1 Department of Nephrology, 3 Biochemistry and 4 Pathology, Pitié-Salpêtrière Hospital, Paris, France, 2 INSERM U423, Necker-Enfants Malades Hospital, Paris, and 5 Laboratoire de Biochimie, Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes, Paris, France
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Abstract |
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Methods. Sprague-Dawley rats were randomized into three groups. In the cisplatin group, animals received one intraperitoneal injection of cisplatin (6 mg/kg) and a daily injection of placebo for 9 days. In the cisplatin+Epo group, animals received intrapertoneal cisplatin and a daily injection of Epo (100 IU/kg) for 9 days. In the control group, animals received both placebo preparations alone. Para-aminohippuric acid and inulin clearances were determined after 4 and 9 days to evaluate renal blood flow and glomerular filtration rate. In addition, light microscopy and immunohistochemistry examinations were performed, and in situ proliferating cell nuclear antigen (PCNA) staining was done to estimate the degree of renal tubular cell regenerative activity. The potential role of epithelial growth factor (EGF) was evaluated by semi-quantitative assessment of EGF immunostaining.
Results. Renal blood flow and glomerular filtration rate decreased significantly in cisplatin and cisplatin+Epo groups versus control group at day 4. However, at day 9, they both were significantly greater in cisplatin+Epo-treated animals than in rats that had received cisplatin alone. Tubular cell regeneration was significantly enhanced at day 4 in cisplatin+Epo group, compared with cisplatin and control groups respectively. EGF immunostaining was not significantly different between the three groups.
Conclusion. Epo significantly enhanced the rate of recovery from acute renal failure induced by cisplatin. PCNA staining indicated that Epo might act directly via stimulation of tubular cell regeneration.
Keywords: acute renal failure; chemotherapy; cisplatin; erythropoietin; tubular necrosis
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Introduction |
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For more than 30 years, the kidney has been known to be the primary site of erythropoietin (Epo) production [3]. Epo is a growth hormone whose effect may not be limited to bone marrow progenitor cells. In vitro, recombinant human Epo stimulates endothelial cell proliferation [4] and angiogenesis [5].
The effects of Epo on cisplatin-induced acute renal failure were therefore investigated in the rat. Quantitative assessment of tubular cell proliferation and epidermal growth factor immunostaining were performed in order to better understand the mechanism of action of Epo on tubular cells.
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Material and methods |
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Animals and experimental groups
Eighty-five Sprague-Dawley male rats weighing 328±5 g (Charles River, Saint Aubin les Elboeufs, France) were fed a standard rat chow (Pietrement, Provins, France) and had free access to water. Pair feeding was accomplished by limiting the daily food available to each pair fed animal to the amount consumed by the control counterpart during the preceding 24 h.
Preliminary experiment: effect of Epo on haematocrit and renal function in control rats
Control rats received daily intraperitoneal Epo administration (100 UI/kg) (n=5) versus placebo (n=10) to evaluate the effect of Epo on haematocrit and renal parameters. In this study, glomerular filtration rate was assessed with endogenous creatinine clearance after 4 days.
Effect of Epo on cisplatin induced acute renal failure
Seventy animals were randomized to receive a unique 6 mg/kg intraperitoneal injection of cisplatin associated with either daily saline (n=30), recombinant Epo 100 UI/kg (n=30) or placebo preparations of both Epo and cisplatin (n=10).
Body weight was recorded daily. Animals from each group were placed into metabolic cages for a 24-h urine collection and functional studies were conducted (after 4 or 9 days of treatment). Some of the animals were then anesthetized to assess glomerular filtration rate (GFR), renal blood flow (RBF) with inulin and para-aminohippuric acid (PAH) clearances respectively and mean arterial blood pressure (MABP) (Gould, Ballainvilliers, France). Kidneys were removed for histological examination and immunostaining.
Histological examination and renal histology
Under general anesthesia using intraperitoneal injection of sinactin (Inactin®, Suzan Bennett, Natick, USA) the abdomen was opened and both kidneys were removed. They were promptly bisected and either fixed in alcoholic Bouin's reagent, or in formol (15%) and then embedded in paraffin; sections were cut at 3 µm and stained with haematoxylin and eosin, PAS, and trichrome Masson with light green.
A pathologist carried out a semiquantitative analysis of the kidney sections in a blinded fashion. Glomeruli and vessels were normal. Changes observed were limited to the tubules, especially to the proximal straight S3 portion, the main site of cisplatin toxicity. Tubular lesions were graded as follows: 0=no damage; 1+=area of tubular epithelial cell swelling, vacuolization, necrosis, desquamation less than 50%; 2+=lesion areas grater than 50% with or without focal involvement of the S3 segment in the medullary rays; 3+=lesion areas 100% with diffuse involvement of the medullary rays.
Immuno-histochemistry
Proliferating cell nuclear antigen (PCNA) is an auxiliary protein to DNA polymerase , and it is expressed in nucleus strongly in late G1 and S phases of the cell cycle. Therefore, PCNA antibodies are used as tools for detecting proliferating cells.
Immunostaining for PCNA was performed using the ABC procedure. Briefly, kidneys were fixed in formalin (15%) overnight, ethanol dehydrated, and paraffin embedded. Sections were cut at 4 µm, mounted on poly-L-lysine-coated glass slides, and either treated for 5 min with 3% hydrogen peroxidase (for EGF labeling) or incubated three times for 5 min in a microwave oven (for PCNA labeling). Then sections were incubated for 5 min with goat non-immune serum to reduce non-specific background staining, followed by incubation for 1 h at room temperature with the specific antibody diluted in 10% goat serum (a murine IgG 2a monoclonal anti-human PCNA antibody (Dako, Trappes, France), diluted 1/200). After a 10-min rinse in PBS, sections were incubated for 10 min at room temperature with a biotinylated goat anti-mouse IgG antibody (Dako, Trappes, France), followed by the avidin/biotin/peroxidase complex (Dako, Trappes, France), and then revealed by 3,3' diaminobenzidine. The sections were counterstained with haematoxylin and mounted in Eukit (Labonord, Villeneuve dAscq, France). Negative controls were obtained by replacing specific antiserum with normal non-immune sera; no labeling was observed, indicating that all the procedure and reagents used resulted in specific labeling. For PCNA, duodenal specimens were used as positive controls.
Control sections of representative tissues were prepared by substitution of the primary antibody with dilutions of normal mouse serum or omission of the primary antibody. The distribution of PCNA staining was compared with the known localization of mitotically active cell population (intestinal crypts).
Immunohistochemical detection of epithelial growth factor (EGF) was performed using chromatography-purified mouse antibodies specific for human EGF (Sigma, Saint Quentin Fallavier, France) at a dilution of 1/100. Immobilized mouse antibodies were detected by the immunoalkaline phosphatase (APAAP) method with an affinity-purified rabbit anti-mouse immunoglobulin serum (1:20 dilution) and APAAP complex (1:50 dilution), (Dako, Trappes, France). Each step was followed by two washes (5 min each) in Tris-HCl buffered saline (pH 7.2). Alkaline phosphatase was developed with a mixture of naphtol AS-BI phosphate and new fuchsin. Levamisole (Sigma, Saint Quentin Fallavier, France) was added to the development solution in order to block endogenous alkaline phosphatase activity.
Quantification of immunostaining
Each tissue sample stained for PCNA was viewed and scored by an observer blinded to the treatment received. Quantitation of tubular cells was performed with an image analyser computer system (Cambridge Instrument Q250 Rueil Malmaison, France) on 20 fields (area field: 0.109 mm2) outside the glomeruli area. Renal tissue samples were divided into corticomedullary junction, cortical and medullary areas for quantitation of staining.
Semi-quantitative assessment of EGF immunostaining in distal tubular sections were graded using a semi-quantitative scoring which took into account the intensity of tubular staining with an individual score: 1+=diffuse weak staining, 2+=diffuse strong staining.
N-Acetyl-ß-D-glucosaminidase (NAG) determination
Fresh urine was collected for NAG concentration. Description of the technique has been reported elsewhere [6].
Analytical procedure
Polyfructosan (Inutest®, Isotec, Saint Quentin, France) and para-aminohippuric acid (PAH®, Serb, Paris, France) clearances were realized as follows: animals were anaesthetized with an intraperitoneal injection of thiobutabarbital sodium (Inactin®, Suzan Bennett, Natick, USA) 100 mg/kg. The jugular vein, carotid artery and left ureter were cannulated with polyethylene tubing. Animals received a bolus dose (5 ml) of a solution containing Inutest® (11%) and PAH® (5.6 10-3%) followed by a continuous infusion of a 6 ml/h solution containing Inutest® (0.03%) and PAH® (4.7 10-3%). After a 60-min equilibration period, two 30-min clearance periods were realized. Blood (at the midpoint of the period) and urine samples were collected.
Statistics
ANOVA were used for statistical evaluation of the data. Data were expressed as means±SEM throughout. P<0.05 was considered as statistically significant. Statistics were performed using Statview 4 running on a Macintosh LCIII computer.
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Results |
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Preliminary experiment: effect of Epo
Five rats received Epo alone for 4 days. Haematocrit increased significantly after 4 days of treatment compared to the control group (n=10) (48±1 vs 41±2%, P<0.05). No significant difference in weight or GFR (assessed with endogenous creatinine clearance) was observed although GFR tended to increase in Epo-treated animals (0.84±0.1 vs 0.59±0.1 ml/min/100 g, P=NS). PCNA immunostaining obtained in two out of five rats treated with Epo showed a significant difference compared to the control group with an increase in the number of stained nuclei in Epo-treated animals. Epo induced no significant effect on renal histology.
Effect of Epo on cisplatin induced acute renal failure
General and biological findings (Figure 1 and Table 2
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Neither cisplatin or Epo did not significantly modify weight, MABP, plasma potassium and total plasma protein.
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Nine days after cisplatin administration, haematocrit was significantly increased in cisplatin+Epo-compared to cisplatin-treated animals and to control group (48±1 vs 41±1 and 42±1%, P<0.05) (Figure 1). No difference in plasma sodium level was observed (147±1, 148±1 and 146±1, P=NS). Plasma magnesium was significantly increased in cisplatin versus cisplatin+Epo and control animals (0.98±0.09 vs 0.63±0.02 and 0.78±0.04 mmol/l respectively, P<0.05) although in the normal range in the three groups.
Renal parameters (Table 3)
Four days after cisplatin administration (Figure 2), a marked, similar and significant decrease in GFR and RBF was observed in cisplatin- and cisplatin+Epo-treated animals versus control group (0.06±0.01 ml/min/100 g and 0.03±0.01 ml/min/100 g vs 0.57±0.1 ml/min/100 g, P<0.05 for GFR and 0.02±0.01 and 0.01±0.01 ml/min/100 g vs 1.62±0.21 ml/min/100 g, P<0.05 for RBF). A significant increase in urinary N-acetyl-ß-D-glucosaminidase excretion (NAG/creatinine) (473±65 and 402±41 vs 108±9 µmol/h/mmol creatinine, P<0.05), proteinuria (0.05±0.01 and 0.08±0.1 vs 0.01±0.003 g/day, P<0.05), sodium fractional excretion (5±1% and 5±1% vs 1±0.5%, P<0.05) and diuresis (25±2 and 34±3 vs 17±3 ml/day, P<0.05) was observed in cisplatin and cisplatin+Epo versus control group.
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Nine days after cisplatin administration, GFR (0.44±0.08 and 0.61±0.12 vs 0.18±0.06 ml/min/100 g, P<0.05) (Figure 2) and RBF (1.06±0.22 and 1.81±0.31 vs 0.48±0.14 ml/min/100 g, P<0.05) were significantly higher in cisplatin+Epo and control group compared to cisplatin treated animals.
Twenty-four-hour diuresis was significantly increased in cisplatin-treated animals compared to control group (36±5 vs 14±2 ml/24 h, P<0.05) and did not differ significantly from cisplatin+Epo-treated animals (30±4 ml/24 h).
Urinary pH, magnesium, NAG excretion, 24 h proteinuria and sodium fractional excretion did not differ between groups.
Histological data (Table 4 and Figures 3
and 4
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Sixty-eight rats were available for histological study. All rats given cisplatin developed marked structural damage, usually involving the entire S3 segments in the outer stripe of the medulla zone. Semi-quantitative assessment of the histological lesions showed a significantly higher score in cisplatin- and cisplatin+Epo-treated animals versus control group at day 4 and day 9. No significant difference was observed between cisplatin- and cisplatin+Epo-treated animals.
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Distribution of PCNA immunoreactivity (Table 5 and Figure 5
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Four days after cisplatin administration, the number of positive cells was significantly higher in the cortical area of the kidney of animals treated by Epo (1790±158) versus cisplatin alone (1191±173) or control group (1110±156, P<0.05). In the cortico-medullary area of the kidney, PCNA stained cells were also significantly higher in Epo-treated animals (2981±230) versus cisplatin alone (2008±287) and control (1065±307, P<0.05). No difference was observed between groups for PCNA staining in the medullary area of the kidney. The overall score (pooled out for the different areas) was significantly higher after 4 days in group cisplatin+Epo compared to cisplatin alone and control groups (7299±588 vs 5091±674 and 3591±968, P<0.05).
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Distribution of EGF immunoreactivity
Semi-quantitative score for EGF staining was not significantly different between cisplatin-, cisplatin+Epo-treated or control animals.
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Discussion |
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In our model, cisplatin induced sub-lethal acute renal failure, with severe alteration of the GFR and the RBF. Urine analysis mainly indicated tubular damage with low proteinuria, decreased urinary pH and increased NAG excretion. Polyuria occurred with severe salt wasting contributing to the concentrating abnormalities and partly to the rise in haematocrit observed after 4 days in cisplatin-treated animals.
Epo in this setting induced a significant increase in RBF and a significant increase in GFR after 9 days. Histological analysis showed acute tubular necrosis predominantly involving the entire S3 segment in the outer stripe of the medulla. Tubular regeneration occurred with significant enhancement of tubular cell proliferation in Epo-treated animals. Recovery of the renal function was significantly enhanced. Our immunohistological studies suggest that Epo may act as a growth factor on tubular cells.
Epo is a cytokine that specifically regulates differentiation and proliferation of erythroid progenitor cells. The proliferating effect of Epo has also been demonstrated in other cellular types like endothelial cells [4]. The rationale for this effect may be that endothelial cells may also possess an Epo receptor [12,13]. Moreover, the expression of specific high-affinity binding sites for Epo has been demonstrated in rat and mouse megakaryocytes [14] explaining the increase in the platelet count observed with high dose Epo in patients [15]. New data recently emerged suggesting that tubular and mesangial cells express authentic Epo receptor mRNA [16]. Epo could then be a currently unrecognized renotropic cytokine.
One alternate hypothesis to explain that Epo enhances renal recovery would involve its haemodynamic effect although it is not clearly demonstrated. In the literature, Epo has been shown to increase single nephron GFR in cortical nephrons without inducing any change in overall filtration rate in rats [17]. In acute experiments, Epo does not seem to induce any haemodynamic change in normal rabbits [18] but may enhance sodium reabsorption possibly by direct tubular action [19]. In chronic studies though, Sprague-Dawley rats exhibited an increase in renal blood flow but after 5 weeks of thrice weekly Epo injections. Haematocrit and MABP in these studies were also dramatically increased [20]. The mechanism for this effect remains unclear since a haemodynamic effect would be expected to occur shortly after the initiation of the treatment. In our hands though, the effect of Epo on filtration rate appears after only 9 days and is not associated with any blood pressure increase.
Different studies demonstrated that EGF may enhance renal tubular cell regeneration and accelerate the recovery of kidney function post-injury [2125]. In ischaemic or toxic situations, immunoreactivity for EGF precursor seems to fall in the kidney, but the breakdown of pre-existing EGF membrane precursor to mature peptide is enhanced. This peptide could then bind to receptors in the proximal tubule and enhance tubular regeneration. In our study EGF immunostaining showed no difference whether or not animals received Epo suggesting that EGF does not mediate Epo's effect on tubular cell regeneration. Alternatively the immunostaining was performed too late after cisplatin administration. Indeed, decreased pre-pro-EGF mRNA expression was found in cells of the distal convoluted tubule and thick ascending limb of Henle's loop 1272 h after injury [26] suggesting that EGF expression varies very quickly after drug insult.
Vaziri et al. previously suggested the potential effect of Epo in enhancing the recovery after cisplatin-induced acute tubular necrosis [27]. In thymidine incorporation studies, Epo promoted cell proliferation in the cortical tissues of the damaged kidney in vivo. This beneficial effect seemed independent from the haemoglobin level since it was also observed in animals treated with Epo whose haematocrit was kept from rising above that of the controls (daily phlebotomies).
In conclusion, we have shown that Epo may accelerate the recovery from acute renal failure induced by cisplatin by stimulation of tubular cell regeneration. Epo may be one of the future therapeutic possibilities especially in the field of anti-cancer drug-induced acute renal failure where its beneficial effect upon anaemia is already recognized.
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Notes |
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References |
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