Pathways of caspase-mediated apoptosis in autosomal-dominant polycystic kidney disease (ADPKD).
BACKGROUND
We have recently demonstrated an increase in apoptosis in Han:SPRD rat kidneys with autosomal-dominant polycystic kidney disease (ADPKD). Caspase-3 and caspase-7 are major mediators of apoptosis. There are two pathways of caspase-3 and caspase-7-mediated apoptosis: (1) the "extrinsic" pathway involving the death receptor Fas, Fas ligand (FasL), and caspase-8 and (2) the "mitochondrial" or "intrinsic" pathway involving Bcl-2 proteins, caspase-2, cytochrome c release, and caspase-9. The aim of the present study was to investigate the pathways of apoptosis in 3-week-old Han:SPRD rats with ADPKD.


METHODS
Fluorescent substrates were used to measure caspase activity. mRNA and protein was determined by ribonuclease protection assays and immunoblotting, respectively. The effect of caspase inhibitors on caspase activity in polycystic kidneys was determined.


RESULTS
Caspase-3 and caspase-7 activity was more than 100% increased in homozygous (Cy/Cy) compared to heterozygous (Cy/+) and normal littermate control (+/+) kidneys. Ribonuclease protection assays demonstrated no difference in caspase-3 mRNA. On immunoblotting, there was an increase in the proform of caspase-3 and caspase-7 in Cy/Cy compared to +/+ and Cy/+ kidneys. Caspase-8 and caspase-9 activity was more than 100% increased in Cy/Cy compared to Cy/+ and +/+ kidneys. On immunoblotting, there was an increase of the proform of both caspase-8 and caspase-9 in Cy/Cy kidneys. There was also an increase in cytochrome c release into the cytosol and an increase in caspase-2 protein and activity in Cy/Cy kidneys. On ribonuclease protection assay there was no difference in FasL mRNA between +/+, Cy/+, and Cy/Cy kidneys. Short-term treatment of Cy/Cy rats with the caspase inhibitor IDN-8050 resulted in inhibition of caspase-3 and caspase-7 activity in the kidney.


CONCLUSIONS
In Cy/Cy kidneys with ADPKD, there was an increase of the proform of caspase-9, an increase in cytochrome c release into the cytosol, and an increase in caspase-2 protein and activity demonstrating involvement of the intrinsic pathway. There was an increase in the proform of caspase-8 demonstrating involvement of the extrinsic pathway. No differences in FasL mRNA were seen suggesting that the extrinsic pathway is independent of the death receptor ligand, FasL.
Tao
Yunxia
Y
Division of Renal Diseases and Hypertension, University of Colorado Health Sciences Center, Denver, Colorado, USA
Kim
Jun
J
Stanley
Melinda
M
He
Zhibin
Z
Faubel
Sarah
S
Schrier
Robert W
RW
Edelstein
Charles L
CL
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D053148:Caspase 3; D053179:Caspase 7; D053181:Caspase 8; D020169:Caspases / Q000502:physiology*; D053222:Fas Ligand Protein; D007150:Immunohistochemistry; D008562:Membrane Glycoproteins / Q000235:genetics; D016891:Polycystic Kidney, Autosomal Dominant / Q000473:pathology* / Q000503:physiopathology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats
D000590:Amino Acid Chloromethyl Ketones; D053222:Fas Ligand Protein; C505662:Faslg protein, rat; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C505410:Casp3 protein, rat; C505528:Casp8 protein, rat; D053148:Caspase 3; D053179:Caspase 7; D053181:Caspase 8; D020169:Caspases