Role of connective tissue growth factor in mediating hypertrophy of human proximal tubular cells induced by angiotensin II.
OBJECTIVE
Cellular hypertrophy is an early important pathological feature of renal diseases such as diabetic nephropathy and remnant kidney. Recent studies have demonstrated that angiotensin II (Ang II) might be a major contributor in regulating cell hypertrophy. However, the exact mechanism is still unclear. The aim of our present work was to investigate the possible role of a newly clarified fibrogenic factor, connective tissue growth factor (CTGF), in mediating the effect of Ang II-induced tubular cell hypertrophy.


METHODS
The cell line HK2 was grown in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal calf serum. After the cells were rested in serum-free medium for 24 h, the dose and time response of CTGF mRNA expression to Ang II stimulation was observed by RT-PCR, and protein synthesis was observed by Western blotting. The effect of anti-CTGF on Ang II (10(-7)M)-induced [(3)H]-leucine incorporation, total protein content (Coomassie brilliant blue G250 method) and change in cellular size [determined by a scanning electronic microscope (SEM)] was also observed. The influence of anti-CTGF antibody on the cell cycle was analyzed by using a fluorescence-activated cell sorter flow cytometer.


RESULTS
The results showed that Ang II induced expression of CTGF mRNA in a time- and dose-dependent manner (p < 0.05 and 0.01, respectively). Stimulation of cells with Ang II (10(-7)M) for 48 h resulted in a 92% increase in [(3)H]-leucine incorporation (5,584 cpm/10(5) cells at 0 h vs. 10,741 cpm/10(5) cells at 48 h; p = 0.01), which was significantly abolished by treatment with anti-CTGF antibody. Ang II (10(-7)M) significantly increased the total protein content in HK2 cells (control: 0.169 +/- 0.011 mg/10(5) cells vs. Ang II: 0.202 +/- 0.010 mg/10(5) cells; p = 0.03), which was markedly inhibited by cotreatment with anti-CTGF antibody. The average cellular diameter determined by SEM showed that the increase in cell size induced by Ang II could be significantly inhibited by anti-CTGF antibody (control: 11.92 +/- 1.62 microm, Ang II group: 20.63 +/- 3.83 microm, Ang II + anti-CTGF group: 16.43 +/- 3.23 microm; p < 0.01, respectively). Furthermore, the flow cytometer study showed that Ang II arrested the cell cycle at G0-G1 phase, which was significantly reversed by treatment with anti-CTGF antibody (G0-G1 percentage: in Ang II group: 76 +/- 1.8%, in Ang II + anti-CTGF group: 71 +/- 1.78%; p = 0.04).


CONCLUSIONS
Our data are the first to clearly demonstrate that CTGF might be an important mediator of Ang II-induced renal hypertrophy, which suggests that inhibiting the production of CTGF might be the new strategy in early prevention of renal fibrosis.
Liu
Bi-Cheng
BC
Institute of Nephrology, Zhong Da Hospital, Southeast University School of Medicine, Nanjing, Jiangsu Province, China. kidney@jlonline.com
Sun
Jing
J
Chen
Qi
Q
Ma
Kun-Ling
KL
Ruan
Xiong-Zhong
XZ
Phillips
Aled O
AO
D000804:Angiotensin II / Q000494:pharmacology*; D015153:Blotting, Western; D002469:Cell Separation; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor; D004305:Dose-Response Relationship, Drug; D005355:Fibrosis / Q000503:physiopathology; D005434:Flow Cytometry; D006801:Humans; D006984:Hypertrophy; D017874:Immediate-Early Proteins / Q000502:physiology*; D036341:Intercellular Signaling Peptides and Proteins / Q000502:physiology*; D007687:Kidney Tubules, Proximal / Q000000981:diagnostic imaging / Q000187:drug effects / Q000473:pathology*; D008855:Microscopy, Electron, Scanning; D008934:Mitogens / Q000276:immunology / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014463:Ultrasonography
C526317:CCN2 protein, human; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008934:Mitogens; D012333:RNA, Messenger; D000804:Angiotensin II; D055513:Connective Tissue Growth Factor