Effects of caspase inhibition on the progression of experimental glomerulonephritis.
BACKGROUND
Caspase-3 has a central role in the execution of apoptosis. In a nephrotoxic nephritis (NTN) model, we previously demonstrated an up-regulation of caspase-3 that was associated with inappropriate renal apoptosis, inflammation, tubular atrophy, and renal scarring.


METHODS
We applied a pan caspase inhibitor, Boc-Asp (OMe)-fluoro-methyl-ketone (B-D-FMK), directly to rat NTN kidney using an intrarenal cannula fed from an osmotic pump. Animals were treated either for the first 7 days (acutely) to determine the effects on renal inflammation (ED-1 staining) and apoptosis (in situ end labeling of fragmented DNA), or for 28 days commencing 15 days after NTN (chronically) to observe the effects on cell death and renal fibrosis. Changes of caspase-3 and caspase-1 activity were detected by fluorometric substrate cleavage assay. Changes in caspase-3 and caspase-1, interleukin-1 beta (IL-1 beta), and collagen I, III, and IV proteins and mRNA were detected by Western blotting and Northern blotting, respectively.


RESULTS
In both treated groups, caspase-3 activity was inhibited, and 17 and 24 kD active caspase-3 proteins were reduced significantly. A compensatory increase of caspase-3 mRNA occurred in the acutely treated group, but decreased in the chronically treated group (P < 0.05). Although there were no significant changes in caspase-1 activity and its active protein, the observed decrease in its precursor in the chronic group was increased by treatment (P < 0.05). Further, IL-1 beta precursor and its mRNA were significantly reduced by treatment only in the chronically treated group. Apoptosis was decreased in the glomeruli of acutely treated rats, and in the tubules and interstitium of chronically treated animals (P < 0.05). Glomerular inflammation was decreased only in the acutely treated group, whereas tubulointerstitial inflammation was lowered in both treated groups (P < 0.05). Glomerulosclerosis was reduced in both inhibitor groups, with a reduction in tubulointerstitial fibrosis and collagen I, III, and IV mRNA restricted to chronically treated animals (P < 0.05). Proteinuria was significantly decreased with caspase inhibition in both treated groups, but not serum creatinine level.


CONCLUSIONS
This study clearly indicates that caspase inhibition reduces renal apoptosis, ameliorates inflammation and fibrosis, and improves proteinuria in experimental glomerulonephritis, which may mainly be related to changes in the caspase enzymatic system.
Yang
Bin
B
Sheffield Kidney Institute, Department of Histopathology and Division of Clinical Sciences, Sheffield Teaching Hospitals, Sheffield University, United Kingdom. by5@le.ac.uk
Johnson
Timothy S
TS
Haylor
John L
JL
Wagner
Bart
B
Watson
Phil F
PF
El Kossi
Mohsen M H
MM
Furness
Peter N
PN
El Nahas
A Meguid
AM
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D020170:Caspase 1 / Q000378:metabolism; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D005355:Fibrosis; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005921:Glomerulonephritis / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D007375:Interleukin-1 / Q000378:metabolism; D007668:Kidney / Q000201:enzymology / Q000276:immunology / Q000473:pathology; D008297:Male; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011921:Rats, Inbred WKY
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D007375:Interleukin-1; D012333:RNA, Messenger; C103192:butyloxycarbonyl-O-methyl-aspartyl-fluoromethyl ketone; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D020170:Caspase 1