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From the Departments of Biochemistry* and Clinical Immunology of Internal Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan
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Abstract |
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The heparin-binding growth factor midkine (MK) and pleiotrophin form a family, which is distinct from ones of other heparin-binding growth factors, such as fibroblast growth factors.11,12 MK is implicated in nephrogenesis,13 and is highly expressed in embryonic as well as adult mouse kidney.14-16 In adult mouse kidney, MK is exclusively expressed in the proximal tubule epithelium.17 It exerts several activities in vitro, including anti-apoptotic and chemotactic ones.18-22 Using MK-deficient (Mdk–/–) mice, a pivotal role of MK in the recruitment of inflammatory cells was revealed in two disease models.17,21 In an artery restenosis model, Mdk–/– mice exhibit significantly less neointima formation as compared with wild-type (Mdk+/+) mice.21 The neointima is the basic lesion of blood vessel restenosis and atherosclerosis, and its formation is induced by recruited inflammatory cells, such as neutrophils and macrophages. In a reperfusion renal injury model, Mdk–/– mice also exhibit less damage.17 In this model, the inflammatory cells are again key players. Consistent with the in vitro chemotactic activity of MK,20,21 inflammatory cell recruitment is significantly suppressed in Mdk–/– mice in both models.17,21
Another important activity of MK is an anti-apoptotic one.18,19 MK inhibits the apoptosis of neuronal cells induced by serum starvation.19 MK prevents degeneration of photoreceptor cells induced by constant light exposure of the eye.23 MK also exerts an anti-apoptotic effect in brain infarction.24 MK induces Bcl-2 in G401 Wilms’ tumor cells, to which, at least in part, anti-apoptotic activity of MK may be attributed.18 Thus, MK has ambivalent activities with respect to CDDP-induced renal damage; an anti-apoptotic one that makes mice resistant to CDDP, and a chemotactic one that makes mice sensitive to CDDP. Therefore, we expected that MK-deficient mice would provide a good model for dissecting the mechanistic sequence of CDDP-induced renal damage, and for evaluating the biological significance of inflammation and apoptosis.
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Materials and Methods |
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CDDP was obtained from Bristol-Myers (Tokyo, Japan). Kits for measuring BUN (blood urea nitrogen) were purchased from Wako (Osaka, Japan). Medium and reagents for cell culture were purchased from Sigma Chemical Co. (St. Louis, MO). Fetal bovine serum, penicillin/streptomycin, and trypsin/ethylenediaminetetraacetic acid solution were from Life Technologies, Inc. (Gaithersburg, MD). OCT compound was from Tissue Tec, Miles Laboratories (Elkhart, IN).
Primary Antibodies and Secondary Reagents
Rabbit anti-MK antibody was prepared as described previously.25 Monoclonal anti-ß-actin antibody was obtained from Sigma-Aldrich (St. Louis, MO). Monoclonal rat anti-mouse neutrophil marker 7/4 antibody was purchased from Serotec Ltd. (Oxford, UK). Fluorescein isothiocyanate-conjugated rabbit anti-rat IgG was from ICN Pharmaceuticals, Inc. (Aurora, OH). Rat antibody against the mouse leukocyte common antigen CD45 was from PharMingen (San Diego, CA). Rat antibody against the mouse macrophage F4/80 antigen was purchased from Serotec Ltd. Fluorescein isothiocyanate anti-mouse CD4 (clone GK1.5) and fluorescein isothiocyanate anti-mouse CD8 (clone 53-6.7) were obtained from eBioscience (San Diego, CA). Anti-mouse Bcl-2 antibody was from Upstate Biotechnology (Lake Placid, NY). Rabbit anti-Bcl-XL antibody, rabbit anti-mouse BAX antibody, rabbit anti-Bak antibody, rabbit anti-Bid antibody, and rabbit anti-caspase3 antibody were purchased from BD Biosciences PharMingen, San Diego, CA. Peroxidase-conjugated rabbit IgG and mouse IgG were from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA).
Animal Treatment and CDDP Administration
Experiments were performed on 8- to 12-week-old male 129SV wild-type (Mdk+/+) mice or MK-deficient (Mdk–/–) mice weighing 25 to 30 g. Mdk–/– mice were generated as described previously.26 After backcrossing of Mdk+/– mice for 12 generations to Mdk+/+ mice, Mdk+/– mice were mated with each other to generate the Mdk+/+ and Mdk–/– mice that were used in this study. Mice were housed under controlled environmental conditions, and maintained with standard food and water. Mice were randomly divided into two groups; one was given a single intraperitoneal injection of saline and the other group of CDDP at the indicated dose (10 to 14 mg/kg of body weight). The mice were sacrificed at 1, 2, 3, and 5 days after injection. Blood samples were collected from the suborbital sinus on the day of sacrifice. Tissue from the kidneys was processed for histology and for protein and mRNA extraction. For measuring CDDP concentration in urine, mice were injected intraperitoneally with 12 mg/kg of cis-platin and housed individually in metabolic cages for 24 hours, and urine was collected in ice-cooled containers for 24 hours after dosing. CDDP concentrations were measured by NAC Co., Ltd. (Tokyo, Japan). The experiments described above were all conducted according to the Animal Experimentation Guide of Nagoya University Graduate School of Medicine.
Survival Course
The survival course of mice given CDDP at 14 mg/kg was examined. After CDDP injection, mice were monitored daily for 7 days. The results were statistically assessed.
Morphology Assessment
The removed kidneys were fixed in 4% paraformaldehyde, embedded in paraffin, and then cut into 2-µm sections. Also, tissues were embedded in OCT and frozen in liquid nitrogen. Four-µm sections were cut with a cryostat and placed on slides coated with 3-aminopropyltriethoxysilane. The tissues were stained with periodic acid-Schiff’s reagent. The degree of morphological changes was determined by light microscopy, and tubulointerstitial damage was scored on a semiquantitative scale as described previously.27 Briefly, the extents of tubular cast formation, tubular dilatation, and tubular degeneration (vascular change, loss of brush border, detachment of tubular epithelial cells, and condensation of tubular nuclei) were scored according to the following criteria by two observers in a blind manner: 0, normal; 1, less than 30% of the pertinent area; 2, 30 to 70%; 3, more than 70%.
Apoptosis Detection and Quantification
The detection of in situ apoptosis using the terminal dUTP nick-end labeling (TUNEL) method was performed following the manufacturer’s technique (Takara Biomedical, Osaka, Japan). Eighteen 10 x 20 fields were examined to count apoptotic cells.
Infiltrating Cell Count
Cryosections were immunohistochemically stained with the antibodies for neutrophils, macrophages, and T cells, respectively. Eighteen 10 x 20 fields were examined to count infiltrating cells.
Primary Culture of Proximal Tubular Epithelial Cells
Proximal tubular epithelial cells were isolated from the kidneys of adult Mdk+/+ or Mdk–/– mice, and then cultured in K1 medium (224.25 ml of Ham’s F12, 226.25 ml of Dulbecco’s modified Eagle’s medium, and 12.5 ml of HEPES) containing 10% fetal bovine serum and hormones, as described previously.28
They were determined to be of proximal tubular origin by immunofluorescent histochemistry: cells were stained positively for cytokeratin (Enzo Diagnostics, Inc., Farmingdale, NY), vimentin (ICN Biomedicals Inc.), and alkaline phosphatase, but negatively for CD31 (BD Biosciences Pharmingen) and 
-smooth muscle actin.
At the confluent state, cells were exposed to 0 to 50 µmol/L CDDP in K1 medium for 24 hours or to 40 µmol/L CDDP for 0, 3, 6, 12, 18, or 24 hours. Cells were then lysed in ice-cold RIPA buffer (comprising 50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1% Nonidet P, 1% deoxycholic acid, and 0.05% sodium dodecyl sulfate) with 0.25 mmol/L phenylmethyl sulfonyl fluoride, kept on ice for 60 minutes, and then centrifuged at 15,000 x g for 10 minutes at 4°C. The supernatants were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting.
Primary Culture of Mouse Embryonic Fibroblasts (MEFs)
MEFs were derived from 15-day-old embryos according to standard protocols.29 MEFs were cultured in a 60-mm-diameter culture dish in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. For 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, 1.0 x 104 cells/well were seeded in 96-well plates and allowed to adhere overnight. Serial dilutions of CDDP were added to wells at concentrations from 1 to 50 µmol/L, and cells were exposed to CDDP for an additional 72 hours. MTT assay was performed according to the manufacturer’s instructions (Cell Counting kit, Wako).
Western Blotting
Mouse kidney tissues were snap-frozen in liquid nitrogen for protein isolation. Proteins were extracted by homogenization of the tissues in ice-cold RIPA buffer (comprising 50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1% Nonidet P, 1% deoxycholic acid, and 0.05% sodium dodecyl sulfate) with 0.25 mol/L phenylmethyl sulfonyl fluoride, and centrifugation at 15,000 x g for 60 minutes at 4°C. The supernatants were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Proteins were visualized using the enhanced chemiluminescence detection system (Amersham Pharmacia, Amersham Biosciences, Piscataway, NJ).
Northern Blotting
Mouse kidney tissues were snap-frozen in liquid nitrogen for total mRNA isolation as described previously.30 To standardize the Northern blots, we used a probe for a housekeeping protein, GAPDH, as the internal control. Chemokine expression including MCP-1, MIP-2, RANTES, and keratinocyte-derived chemokine (KC) were assessed.
KC Protein Enzyme-Linked Immunosorbent Assay (ELISA)
After protein extraction from mouse kidney tissue, KC levels were measured using an ELISA kit (IBL Co., Ltd., Gunma, Japan) according to the manufacturer’s instructions. Results were normalized to total protein concentration.
RNase Protection Assay
Total mRNA was subjected to RNase protection assay using the multiprobe ribonuclease protection assay system (mouse CK-5c probe set; BD Biosciences Pharmingen) according to the manufacturer’s instructions. Multiprobe includes cDNAs for nine proteins, lymphotactin, RANTES, MIP-1, MIP-1ß, MIP-2, IP-10, MCP-1, TCA-3, and eotaxin, and two cDNAs for housekeeping proteins, L32 and GAPDH. The bands were quantified with a PhosphorImager using ImageQuant software (Amersham Biosciences K.K., Tokyo, Japan), and the chemokine mRNA levels were normalized as to the L32 mRNA levels.
In Vivo Administration of Anti-Sense Oligodeoxyribonucleotide (ODN) Targeted to MK
Mouse MK anti-sense (AS)- and reverse (R)-ODNs were prepared as described previously.31 The sequences were as follows. AS, 5'-AGGGCGAGAAGGAAGAAG-3'; R, 5'-GAAGAAGGAAGAGCGGGA-3'. AS or R ODN (1 mg/kg of body weight) were dissolved in 150 µl of saline and injected intravenously via tail vein before administration of cis-platin. For evaluation of AS effect on CDDP nephrotoxicity, we counted the infiltrating neutrophils on day 1.
Statistical Analysis
All data are expressed as means ± SE. Statistical analysis was performed with unpaired, two-tailed Student’s t-test for single comparisons or analysis of variance for multiple comparisons. In the survival course experiment, statistical significance was assessed with log rank. A P value <0.05 was taken to indicate a significant difference.
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Results |
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We examined the survival courses of Mdk+/+ and Mdk–/– mice by injecting 14 mg of CDDP/kg of body weight. Almost 90% of the Mdk–/– mice exhibited 7-day survival. In contrast, only 30% of the Mdk+/+ mice survived (Figure 1)
. There was a statistically significant difference between the two genotypes (P < 0.01).
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Because most mice injected with 14 mg/kg of CDDP died of acute renal failure by day 7 (Figure 1)
, we intended to determine the appropriate dose for monitoring changes in the kidney. The histology of Mdk+/+ mice 5 days after CDDP injection is shown in Figure 2
. Renal damage was manifested as cast formation, tubular dilation, and tubular degeneration. Although there was negligible damage with 10 mg/kg (Figure 2B)
, damage became more severe with increasing dose of CDDP more than 11 mg (Figure 2; C to F)
. The MK protein expression profile also showed an apparent threshold. MK protein expression did not show any changes at day 5 with 10 mg/kg as compared with an untreated control, but was dramatically suppressed with more than 11 mg/kg (Figure 3, A and B)
. In contrast to histological changes that became progressively worse with increasing doses, MK protein expression was suppressed to a similar extent if the dose was more than 11 mg/kg (Figure 3, A and B)
. This suggests that a lower dose (eg, 11 or 12 mg/kg) is sufficient to determine the effect of MK protein expression (eg, Mdk+/+ versus Mdk–/–), and has the advantage of reducing the background pathological changes induced by high-dose CDDP. In addition, a lower dose, unlike 14 mg/kg, was not lethal (data not shown), so we were able to monitor the kidney without postmortem changes. Therefore, we chose the dose of 12 mg/kg for further experiments.
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. MK mRNA expression showed a profile similar to that of protein, but exhibited a statistical difference at day 1 as compared with day 0 (Figure 3, G and H)
, suggesting that posttranscriptional regulation is also involved in MK protein expression.
Renal function and tissue morphology were then evaluated by means of BUN and histology, respectively. In Mdk+/+ mice, BUN elevation became prominent at day 3 as compared with at day 0 (Figure 4A)
. Mdk+/+ mice showed a worse phenotype than Mdk–/– mice in terms of BUN and histology (Figure 4)
. There were significant differences between these genotypes on day 5 in BUN, tubular degeneration, and cast formation (Figure 4, A and C)
. However, a critical event was expected to take place earlier, because, at a later stage (later than day 2), MK expression was strongly suppressed in Mdk+/+ mice (Figure 3; E to H)
, and was negative in Mdk–/– mice.
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To elucidate the mechanisms underlying the different sensitivities of Mdk+/+ and Mdk–/– mice to CDDP, we next examined apoptosis. As compared with on day 0, apoptotic cells became conspicuous on day 2 in both Mdk+/+ and Mdk–/– mice (P < 0.05) (Figure 5B)
. The apoptotic cell count was significantly less in Mdk–/– mice on day 5 (P < 0.001) (Figure 5, A and B)
. This result was contrary to that expected from the anti-apoptotic activity of MK.18,19
As shown in Figure 5C
, most of the apoptotic cells were not positive with the anti-CD45 antibody, which is a marker of inflammatory cells, indicating that apoptotic cells were probably proximal tubular cells but not inflammatory cells.
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. However, there was no significant difference between Mdk+/+ and Mdk–/– mice (Figure 5E)
. Bcl-XL decreased gradually with time after CDDP treatment (Figure 5D)
. BAX and Bid exhibited peaks at 3 to 6 hours, and that of Bak was at 12 hours after CDDP treatment (Figure 5D)
. Also there were no differences between Mdk+/+ and Mdk–/– mice in these expressions (Figure 5E)
. Thus, we could not find any relation between the anti-apoptotic effect of MK and Bcl-2 family proteins in primarily cultured tubular epithelial cells. No Differences in Susceptibility to CDDP and Delivery of CDDP in Mdk+/+ and Mdk–/– Mice
The differences of CDDP nephrotoxicity between Mdk+/+ and Mdk–/– mice might be because of a difference in general susceptibility to CDDP or delivery of CDDP to tubule cells. To address this question, we examined cell survival of MEFs derived from Mdk+/+ and Mdk–/– mice against several doses of CDDP and measured the CDDP in urine. There were no differences in susceptibility at any doses (1 to 50 µmol/L) of CDDP (Figure 6A)
. With regards to the delivery of CDDP to tubule cells, we found no differences of CDDP in urine between Mdk+/+ and Mdk–/– mice (Figure 6B)
.
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Another important activity of MK is a chemotactic one. Therefore, we determined the number of neutrophils that invaded the tubulointerstitium. Although detectable renal damage, as assessed by means of histology and BUN, appeared on day 3 (Figure 4)
, and apoptosis became prominent on day 2 (Figure 5)
, neutrophil infiltration was recognized on day 1 in Mdk+/+ mice (P < 0.005 versus day 0) (Figure 7, A and B)
. In Mdk+/+ mice, neutrophil infiltration reached the maximum level on day 5, and then decreased. In contrast, it became apparent on day 2 (P < 0.005 versus day 0), reaching the maximum level on day 3 in Mdk–/– mice. Thus, Mdk+/+ mice showed rapid induction and prolonged persistence of neutrophil infiltration, whereas Mdk–/– mice exhibited slow induction and rapid fading. Consequently, the number of infiltrating neutrophils was significantly lower in Mdk–/– as compared with in Mdk+/+ mice on days 1 and 5 (Figure 7B)
.
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, macrophage; Figure 7Da
, CD4-positive T cells; Figure 7Db
, CD8-positive T cells). We also found statistical differences between Mdk+/+ and Mdk–/– mice on days 5, 3, and 7, respectively. The results also indicated that neutrophil infiltration was the earliest event in inflammatory cell infiltration (Figure 7B)
.
MK is known to induce chemokines in the ischemic reperfusion injury model of the kidney.17
We thus investigated whether or not chemokines were induced in this CDDP-induced renal damage model. It is well known that KC acts as chemoattractant for neutrophils. ELISA assay showed that KC protein was slowly induced in damaged kidney, whereas we could not find any differences between Mdk+/+ and Mdk–/– mice (Figure 7Ea)
. KC mRNA expression as revealed by Northern blotting showed results similar to those of protein (Figure 7Eb)
. The expression of chemokines, such as MCP-1 and MIP-2, was not detectable on Northern blot analysis in the CDDP model (data not shown), which was in contrast to in the case of the ischemic reperfusion injury model.17
To examine induction of other chemokines, we performed the RNase protection assay. Lymphotactin and MIP-2 were induced from day 2 and reached peaks on day 3 (Figure 7E, c and d)
. However, there were no significant differences in these expression between Mdk–/– and Mdk+/+ mice. The expression of other chemokines, including RANTES, MIP-1, MIP-1ß, IP-10, MCP-1, TCA-3, and eotaxin, was also low and was not different between the two genotypes (data not shown). These results suggest that, even though low expression of chemokines would contribute to neutrophil infiltration, MK plays a direct critical role in the inflammation in the CDDP model.
MK AS ODN Ameliorates Neutrophil Infiltration
To further prove the importance of MK in this model, we used mouse MK AS ODN that had been established in our laboratory.31
MK protein expression was most suppressed when AS ODN was injected via tail vein 16 to 24 hours before sacrifice (Figure 8, A and B)
. In contrast to AS ODN, reverse control (R) did not suppress MK protein expression 24 hours after ODN administration (Figure 8C)
. We speculated that MK was involved in early event of CDDP nephrotoxicity, especially in neutrophil recruitment. Thus, we decided to inject AS ODN before CDDP administration. MK AS ODN injected 24 hours before CDDP administration significantly suppressed the numbers of infiltrating neutrophils on day 1 (24 hours after CDDP administration), as compared with MK R ODN (Figure 8E)
.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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. We speculate that molecular circuits involving MK participate in early phase to induce inflammation. Such molecular circuits may initiate chain reactions, which make the inflammation worse and consequently enhance the death of the proximal tubule epithelial cells. This idea explains why we observed significant differences in apoptosis, BUN, and histological damages in late phase, on day 5, between Mdk+/+ and Mdk–/– mice in this study. Although it is known that MK has chemotactic activity to neutrophils and macrophages,20,21 there are no reports for T cells. No matter how MK exhibits effects on chemotaxis of macrophages and T cells, differences of infiltration of these cells between Mdk+/+ and Mdk–/– mice became apparent in later time points, when MK expression is lower than the normal level in Mdk+/+ mice. Therefore, the above-mentioned chain reactions initiated by neutrophil infiltration in the early stage may eventually influence the recruitment of other leukocytes in later stages. However, we do not exclude the possibility that MK expressed in a minimal amount in later stages might collaborate with inflammatory reactions initiated by early phase neutrophil infiltration, and contribute to the infiltration of macrophages and T cells in later stages, which could influence renal damages as estimated by apoptosis, BUN, and histological changes.
MK exhibited anti-apoptotic activity toward Wilms’ tumor cells, which were used as a model of tubular epithelial cells.18 However, these experiments were performed in vitro. We consider that, in vivo, the anti-apoptotic activity of endogenous MK was overwhelmed by enhanced leukocyte migration caused by MK. Concerning the in vitro anti-apoptotic activity of MK toward neuronal cells,19 we confirmed the activity in vivo: exogenously supplied MK inhibits retinal cell degeneration caused by exposure to constant light23 and delayed death of hippocampal neurons.24 With a large dose of exogenously supplied MK, the anti-apoptotic activity might become more conspicuous. Therefore, we cannot exclude the possibility that MK prevents the damage of tubular epithelial cells, when it is supplied in a large quantity by means of a suitable method.
One of the direct actions of CDDP is the formation of CDDP-glutathione conjugate,32,33
which is subsequently metabolized to CDDP-cysteinyl-glycine conjugates via 
-glutamyl transpeptidase in the proximal tubules. The conjugates may be further cleaved to cysteine-conjugates by aminopeptidase, which is also on the surface of the cells. CDDP-glutathione conjugates, CDDP-cysteinyl-glycine conjugates, and CDDP-cysteine conjugates are more toxic to proximal tubule cells than CDDP.34
Indeed, -glutamyl transpeptidase-deficient mice exhibit resistance to the nephrotoxic effects of CDDP.35
In many studies on CDDP nephrotoxicity, much attention was focused on such direct effects of CDDP. Even though the inflammatory mechanism plays a critical role in the pathogenesis in ischemic acute renal injury,17,36
little information has been available regarding the role of this mechanism in renal damage induced by toxic reagents such as CDDP. However, recent reports demonstrated that inflammation is important in CDDP-induced renal damage.8-10
Ramesh and Reeves10
reported that inhibitors of tumor necrosis factor- ameliorate CDDP-induced renal dysfunction. Therefore, the present study not only supports the importance of inflammation in this model, but also provides an interesting molecular target for prevention of CDDP-induced renal dysfunction.
Because the ODN is preferentially absorbed by the proximal tubule epithelial cells,37,38 preventive administration of the MK anti-sense ODN would be a candidate therapy for CDDP-induced renal dysfunction. The present data support the potential of MK as a candidate molecular target for the prevention of CDDP-induced renal dysfunction.
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Footnotes |
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Supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan (15390103 to T.M.; 14580647 to K.K.).
Accepted for publication July 14, 2004.
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90%. The solid line indicates Mdk+/+ and the dotted line indicates Mdk–/– mice. n = 34; P < 0.01 (log rank test).
, Mdk+/+ mice; 
, Mdk–/– mice. *, P < 0.05 versus wild normal control; **, P < 0.001. B: Tubulointerstitial damage on days 0, 3, and 5 (periodic acid-Schiff staining) is shown. a, Mdk+/+ day 0; b, Mdk+/+ day 3; c, Mdk+/+ day 5; d, Mdk–/– day 0; e, Mdk–/– day 3; f, Mdk–/– day 5. Arrow, cast formation; open arrow, tubular degeneration. C: Semiquantitative analysis of tubulointerstitial damage. The degrees of damage including cast formation, tubular dilation, and tubular degeneration were scored according to the method described in Materials and Methods. These are the results on day 5. More severe damage is indicated by higher values. Data are means ± SE (n = 6). *, P < 0.05; **, P < 0.005. Scale bar, 50 µm (a–e).
