Caspase-3 and apoptosis in experimental chronic renal scarring.
BACKGROUND
Caspase-3 is a member of the caspase enzyme family, having a central role in the execution of apoptosis. However, the significance of Caspase-3 in the inappropriate and excessive apoptosis that contributes to the progression of non-immune-mediated renal scarring has not been established.


METHODS
Kidneys from sham-operated and subtotal nephrectomized (SNx) rats were harvested on days 7, 15, 30, 60, 90 and 120 post-surgery. These were analyzed for apoptosis (in situ end labeling of DNA, light and electron microscopy), Caspase-3 activity (fluorometric substrate cleavage assay), protein and mRNA (Western and Northern blotting), as well as distribution (immunohistochemistry), inflammation (ED-1 immunohistochemistry) and fibrosis (Masson's Trichrome staining).


RESULTS
Apoptosis, inflammation and fibrosis gradually increased in glomeruli, tubules and interstitium of SNx rats. Caspase-3 was mainly located in damaged tubules, but also was found in some glomerular and interstitial cells. Little or no staining was noted in sham-operated kidneys. In SNx kidneys, Caspase-3 activity was significantly increased from day 30 and peaked on day 120 (2.5-fold). This resulted from increases in the 17 and 24 kD active protein subunits. The 32 kD precursor was increased at all time points (1861% on day 120, P < 0.01). Caspase-3 changes were transcription-dependent with the 2.7 kb caspase-3 mRNA significantly increased at all time points (287% on day 120). Caspase-3 activity was a better predictor of apoptosis (Std beta coefficient = 0.347, P < 0.05) than Caspase-3 proteins or mRNA; however, Caspase-3 at all levels correlated with apoptosis, inflammation and fibrosis (all P < 0.01).


CONCLUSIONS
Up-regulation of apoptosis in remnant kidneys is likely to be Caspase-3-dependent as it is associated with increases in Caspase-3 at the activity, protein and mRNA levels. Therefore, Caspase-3 is a potential therapeutic target for the modification of renal cell apoptosis and subsequently renal fibrosis.
Yang
B
B
Sheffield Kidney Institute, Northern General Hospital Trust, Sheffield University, Sheffield S5 7AU, England, UK. Bin.Yang@Sheffield.ac.uk
El Nahas
A M
AM
Thomas
G L
GL
Haylor
J L
JL
Watson
P F
PF
Wagner
B
B
Johnson
T S
TS
D000818:Animals; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D002908:Chronic Disease; D005355:Fibrosis; D005921:Glomerulonephritis / Q000473:pathology*; D007249:Inflammation / Q000175:diagnosis; D008297:Male; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar
D012333:RNA, Messenger; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases