[Hypoxia induces the expression and secretion of connective tissue growth factor and fibronectin by cultured renal cortical myofibroblasts].
OBJECTIVE
To investigate whether hypoxia can affect the expression and secretion of connective tissue growth factor(CTGF) and fibronectin(FN) in primary cultured rat renal cortical myofibroblasts .


METHODS
The primary cultured rat renal cortical myofibroblasts were subjected to hypoxic (1%O(2)) or normoxic (21% O(2)) conditions for a variety of times. The protein levels of HIF-1alpha, CTGF and FN protein were analyzed by Western blotting in both the whole cell lysates and supernatant culture medium 6 h, 12 h and 24 h after incubation, respectively. RT-PCR was carried out to measure the levels of FN mRNA at different time points (2 h,3 h,6 h and 12 h). The activity of gelatinase MMP-2 and MMP-9 in the supernatant from the cultured cell medium was assayed by gelatin zymography.


RESULTS
The expression of HIF-1alpha was induced at h6 in cells under hypoxia incubation. The levels of cellular CTGF protein were increased in hypoxia treated myofibroblasts at h6 (175%+/-52%),significantly elevated at h12 (347%+/-67%, P<0.05 ) , and sustained the high levels by 24 h (143%+/-27%). The protein level of CTGF in supernatant culture medium reached 3.48 times higher than that in normoxic group of cells at h24 (348%+/-99% , P<0.05 ). The levels of secreted FN by myofibroblasts were elevated under hypoxia at h6 (187%+/-42%), h12 (199%+/-51%) and reached the peak level at h24 (210%+/-29%, P<0.05), whereas the levels of cellular FN was declined at the same time points. Furthermore, we found the expression of FN mRNA was increased in cells under hypoxia condition at h3, reached the peak level at h6(135%+/-13%, P<0.05), and then decreased to the comparable level of cells in normoxic group at h12. The activities of MMP-2 and MMP-9 in the supernatant cultured medium were not significantly changed along with the experimental time points.


CONCLUSIONS
Hypoxia could potentiate renal interstitial fibrosis through stimulating the expression and secretion of CTGF and FN in cultured cortical myofibroblasts.
Guo
Li-ping
LP
Department of Nephrology, Peking University First Hospital, Institute of Nephrology, Peking University, Beijing 100034, China.
Huang
Hai-chang
HC
Li
Jing-zi
JZ
D000818:Animals; D015153:Blotting, Western; D015687:Cell Hypoxia; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005353:Fibronectins / Q000235:genetics*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis; D017874:Immediate-Early Proteins / Q000096:biosynthesis*; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis*; D007672:Kidney Cortex / Q000166:cytology / Q000378:metabolism*; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
C526316:CCN2 protein, rat; D005353:Fibronectins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D055513:Connective Tissue Growth Factor