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. The 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: Severe acute renal failure was observed, with maximum functional impairment 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 severely damaged. Proliferation (proliferating cell nuclear antigen (PCNA) staining) 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 tubules had regenerated completely. Interstitial leukocytes (OX-1 immunohistochemical staining) were already prominent at day 1, thereafter gradually increasing with time. The so-called neutrophil-specific identification methods (myeloperoxidase (MPO), chloroacetate esterase, mAb HIS-48) proved to be non-specific, since they also stained for macrophages, as demonstrated by flow cytometry and the combination of these stainings with the macrophage-specific 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 hand, neutrophil presence investigated by H&E staining revealed only a few neutrophils in glomeruli, medullary rays, and OSOM at 24 h after the ischaemic 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, significant 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 cells/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, reaching 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 cellular infiltrate, but with an important renal MPO activity, reflecting the activation of the adhering inflammatory cells (polymorphonuclear cells (PMNs) and mainly monocytes/macrophages). Only later at the time and site (OSOM) of regeneration a sequential accumulation of monocytes/macrophages and T cells 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-reacting with monocytes/macrophages, explains the controversy in literature concerning the number of PMNs in post-ischaemic injury.

Antibodies to both ICAM-1 and LFA-1 do not protect the kidney against toxic (HgCl2) injury.

BACKGROUND: The role of inflammatory leukocytes in acute renal failure (ARF) remains controversial and appears largely uninvestigated in toxic (in contrast to ischemic) ARF. METHODS: Female Wistar rats were injected with monoclonal antibodies (mAbs) directed to both the leukocyte function-associated antigen 1 (LFA-1) and the intercellular adhesion molecule 1 (ICAM-1). Doses (6 mg/kg of each mAb) were given 24 hours prior to the induction of acute tubular necrosis (ATN) by mercuric chloride administration (2 mg/kg, subcutaneously, day 0) and subsequently every 48 hours. Control rats similarly received either control antibody (12 mg/kg) or vehicle prior to and following the induction of ATN. Renal function was also measured from male Lewis rats that were similarly treated with anti-adhesion antibodies during exposure to 30 minutes of unilateral renal ischemia. RESULTS: Injected antibodies were demonstrated on peripheral blood leukocytes (flow cytometrical detection of mouse anti-LFA-1) and on endothelium (immunohistochemical staining of mouse anti-ICAM-1) and were measured in serum (enzyme-linked immunosorbent assay). Macrophages and T cells were prominent in the kidney of control treatment rats after HgCl2 injection, but anti-adhesion treatment clearly had prevented their infiltration. Notwithstanding, renal tubular injury was equally pronounced in all mercuric chloride treatment groups and so was the decline in renal function (serum creatinine, proteinuria). Tubular epithelial cell proliferation seemed slightly less pronounced and delayed in anti-adhesion treated rats. Kidneys from ischemia exposed rats were, however, functionally protected by identical anti-ICAM-1/anti-LFA-1 treatment. CONCLUSION: Prevention of cellular infiltration by mAbs to LFA-1 and ICAM-1 has no effect on renal morphology, function, or regeneration following mercuric chloride-induced ARF in the rat. This result contrasts with the functional protection of the rat kidney to ischemia/reperfusion injury by virtue of an identical antibody treatment protocol. Resolving that controversy should bring better insight in fundamental processes underlying different types of ARF, and will be the subject of further study.

Regeneration processes in the kidney after acute injury: role of infiltrating cells.

The transient presence of infiltrated leukocytes in the kidney during acute renal failure as well as the location of these cells within the renal interstitium suggest their association with tubular injury and/or regeneration. To date, however, neither a positive nor a negative contribution of these cells to the pathophysiology of this disease could be unambiguously demonstrated. Ill-defined methods for identifying interstitial leukocytes have added to the controversy concerning the role of inflammatory cells in renal regeneration. The current literature survey presents a qualitative description of the renal interstitial accumulation of leukocytes as observed in some acute renal failure models, with special attention to those displaying acute tubular necrosis of particular nephron subsegments. We conclude that lethal or sublethal injury to renal tubular epithelial cells following toxic or ischemic insults leads to the manifestation of an interstitial mononuclear cell infiltrate. Whereas macrophages and T lymphocytes almost invariably take part, the former being the dominant cell population with respect to both magnitude and presence over time, polymorphonuclear cells seem to be significantly increased only in the case of pyelonephritis. Infiltrating cells have often been regarded rather harmful to the tissue, mainly due to the quite well understood injuring capacity of the latter. On the other hand, we speculate mononuclear leukocytes through their potential of producing different cytokines and growth factors (FGF, TGF-alpha, EGF-like, IL-2, etc.) might well play an initiating and mediating role in renal regeneration after acute tubular necrosis. Therefore, the role of infiltrating leukocytes in the injury/regeneration process during acute renal failure remains highly controversial and should be further elucidated.

Neutrophils and acute ischemia-reperfusion injury.

Leukocyte infiltration in response to I/R injury is a well-known but poorly understood phenomenon. The contribution of neutrophils in this process is still controversial. Despite numerous data, little is known about exact numbers of infiltrating neutrophils. The role of monocytes/macrophages in this process is even more unclear. The role neutrophils in the kidney and other organs was reviewed. The variability in models and methods for neutrophil quantification were examined, along with carrying out a critical overview of depletion and anti-adhesion approaches. Nevertheless, the exact role attributed to neutrophils in the I/R process remains unclear.

Inflammatory cells in renal pathology.

To gain better insight in the regeneration process responsible for the functional and morphological recovery after acute renal failure (ARF) is one major goal in the field of Nephrology. Results opposing endogeneously produced polypeptide growth factors to be the mediators of this process accumulate. Indeed, the early decrease of renal epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1) in different experimental models of ARF has been frequently shown at both the mRNA and protein level, while other growth factors could not be shown to increase. Moreover, the inaccessability of the upregulated receptors for endogeneously produced growth factors, has encouraged research to seek alternative origins for the signals inducing renal regeneration. The accumulation of mononuclear leukocytes in the renal interstitium is a striking observation in renal failure. The existence of a regenerative potential provided by the network of inflammatory mononuclear leukocytes is supported by studies on tissue repair in different fields. This review discusses the infiltrating network of mononuclear leukocytes as a major participant in the regeneration process after acute renal failure, including the approach which can be followed to investigate this hypothesis.

Selective depletion of CD8-positive leukocytes does not alter mercuric chloride induced acute renal failure in the rat.

The process of injury and regeneration in different models of acute renal failure is accompanied by the transient interstitial accumulation of mononuclear leukocytes. The relationship between these accumulated cells and the onset and progression of the regeneration process resulting in the complete functional and morphological recovery is still a matter of debate. In this process cell subsets may either be selectively important or combine to a communicative network, signalling the cells at the site of injury. In a first trial to investigate this hypothesis, the CD8-positive subset of leukocytes, consisting mainly of cytotoxic and suppressor T lymphocytes and to a lesser extent of natural killer cells, was depleted in vivo in rats by means of a monoclonal antibody directed against CD8. Although the depletion obtained evidently prevented the infiltration of these cells into the renal interstitium, it could not influence neither the development nor the resolution of renal insufficiency in response to mercuric chloride administration as compared with control animals who had received an irrelevant isotype-matched monoclonal antibody. The extent of renal damage was unaffected as were onset and duration of renal epithelial cell proliferation. Consequently, these data do not support a major role for the CD8-positive cell subset per se in the development of acute nephrotoxic injury and subsequent regeneration.

Inflammatory cells in renal regeneration.

Much research has been performed to gain better insight into the regeneration process, responsible for the functional and morphological recovery after acute renal failure (ARF). Many investigators focused on endogenously produced polypeptide growth factors as the major mediators of tubular epithelial cell proliferation. However, arguments contradicting this hypothesis have recently gained more support. Indeed, the early decrease of renal epidermal growth factor (EGF) and insulinlike growth factor-1 (IGF-1) in different experimental models of ARF has been frequently shown at both the mRNA and protein level, while other growth factors could not be shown to increase. Moreover, the inaccessibility of the upregulated receptors for endogenously produced growth factors has encouraged research to seek alternative origins for the signals inducing renal regeneration. The accumulation of mononuclear leukocytes in the renal interstitium is a striking observation in renal failure. Where the interstitial disease, recognized by the persistent interstitial accumulation of leukocytes, is a better predictor of chronic renal failure and developing fibrosis, ARF distinguishes itself by the disappearance of the infiltrate when regeneration is complete. The existence of a regenerative potential provided by the network of inflammatory mononuclear leukocytes is supported by studies on tissue repair in different fields. This review discusses the infiltrating network of mononuclear leukocytes as a major participant in the regeneration process after acute renal failure, including the approach which can be followed to investigate this hypothesis.

Time course of growth factor expression in mercuric chloride acute renal failure.

BACKGROUND: Renal EGF expression decreases in varying models of acute renal failure (ARF). We found previously that the loss of distal tubular EGF during gentamicin ARF is strongest in the cortex, where proximal tubular injury was most severe. To gain more insight into the mechanism underlying this apparent anatomical association, renal growth factor expression was investigated during mercuric chloride ARF, in which proximal tubular injury is most severe in the outer stripe of the outer medulla (OSOM). METHODS: Endogenous renal growth factor expression was investigated by RNA hybridization and by immunohistochemistry in a rat model of mercuric chloride ARF. In addition we determined temporal and spatial profiles of tubular injury, cell proliferation, and mononuclear cell infiltration during the 3-week observation period. RESULTS: Serum creatinine values were maximal 2 days after treatment and were again normalized at day 6. Tubular injury was most severe in the PST and maximal at day 2. Cell proliferation was also higher in the PST and maximal at day 4. Three weeks after treatment, normal renal morphology was restored. Increased numbers of mononuclear cells appeared transiently in the renal interstitium from day 1 on. Most of these cells were macrophages and T lymphocytes; macrophages surrounded preferentially the severely injured PST in the OSOM. In analogy to gentamicin ARF, renal EGF and IGF-I gene expression were decreased early in the setting of mercuric chloride ARF. The decrease in distal tubular EGF staining was most pronounced in the OSOM, i.e. the anatomical area where mercuric-chloride-induced proximal tubular injury was most severe. CONCLUSIONS: Renal EGF and IGF-I gene expression decreases strongly during mercuric chloride ARF. The spatial association between the initial decrease of distal tubular EGF expression and the zone of major proximal tubular injury could originate from metabolic alterations secondary to oxygen starvation. A possible role of mononuclear cells remains to be determined.