Dk. Ysebaert et al., Identification and kinetics of leukocytes after severe ischaemia/reperfusion renal injury, NEPH DIAL T, 15(10), 2000, pp. 1562-1574
Background. Leukocyte adhesion/infiltration in response to renal ischaemia/
reperfusion (I/R) injury is a well-known but poorly understood phenomenon T
he identification? kinetics, and exact role of these inflammatory cells in
I/R injury and regeneration are still matters of debate.
Methods. Uninephrectomized rats were submitted to 60 min renal ischaemia by
clamping of renal vessels.
Results. Seven acute renal failure was observed, with maximum functional im
pairment on day 2. By 12 h after the ischaemic event, up to 80% of proximal
tubular cells in the outer stripe of outer medulla (OSOM) were already sev
erely damaged. Proliferation (proliferating cell nuclear antigen (PCNA) sta
ining) started after 24 h, reaching maximum activity on day 3. Regeneration
of tubular morphology started on the 3rd day, and after 10 days 50% of tub
ules had regenerated completely. Interstitial leukocytes (OX-1 immunohistoc
hemical staining) were already prominent at day 1, thereafter gradually inc
reasing with time. The so-called neutrophil-specific identification methods
(myeloperoxidase (MPO), chloroacetate esterase, mAb HIS48) proved to be no
n-specific, since they also stained for macrophages, as demonstrated by flo
w cytometry and the combination of these stainings with the macrophage-spec
ific ED-1 staining, MPO activity was already significantly increased at 1 h
post-I/R (439 +/- 34%, P < 0.005), reaching its maximum activity after 12
h of I/R ( 1159 +/- 138%, P < 0.0005), declining thereafter. On the other h
and, neutrophil presence investigated by H&E staining revealed only a few n
eutrophils in glomeruli, medullary rays, and OSOM at 24 h after the ischaem
ic event (4.7 +/- 4.2 cells/mm(2) vs controls = 2.3 +/- 2.0 cells/mm(2) (n.
s.)), and remained unchanged over the next 10 days. In contrast, significan
t monocyte/macrophage adhesion/infiltration (ED-1 staining) occurred at the
OSOM at 24 h post-ischaemia (at 24 h, 120 +/- 46 cells/mm(2) vs sham = 18
+/- 4 cells/mm(2) (P < 0.05)), became prominent at day 5 (1034 +/- 161 cell
s/mm(2) vs sham = 18 +/- 18 cells/mm(2) (P< 0.05)), and almost disappeared
after 10 days. CD4(+) cells (W3/25) gradually increased from day 5, reachin
g a maximum at day 10. A few CD8(+) cells (OX-8) were apparent from days 3
until 10, but no B-cells (OX-33) were observed.
Conclusions. After severe warm I/R renal injury, a pronounced acute tubular
necrosis occurs during the first 12-24 h in the absence of a marked cellul
ar infiltrate, but with an important renal MPO activity, reflecting the act
ivation of the adhering inflammatory cells (polymorphonuclear cells (PMNs)
and mainly monocytes/macrophages). Only later at the time and site (OSOM) o
f regeneration a sequential accumulation of monocytes;macrophages and T cel
ls becomes prominent, in contrast with the low number of neutrophils found
in the kidney during the 10-day post-ischaemic period. The non-specificity
of the so-called neutrophil-specific identification methods (MPO activity,
naphthol AS-D chloroacetate esterase, or mAb HIS-48 staining), cross-reacti
ng with monocytes/macrophages, explains the controversy in literature conce
rning the number of PMNs in post-ischaemic injury.