Inhibitors directed towards caspase-1 and -3 are less effective than pan caspase inhibition in preventing renal proximal tubular cell apoptosis.
BACKGROUND
Uncontrolled apoptosis contributes to tubular cell deletion in renal scarring. Caspase-3 has a central role in the execution of apoptosis and may provide a target for regulating cell death. Here we evaluate three caspase inhibitors: B-D-FMK (pan caspase inhibitor), Z-DEVDFMK (predominantly Caspase-3 inhibitor) and Z-VAD-FMK (predominantly Caspase-1 and -3 inhibitor) to ameliorate apoptosis induced by cisplatin in rat proximal tubular (RPT) cells.


METHODS
Caspase-3 activity (substrate cleavage assay) and protein (immunocytochemistry and Western blotting), apoptosis (Annexin V flow cytometry, in situend labelling of fragmented DNA, light/electron microscopy and DNA laddering) and cell survival (trypan blue exclusion and propidium iodide flow cytometry) were determined in RPT cells exposed to cisplatin with and without caspase inhibitors.


RESULTS
Cisplatin induced a dose-dependent increase in Caspase-3 activity and 8-fold of increase in apoptosis (p < 0.01) when applied for 24 h at 100 microM. B-D-FMK (40 microM), Z-DEVD-FMK (15 microM) and Z-VAD-FMK (22 microM) almost completely inhibited the 25-fold increase in Caspase-3 activity and decreased apoptosis from 15.9 +/- 4.4 to 2.0 +/- 0.6% (p < 0.01), 15.0 +/- 2.2 and 15.0 +/- 2.2% respectively. DNA ladders were visible in cisplatin-treated cells, which disappeared following addition of B-D-FMK and decreased with Z-VAD-FMK and Z-DEVD-FMK. Cisplatin reduced cell survival to 61% by trypan blue exclusion. B-D-FMK and Z-VAD-FMK increased this to 87 and 75%, but Z-DEVD-FMK had no significant effect.


CONCLUSIONS
Cisplatin causes an increase in RPT apoptosis that is associated with increased Caspase-3 activity. All caspase inhibitors were equally effective at reducing Caspase-3 activity, however the pan caspase inhibitor B-D-FMK was more effective at preventing apoptosis and increasing cell survival. Therefore, pan caspase inhibition offers the greatest potential for the prevention of renal tubular cell deletion by uncontrolled apoptosis.
Yang
Bin
B
Sheffield Kidney Institute, Sheffield University Division of Clinical Sciences, Sheffield, UK.
El Nahas
A Meguid
AM
Fisher
Marie
M
Wagner
Bart
B
Huang
Linhong
L
Storie
Ian
I
Barnett
David
D
Barratt
Jonathan
J
Smith
Alice C
AC
Johnson
Timothy S
TS
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000032:analysis / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000037:antagonists & inhibitors; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D053938:DNA Fragmentation / Q000187:drug effects; D005434:Flow Cytometry; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D009336:Necrosis; D009842:Oligopeptides / Q000494:pharmacology; D051381:Rats
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C103192:butyloxycarbonyl-O-methyl-aspartyl-fluoromethyl ketone; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D002945:Cisplatin