* First Department of Pathology, Second Department of Internal Medicine, Regeneration Research Center for Intractable Diseases, and Department of Clinical Sciences and Laboratory Medicine, Kansai Medical University, Moriguchi, Osaka; and || Department of Biotechnology, Kyoto Institute of Technology, Sakyo-ku, Kyoto, Japan
Address correspondence to: Dr. Susumu Ikehara, First Department of Pathology, Kansai Medical University, Moriguchi, Osaka, Japan, 570-8506. Phone: 81-6-6993-9429; Fax: 81-6-6994-8283; E-mail: ikehara{at}takii.kmu.ac.jp
Cisplatin, which is a broadly used anticancer drug, is widelyknown to induce acute renal failure as a result of renal tubularinjury. This article examines whether G-CSF and/or M-CSF rescuesmice from renal failure induced by cisplatin. BALB/c mice receivedintraperitoneal injections with or without G-CSF and/or M-CSFfor 5 d (from day –5 to day –1). The day after thelast injection of G-CSF and/or M-CSF (day 0), the mice receivedan intraperitoneal injection of cisplatin. When pretreated withG-CSF or G-CSF + M-CSF, the mice showed longer survival andlower serum creatinine and blood urea nitrogen levels than micethat had been received injections of M-CSF or saline. Histologically,pretreatment with G-CSF or G-CSF + M-CSF attenuated the damageto renal tubules induced by cisplatin. BALB/c mice that hadreceived a transplant of bone marrow cells of enhanced greenfluorescent protein (EGFP)-transgenic mice ([EGFPBALB/c] mice)were treated with or without G-CSF and/or M-CSF, followed byinjection of cisplatin as well as above. [EGFPBALB/c] mice thatwere treated with G-CSF or G-CSF + M-CSF showed a significantlyhigher number of EGFP+ tubular epithelial cells in the kidneythan mice that were treated with only M-CSF or saline. Theseresults suggest that bone marrow cells mobilized by G-CSF acceleratethe improvement in renal functions and prevent the renal tubularinjury induced by cisplatin and that M-CSF enhances the effectsof G-CSF.
Cisplatin, an anticancer drug, is broadly used for the therapyof cancers such as ovarian, head and neck carcinomas, and germcell tumors (1). It has been reported that cisplatin inducesinjury to renal tubular epithelial cells (RTEC), leading torenal failure (2). The cytotoxicity of cisplatin is consideredto be due to several factors, including peroxidation of cellmembrane (3), mitochondrial dysfunction (4), inhibition of proteinsynthesis (5), and DNA injury (6)
Recently, it was reported that bone marrow cells (BMC) can differentiateinto not only hematopoietic tissue but also nonhematopoietictissues (7–13). BMC have also been reported to differentiateinto RTEC (7,14–16) and to repair renal tubules afterischemic injury (15,16). In the experiment of Kale et al. (15),Lin–Sca-1+ bone marrow–derived cells were mobilizedinto the peripheral blood by transient renal ischemia and seemedto migrate specifically to injured regions of the renal tubule,followed by transdifferentiation into RTEC. These data suggestthat mobilized BMC might differentiate into RTEC, resultingin recovery from ischemic injury. However, very recently, itwas also reported that at least some part of the phenomenonof transdifferentiation from BMC can be attributed to cell fusion(17–20)
In 1999, Takahashi et al. (8) reported that GM-CSF mobilizednot only hemopoietic stem cells but also endothelial progenitorcells from the bone marrow into the peripheral blood. It isalso widely known that G-CSF mobilizes hemopoietic stem cellsfrom the bone marrow into the peripheral blood, and this effectis now clinically utilized for allogeneic and syngeneic peripheralblood stem cell transplantation (21). Similar to GM-CSF, G-CSFmobilizes BMC into an ischemic heart, repairing the infarctedheart and improving the heart’s function (12)
In this article, we demonstrate that G-CSF mobilizes BMC intothe peripheral blood and rescues the mice from cisplatin-inducedrenal failure. M-CSF itself does not rescue the mice from therenal failure induced by cisplatin but enhances the effectsof G-CSF
Mouse Model of Cisplatin-Induced Acute Renal Failure and Cytokine Administration
BALB/c male mice at 7 to 8 wk of age were obtained from SLC(Shizuoka, Japan). The mice received an injection of G-CSF (250µg/kg per d; donated by Chugai Pharmaceutical Co. Ltd.,Tokyo, Japan), M-CSF (250 µg/kg per d; donated by KyowaHakko Kogyo, Tokyo, Japan), or G-CSF (250 µg/kg per d)+ M-CSF (250 µg/kg per d) into the intraperitoneal spaceonce a day for 5 consecutive days. The day after the last injectionof cytokines, single intraperitoneal injections of cisplatin(20 mg/kg body wt) were given to these mice. This dose of cisplatininduces severe renal failure in mice (22). As a control, salinewas injected instead of G-CSF and/or M-CSF once a day for 5consecutive days before the injection of cisplatin. The micewere maintained on a standard diet, and water was freely available
Bone Marrow Transplantation
Intrabone marrow–bone marrow transplantation (IBM-BMT)from enhanced green fluorescent protein (EGFP)-transgenic miceinto BALB/c mice was carried out as described previously (23).Briefly, BALB/c mice at 7 to 8 wk of age were irradiated witha single dose at 7.5 Gy by a 137Cs source. One day after theirradiation, BMC were collected from the femurs and tibias ofEGFP-transgenic mice (24), which were donated by Dr. Okabe (OsakaUniversity, Osaka, Japan). The BMC from the EGFP-transgenicmice were transplanted into the tibias of the irradiated BALB/cmice ([EGFPBALB/c] mice). EGFP-transgenic mice are derived fromC56BL/6 mice. However, this method of BMT (IBM-BMT) does notreadily induce graft-versus-host disease, as described previously(23). Actually, in this experiment, no mice showed any symptomsof graft-versus-host disease. One month after the BMT, [EGFPBALB/c]mice were used for experiments after confirmation that >90%of the peripheral blood nuclear cells were derived from EGFP-transgenicmice
Numbers of White Blood Cells and Neutrophils
The peripheral blood of the mice was collected using EDTA-coatedtubes. The numbers of white blood cells (WBC) and neutrophilsin the peripheral blood were examined using an SF-3000 autoanalyzerfor the peripheral blood (Sysmex, Kobe, Japan)
Histologic Analysis
The kidneys of the BALB/c mice were removed and fixed in 10%buffered formalin and embedded in paraffin, processed for lightmicroscopy, and stained with hematoxylin and eosin. For detectingapoptotic cells, the TdT-mediated dUTP nick end labeling (TUNEL)method was performed using the Takara In Situ Apoptosis DetectionKit (Takara, Otsu, Japan). The kidneys of the [EGFPBALB/c] micewere removed and embedded in optimal cutting temperature compound(Sakura, Tokyo, Japan) and quickly frozen in acetone cooledby dry ice. After adjustment of their horizontal planes parallelto the cutting plane, 2-µm frozen sections were made ina cryostat
Antibodies
The antibodies (Ab) used in this study were as follows: rabbitpolyclonal anti–pan-cytokeratin Ab (1:100; Santa CruzBiotechnology, Santa Cruz, CA), anti–aquaporin-1 Ab (1:100;Santa Cruz Biotechnology), and R-PE–conjugated goat anti-rabbitAb (1:50; Southern Biotechnology Associates, Birmingham, AL)for immunocytochemistry and biotin-labeled mAb (anti-CD3, anti-B220,anti-CD11b, anti-CD11c, anti-NK1.1, anti-Gr1 and anti-Ter 119;Pharmingen), PE-labeled mAb (anti–Sca-1, anti-CD34, andanti–c-kit; Pharmingen), and PE-Cy5.5–labeled avidin(Pharmingen) for flow cytometry
Immunocytochemistry
The specimens, which had been fixed with 4% paraformaldehyde,were stained with rabbit Ab (anti–pan-cytokeratin Ab oranti–aquaporin-1 Ab) and then stained with PE-labeledgoat anti-rabbit Ab. The stained specimens were observed usinga confocal microscope (LSM510-META, Carl Zeiss, Oberkochen,Germany; or Fluoview, Olympus, Tokyo, Japan)
Flow Cytometry
The peripheral blood was stained with (1) biotin-labeled mAb(anti-CD3, anti-B220, anti-CD11b, anti-CD11c, anti-NK1.1, anti-Gr1,and anti-Ter 119) and PE-labeled anti–Sca-1 mAb followedby staining with PE Cy5.5-labeled avidin, (2) biotin-labeledmAb (anti-CD3, anti-B220, anti-CD11b, anti-CD11c, anti-NK1.1,anti-Gr1, and anti-Ter 119) and PE-labeled anti-CD34 mAb followedby staining with PE Cy5.5-labeled avidin, and (3) biotin-labeledmAb (anti-CD3, anti-B220, anti-CD11b, anti-CD11c, anti-NK1.1,anti-Gr1, and anti-Ter 119) and PE-labeled anti–c-kitmAb followed by staining with PE Cy5.5-labeled avidin, followedby hemolysis with BD PharM Lyse (BD Bioscience Pharmingen).The samples were analyzed by a flow cytometer, BD LSR (BD BiosciencePharmingen). Absolute numbers of lineage– (Lin–)CD34+ cells, Lin– c-kit+ cells, and Lin–Sca-1+ cellswere calculated with percentage of each fraction and numberof WBC
Measurement of Blood Urea Nitrogen and Serum Creatinine Levels
Serum was obtained from the mice 2 to 4 d after injection ofcisplatin. Blood urea nitrogen (BUN) and creatinine levels ofthe serum were measured using an autoanalyzer (Hitachi 7150auto-analyzer; Hitachi, Tokyo, Japan)
Bone Marrow Ablation
Bone marrow ablation (BMA) was performed by irradiation. BALB/cmice were irradiated at 9.5 Gy for BMA. From 1 d after irradiation,the mice received injections of G-CSF (250 µg/kg per d)+ M-CSF (250 µg/kg per d) into the intraperitoneal spaceonce a day for 5 consecutive days. The day after the last injectionof cytokines, single intraperitoneal injections of cisplatin(20 mg/kg body wt) were given to these mice. As a control ofcytokine injection, saline was injected instead of G-CSF + M-CSFonce a day for 5 consecutive days before the injection of cisplatin.Serum was collected from the mice 4 d after the injection ofcisplatin, followed by measurement of BUN
Platinum Uptake by the Kidneys
BALB/c mice, which had been pretreated with or without G-CSF(250 µg/kg) and/or M-CSF (250 µg/kg) for 5 consecutivedays, were killed 4 d after the injection of cisplatin, andthe kidneys were collected. The kidneys of mice are too lightto be measured individually, and we therefore asked the NACCo. Ltd. (Tokyo, Japan) to measure the platinum concentrationsof four kidneys (from two mice)
Statistical Analyses
The results are represented as mean ± SD. The significanceof the data was determined by a two-tailed t test, except forthe significance of survival rate. The significance of survivalrate was computed with a log-rank test. P < 0.05 was significant
Pretreatment with G-CSF or G-CSF + M-CSF Prolongs Survival of Cisplatin-Treated Mice
It has been reported that bone marrow stem cells help repairischemically injured renal tubules and that G-CSF and othercytokines have the ability to mobilize bone marrow stem cellsinto the peripheral blood. Therefore, we examined whether pretreatmentwith G-CSF and/or M-CSF could prolong the survival of cisplatin-treatedmice. As shown in Figure 1, treatment with G-CSF or G-CSF +M-CSF prolonged the survival of cisplatin-treated mice, whereastreatment with M-CSF or saline did not; almost all of the micethat were pretreated with only M-CSF or saline died within 10d of the cisplatin injection. However, approximately 45% ofthe mice that were pretreated only with G-CSF and 55% of thosethat were pretreated with G-CSF + M-CSF survived up to 30 dafter cisplatin injection. These results suggest that G-CSFhas the ability to rescue mice from the renal tubular injuryinduced by cisplatin. Although M-CSF itself cannot rescue cisplatin-treatedmice, it enhances the ability of G-CSF: The mice that were pretreatedwith G-CSF + M-CSF showed a better survival rate than the micethat were pretreated with only G-CSF, even though there wasno significant difference between the groups
Figure 1. Survival rates of cisplatin-administered mice that were pretreated with or without G-CSF and/or M-CSF. BALB/c mice received an injection of G-CSF (250 µg/kg per d), M-CSF (250 µg/kg per d), G-CSF (250 µg/kg per d) + M-CSF (250 µg/kg per d), or saline (as a control of cytokines) into the intraperitoneal space once a day for 5 consecutive days. The day after the last injection of cytokines, single intraperitoneal injections of cisplatin (20 mg/kg body wt) were given to these mice. The mice were maintained on a standard diet, and water was freely available. Mice were observed to determine survival rate (n = 23, 10, 11, and 11 for saline pretreatment group, M-CSF pretreatment group, G-CSF pretreatment group, and G-CSF + M-CSF pretreatment group).
Examination of Renal Functions in Cisplatin-Treated Mice
Cisplatin causes renal tubular damage, which induces renal failure,and G-CSF or G-CSF + M-CSF rescues the cisplatin-treated mice.Therefore, it is conceivable that G-CSF or G-CSF + M-CSF protectsthe kidney from the injury caused by cisplatin. To clarify thisquestion, we measured serum BUN and creatinine levels afterinjecting the cisplatin. As shown in Figure 2, A and B, allgroups showed similar serum BUN and creatinine patterns until3 d after cisplatin injection. Namely, 2 d after cisplatin injection,the serum BUN and creatinine levels of all groups had alreadyincreased in comparison with untreated control mice. Three daysafter the cisplatin injection, the serum BUN and creatininelevels of all groups had increased further. Four days afterthe cisplatin injection, the BUN levels of the mice that receivedinjections of saline or M-CSF had increased further, whereasthose of the mice that received injections of G-CSF remainedunchanged in comparison with day 3. Surprising, the BUN andcreatinine levels of the mice that received injections of G-CSF+ M-CSF had decreased in comparison with day 3. We show thedata from day 4 in detail in Figure 2, C and D. The mice thatwere pretreated with G-CSF or G-CSF + M-CSF showed significantlylower serum levels of creatinine than the mice that were treatedwith saline. The M-CSF–pretreated mice showed slightlylower levels of BUN than the saline-treated mice, but therewas no significant difference between the M-CSF–treatedmice and the saline-treated mice. The BUN levels showed a similartendency to the serum creatinine levels, but a significant differencewas seen only between the saline-treated mice and the G-CSF+ M-CSF–treated mice. These results suggest that pretreatmentwith G-CSF and/or M-CSF improves the renal function of cisplatin-treatedmice but that G-CSF is more effective than M-CSF. Moreover,the combination of G-CSF and M-CSF is more effective than theuse of G-CSF or M-CSF alone
Figure 2. Examination of serum creatinine and blood urea nitrogen (BUN) levels. BALB/c mice received an injection of G-CSF (250 µg/kg per d), M-CSF (250 µg/kg per d), G-CSF (250 µg/kg per d) + M-CSF (250 µg/kg per d), or saline (as a control of cytokines) into the intraperitoneal space once a day for 5 consecutive days. The day after the last injection of cytokines, single intraperitoneal injections of cisplatin (20 mg/kg body wt) were given to these mice. The mice were maintained on a standard diet, and water was freely available. From 2 to 4 d after cisplatin injection, mice were killed to examine serum creatinine and BUN levels. Mean data of BUN (A) and creatinine (B) levels of time course from day 2 to day 4 after cisplatin injection are shown. Serum BUN (C) and creatinine (D) levels of the mice 4 d after cisplatin injection are shown. "Untreated mice" means mice without cytokine and cisplatin (n = 5, 23, 14, 13, and 10 for untreated group, saline pretreatment group, M-CSF pretreatment group, G-CSF pretreatment group, and G-CSF + M-CSF pretreatment group).
Histologic Examination of Kidneys of Cisplatin-Treated Mice
We next examined the histologic changes in the kidney afterthe treatment with cisplatin. The renal tubules of the micethat were treated with saline showed severe damage (Figure 3),namely, significant numbers of RTEC had died or had detachedand disappeared, whereas the renal tubules of the mice thatwere treated with G-CSF or G-CSF + M-CSF showed significantevidence of recovery from the renal tubular damage. The M-CSF–pretreatedmice showed only slight amelioration of the renal tubular damage.To evaluate the damage to RTEC, we examined the percentagesof apoptotic RTEC using the TUNEL method. The percentages ofTUNEL-positive RTEC were 12.3 ± 2.4, 10.9 ± 3.3,4.5 ± 2.2, and 1.4 ± 1.7 in saline-, M-CSF–,G-CSF–, and G-CSF + M-CSF–treated mice, respectively.These results paralleled the changes in the serum creatinineor BUN levels. In our experiment, very few inflammatory cellswere observed in the kidney even after the cisplatin injection
Figure 3. Histologic analyses of kidneys of cisplatin-treated mice. BALB/c mice received an injection of G-CSF (250 µg/kg per d), M-CSF (250 µg/kg per d), G-CSF (250 µg/kg per d) + M-CSF (250 µg/kg per d), or saline (as a control of cytokines) into the intraperitoneal space once a day for 5 consecutive days. The day after the last injection of cytokines, single intraperitoneal injections of cisplatin (20 mg/kg body wt) were given to these mice. The mice were maintained on a standard diet, and water was freely available. Four days after cisplatin injection, the mice were killed for histologic examination of the kidneys. Morphologic changes in hematoxylin and eosin staining are shown. The figures indicate renal tubules of saline-pretreated mice (A), M-CSF–pretreated mice (B), G-CSF–pretreated mice (C), and G-CSF + M-CSF–pretreated mice (D). Representative data are shown for three independent experiments. Magnification, x50.
Mobilization of Stem Cells and/or Immature Precursor Cells by G-CSF or G-CSF + M-CSF
Because it has been reported that G-CSF mobilizes hematopoieticprecursor cells from bone marrow into the peripheral blood andthat the induction of ischemia of the kidney also mobilizesprecursor cells from the bone marrow (15), we attempted to ascertainwhether G-CSF and/or M-CSF could mobilize precursor cells fromthe bone marrow into the peripheral blood in our system. Asshown in Figure 4, the numbers of WBC increased after the injectionof G-CSF or G-CSF + M-CSF for 5 consecutive days, but 4 d afterthe injection of cisplatin, the number of WBC had decreasedin all groups and there was no difference between any of therespective groups. It has been reported that CD34, c-kit, andSca-1 are markers of stem cells and precursor cells. Therefore,we examined whether the number of Lin– CD34+ cells, Lin–c-kit+cells, and Lin–Sca-1+ cells increased as a result of theadministration of G-CSF and/or M-CSF. In the peripheral bloodof G-CSF- and/or M-CSF–treated mice, Lin–CD34+ cells,Lin–c-kit+ cells, and Lin–Sca-1+ cells increased,whereas they decreased after cisplatin injection
Figure 4. Effects of G-CSF and/or M-CSF on mobilization of stem cells and effect of cisplatin on the number of stem cells in the peripheral blood. BALB/c mice received an injection of G-CSF (250 µg/kg/ per d) and/or M-CSF (250 µg/kg per d) for 5 consecutive days. As a control, saline was injected instead of the cytokines. On the day after the last injection of cytokines, peripheral blood was obtained from the mice and the numbers of white blood cells (WBC), neutrophils, Lin–CD34+ cells, Lin–Sca-1+ cells, and Lin–c-kit+ cells were examined as described in the Materials and Methods section (black bars). One day after the last injection of cytokines, cisplatin (20 mg/kg) was injected peritoneally into the mice, as described in the Materials and Methods section. Four days after cisplatin injection, the peripheral blood of the mice was obtained and numbers of WBC, neutrophils, Lin–CD34+ cells, Lin–c-kit+ cells and Lin–Sca-1+ cells were examined ("Cis" in the figure, hatched bars; n = 4).
BMA Prevents Improvement in Renal Functions by G-CSF and M-CSF
Next, we examined whether BMC really contribute to the improvementin renal functions induced by G-CSF + M-CSF. As shown in Figure 5,when the bone marrow was injured by irradiation, the renalfunctions were not improved by the administration of G-CSF +M-CSF. This result suggests that BMC mobilized by G-CSF andG-CSF + M-CSF can be attributed to the prevention of the renalfailure induced by cisplatin
Figure 5. Effect of bone marrow ablation (BMA) on renal functions. Irradiated BALB/c mice (9.5 Gy) received an injection of G-CSF + M-CSF into the intraperitoneal space once a day for 5 consecutive days. The day after the last injection of cytokines, single intraperitoneal injections of cisplatin were given to these mice, as described in the Materials and Methods section. As a control of cytokine injection, saline was injected instead of G-CSF + M-CSF once a day for 5 consecutive days before the injection of cisplatin. Serum was collected from the mice 4 d after the injection of cisplatin, followed by measurement of BUN levels (n = 4).
Mobilized BMC by G-CSF or G-CSF + M-CSF Migrate to RTEC, Inhibiting Deterioration of Renal Functions
It has been reported that bone marrow–derived cells developinto RTEC (7,14–16) and rescue the mice from renal failurein the ischemia-induced model (15). Therefore, we examined whetherG-CSF and/or M-CSF can mobilize the precursors of RTEC fromthe bone marrow into the peripheral blood. BMC from EGFP-transgenicmice were transplanted into preirradiated BALB/c mice, as describedin the Materials and Methods section. One month after the BMT,we initiated the administration of G-CSF and/or M-CSF to themice that had received bone marrow transplantation, followedby the injection of cisplatin. Four days after cisplatin injection,the mice were killed for histologic examination; frozen sectionsof the kidney were stained with anti–pan-cytokeratin Abor anti–aquaporin-1 Ab followed by R-PE–conjugatedgoat anti-rabbit Ab, because pan-cytokeratin is a marker forepithelial cells, whereas aquaporin-1 is a marker for renalproximal tubule, which cisplatin mainly injures (25). As shownin Figures 6 and 7, there was a much greater number of EGFP+and pan-cytokeratin+ cells in the renal tubules in the micethat were pretreated with G-CSF or G-CSF + M-CSF than in themice that were pretreated with M-CSF or saline. Moreover, thecells that expressed both EGFP and pan-cytokeratin in G-CSF+ M-CSF–treated mice were more than those in only G-CSF–treatedmice. The pattern of the distribution of aquaporin-1+EGFP+ cellswas similar to that of pan-cytokeratin+EGFP+ cells (data notshown). These results suggest that G-CSF can mobilize BMC, whichrescue RTEC from damage by cisplatin, into the peripheral bloodfollowed by migration to the damaged kidneys and that M-CSFaugments the effects of G-CSF
Figure 6. Bone marrow–derived cells in renal tubules. Cisplatin was injected peritoneally into [enhanced green fluorescent protein (EGFP)BALB/c] mice that had been pretreated with or without G-CSF and/or M-CSF. Four days after cisplatin injection, the mice were killed to obtain frozen specimens of the kidneys. Frozen sections of the kidneys were stained with anti–pan-cytokeratin (rabbit antibody [Ab]) followed by staining with PE-labeled goat anti-rabbit Ab. Therefore, in A through D, orange indicates pancytokeratin-positive cells, green indicates EGFP-positive cells, and yellow shows both pancytokeratin-positive and EGFP-positive cells. The figures indicate renal tubules of saline-pretreated mice (A), M-CSF–pretreated mice (B), G-CSF–pretreated mice (C), and G-CSF + M-CSF–pretreated mice (D). Magnification, x100. Representative data are shown for five independent experiments. Enlargements showing independent colors and merged colors are shown on the right side of the figure.
Figure 7. EGFP+ renal tubular epithelial cells (RTEC) increase in kidney of cytokine-treated mice with cisplatin. Cisplatin was injected peritoneally into [EGFPBALB/c] mice that had been pretreated with or without G-CSF and/or M-CSF as described in the Materials and Methods section. Four days after cisplatin injection, the mice were killed to obtain frozen specimens of the kidneys. Frozen sections of the kidneys were stained with anti–pan-cytokeratin (rabbit Ab) followed by staining with PE-labeled goat anti-rabbit Ab. Percentages of renal tubules that contained EGFP+ RTEC per total renal tubules are shown.
Recently, bone marrow has become extremely popular as a sourceof most body tissues; BMC have been reported to be able to differentiateinto multilineage mature cells, i.e., epithelial cells of theskin, bronchus, lung and intestine, nerve cells, muscle cells(including cardiomyocytes), and hepatocytes. Very recently,it was reported that the phenomenon of transdifferentiationfrom BMC into other tissues can be attributed to cell fusion(but not to real transdifferentiation). In the present study,we have shown that G-CSF mobilizes BMC, which migrate to theinjured RTEC, and that the epithelial cells functionally work,resulting in low creatinine and low BUN levels and an improvementin survival rates in cisplatin-treated mice. In our experiment,M-CSF thus improved renal function slightly but did not prolongthe survival of the cisplatin-treated mice, although it enhancedthe effect of the G-CSF
Krause et al. (7,14–16) showed using BMT experiments thatBMC can differentiate into RTEC in mice. It has also been demonstratedthat BMC contribute to the recovery of renal tubules injuredby ischemia (15,16). However, our experiment shows that G-CSF,which is already clinically used for the mobilization of hemopoieticstem cells, mobilizes BMC and rescues the cisplatin-treatedmice from renal tubular failure. Until recently, it has generallybeen accepted that undifferentiated "stem cells" reside in themonolayer of epithelial cells in the tubular wall of the kidneyand that, under selected circumstances, these cells may committo a differentiation program that leads to a specialized epithelialphenotype (26). If the differentiation of the "stem cells" intoRTEC is the only way to restore the injured RTEC, then cisplatin-treatedmice should survive or die independent of mobilization by G-CSF
It was reported recently that cell fusion is a major mechanismunderlying organ repair (17–20), although many reportshave indicated that it is due to transdifferentiation (27–29).Thus, it is controversial whether it is due to fusion or thetransdifferentiation of BMC into other tissues. In this study,we attempted to elucidate this but failed. We are now in theprocess of elucidating the exact mechanism underlying the restorationof renal functions by pretreatment with G-CSF and/or M-CSF
When mice received an injection of cisplatin, BMC were alreadymobilized into the peripheral blood. However, until 3 d afterthe cisplatin injection, renal function deteriorated even inthe G-CSF + M-CSF–treated mice. Moreover, there was nodifference in platinum uptake by the kidneys between any ofthe respective groups (data not shown). These results suggestthat RTEC in all groups absorbed similar amounts of platinumand were injured similarly at first. However, later, bone marrow–derivedcells migrated to the injured RTEC and improved the renal functions.It has been reported that G-CSF increases neutrophils and augmentsinflammation (30). Azoulay et al. (31) described how G-CSF augmentedalveolar neutrophil recruitment and enhanced bleomycin-inducedacute lung injury. Very recently, Togel et al. (32) showed thatthe administration of G-CSF impairs renal function in a murineischemic acute renal failure model. In the study, G-CSF augmentsthe number of not only circulating progenitor cells but alsoneutrophils, followed by the infiltration of neutrophils intothe injured kidneys, which results in the deterioration of therenal function. However, in our experiment, very few inflammatorycells existed even in the kidneys of mice that were treatedwith G-CSF, and the renal functions of the G-CSF–treatedmice were better than those of the saline-treated control mice.Because it has been shown that neutrophils have toxic effectson various tissues (31,32), it is conceivable that the differentresults between Togel et al. and us are attributable to thedifference in the numbers between progenitor cells and neutrophilsin the kidneys
Acute renal failure based on acute renal tubular dysfunctionis a common disease, and a number of strategies are used fortreating acute renal tubular dysfunction (33). Hemodialysisand peritoneal dialysis are the most effective tools for treatingacute renal failure because they can compensate for the lossof renal functions. However, dialysis itself cannot repair theRTEC. Some reagents, such as fosfomycin (34,35), anti-TNF (36),and antioxidants (37), have been reported to have the abilityto protect RTEC from noxious substances. From our results, wesuggest that the mobilization of BMC by G-CSF, etc., could becomea new strategy for preventing not only acute renal failure asa result of the necrosis of RTEC but also the side effects ofdrugs on various organs
Acknowledgments
This work was supported by a grant from the Haiteku ResearchCenter of the Ministry of Education; a grant from Millenniumof the Ministry of Education, Culture, Sports, Science and Technology;a grant-in-aid for scientific research (B)11470062; grants-in-aidfor scientific research on priority areas (A)10181225 and (A)1162221;a grant-in-aid for scientific research (Hoga) 16659107; Healthand Labor Science research grants (Research on Human Genome,Tissue Engineering Food Biotechnology); a grant from the ScienceFrontier program of the Ministry of Education, Culture, Sports,Science and Technology; a grant from The 21st Century COE Programof the Ministry of Education, Culture, Sports, Science and Technology;a grant from the Department of Transplantation for RegenerationTherapy (Sponsored by Otsuka Pharmaceutical Company, Ltd.);a grant from Molecular Medical Science Institute, Otsuka PharmaceuticalCo., Ltd.; and a grant from Japan Immunoresearch LaboratoriesCo., Ltd
We thank Mr. Kadosaka, Ms. Tokuyama, Ms. Murakami-Shinkawa,and Ms. Miura for expert technical assistance, and also Mr.Hilary Eastwick-Field and Ms. Ando for the preparation of thismanuscript
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Received for publication January 22, 2004.
Accepted for publication December 18, 2004.
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Top
Abstract
Introduction
BALB/c] mice) were treated with or without G-CSF and/or M-CSF, followed by injection of cisplatin as well as above. [EGFP
mediates chemokine and cytokine expression and renal injury in cisplatin nephrotoxicity.
J Clin Invest 110
: 835
–842, 2002[CrossRef][Medline]
