Cell Biology – Immunology – Pathology

Kidney International (2003) 64, 864–873; doi:10.1046/j.1523-1755.2003.00150.x

Effect of immunosuppression on damage, leukocyte infiltration, and regeneration after severe warm ischemia/reperfusion renal injury

Dirk K Ysebaert, Kathleen E De Greef, Sven R Vercauteren, Anja Verhulst, Marc Kockx, Gert A Verpooten and Marc E De Broe

Departments of Experimental Surgery, Pharmacology, and Nephrology, University of Antwerp, Belgium

Correspondence: Marc E. DeBroe, M.D., Ph.D., University of Antwerp, Department of Nephrology-Hypertension p/a, University Hospital Antwerp, Wilrijkstraat 10 B-2650, Edegem/Antwerpen, Belgium. E-mail: debroe@uia.ua.ac.be

Received 29 January 2001; Revised 6 March 2002; Re-revised 10 April 2003; Accepted 14 April 2003.

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Abstract

Effect of immunosuppression on damage, leukocyte infiltration, and regeneration after severe warm ischemia/reperfusion renal injury.

Background

 

Post-ischemia/reperfusion (I/R) damage, accompanied by leukocyte infiltration, is unavoidable in renal transplantation, as is the need for immunosuppressive treatment. Influence of immunosuppressive treatment on post-I/R renal damage, nonalloimmune cellular infiltration, and regeneration is not well studied.

Methods

 

Uninephrectomized inbred LEW rats were submitted to warm renal ischemia of 45 minutes/60 minutes, and received different immunosuppressive regimens: cyclosporine (CsA) 10 mg/kg/day subcutaneously in the neck daily, or mycophenolate mofetil (MMF) 20 mg/kg/day by daily oral gavage. Control animals underwent sham operation (unilateral nephrectomy) with immunosuppressive treatment or ischemia with vehicle administration. In addition the effect of MMF/mycophenolic acid (MPA) on renal tubule cell proliferation in culture was studied with bromodeoxyuridine incorporation.

Results

 

The post-I/R interstitial cellular infiltration/proliferation consisted mainly of mononuclear leukocytes [first monocytes/macrophages (Mo/MPhi) followed by CD4+ cells]. This mononuclear cell infiltration became apparent 24 hours after injury at the time of acute tubular necrosis, and was most prominent during the phase of regeneration. Severe I/R combined with CsA aggravated morphologic damage and dysfunction, without effect on tubular cell proliferation and tubular regeneration. Early leukocyte infiltration was qualitatively and quantitatively comparable to control animals, yet decreased moderately later in time. In contrast, MMF in combination with severe I/R did not influence initial morphologic damage and dysfunction. Although the initial leukocyte infiltration was comparable to control animals, the subsequent mononuclear cell accumulation, especially CD4 T cells decreased dramatically during MMF treatment. This was concomitant with a decrease of tubular cell proliferation and hence tubular regeneration. Increasing MPA concentrations in renal tubular cell culture caused a significant decrease in total cell number, and an almost arrest of bromodeoxyuridine incorporation, as measurement of cell proliferation.

Conclusion

 

Immunosuppressive treatment with CsA or MMF affected significantly and in a different manner post-I/R renal morphologic damage, interstitial leukocyte, accumulation and regeneration.

Keywords:

ischemia, reperfusion, damage, regeneration, rat, kidney, neutrophils, macrophages, lymphocytes, cyclosporine, mycophenolate mofetil, immunosuppression

A certain degree of ischemia/reperfusion (I/R) injury is always present in renal transplantation, as is the need for immunosuppressive treatment. The highly perfused kidney is particularly vulnerable to the deleterious effects of renal hypoperfusion (ischemia) and to many toxic substances, or a combination of both. Delayed graft function (DGF) of renal allografts is still frequently observed in the immediate posttransplant period, very often as a result from postischemic acute tubular necrosis (ATN), drug nephrotoxicity, acute rejection or combinations of these factors1,2. It is becoming increasingly apparent that DGF can also be associated with more ominous consequences like acute and chronic rejection, and particular poor long-term renal graft survival2,3. Although the number of acute rejections have been dramatically reduced over the last 10 years by optimization of prophylactic immunosuppressive regimens, the frequency of DGF has remained. In the Eurotransplant area it is still 33%4. Moreover, due to increased organ shortage, increased use of marginal donors and nonheart-beating donors, the problem of DGF and its related problems will persist. The clinical impression exists that the structural and functional recovery from the unavoidable posttransplant ischemia seems to be delayed, compared to nontransplant-related acute renal failure (ARF)5. What kind of role, if any, immunosuppressive drugs play in this recovery remains unexplored.

A manifest and intriguing effect during ATN and repair is the prompt appearance of inflammatory cells, immediately following injury, disappearing when the regeneration is complete. This post-I/R infiltrate consists of an accumulation of monocytes /macrophages (Mo/MPhi) followed by helper T cells, most prominent at the time and place of regeneration, when functional recovery has already taken place6,7. Although the literature on the possible role of these interstitial leukocytes is abundant, their exact initiating and resolving mechanisms, and their effect on early and final outcome of ARF, are still unclear8. It is reasonable to consider a possible role of immunosuppressive drugs, by their direct action on leukocytes (especially lymphocytes) and their function, in the post-I/R renal injury and repair.

Current immunosuppressive therapy is rather unspecific and affects defense mechanisms. Traditional initial immunosuppressive regimens in nonimmunized cadaveric first kidney transplant recipients implies steroids, calcineuri ninhibitors [cyclosporine A (CsA), tacrolimus] and antiproliferative agents [mycophenolate mofetil (MMF), azathioprine]. Calcineurin inhibition by CsA, still one of the most widely used immunosuppressive drug in renal transplantation, prevents interleukin-2 (IL-2) gene transcription, a cytokine with a pivotal role in helper T-cell activation and clonal expansion of helper and cytotoxic T cells9. CsA also interferes with the T cell–epithelial cell adhesion by altering leukocyte function-associated molecule-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) expression10. Among many side effects, acute cyclosporine nephrotoxicity, resulting from vasoconstriction of the afferent arteriole may exacerbate ATN in cadaveric renal transplantation11.

MMF is a new immunosuppressive agent recently released for clinical use, in conjunction with steroids and cyclosporine, for the prevention of acute rejection in renal transplant patients12. MMF is a prodrug, the active drug being mycophenolic acid (MPA), which itself is a noncompetitive reversible inhibitor of the enzyme inosine monophosphate dehydrogenase (IMPDH). This enzyme is a key enzyme in the de novo purine synthesis pathway, to which lymphocytes are more dependent than other cells. Inhibition of IMPDH has little impact on proliferation in most eukaryotic cells due to the existence of a salvage pathway, in contrast to lymphocytes where MPA specifically inhibits their proliferation, thus limiting cell-mediated immune mechanisms13. In vitro, MMF inhibits de novo B and T lymphocyte proliferation in response to polyclonal T- and B-cell mitogens, and decreases the generation of cytotoxic T cells14. MMF also decreases homing of monocytes to the endothelium15. Additional anti-inflammatory actions may derive from inhibition of adhesion molecules, limiting the migration of lymphocytes and other inflammatory cells16. MMF inhibits smooth muscle cell proliferation in rat aortic allografts17, and in rat remnant kidney18. Among side effects gastrointestinal toxicity is frequently observed, but nephrotoxicity is not reported.

In this paper, the functional and morphologic evolution of ATN, the rate of regeneration, and the pattern and degree of interstitial leukocyte infiltration/proliferation after severe warm I/R-induced renal injury is studied in rats treated with two different immunosuppressive drug regimens (CsA and MMF).

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METHODS

Animals

After overnight fasting, the surgical procedures were carried out under anesthesia with sodium penthobarbitone (60 mg/kg) in inbred male LEW rats (220 to 260 g). A midline abdominal incision was made and heparin (50 IU, intraperitoneally) was administered. In order to help to maintain thermoregulation during ischemia, the abdominal contents were repositioned and covered with a warm wet dressing. Left renal ischemia was performed by cross-clamping the left renal pedicle for 60 minutes with a microvascular clamp, followed by right nephrectomy at the end of the ischemia period. In nonischemic control animals, the left renal pedicle was dissected, but not clamped, followed by a right nephrectomy. Careful dissection was carried out to preserve the blood supply to the adrenal glands. The kidneys were inspected for ischemia as well for adequate reperfusion for 2 minutes. Postoperative animals were allowed to recover, each in a separate cage, at constant temperature (18°C) and humidity (45%) on a 12-hour light/dark cycle. They received free tap water ad libitum and paired feeding using standard laboratory rat pellets. This paired feeding is of particular importance since CsA induces anorexia and MMF may have gastrointestinal side effects. Animals were weighed and inspected daily. For each experimental time point, four animals were sacrificed (day 1, 2, 3, 5, 7, and 10). Blood samples were taken by heart puncture. All procedures were carried out in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals No. 85-23 (1985), and with approval of the Ethical Committee of the University of Antwerp.

Experimental groups: Immunosuppression

The daily administration of CsA (Sandimmun®, Novartis, Basel, Switzerland) (10 mg/kg/day) was given subcutaneously in the neck, starting at the end of the warm ischemic period of 60 minutes. Animals were randomly assigned to four groups: (1) nephrectomy + vehicle (cremophore) (N = 24), (2) nephrectomy and CsA (N = 24), (3) nephrectomy and renal ischemia + vehicle (N = 24), and (4) nephrectomy, renal ischemia and CsA (N = 24).

In a pilot experiment, the use of a previous proven immunosuppressive dose of 20 mg/kg/day MMF in combination with 60 minutes I/R proved to be lethal for a majority of animals, due to gastrointestinal toxicity (ulcers, diarrhea). Control animals in this experiments sustained the dose very well. As a drug dose of less than 20 mg/kg/day seemed not immunosuppressive in literature, we decided to conduct the final experiment using 45 minutes I/R: (1) nephrectomy + vehicle (N = 24), (2) nephrectomy and MMF (N = 24), (3) nephrectomy and renal ischemia + vehicle (N = 24), and (4) nephrectomy, renal ischemia and MMF (N = 24). MMF was given daily by oral gavage, starting as soon the animals had recovered from anesthesia. The experiments with CsA either MMF were conducted as separate experiments, each with their own control groups. Accordingly, the statistical, descriptive analysis, and conclusions were performed separately.

Biochemical determinations

Serum was obtained at sacrifice and stored at -20°C until use. Serum creatinine values were determined in duplicate using a colorimetric method as modified by Jaffé (Creatinine Merckotest, Diagnostica Merck, Darmstadt, Germany).

Tissue collection and fixation

Immediately after sacrifice, five sagittal tissue sections (1 mm thick) were collected from the left kidney and fixed in formalin calcium, methacarn, and Dubosq Brazil fixative. Two sections were stored in liquid nitrogen.

Morphologic analysis of tubular injury, regeneration, cell proliferation, and apoptosis

The degree of injury in different tubular compartments was established on periodic acid-Schiff reagent (PAS)-hematoxylin-proliferating cell nuclear antigen (PCNA)–stained sections of methacarn-fixed and paraffin-embedded renal tissue. Proliferation was determined by immunohistochemical staining for the PCNA using the PC10 monoclonal antibody (Dako, Glostrup, Denmark). This PCNA is expressed when cells are going into DNA synthesis (cell division, DNA repair, apoptosis). Sections were counterstained with PAS. Nuclei were stained with methyl green. Histologic damage of the kidney was scored semiquantitatively, as previously described19,20. Fifty proximal tubules in the outer stripe of the outer medulla (most sensitive zone for ischemic injury) were assigned as (1) tubules with normal appearance; (2) tubules with signs of sublethal injury (loss of apical brush border); (3) tubules with 25% to 75% tubular cell loss; (4) totally (100%) necrotic tubules (total destruction of all epithelial cells, naked basement membrane); and (5) tubules with signs of early regeneration (tubules without brush border, lined with a flat epithelium containing cells with large dark nuclei (PCNA+) and a varying degree of cytoplasmatic volume. The proliferation was measured by counting the number of PCNA-immunoreactive nuclei in 25 circular-shaped proximal tubules.

Apoptosis was investigated tissue samples of day 2 and 10 after I/R with different methods: terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick end labeling (TUNEL)-reaction, caspase-3 immunohistochemistry, and direct visualization of apoptotic bodies in tubular cell by hematoxylin-eosin (H&E) staining.

Renal adhesion/ infiltration: Leukocyte cell markers

Immunohistochemical detection of Mo/MPhi was performed on methacarn-fixed, paraffin-embedded renal tissue sections using the ED-1 monoclonal antibody (Serotec, Oxford, UK). This antibody is directed to a cytoplasmatic antigen of tissue macrophages and monocytes21. Sections (4 mum) were mounted on poly-L-lysine–coated microscopic slides and treated for 5 minutes with 0.003% trypsin III (Sigma Chemical Co., St. Louis, MO, USA) in 10 mmol/L Tris-HCl buffer, pH7.3. After washing in Tris saline buffer (TSB) (0.01 mol/L Tris-HCl, pH 7.6, in 0.9% NaCl) and treatment with normal horse serum (1/5), the sections were incubated overnight with the primary antibody ED-1.

T lymphocytes (helper T cells, W3/25; suppressor/cytotoxic T cells, OX-8) and B lymphocytes (OX-33) were demonstrated on 5 mum renal tissue cryosections prefixed in 4% formaldehyde (BDH Chemical Ltd., Poole, UK) buffered with 0.1 mol/L Na-cacodylate, pH 7.4, containing 1% CaCl2. Sections were incubated overnight with W3/25 (1/800), OX-8 (1/400), or OX-33 (1/100) (Serotec). Appropriate antibody dilutions were determined in preliminary experiments. The W3/25 monoclonal antibody recognizes the rat CD4-equivalent present on T-helper cells22 and on macrophages23. The OX-8 monoclonal antibody reacts with T-suppressor/cytotoxic and natural killer cells, and is directed to the rat CD8-equivalent24. The OX-33 monoclonal antibody reacts with only peripheral B cells25. Endogenous tissue peroxidase activity was inhibited by immersion in methanol for 15 minutes, followed by 30 minutes incubation with 0.03% H2O2 in TSB. After washing, subsequent incubations were performed with the avidin-biotin peroxidase complex (Vectastain, Vector Laboratories, Inc., Burlingame, CA, USA) and 9-amino-ethylcarbazole as the chromogen supplemented with the H2O2 as substrate. As controls, a spleen section of the rat was also stained. Nuclei were counterstained with methyl green. Positive cells were counted in 20 fields of 0.16 mm2 randomly chosen outer stripe of the outer medulla microscopic fields (magnification, times125) in each animal and data were expressed as positive cells per mm2.

Histochemical detection of polymorphonuclear (PMN) cells was performed on methacarn-fixed, paraffin-embedded renal tissue sections. The H&E staining was used to identify and to quantify the infiltration of PMN cells, based upon the localization of the cell and morphology of the nucleus of the cell. Positively stained cells were quantified in 75 randomly chosen microscopic fields (magnification, times1000) in cortex, outer stripe of the outer medulla (+medullary rays), and inner medulla, expressed as positive cells per mm2.

Human renal tubule cell proliferation in culture

Human proximal and distal tubular cells were prepared after tumor nephrectomy from those portions of the human kidney not involved in renal cell carcinoma and processed in a sterile manner. Cortex and outer stripe of the outer medulla were dissected, cut into pieces of plusminus1 mm3 and digested in RPMI 1640 containing collagenase D solution (Roche, Ottweiler, Germany), supplemented with DNAse (Sigma Chemical Co.). The suspension was shaken vigorously for 2 hours at 37°C and sieved through a 120 mum sieve. The resulting single cell suspension was loaded on top of a discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradient with densities 1.04 and 1.07 g/mL. After centrifugation, cells from the intersection were carefully aspirated, washed and brought into culture as a mixed population of proximal tubular, distal tubular, and collecting duct cells. Cells were seeded in 48-well plates (12,500 cells/well) in alpha-minimal essential medium (MEM) (Life Technologies, Rockville, MD, USA), modified according to Gibson d'Ambrosio, supplemented with 10% fetal calf serum and different concentrations (50, 25, 2.5, 0.25 and 0 mumol/L) of MPA (Sigma Chemical Co.). The control cells (0 mumol/L MPA) were incubated with the same amount of methanol. The cells were grown at 37°C and 5% CO2 in a humidified atmosphere for 5 days.

For cell count, cells of two wells per MPA concentration were trypsinized and counted in a hemocytometer on day 3 and day 4. Proliferation was measured with a cell proliferation enzyme-linked immunosorbent assay (ELISA) based on bromodeoxyuridine incorporation (Roche, Mannheim, Germany). Cells of 5 wells per MPA concentration were incubated with the bromodeoxyuridine label for 24 hours between the two cell counts. The ELISA was performed according to manufacturer instructions. Proliferation in the presence of different concentrations of MPA was expressed as percentage of the proliferation in the control cultures.

Statistics

Data are presented as means plusminus standard deviation. They were compared with a one-way analysis of variance (ANOVA) analysis and a Student-Newman-Keuls test was used to prove qualitative differences by using the software package SPSS. Significant differences were anticipated when P < 0.05.

Correlation testing was performed between functional, morphologic, and severity of leukocyte infiltrate. Bivariate correlation testing was performed using SPSS 10.1 software. The complete data set was analyzed with parametric (Pearson) and nonparametric (Spearman's rho) correlation coefficient testing. Test of significance was two-tailed.

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RESULTS

Cyclosporine: Kidney function and morphology

Nonischemic uninephrectomized rats maintained normal renal function during the whole study period, unaffected by CsA or vehicle (data not shown). Uninephrectomized rats submitted to 60 minutes warm renal ischemia without immunosuppression developed sustained severe ARF, which was significantly more pronounced in CsA group after 48 hours (serum creatineischemiaday2 = 4.0 plusminus 1.98 mg/dL vs. serum creatinineischemia+CsAday2 = 7.2 plusminus 0.31 mg/dL; P < 0.05). In the ischemia group, in the absence of CsA, functional recovery of the kidney started after day 2 and was complete at day 7 (serum creatinineischemiaday7 = 1.06 plusminus 0.11 mg/dL; P < 0.05). In the group with a daily administration of CsA, 10 mg/kg/day, recovery of renal function also started after day 2, but was not complete at the end of the study period (serum creatinineischemiaday10 = 1.05 plusminus 0.25 mg/dL vs. serum creatinineischemia+CsAday10 = 1.65 plusminus 0.5 mg/dL; P < 0.05) Figure 1a.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Effect of immunosuppressive dose of cyclosporine A (CsA) on post-ischemia/reperfusion (I/R) renal function (A), tubular proliferative activity [proliferating cell nuclear antigen (PCNA)] (B), and tubular damage and regeneration (different degrees of tubular necrosis and regeneration) (C). Values are expressed as mean plusminus SD. *P < 0.05. Control is uninephrectomy without ischemia on the remaining kidney but with CsA.

Full figure and legend (30K)

Nonischemic uninephrectomized rats maintained normal, very modest tubular proliferation activity (PCNA positivity) during the whole study period, unaffected by CsA or vehicle (data not shown). In Figure 1, their mean is presented as controls. In uninephrectomized rats submitted to warm renal ischemia for 60 minutes without immunosuppression, proliferation started already on day 1 with a maximum on day 3. Although initially unaffected, tubular cell proliferation (PCNA) was significantly increased from day 5 to day 10 in CsA-treated post-I/R rats [PCNA number positive nuclei per cross-sectionischemiaday5 = 2.4 plusminus 1.8 vs. PCNAischemia+CsAday5 = 4.4 plusminus 0.2 (P < 0.05); PCNAischemiaday7 = 0.2 plusminus 0.4 vs. PCNAischemia+CsAday7 = 2.0 plusminus 1.0 (P < 0.05); PCNAischemiaday10 = 0.1 plusminus 0.1 vs. PCNAischemia+CsAday10 = 0.4 plusminus 0.2 (P < 0.05)]Figure 1b.

In nonischemic uninephrectomized rats treated with CsA or vehicle, no damaged, hence no regenerating, tubules were demonstrated (data not shown). Uninephrectomized rats submitted to warm renal ischemia for 60 minutes without immunosuppression sustained severe ATN at 24 hours post-ischemia with more than 60% of proximal tubular cells in the outer stripe of outer medulla completely damaged Figure 1c. CsA treatment resulted in even greater and sustained damage [% totally necrotic tubulesischemiaday1 = 59.5 plusminus 13.1% vs. ischemia+CsAday1 = 90.0 plusminus 5.3% (P < 0.05); ischemiaday2 = 20.7 plusminus 22.3% vs. ischemia+CsAday2 = 94.0 plusminus 4.1% (P < 0.05)], but unimpaired regeneration, even enhanced [% of tubules with signs of early regenerationischemiaday5 = 56.0 plusminus 10.2% vs. ischemia+CsAday5 = 82.0 plusminus 8.1% (P < 0.05)]. The number of tubules with complete brush border after 10 days was less compared to control animals [% of tubules with normal appearanceischemiaday10 = 47.0 plusminus 9.8% vs. ischemia+CsAday10 = 21.0 plusminus 5.3% (P < 0.05)].

With either method used, it was difficult to separate apoptosis from the overwhelming necrosis on day 2 post-I/R. However, no differences were found in apoptosis with either method, not on day 2 and not on day 10 post-I/R.

Mycophenolate mofetil: Kidney function and morphology

Nonischemic uninephrectomized rats maintained normal renal function during the whole study period, unaffected by MMF or vehicle (data not shown). Uninephrectomized rats submitted to warm renal ischemia for 45 minutes without immunosuppression developed severe ARF, but in contrast to CsA, ARF and functional recovery in MMF group were not different from post-I/R controls Figure 2a.

Figure 2.
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Effect of immunosuppressive dose of mycophenolate mofetil (MMF) on post-ischemia/reperfusion (I/R) renal function (A), tubular proliferative activity [proliferating cell nuclear antigen (PCNA)] (B), and tubular damage and regeneration (different degrees of tubular necrosis and regeneration) (C). Values are expressed as mean plusminus SD. *P < 0.05. Control is uninephrectomy without ischemia on the remaining kidney but with MMF.

Full figure and legend (26K)

The proliferation activity (PCNA), however, was significantly diminished at a time of expected highest activity in MMF-treated rats [PCNAischemiaday2 = 25.3 plusminus 7.4 vs. PCNAischemia+MMFday2 = 6.3 plusminus 2.5 (P < 0.05); PCNAischemiaday3 = 50.0 plusminus 12.8 vs. PCNAischemia+MMFday3 = 23.5 plusminus 11.7 (P < 0.05)], resulting in delayed tubular regeneration Figure 2b.

In nonischemic uninephrectomized rats treated with MMF or vehicle, no damaged, hence no regenerating, tubules were demonstrated (data not shown). Uninephrectomized rats submitted to warm renal ischemia for 45 minutes without immunosuppression sustained severe ATN at 24 hours post-ischemia with almost 70% of proximal tubuluar cells in the outer stripe of the medulla completely damaged Figure 2c. In MMF-treated rats, initial damage (ATN) was similar to non-MMF post-I/R kidneys at day 1, but the impaired proliferation activity (PCNA) resulted in delayed tubular regeneration, so that after 2 to 3 days, significant more tubules remained totally necrotic [% totally necrotic tubules ischemia day 2 = 25.0 plusminus 9.6% vs. ischemia + MMF day 2 = 40.0 plusminus 17.3% (P < 0.05); ischemia day 3 = 0.0 plusminus 0.0% vs. ischemia + MMF day 3 = 7.0 plusminus 4.0% (P < 0.05)]. After 7 to 10 days, fewer tubules were completely regenerated [% of tubules with normal appearance ischemia day 7 = 62.5 plusminus 5.4% vs. ischemia + MMF day 7 = 18.0 plusminus 7.5% (P < 0.05); ischemia day 10 = 60.0 plusminus 9.5% vs. ischemia + MMF day 10 = 34.0 plusminus 7.6% (P < 0.05)].

With either method used, it was difficult to separate apoptosis from the overwhelming necrosis on day 2 post-I/R. However, no differences in apoptosis were found with either method, not on day 2, not on day 10 post-I/R.

Cyclosporine: Intrarenal leucocytes accumulation

Polymorphonuclear cells (H&E staining)
 

The number of PMNs in post-I/R kidneys with immunosuppression was comparable to their post-I/R controls. They remain scarce and their number was even never significantly different from nonischemic controls Figure 3a.

Figure 3.
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Effect of cyclosporine A (CsA) on renal interstitial leukocyte infiltration in the outer stripe of the medulla of postischemic rats (expressed as number/mm2). (A) Polymorphonuclear (PMN) cells. (B) ED-1, monocytes/macrophages. (C) W3/25, CD4+ helper T cells (and to some extent monocytes/ macrophages). (D) OX-8, suppressor/cytotoxic T cells. Values are expressed as mean plusminus SD. *P < 0.05; **P < 0.01. Control is uninephrectomy without ischemia on the remaining kidney but with CsA.

Full figure and legend (44K)

Monocytes/macrophages (ED-1 staining)
 

In uninephrectomized rats submitted to warm renal ischemia for 60 minutes without CsA, Mo/MPhi were already observed after 24 hours, most prominent at day 5 and tended to disappear after 10 days. Although the early (<day 2) infiltration was unaffected, treatment with CsA resulted in significant diminished Mo/MPhi infiltration at day 5 [ED-1ischemiaday5 = 1034 plusminus 160 cells/mm2 vs. ED-1ischemiaday5+CsA = 261 plusminus 27 cells/mm2 (P < 0.05)]Figure 3b.

Lymphocytes (W3/25, OX-8, OX-33 staining)
 

Although the early (<day2) appearance of the lymphocytes in the interstitium was unaffected, the number of intrarenal helper T cells (W3/25) was significantly reduced in CsA-group [W3/25ischemiaday5 = 1603 plusminus 201 cells/mm2 vs. W3/25ischemiaday5+CsA 201 plusminus 67 cells/mm2 (P < 0.01); W3/25ischemiaday10 = 1889 plusminus 603 cells/mm2 vs. W3/25ischemiaday10+CsA 651 plusminus 105 cells/mm2 (P < 0.05)], while T-suppressor/cytotoxic cells (OX-8) were not different from controls Figure 3c and d.

No B cells (OX-33) were present in controls nor in the CsA-treated group (data not shown).

Mycophenolate mofetil: Intrarenal leukocytes accumulation

Polymorphonuclear cells (H&E staining)
 

The number of PMNs in post-I/R kidneys with immunosuppression was comparable to their post-I/R controls. They remain scarce and their number was even never significantly different from nonischemic controls Figure 4a.

Figure 4.
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Effect of mycophenolate mofetil (MMF) on renal interstitial leukocyte infiltration in the outer stripe of the medulla of postischemic rats (expressed as number/mm2). (A) Polymorphonuclear (PMN) cells. (B) ED-1, monocytes/macrophages. (C) W3/25, CD4+ helper T cells. (D) OX-8, suppressor/cytotoxic T cells. Values are expressed as mean plusminus SD. *P < 0.05; **P < 0.01. Control is uninephrectomy without ischemia on the remaining kidney but with MMF.

Full figure and legend (27K)

Monocytes/macrophages (ED-1 staining)
 

In uninephrectomized rats submitted to warm renal ischemia for 45 minutes without MMF, Mo/MPhi were already observed after 24 hours, most prominent at day 5 and tended to disappear after 10 days. Although the early (<day 2) infiltration was unaffected, immunosuppressive treatment resulted in significant diminished Mo/MPhi infiltration [ED-1ischemiaday3 = 401 plusminus 123 cells/mm2 vs. ED-1ischemia+MMFday3 = 94 plusminus 29 cells/mm2 (P < 0.05) (ED-1ischemiaday5 = 521 plusminus 84 cells/mm2 vs. ED-1ischemiaday5+MMF = 85 plusminus 32 cells/mm2 (P < 0.01)]Figure 4b.

Lymphocytes (W3/25, OX-8, OX-33 staining)
 

Under MMF treatment, the number of intrarenal T cells in the interstitium remained unchanged compared to the nonischemic controls. Indeed, total blockade of T-cell accumulation was observed, both helper T cells (from day 5) and suppressor/cytotoxic T cells (from day 2) within the post-I/R kidney [W3/25 ischemiaday10 = 1632 plusminus 122 cells/mm2 vs. W3/25ischemiaday10+MMF 136 plusminus 2 cells/mm2 (P < 0.01); OX-8 ischemia day 10 = 60.2 plusminus 11.7 cells/mm2 vs. OX-8ischemiaday10+MMF 3.8 plusminus 2.9 cells/mm2 (P < 0.001)]Figure 4c. The number of T cells under MMF treatment remained even identical to nonischemic controls.

No B cells (OX-33) were present in controls nor in the MMF-treated group (data not shown).

Correlation testing between functional, morphologic and severity of leukocyte infiltrate showed no clear correlation between different types of infiltrating cells and damage, and an inverse correlation between infiltrating cells and regeneration. The results of the parametric correlation testing were similar to the non-parametric testing.

Effect of mycophenolate mofetil on tubular cell culture
 

A significant decrease in total cell number with increasing MPA concentrations was observed. Cell proliferation was almost abolished using 25 to 50 mumol/L MPA concentrations. Bromodeoxyuridine incorporation was used as measurement of cell proliferation. With the highest concentrations (25 to 50 mumol/L) the proliferation dropped to 20% of the proliferation rate in fetal calf serum 10% medium Figure 5.

Figure 5.
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Effect of mycophenolate mofetil (MMF) on tubular cell culture. (A) Cell count of human tubular cells in culture in the presence of different mycophenolic acid (MPA) concentrations on day 3 and day 4. (B) Proliferation measured by bromodeoxyuridine incorporation in the presence of different concentrations MPA, expressed as percentage of the proliferation in the control cultures.

Full figure and legend (37K)

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DISCUSSION

Renal transplantation results in multiple proinflammatory insults to the kidney, including alloantigen-independent I/R and alloantigen-dependent acute and chronic rejection. All these insults are associated with endothelial activation, leukocyte adhesion, and subsequent infiltration of the kidney, resulting in DGF, of which post I/R ATN is the most prominent feature. Most kidneys that exhibit DGF eventually recover, but the clinical impression exists that this structural and functional recovery is delayed after cadaveric renal transplantation compared to the nontransplant related ARF. In the setting of posttransplant ATN, many transplant centers use induction therapy with potent anti-T-cell antibody preparations, which allows for delayed introduction of full-dose cyclosporine or tacrolimus. The nephrotoxic effects of high-dose cyclosporine or tacrolimus may extend the duration of this ATN, although the cell histologic basis of this hypothesis has not been studied yet.

The functional and morphologic approach of damage and regeneration used in this nonalloimmune model of ATN provides arguments that two commonly used immunosuppressive drugs, CsA and MMF, given at the time of I/R and at a clinical immunosuppressive dose, affected this damage and recovery, each in a different manner. CsA treatment was found to aggravate functional and morphologic I/R-induced damage while MMF treatment did not. Tubular cell proliferation under CsA treatment was intact while under MMF treatment it was clearly suppressed. Hence, proximal tubular regeneration activity remained intact under CsA treatment but ultimate differentiation after 10 days was delayed, most likely because of the more pronounced immediate post-I/R damage compared to their controls. In contrast, although initial damage under MMF treatment was comparable to controls, the proximal tubular regeneration was clearly delayed, resulting in decreased differentiation after 10 days. Both immunosuppressive treatments did not affect the initial accumulation of intrarenal leukocytes. However, between days 5 to10 after warm I/R, the mononuclear cell infiltration/proliferation (first MPhi and later CD4+ cells) was moderately depressed under CsA treatment, in accordance with the finding of an analog inhibition of leukocyte infiltration by CsA after 24 hours cold ischemia26. The combination of I/R with MMF, however, resulted in an almost complete arrest of the CD4+ and CD8+ cell proliferation at days 5 to 10 after warm I/R.

It is well known that a dose-related CsA-induced acute renal dysfunction is a frequent adverse effect noted with this effective immunosuppressive drug. Our experiments of CsA administration in an immunosuppressive dose of 10 mg/kg/day on a unilateral nephrectomy without ischemia of the remaining native kidney showed no tubular damage. No signs of cell proliferation (PCNA positivity) or interstitial leukocyte infiltration were observed during 10 days. On the contrary, the ischemic kidney has been reported to be more susceptible to CsA, as result of an additional (mediated by endothelin and other vasoactive substances) vasoconstriction upon the existing postischemic intrarenal vasoconstriction, rather than direct tubular injury11,27. The resulting ARF has shown to be gradually enhanced after prolonged periods of ischemia (30 right arrow 60 minutes) and increasing doses of CsA (5right arrow10right arrow25 mg/kg/day)27. Since the introduction of CsA in the 1980s, patients with ATN are thought to benefit from conversion from CsA to polyclonal antilymphocytic preparations, since CsA has been shown to increase the recovery time from transplant ATN28,29,30,31. In this study, however, no arguments for delayed functional recovery or morphologic regeneration were found. Indeed, proliferation activity post-ATN was not hampered by CsA, in fact, it was slightly increased, most likely related to the fact that there was more damage to be repaired.

MMF had a negative impact on initial tubular cell proliferation and consequently tubular regeneration. Our in vitro results confirm our in vivo results using clinically relevant MPA concentrations, which caused an almost arrest of tubular cell proliferation. Meanwhile, other investigators have made similar observations of the adverse effect of MMF/MPA on cell growth and chemokine release of tubular epithelial cells32. This is an unreported and unexpected finding since MPA is considered to be a lymphocyte-selective antiproliferative agent, due to the potent inhibition of the de novo synthesis of guanosine 5'-triphosphate (GTP) upon which lymphocytes rely heavily14. Apparently, the possibility exists that MMF also affects cells with high proliferative capacity and activity like proximal tubular cells recovering from ATN. The inhibition of smooth muscle cell proliferation in rat aortic allografts by MMF33 corroborates the nonexclusive antiproliferative mode of action of MMF on lymphocytes. In this respect, the gastrointestinal side effects are probably also related to the action of MPA on the fast-dividing cells enterocytes are. If the thesis stands that T cells are important in damage and regeneration34, the negative effect of MMF on tubular cell proliferation could also be explained as a consequence of depressed lymphocyte proliferation, hence, loss of regenerative potential. Indeed, it was previously suggested35,36 that these infiltrating inflammatory cells might be a source of growth-stimulating substances, implying a role in the repair process after ARF.

Because of its performant immunosuppressive capacity and because MMF does not seems to have a nephrotoxic potential, its safe introduction immediately after transplantation of even severe I/R damaged kidneys could be considered. Indeed, the functional recovery of the ischemic kidney seems not affected by MMF. In view, however, of the negative impact of MMF on tubular proliferation, hence, tissue regeneration, the results of our study suggest to postpone the use of MMF until the complete regeneration and recovery of kidney function in severe I/R damaged kidneys. The use of MMF during ATN may have a negative impact on long-term organ function due to nonimmunologic reasons. How far the negative impact on tubular cell proliferation and, hence, tubular regeneration will affect long-term graft function is an open question37. Literature, however, is abundant on the detrimental effect of affected initial post I/R morphology and function on ultimate graft function and survival2,3,38,39.

As far as animal data can be extrapolated to humans, the results of this study provide arguments for the more optimal use of two important immunosuppressive drugs in case of transplantation of severe I/R damaged kidneys. The interference of MMF with regeneration of I/R damaged renal allografts in rats, not being the case with CsA, justify testing these observations in human renal allotransplantation.

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