Connective Tissue Growth Factor Plays an Important Role in Advanced Glycation End Product–Induced Tubular Epithelial-to-Mesenchymal Transition: Implications for Diabetic Renal Disease

TGF-β1 (10 ng/ml), AGE-BSA, and BSA (40 μM) induce the gene and protein expression of α-SMA in NRK-52E cells, as shown by real-time reverse transcription–PCR (RT-PCR; A) and a representative Western blot (B). Each bar represents the mean ± SEM of six samples per group. *P < 0.01 versus control; ^#P < 0.05 versus BSA.

TGF-β1, AGE-BSA, and BSA reduce the gene and protein expression of E-cadherin in NRK-52E cells, as demonstrated by real-time RT-PCR (A) and a representative Western blot (B). Each bar represents the mean ± SEM of six (mRNA) or three (protein) samples per group. *P < 0.05 versus control; ^#P < 0.01 versus BSA.

TGF-β1 and AGE-BSA induce the gene and protein expression of connective tissue growth factor (CTGF) in NRK-52E cells, as demonstrated by real-time RT-PCR (A) and representative Western blot analysis of whole-cell lysates (B). (C) Western blot of control and TGF-β1 samples demonstrating expression of CTGF protein. To enable the quantification of CTGF, loading in B was adjusted for TGF-β1 samples to prevent saturation of signal (lanes 4 through 6). Even loading of the control and TGF-β1 samples is shown in C. Each bar represents the mean ± SEM of six (mRNA) or three (protein) samples per group. *P < 0.01 versus control; ^#P < 0.01 versus BSA.

TGF-β1, AGE-BSA, and BSA induce the gene expression of the matrix molecules collagen IV and fibronectin in NRK-52E cells, as demonstrated by real-time RT-PCR. Each bar represents the mean ± SEM of three to six samples per group *P < 0.01 versus control; ^#P < 0.05 versus BSA.

NRK-52E cells that were stimulated with CTGF sense adenovirus for 3 (A) or 6 d (B) undergo EMT, as demonstrated by cellular morphology, de novo expression of α-SMA, and reduced expression of E-cadherin at membrane junctions, with relocalization to the perinuclear region (arrow).

NRK-52E cells that were infected with CTGF sense adenovirus for 3 or 6 d alter the gene expression of the EMT markers α-SMA vimentin, and E-cadherin (A) and the ECM molecules collagen IV and fibronectin (C) in NRK-52E cells, as demonstrated by real-time RT-PCR. (B) NRK-52E cells that were infected with CTGF sense adenovirus for 3 d show decreased expression of E-cadherin protein as assessed by Western blot. Each bar represents the mean ± SEM of six samples per group. *P < 0.01 versus vector control.

EMT in NRK-52E cells associated with BSA and AGE-BSA (A) or control and TGF-β1 treatments (B) was investigated after transfection with CTGF-286; this was assessed by light microscopy and confocal microscopy of cells immunostained for α-SMA, vimentin, and E-cadherin. α-SMA, vimentin, and E-cadherin are seen in green. Cell nuclei were counterstained with propidium iodide (red). Magnifications: light microscopy ×200; confocal ×440.

Renal expression of TGF-β1, CTGF, α-SMA, and collagen IV mRNA, as assessed by in situ hybridization. Study groups are control, diabetic for 32 wk, and diabetes + alagebrium treatment (Diab+ALT) from 16 to 32 wk.