1. Volume 64, Issue 5, Pages 1632-1642 (November 2003)
Vitamin D3 up-regulated protein-1 regulates collagen expression in mesangial cells 
Tsutomu Kobayashi, Sayuri Uehara, Takanori Ikeda, Hiraku Itadani, Hidehito Kotani 
Kidney International 
Volume 64, Issue 5, Pages (November 2003)
DOI: /j x
Copyright © 2003 International Society of Nephrology Terms and Conditions

2. Figure 1
Induction of vitamin D3 up-regulated protein-1 (VDUP-1) expression and type IV collagen α1 (COL4A1) expression in human mesangial cells. Human mesangial cells were exposed to normal glucose (control), and high glucose (HG) for 1, 3, and 7 days. Gene expression was analyzed by quantitative polymerase chain reaction (PCR) with corresponding the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control for loading. (A) The expressions of VDUP-1 mRNA. (B) The expression levels of COL4A1 mRNA. The mean ± SD (N = 3) of obtained values are shown with the relative ratio in control cells assigned a value of 1. *P < 0.05 vs. control; †P < 0.01 vs. control.
Kidney International  , DOI: ( /j x)
Copyright © 2003 International Society of Nephrology Terms and Conditions

3. Figure 2
Induction of vitamin D3 up-regulated protein-1 (VDUP-1) expression and type IV collagen α1 (COL4A1) expression in MES13 cells. MES13 cells were incubated for 4 days in normal glucose or high glucose condition. Gene expressions were analyzed by quantitative polymerase chain reaction (PCR) with corresponding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control for loading. (A) Expression levels of VDUP-1 mRNA. (B) Expression levels of COL4A1 mRNA. The values are indicated with the relative ratio to each gene expression in the samples at day 0 assigned a value of 1. Each point represents the mean ± SD (N = 4). *P < 0.05 vs. control; †P < 0.01 vs. control.
Kidney International  , DOI: ( /j x)
Copyright © 2003 International Society of Nephrology Terms and Conditions

4. Figure 3
Up-regulation of type IV collagen α1 (COL4A1) expression by vitamin D3 up-regulated protein-1 (VDUP-1) in MES13 cells. (A) The scheme of ecdysone-inducible expression system. Abbreviations are: EcR, ecdysione receptor; RXR, retinoid X receptor; P, ponasterone A (synthetic ecdysone analog); E/GRE, ecdysone response element. (B) The induction of VDUP-1 expression in MES13 cells with the ecdysone-inducible expression system. Cells were incubated for 24 hours in normal glucose condition with (▪) or without (□) ponasterone A. Gene expressions were analyzed by quantitative PCR with corresponding that of GAPDH as an internal control. The values are indicated with the relative ratio to the expression of VDUP-1 in the condition without ponasterone A assigned a value of 1. (C) The expression of COL4A1 mRNA in MES13 cells. The values are indicated with the relative ratio to the expression of COL4A1 mRNA in the condition without ponasterone A assigned a value of 1. (D) The accumulation of type IV collagen protein in the culture medium. Cells containing pIND-VDUP were incubated for 24h in normal glucose condition with (▪) or without (□) ponasterone A. The quantities of type IV collagen contained in the media at the end of incubation were determined by enzyme-linked immunosorbent assay (ELISA). Each point represents the mean ± SD (N = 3). *P < 0.05 vs. without ponasterone A; †P < 0.01 vs. without ponasterone A.
Kidney International  , DOI: ( /j x)
Copyright © 2003 International Society of Nephrology Terms and Conditions

5. Figure 4
Effects of truncated vitamin D3 up-regulated protein-1 (VDUP-1) on expression levels of type IV collagen α1 (COL4A1) in MES13. (A) Constructs of truncated VDUP-1. The numbers above the closed bars indicate the amino acid numbers of VDUP-1 protein. Each construct was transfected into MES13 cells with ecdysone-inducible systems, and the expressions of transfected genes were induced by ponasterone A. (B) The gene expressions of VDUP-1 and COL4A1 in the cells (upper, full-length transfected; middle, DEL155 transfected; bottom, DEL226 transfected). Cells were treated with (▪) or without (□) ponasterone A for 24 hours. Left graphs show the expression levels of VDUP-1, and right graphs show the expression levels of COL4A1. The values of gene expressions are indicated with the relative ratio to each gene expression in each clone without ponasterone A assigned a value of 1. Each point represents the mean ± SD (N = 3). †P < 0.01 vs. control.
Kidney International  , DOI: ( /j x)
Copyright © 2003 International Society of Nephrology Terms and Conditions

6. Figure 5
Vitamin D3 up-regulated protein-1 (VDUP-1) expression in streptozotocin-induced diabetic mice. (A) Blood glucose concentrations of streptozotocin-induced diabetic mice. (□) indicates blood glucose levels of control mice and (▪) indicates that of diabetic mice. (B) The expression levels of type IV collagen α1 (COL4A1) and VDUP-1 in kidneys of diabetic mice. The gene expression levels were analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) with corresponding that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control for loading. Left graph shows the levels of COL4A1 mRNA, and right graph shows the levels of VDUP-1 mRNA. The values are indicated with the relative ratio to each gene expression in control mice assigned a value of 1. Each point represents the mean ± SE (N = 4). (C) The gene expression levels of VDUP-1 in glomeruli of diabetic mice. The glomeruli were collected by laser capture microdissection (LCM), and the expression levels of VDUP-1 were analyzed by quantitative RT-PCR. The values are indicated with the relative ratio to the gene expression in control mice assigned a value of 1. Each point represents the mean ± SE (N = 3). *P < 0.05 vs. control; †P < 0.01 vs. control.
Kidney International  , DOI: ( /j x)
Copyright © 2003 International Society of Nephrology Terms and Conditions

7. Figure 6
Effect of transforming growth factor- β (TGF- β) blockade on vitamin D3 up-regulated protein-1 (VDUP-1)-induced type IV collagen α1 (COL4A1) expression. MES13 cells containing pIND-VDUP were incubated for 24 hours in normal glucose condition (□), with 5 μmol/L ponasterone A (▪), or with 5 μmol/L ponasterone A+20μg/mL TGF- β-neutralizing antibody (▨). (A) The expression of VDUP-1 mRNA in MES13 cells. (B) The expression of COL4A1 mRNA in MES13 cells. The values are indicated with the relative ratio to the expression of each gene in the condition without ponasterone A and anti-TGF- β antibody assigned a value of 1. Each point represents the mean ± SD (N = 3). *P < 0.05 vs. normal condition; †P < 0.01 vs. normal condition; ‡P < 0.05 vs. ponasterone A without anti-TGF- β antibody.
Kidney International  , DOI: ( /j x)
Copyright © 2003 International Society of Nephrology Terms and Conditions