressing siRNA m957 and 1562 to silence CDA1 (siRNA). These cells were then treated or not treated with TGF-β (0.2 ng/ml) for a further 30 h. Total RNA was extracted and real-time PCR was performed to measure the relative mRNA levels for CDA1 (c), collagen III (d), and collagen I (e). Values are means±s.e. s.e. (n=6) is shown as error bar. *P<0.05 and ‡P<0.001 vs no TGF-β treatment; §P<0.05 and †P<0.001 vs HK-2 cells with no virus and no TGF-β treatment, or with control adenovirus and no TGF-β treatment.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

8. Figure 7
Cell division autoantigen 1 (CDA1) modulates the transforming growth factor (TGF)-β type I but not the type II receptor to affect TGF-β signaling in CDA1 Tet-Off HeLa cells. (a) CDA1 overexpression alone induces phosphorylation of Smad3. The CDA1 Tet-off HeLa cells were cultured with decreasing concentrations of doxycycline as specified to regulate expression of the CDA1 transgene for 72 h. Western blot analysis shows CDA1, phospho-Smad3 (Ser433/435) (p-Smad3), Smad2/3, and α-tubulin. (b) CDA1 enhances TGF-β-responsive luciferase reporter activity. CDA1 Tet-off HeLa cells were co-transfected with the cytomegalovirus promoter-driven β-galactosidase expression plasmid and the TGF-β-responsive luciferase reporter construct p3TP-Lux (3TP), or Smad3-specific reporter construct p(CAGA)12-Luc ((CAGA)12). The cells were cultured in the absence of doxycycline to turn on, or in the presence of 5 ng/ml doxycycline to turn off, the CDA1 transgene for 48 h. Luciferase activities normalized against the co-transfected β-galactosidase activities are shown as fold induction by CDA1 overexpression (black bars) compared with control cells with the CDA1 transgene turned off (white bars). Values are means±s.e. s.e. (n=6) is shown as error bar. *P<0.001 vs CDA1 transgene turned off (white bars). (c and d) CDA1-induced phosphorylation of Smad3 and ERK1/2(p44/42) is TβRI-dependent. The TβRI inhibitor, SB (SB), was added to the CDA1 Tet-off HeLa cells at the specified concentrations when doxycycline was withdrawn from the medium to turn on the CDA1 transgene (+), or when doxycycline (5 ng/ml) was added to the medium to turn off the CDA1 transgene (-), for 24 h. Western blots are shown for protein expression levels of (c) CDA1, phospho-Smad3 (Ser435/455) (p-Smad3), Smad2/3, and GAPDH and (d) CDA1, phospho-ERK1/2(p44/42) (Thr202/Tyr204) (p-ERK1/2), and total ERK1/2. (e and f) mRNA levels of CDA1 (left panels), TβRI (central panels), and TβRII (right panels) in CDA1 Tet-off HeLa cells were determined by quantitative real-time reverse transcription-PCR. CDA1 transgene expression was either fully turned on by doxycycline withdrawal for certain periods of time as specified (e), or regulated by doxycycline at various concentrations as specified for 72 h (f). The mRNA levels relative to the cells with no CDA1 transgene expression, 0 h (e) or 5 ng/ml doxycycline (f) are shown as fold changes. Values are means±s.e. s.e. (n=6) is shown as error bar. *P<0.001 vs CDA1 transgene turned off.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

9. Figure 8
Adenovirus-delivered cell division autoantigen 1 (CDA1) overexpression in HK-2 cells resulted in Smad3 phosphorylation. (a) HK-2 cells were treated with transforming growth factor (TGF)-β at specified concentrations for 30 min. Western blot analysis shows CDA1 and GAPDH as an appropriate control for protein loading. (b) HK-2 cells were infected by a low dose (250 multiplicity of infection, MOI) (L) or a high dose (1000 MOI) (H) adenovirus, or by no virus (NV), for 24 h and then the cells were cultured for a further 30 h in fresh medium. (c) HK-2 cells were untreated (-) or treated (+) with TGF-β at 0.2 ng/ml, or with SB at 2 μM (SB), or with vehicle dimethylsulfoxide for 30 min, or pretreated with SB at 2 μM followed by TGF-β treatment at 0.2 ng/ml for 30 min. (d) HK-2 cells were infected by a low dose (250 MOI) (L) or a high dose (1000 MOI) adenovirus, or by no virus (NV), for 24 h and the cells were then untreated (-) or treated (+) with TGF-β at 0.2 ng/ml for a further 30 h. Western blots show CDA1, phospho-Smad3 (pSmad3), total Smad3, and β-actin or GAPDH.
Kidney International  , DOI: ( /ki )
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10. Figure 9
Effect of cell division autoantigen 1 (CDA1) on transforming growth factor (TGF)-β signaling and extracellular matrix gene expression is dose-dependently inhibited by the Smad3 inhibitor, SIS3. HK-2 cells were infected by CDA1 expressing adenovirus (Ad-CDA1) and a control adenovirus (Ad-Con) at 250 multiplicity of infection (MOI) in the presence of SIS3 at specified concentrations for 24 h, washed with phosphate-buffered saline, and then incubated for a further 30 h in medium containing SIS3 at the specified concentrations. (a) Western blotting analysis showing CDA1, phospho-Smad3, and α-tubulin. (b) mRNA levels of TGF-β type I receptor (TβRI) as determined by real-time PCR in the HK-2 cells. (c, d) Luciferase activities of p3TP-Lux (c) and p(CAGA)12-Luc (d). Before the adenoviral infection, the HK-2 cells were transfected with the reporter and control plasmids as described in the legend of Figure 5b and then infected with adenoviruses and treated with SIS3 as described above. (e–g) mRNA levels of collagens I (e), III (f), and IV (g), which were determined by real-time PCR. Values are means±s.e. s.e. (n=6) is shown as error bar. *P<0.001 vs Ad-Con-infected HK-2 cells; ‡P<0.01 for Ad-CDA1 groups treated with SIS3 at 1 vs 0 μM, 5 vs 1 μM, or 10 vs 5 μM.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions