Expression of apoptosis-related genes in chronic cyclosporine nephrotoxicity in mice.
To define the mechanism of cyclosporine (CsA)-induced apoptosis, we investigated the expression of apoptosis-related genes in experimental chronic CsA nephrotoxicity. Mice on a low-salt (0.01%) diet were given vehicle (VH, olive oil, 1 mg/kg/day), or CsA (30 mg/kg/day), and sacrificed at 1 and 4 weeks. Apoptosis was detected with deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) stain, and the expressions of apoptosis-related genes were evaluated by reverse transcription-polymerase chain reaction, immunoblot or immunohistochemistry. The activity of caspase 1 and 3 was also evaluated. The CsA group showed increases in apoptotic cells compared with the VH group (54 +/- 41 vs. 3 +/- 3, p < 0.05), and the number of apoptotic cells correlated well with interstitial fibrosis scores (r = 0.83, p < 0.01). The CsA group showed a significant increase in Fas-ligand mRNA (0.20 vs. 0.02 amol/microgram total RNA, p < 0.05) and Fas protein expression (146% vs. 95%, p < 0.05), compared with the VH group. The CsA group showed significant increases in ICE mRNA (0.21 vs. 0.03 amol/microgram total RNA at 4 weeks, p < 0.05) and CPP32 mRNA (0.18 vs. 0.03 amol/microgram total RNA at 4 weeks, p < 0.05), compared with the VH group. The enzymatic activity of ICE (16.6 vs. 7.9 rho mol/microgram/h, p < 0.05) and CPP32 protease (15.6 vs. 2.7 rho mol/microgram/h, p < 0.05) proteases were increased in the CsA group, compared with the VH group. The ratio between bax and bcl-2 protein increased significantly in the CsA group (5.3-fold), compared with the VH group. Levels of p53 protein also increased in the CsA group. Immunohistochemical detection of Fas, Fas-ligand, ICE and CPP32 revealed strong immunoreactivity in renal tubular cells in areas of structural injury. These findings suggest that local activation of the apoptosis-related genes is associated with CsA-induced apoptotic cell death.
Division of Nephrology, Catholic University of Korea, Seoul, Korea. yangch@cmc.cuk.ac.kr
Chul Woo
CW
Yang
Gregory R
GR
Faulkner
Ihab M
IM
Wahba
Tracy A
TA
Christianson
Grover C
GC
Bagby
Dong Chan
DC
Jin
Hanna E
HE
Abboud
Takeshi F
TF
Andoh
William M
WM
Bennett
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D001794:Blood Pressure; D001835:Body Weight; D020170:Caspase 1 / Q000235:genetics / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D002908:Chronic Disease; D016572:Cyclosporine / Q000633:toxicity*; D053222:Fas Ligand Protein; D007150:Immunohistochemistry; D007166:Immunosuppressive Agents / Q000633:toxicity*; D007674:Kidney Diseases / Q000139:chemically induced* / Q000378:metabolism / Q000473:pathology*; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008813:Mice, Inbred ICR; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D051028:bcl-2-Associated X Protein; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism
C494827:Bax protein, mouse; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D007166:Immunosuppressive Agents; D008562:Membrane Glycoproteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D019014:fas Receptor; C052617:Bcl2 protein, mouse; D016572:Cyclosporine; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D020170:Caspase 1