A shift in the Bax/Bcl-2 balance may activate caspase-3 and modulate apoptosis in experimental glomerulonephritis.
BACKGROUND
Although apoptosis has been linked to the renal cell deletion and ensuing renal fibrosis, its regulating mechanisms remain obscure. Of the known regulators of apoptosis, the best characterized is the Bax to Bcl-2 ratio. However, its importance in controlling apoptosis in glomerulonephritis is unclear. Here, using the nephrotoxic nephritis (NTN) model, we evaluated Bax/Bcl-2 in relation to changes in the apoptosis co-ordination enzyme, caspase-3.


METHODS
Kidneys were harvested at days 7, 15, 30 and 45 post-injection of anti-glomerular basement membrane antibody into Wistar Kyoto rats. These were analyzed for apoptosis (in situ end labeling of fragmented DNA, light and electron microscopy), Bax/Bcl-2 protein (Western blotting), mRNA (Northern blotting) and distribution (immunohistochemistry), as well as caspase-3 activity (substrate cleavage assay), inflammation (ED1 staining), proliferation (proliferating cell nuclear antigen staining) and fibrosis (Masson's Trichrome staining).


RESULTS
Bax mRNA was significantly increased while that of Bcl-2 was decreased throughout the time course (+265% and -62% by day 45). Increased Bax and decreased Bcl-2 protein were noted, significantly so on day 7 (+177% and -21%) and day 45 (+363% and -17%). Bax protein was observed in dilated and atrophic tubules, sclerotic glomeruli and inflamed interstitium, while Bcl-2 was only visible in atrophic tubules. The ratios of Bax to Bcl-2 mRNA and protein were significantly increased at all time points. These correlated (P < 0.05) with up-regulated caspase-3 activity (r = 0.742 and 0.531), apoptosis (r = 0.712 and 0.540), proliferation (r = 0.611, mRNA only), inflammation (ED1+, r = 0.474 and 0.419) and fibrosis (r = 0.474 and 0.729).


CONCLUSIONS
Our findings suggest that the changes in the ratio of Bax to Bcl-2 may contribute to the caspase-3 activation and the modulation of renal apoptosis associated with renal inflammation, tubular atrophy and renal fibrosis in experimental glomerulonephritis.
Yang
Bin
B
Sheffield Kidney Institute, Division of Clinical Sciences, Department of Histopathology, Northern General Hospital Trust, Sheffield University, Herries Road, Sheffield, England S5 7AU, UK.
Johnson
Timothy S
TS
Thomas
Graham L
GL
Watson
Philip F
PF
Wagner
Bart
B
Furness
Peter N
PN
El Nahas
A Meguid
AM
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D005355:Fibrosis; D015870:Gene Expression; D005921:Glomerulonephritis / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D007668:Kidney / Q000737:chemistry / Q000201:enzymology / Q000473:pathology; D008297:Male; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000235:genetics*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis / Q000235:genetics*; D051381:Rats; D011921:Rats, Inbred WKY; D051028:bcl-2-Associated X Protein
C494828:Bax protein, rat; D018809:Proliferating Cell Nuclear Antigen; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases