Identification and kinetics of leukocytes after severe ischaemia/reperfusion renal injury

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.