Erythropoietin enhances recovery after cisplatin-induced acute renal failure in the rat

Corinne Bagnis1,, Hélène Beaufils2, Claude Jacquiaud3, Yvette Adabra1, Chantal Jouanneau2, Gilles Le Nahour4, Marie Chantal Jaudon3, Richard Bourbouze5, Claude Jacobs1 and Gilbert Deray1

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



   Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Background. Erythropoietin (Epo) is a growth factor whose synthesis mainly takes place in the kidney. Epo has been shown to support the growth not only of erythroid progenitor cells but also of certain other cell types. We attempted to establish whether Epo enhances the recovery from acute renal failure induced by cisplatin.

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



   Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
The anticancer drug cisplatin, a heavy metal complex, is one of the most active drugs used in the treatment of tumours, including advanced ovarian and non-small cell lung cancer [1,2]. The full therapeutic potential of cisplatin is limited by long-lasting and potentially debilitating toxicity, most notably renal. This drawback is of importance due to its high incidence and its impact on patient morbidity and mortality.

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.



   Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Drugs
Cisplatin (Cisplatine®, Lilly, Saint Cloud, France) was provided as a powder dissolved in saline (1 mg/ml). Epo was a gift from Cilag Laboratories (Eprex®, Laboratoires Cilag, Levallois Perret, France) and was provided as a solution containing 2000 UI/ml.

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 {delta}, 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 d’Ascq, 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.



   Results
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
At the beginning of the protocol, all groups were comparable for weight, creatinine clearance, diuresis and haematocrit (Table 1Go ).


View this table:
[in this window]
[in a new window]
 
Table 1. Basal weight (g), 24-h diuresis (ml), haematocrit (%) and creatinine clearance (ml/min/100 g) in the three experimental groups (cisplatin, cisplatin+Epo, and control group)

 

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 1Go and Table 2Go)
Neither cisplatin or Epo did not significantly modify weight, MABP, plasma potassium and total plasma protein.



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 1. Effect of cisplatin with and without Epo on haematocrit expressed as mean±SEM. *P<0.05 versus control group, °P<0.05 versus cisplatin group.

 

View this table:
[in this window]
[in a new window]
 
Table 2. Body weight, mean arterial blood pressure, haematocrit, and plasma biochemistry in the three experimental groups expressed as means±SEM.

 
Four days after cisplatin administration, no difference in haematocrit was observed between cisplatin and cisplatin+Epo groups, both being significantly higher than in control group (46±1, 48±2 vs 41±2% in cisplatin-, cisplatin+Epo- and placebo-treated animal respectively, P<0.05). Plasma sodium level was significantly increased in cisplatin versus cisplatin+Epo-treated animals (147±1 vs 143±1 mmol/l, P<0.05) and versus control group (145±0.5 mmol/l, P<0.05). Plasma magnesium was significantly different in the three groups (respectively cisplatin, cisplatin+Epo and control groups: 0.96±0.03, 1.06±0.04 and 0.66±0.03 mmol/l, P<0.05).

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 1Go). 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 3Go)
Four days after cisplatin administration (Figure 2Go), 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.


View this table:
[in this window]
[in a new window]
 
Table 3. Renal and urinary functional and biochemical parameters in the three experimental groups expressed as means±SEM

 


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 2. Effect of cisplatin with and without Epo on GFR (ml/min/100 g) expressed as means±SEM. *P<0.05 versus control group, °P<0.05 versus cisplatin group.

 
Urinary pH (6.91±0.2 and 6.79±0.1 vs 7.99±0.2, P<0.05) was significantly lower in cisplatin and cisplatin+Epo-treated animals versus control group. No difference in urinary magnesium was observed.

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 2Go) 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 4Go and Figures 3Go and 4Go)
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.


View this table:
[in this window]
[in a new window]
 
Table 4. Semi-quantitative evaluation of histological lesions

 


View larger version (153K):
[in this window]
[in a new window]
 
Fig. 3. Histological aspects of the rat corticomedullary junction four days after cisplatin administration showing acute tubular necrosis affecting primarily the proximal tubule. No glomerular or vascular lesion is observed. Epo did not modify the effects of cisplatin on renal histology (G x300, haematein-eosin).

 


View larger version (144K):
[in this window]
[in a new window]
 
Fig. 4. Histological aspects of the rat corticomedullary junction 9 days after cisplatin administration (G x300, haematein-eosin).

 

Distribution of PCNA immunoreactivity (Table 5Go and Figure 5Go)
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).


View this table:
[in this window]
[in a new window]
 
Table 5. Number of tubular cell nuclei stained with proliferating cell nuclear antigen (PCNA) four and nine days after cisplatin administration expressed as mean±SEM

 


View larger version (127K):
[in this window]
[in a new window]
 
Fig. 5. PCNA staining showing large nuclei of regenerating cells (day 4) (G x300).

 
Nine days after cisplatin administration, no difference was observed in the different groups for the overall score, the cortical and medullary scores. In the cortico-medullary area, PCNA staining was significantly higher in cisplatin-treated animals (3025±227) versus Epo+cisplatin treated animals (2226±210, P<0.05) and versus control group (1173±261, P<0.05).

Distribution of EGF immunoreactivity
Semi-quantitative score for EGF staining was not significantly different between cisplatin-, cisplatin+Epo-treated or control animals.



   Discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Various hypotheses have been proposed to explain cisplatin acute renal failure pathogenesis. There is convincing evidence that the primary biochemical lesion induced by cisplatin in cancer cells is inhibition of DNA synthesis (7) but the relationship with such DNA-binding in kidney cells to its ultimate toxicity is unknown. Cisplatin accumulates in the renal tubular cells approximately five times its extracellular concentration, with most prominent uptake in the S3 segment of the proximal tubule. Experimental prevention of acute cisplatin nephrotoxicity may be obtained either with intensive hyperhydration (8), mainly containing chloride salts (9), diuretics (10) or radical scavengers administration (11). In the clinical setting, despite hyperhydration, a well-recognized protective measure, acute renal failure occurs in 5–10% of the cases. Accelerating the recovery of renal function, while shortening the necessity of haemodialysis, may positively influence prognosis in those patients.

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 12–72 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.



   Notes
 
Correspondence and offprint requests to: Corinne Bagnis, Massachusetts General Hospital East, Department of Nephrology, Renal Unit 8th floor, 149 13th Street, Charlestown, MA 02129, USA. Back



   References
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 

  1. Loehrer PJ, Einhorn LH. Cisplatin. Ann Intern Med1984; 100: 704–713[ISI][Medline]
  2. Dillman RO, Seagren SL, Propert KJ. A randomized trial of induction chemotherapy plus high dose radiation versus radiation alone in stage III non-small cell lung cancer. N Engl J Med1990; 323: 940–945[Abstract]
  3. Jacobson LO, Goldwasser E, Fried W, Plzak L. Role of the kidney in erythropoiesis. Nature1957; 170: 633–634
  4. Nagai T, Akizawa T, Nakashima Y, Kohjiro S, Nabeshima K, Kanamori N et al. Effects of rHuEpo on cellular proliferation and endothelin-1 production in cultured endothelial cells. Nephrol Dial Transplant1995; 10: 1814–1819[Abstract]
  5. Carlini RG, Reyes AA, Rothstein M. Recombinant human erythropoietin stimulates angiogenesis in vitro. Kidney Int1995; 47: 740–745[ISI][Medline]
  6. Bourbouze R, Dubois M, Gluckman JC, Legrain M. Excretion of urinary N-acetyl-{alpha}-D-glucosaminidase isoenzymes after renal tranplantation in the rat. J Clin Chem Clin Biochem1987; 25: 71–76[ISI][Medline]
  7. Harder HC, Rosenberg B. Inhibitory effects of antitumor platinum compounds on DNA, RNA and protein synthesis in mammalian cells in vitro. Cancer1970; 6: 207–216
  8. Heidemann TH, Gerkens JF, Jackson EK, Branch RA. Attenuation of cis-platinium-induced nephrotoxicity in the rat by high-salt diet, furosemide and acetazolamide. Arch Pharmacol1985; 329: 201–205
  9. Daley-Yates PT, McBrien DC. A study of the protective effect of chloride salts on cisplatin nephrotoxicity. Biochem Pharmacol1985; 34: 2363–2369[ISI][Medline]
  10. Cvitkovic E, Szaulding L, Bethune V, Martin J, Whitmore WF. Improvement of Cis dichlorodiammine platinium (NCS 119875): therapeutic index in an animal model. Cancer1977; 39: 1357–136[ISI][Medline]
  11. Gaedeke J, Fels LM, Bokemeyer C, Mengs U, Stolte H, Lentzen H. Cis-platin nephrotoxicity and protection by silibinin. Nephrol Dial Transplant1996; 11: 55–62[Abstract]
  12. Anagnostou A, Lee ES, Kessimian N, Levinson R, Steiner M. Erythropoietin has a mitogenic effect and a positive chemotactic effect on endothelial cells. Proc Natl Acad Sci USA1990; 87: 5978–5982[Abstract]
  13. Anagnostou A, Liu Z, Steiner M, Chin K, Lee ES, Kessimian N et al. Erythropoietin receptor mRNA expression in human endothelial cells. Proc Natl Acad Sci USA1994; 91: 3974–3978[Abstract]
  14. Fraser JK, Tan AS, Lin FK, Berridge MV. Expression of specific high-affinity binding sites for erythropoietin on rat and mouse megacaryocytes. Exp Hematol1989; 17: 10–16[ISI][Medline]
  15. Kitagawa S, Masaki S, Miura Y. Recombinant human erythropoietin at high doses stimulates thrombopoiesis: treatment for protracted severe myelosuppression complicating interferon-alpha and busulfan therapy for chronic myelogenous leukemia. Eur J Haematol1995; 55: 285–286[ISI][Medline]
  16. Westenfelder C. Tubular and mesangial cells of human, rat and mouse kidney express authentic erythropoietin receptor mRNA. Nephrology1997; S22
  17. Paixao AD, Ferreira AT, Oshiro ME, Razvickas CV, Boim MA, Schor N. Renal hemodynamic response to erythropoietin-induced polycythemia in 5/6 nephrectomised rats is different from normal rats. Exp Nephrol1998; 3: 245–252
  18. Nushiro N, Sakamaki T, Misawa S, Seino M, Omata K, Imai Y, Sekino H, Murata K, Abe K. The effects of intrarenal infusion of recombinant human erythropoietin on renal hemodynamics and renal function in anesthesized rabbits. Nippon Jinzo Gakhai Shi1993; 35: 125–131
  19. Nushiro N, Sakamaki T, Hoshino J, Nakamura T, Sakamoto H, Imai Y, Seino M, Omata K, Sekino H, Abe K. Recombinant human erythropoietin stimulates tubular reabsorption of sodium in anesthesized rabbits. Hypertens Res1995; 18: 203–207[Medline]
  20. Wilcox CS, Deng X, Doll AH, Snellen H, Welch WJ. Nitric oxide mediates renal vasodilation during erythropoietin-induced polycythemia. Kidney Int1993; 44: 430–435[ISI][Medline]
  21. Humes HD, Cieslinski DA, Coimbra TM, Messana JM, Glavao C. Epidermal growth factor enhances renal tubule cell regeneration and repair and accelerates the recovery of renal failure. J Clin Invest1989; 84: 1757–1767[ISI][Medline]
  22. Norman J, Tsau YK, Bacay A, Fine LG. Epidermal growth factor accelerates functional recovery from ischaemic acute tubular necrosis in the rat: Role of the epidermal growth factor receptor. Clin Sci1990; 78: 445–450[ISI][Medline]
  23. Miller SB, Martin DR, Kissane J, Hammerman MR. Insulin-like growth factor I accelerates recovery from ischemic acute tubular necrosis in the rat. Proc Natl Acad Sci USA1992; 89: 11876–11880[Abstract]
  24. Coimbra TM, Cielinski DA, Humes HD. Epidermal growth factor accelerates renal repair in mercuric chloride nephrotoxicity. Am J Physiol1990; 59: F438–F443
  25. Morin NL, Laurent G, Nonclerq D et al. Epidermal growth factor accelerates renal tissue repair in a model of gentamycin nephrotoxicity in rats. Am J Physiol1992; 263: F806–F811[Abstract/Free Full Text]
  26. Saferstein R, Zelent AZ, Price PM. Reduced pre-pro-epidermal growth factor mRNA and decreased EGF expression in ARF. Kidney Int1989; 36: 810–815[ISI][Medline]
  27. Vaziri ND, Zhou XJ, Liao SY. Erythropoietin enhances recovery from cis-platin induced acute renal failure. Am J Physiol1994; 266: F360–F366[Abstract/Free Full Text]
Received for publication: 11. 6.00
Revision received 28. 8.00.