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Published ahead of print on December 28, 2005
J Am Soc Nephrol 17: 442-453, 2006
© 2006 American Society of Nephrology
doi: 10.1681/ASN.2004090755
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Basic Immunology and Pathology

IFN-Inducible Protein-10 Plays a Pivotal Role in Maintaining Slit-Diaphragm Function by Regulating Podocyte Cell-Cycle Balance

Gi Dong Han*,{dagger}, Koichi Suzuki*, Hiroko Koike*, Kenji Suzuki{ddagger}, Hiroyuki Yoneyama§, Shosaku Narumi§, Fujio Shimizu* and Hiroshi Kawachi*

* Department of Cell Biology, Institute of Nephrology; {ddagger} Department of Gastroenterology and Hepatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan, {dagger} Department of Food Science and Technology, Yeungnam University, Gyeongsan, Republic of Korea; and § Department of Molecular Preventive Medicine, School of Medicine and Core Research and Evolutional Science and Technology, University of Tokyo, Tokyo, Japan

Address correspondence to: Dr. Hiroshi Kawachi, Department of Cell Biology, Institute of Nephrology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Niigata, 951-8510, Japan. Phone: +81-25-227-2160; Fax: +81-25-227-0770; E-mail: kawachi{at}med.niigata-u.ac.jp

Received for publication September 10, 2004. Accepted for publication October 25, 2005.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IFN-inducible protein-10 (IP-10/CXCL10) is a potent chemoattractant for activated T lymphocytes and was reported recently to have several additional biologic activities. In this study, the pathophysiologic role of IP-10 in the glomerular visceral epithelial cell (podocyte) was investigated. In cultured podocytes subjected to recombinant IP-10 treatment, the expression of slit-diaphragm (SD) components nephrin and podocin clearly was heightened. Rats that had puromycin aminonucleoside nephropathy and anti–nephrin antibody–induced nephropathy and were subjected to anti–IP-10 function-blocking antibody (anti–IP-10 mAb) treatment displayed a decrease in the protein level of SD components, as well as exacerbated proteinuria. For exploration of the mechanisms of this process, the interaction between IP-10 and the cell-cycle regulatory proteins was investigated. Cultured podocytes subjected to recombinant IP-10 treatment displayed an increase in the protein level of p27Kip1, whereas the levels of cyclins E and A decreased. The expression of IP-10 and SD components was heightened by the treatment of siRNA of cyclin A, whereas these expressions were lowered by the treatment of siRNA of p27Kip1. Proteinuric rats subjected to anti–IP-10 mAb treatment displayed a heightened expression of cyclin A from the early phase of the disease, which indicates that the anti–IP-10 mAb treatment exacerbates podocyte injury by disturbing the cell-cycle balance. These results raise the possibility that IP-10 could become a novel therapeutic target in nephrotic syndrome and several diseases with altered cell-cycle balance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IFN-inducible protein of 10 kD (IP-10/CXCL10), identified as a member of the CXC chemokine family (1), is reported to be expressed in a variety of cells (2–6) and to have several additional biologic activities, such as the modulation of the expression of adhesion molecules and the inhibition of cell proliferation and angiogenesis (7). We have demonstrated that IP-10 can inhibit directly the proliferation of epithelial cells in the murine acute colitis model (8). In our previous study, we reported that IP-10 and its receptor CXCR3 are expressed in glomerular visceral epithelial cells (podocytes) and that IP-10 in the podocyte maintains the differentiated structure and function of the podocyte in an autocrine manner (9).

Podocytes are highly specialized cells that are characterized by interdigitating foot processes and the slit diaphragm (SD) connecting the adjacent foot processes. Because the adjacent foot processes arise from the cell bodies of the neighboring cells, the SD is a highly differentiated intercellular junction. The SD is thought to function as a size-selective permeability barrier in the glomerular capillary wall, preventing the leak of plasma proteins into primary urine (10–12). During the past several years, some molecules have been reported to be associated with SD. Nephrin, identified as a gene product of NPHS1 (the mutated gene of the Finnish type congenital nephrotic syndrome), is considered to be a component of the SD critical for maintaining the barrier function. Following nephrin (13,14), podocin (15,16) and CD2-associated protein (CD2AP) (17) are reported to be functional molecules of the SD. Some recent reports have shown that the expression of these SD components is affected in a variety of genetic and acquired diseases that manifest proteinuria (10,18). Investigations into the precise function of the SD and the regulatory mechanism that maintains the SD function surely will lead to the development of new, effective therapeutic strategies for treating nephrotic syndrome. Another important characteristic of the podocyte is that it is a terminally differentiated cell. Studies on the mechanism of the arrested podocyte cell cycle seem to be worth pursuing not only in the field of nephrology but also in that of cell biology.

The purpose of this study was to investigate whether IP-10 can be a therapeutic target in nephrotic syndrome. We analyzed the kinetics of the expression of IP-10/CXCR3 and the role of IP-10 in two experimental models of nephrotic syndrome, puromycin aminonucleoside (PAN) nephropathy and anti–nephrin antibody (ANA)–induced nephropathy, both with SD dysfunction resulting in proteinuria. The second purpose of this study was to investigate the mechanism by which IP-10 maintains the differentiated podocyte phenotype. It was reported recently that the differentiated podocyte phenotype is maintained by the cell-cycle balance (19). The mammalian cell cycle is governed by the balance of positive and negative cell-cycle regulatory proteins, namely, the cyclins and cyclin-dependent kinase inhibitors (CKI), respectively. Cyclin E is responsible for the progression of the G1/S phase, whereas the S/G2/M phase is promoted by cyclins A and B. The phosphorylation of the retinoblastoma protein (pRb) contributes to the proliferation of cells in the late G1 phase (20,21). These cell-cycle activators are negatively regulated by CKI p27Kip1 and p57Kip2. Some studies have shown that the expression of CKI is lowered in podocyte diseases, such as focal segmental glomerulosclerosis, collapsing glomerulonephropathy, and nephrotic syndrome (22–24). In this study, we analyzed whether IP-10 contributes to the regulation of the expression of the cell-cycle regulatory proteins.

This study shows that IP-10 contributes to the regulation of the expression of SD components not only in the physiologic state but also in pathologic states. It is also demonstrated here that IP-10 regulates the cell-cycle balance of the podocyte. We propose here the heightening of the IP-10 function as one of the attractive therapeutic target candidates in nephrotic syndrome and a variety of diseases in which the negative regulation of the cell-cycle balance is altered.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All experiments were performed using specific pathogen-free female Wistar rats (6 wk old) that weighed 140 to 180 g (purchased from Charles River Japan, Atsugi, Japan). All animal experiments conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Culture of Podocyte
The conditionally immortalized mouse podocyte cell line was provided by Dr. Peter Mundel (Albert Einstein College of Medicine, Bronx, NY). Cultivation of differentiated immortalized mouse podocytes was conducted as reported previously (25). In brief, podocytes were maintained in RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% FBS (Life Technologies Inc., Grand Island, NY), 100 U/ml penicillin (Banyu Pharmaceutical, Tokyo, Japan), and 0.1 mg/ml streptomycin (Meiji Seika Kaisha, Tokyo, Japan). To propagate podocytes, we cultivated cells at 33°C (permissive conditions), and the culture medium was supplemented with 10 U/ml mouse recombinant IFN-{gamma} (rIFN-{gamma}) (Pepro Tech EC, London, England) to enhance expression of a thermosensitive T-antigen. To induce differentiation, we maintained podocytes at 37°C without IFN-{gamma} (nonpermissive conditions) for at least 1 wk before using in the experiment.

Immunohistochemical Studies
Tissue samples for the immunofluorescence (IF) studies were prepared as described previously (9). The frozen sections, 3 µm thick, were cut with a cryostat and stained with the following antibodies. The rabbit anti–cyclin E, rabbit anti–cyclin A, goat anti–IP-10, and goat anti-CXCR3 antibodies were commercially purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The rabbit anti-nephrin antibody (intracellular site) (26) and rabbit anti-podocin antibody (N-terminal site) (16) were prepared as reported previously. FITC-conjugated swine anti-rabbit IgG was used for anti–cyclin E, anti–cyclin A, anti-nephrin, and anti-podocin antibodies. These secondary antibodies were purchased from DAKO (Glostrup, Denmark). FITC-conjugated anti-goat IgG was used for anti–IP-10 and anti-CXCR3 antibodies. These secondary antibodies were purchased from Southern Biotechnology Associates (Birmingham, AL).

Western Blot Analysis
The rat glomeruli and conditionally immortalized podocyte were isolated with PBS that contained protease inhibitors and solubilized with SDS sample buffer (consisting of 5% SDS, 6% beta-mercaptoethanol, 150 mmol/L NaCl, 10% glycerol, and 0.001% bromphenol blue in 250 mol/L Tris-HCl [pH 6.8]) with protease inhibitors. The insoluble material was removed by centrifugation at 15,000 x g for 10 min. The concentration was measured by the bicinchoninic acid method (Pierce Chemical, Rockford, IL), and the solubilized material was subjected to SDS-PAGE with 5, 10, or 12% acrylamide gel according to the method of Laemmli et al. (27) and transferred to a polyvinyl difluoride membrane (Bio-Rad, Hercules, CA) by electrophoretic transblotting for 30 min using Trans-Blot SD (Bio-Rad). After blocking with bovine skim milk, strips of the membranes were exposed to the primary antibodies as described above and additionally rabbit anti-p27Kip1, rabbit anti-p57Kip2, rabbit anti-pRb antibodies, and mouse anti-Rb mAb (Cell Signaling Technology Inc., Danvers, MA) and rabbit anti–beta-actin antibody (Sigma, St. Louis, MO). After overnight incubation, the membranes were washed three times and then incubated with alkaline phosphatase–conjugated goat anti-rabbit IgG (Bio Source International, Tago Immunologicals, Camarillo, CA) or with alkaline phosphatase–conjugated anti-mouse IgG (Bio Source International, Tago Immunologicals). The reaction was developed with an alkaline phosphatase chromogen kit (5-bromo-4-chloro-3-indolil phosphate p-toluidine salt/nitro blue tetrazolium; Biomedica, Foster City, CA).

Reverse Transcription–PCR Analysis
Semiquantitative reverse transcription–PCR (RT-PCR) with glomerular total RNA and conditionally immortalized podocyte total RNA was performed basically according to the method described previously (9). The primers were designed according to the published sequences (Table 1). Negative controls without cDNA and positive controls of cDNA from Con-A–stimulated rat spleen cells were included.


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  		Table 1. PCR primers used in this studya

 
Experimental Design
rIP-10 and Anti–IP-10 mAb Treatment of Cultured Podocytes.
Podocytes cultured under nonpermissive conditions were treated as follows: Treatment 1, the cells were treated with medium that contained 10 ng/ml rIP-10 (RELIA tech GmbH, Braunschweig, Germany); treatment 2, the cells were treated with medium that contained 10 ng/ml rIP-10 after preincubation with 0.2 mg/ml anti–IP-10 mAb for 1 h; treatment 3, The cells were treated with medium without rIP-10 after preincubation with 0.2 mg/ml anti–IP-10 mAb; and treatment 4, the cells were treated with medium without both rIP-10 and anti–IP-10 mAb.

The cells were harvested after incubation with rIP-10 for 24 h. The optimal concentrations of rIP-10 and anti–IP-10 mAb were determined in preliminary experiments. mRNA expression of nephrin and podocin in the harvested cells was analyzed by semiquantitative RT-PCR, and the protein levels of cell-cycle regulatory proteins, including cyclins E and A, CKI p27Kip1 and p57Kip2, retinoblastoma gene product (Rb), and phosphorylated Rb (pRb), were analyzed by Western blotting. Each set of experiments was repeated at least five times. The anti–IP-10 mAb was obtained by immunizing mice with rat CXCL10/Fc fusion protein as described previously (28).

Podocytes cultured under permissive conditions were also treated with rIP-10 or anti–IP-10 mAb for 24 or 48 h. After the treatment, the numbers of the cells were counted using a hemacytometer.

Small Interfering RNA.
The small interfering RNA (siRNA) sequences that target cyclin A (National Center for Biotechnology Information accession no. Z26580; 25 nucleotides in length corresponding to positions 948 to 972 of open reading frame) and p27Kip1 (National Center for Biotechnology Information accession no. BC014296; 21 nucleotides in length corresponding to positions 73 to 93 of open reading frame) were synthesized by iGENE Inc. (Tsukuba, Japan) and Qiagen Inc. (Dusseldorf, Germany), respectively. Control siRNA were purchased from Qiagen Inc. Before transfection, podocytes were cultured to a density of 70 to 80% at 37°C as described above and then were transfected with the siRNA using a Trans IT-TKO transfection reagent (Mirus, WI) or HiPerFect Transfection Reagent (Qiagen Inc.) protocols. Cells were harvested 48 h after siRNA treatment for RT-PCR and Western blots analyses. Podocytes cultured under permissive conditions were also treated with those siRNA and harvested in 48 h for counting the numbers of cells.

Studies in PAN and ANA Nephropathy.
PAN nephropathy was induced in rats by the intravenous injection of 10 mg/100 g body wt PAN. ANA nephropathy was induced in rats by the intravenous injection of 8 mg/rat ANA (anti-nephrin antibody 5-1-6). Anti-nephrin mAb 5-1-6 was prepared as described previously (29). The rats were killed at 1 h after the induction of the disease, on days 1, 4, 9, and 28 of PAN nephropathy and at 1 h and days 1, 5, and 14 of ANA nephropathy (n = 5 per each time point). The right kidney was removed, weighed, cut into portions, and used for the assessment of IF. The left kidney and remaining portion of the right kidney were pooled in each group and used to prepare total glomerular RNA. Twenty-four-hour urine samples were collected just before the rats were killed. Urine protein concentrations were determined by colorimetric assay (Bio-Rad, Oakland, CA) using BSA as a standard. The kinetics of the expression of IP-10, CXCR3, nephrin, and podocin were analyzed by IF and semiquantitative RT-PCR. The staining of cyclins A and E was also analyzed.

In Vivo Anti–IP-10 mAb Function-Blocking Study.
Anti–IP-10 mAb (3 mg/100 g body wt) was injected intravenously into normal Wistar rats daily (n = 3). As a control, RVG1 was injected instead of anti–IP-10 mAb (n = 3). The kidneys of these rats were removed and used for the assessment of IF on day 5. The expression of p27Kip1, cyclin A, and cyclin E was analyzed by IF.

Anti–IP-10 mAb (3 mg/100 g body wt) was injected intravenously into rats with PAN nephropathy and ANA nephropathy at 5 h after disease induction. The rats were treated daily with anti–IP-10 mAb until the day they were killed. As a control, RVG1 was injected instead of anti–IP-10 mAb. The kidneys of these rats were removed on days 9 and 21 for PAN nephropathy and on days 5 and 14 for ANA nephropathy (n = 5 per each time point of the model). The right kidney was weighed, cut into portions, and used for the assessment of IF. The glomeruli were isolated from the left kidneys, and the remaining portion of the right kidneys were pooled in each group and were placed into two tubes. One was used to prepare total glomerular RNA, and the other was used for glomerular lysate. Glomerular mRNA expression of podocyte-associated proteins (nephrin, podocin, podoplanin, and podocalyxin) and IP-10 was analyzed by RT-PCR. The protein level of nephrin and podocin was analyzed by Western blotting with glomerular lysate. Twenty-four-hour urine samples were collected on days 3, 5, 7, 9, 14, 21, and 28 in PAN nephropathy and on days 1, 3, 5, 7, 10, and 14 in ANA nephropathy. Urine protein concentrations were determined as described above. The expression of cyclin A on day 9 of PAN nephropathy and on day 5 of ANA nephropathy was analyzed by IF.

Statistical Analyses
All values are expressed as means ± SD. The statistical significance (defined as P < 0.05) was evaluated using the unpaired t test or Mann Whitney U test. Data were analyzed using the GraphPad InStat 3.05 (GraphPad Software Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
rIP-10 Treatment Heightened Expression of SD Components in Cultured Podocytes
Heightened mRNA of the podocin and nephrin and heightened protein level of podocin were detected in the cultured podocytes treated with rIP-10 for 24 h. This effect was interfered with by the preincubation treatment with anti–IP-10 mAb. The expression of podocin and nephrin in cultured podocytes treated with anti–IP-10 mAb, without subsequent incubation of rIP-10, was lower compared with the control level (Figure 1).


Figure 1
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  		Figure 1. Reverse transcription–PCR (RT-PCR) and Western blot analyses of podocin and nephrin in murine cultured podocytes treated with recombinant IFN-inducible protein-10 (rIP-10). The cultured podocytes were incubated with rIP-10 for 24 h after preincubation with or without anti–IP-10 mAb. The mRNA expressions of podocin and nephrin (A) and protein level of podocin (B) were heightened as a result of the administration of rIP-10, and the effect was inhibited by preincubation with anti–IP-10 mAb. The expression of podocin and nephrin in cultured podocytes treated with anti–IP-10 mAb without subsequent incubation of rIP-10 was lower compared with the control level. The ratios of the densitometric signal of podocin and nephrin to that of the internal control (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], beta-actin) were analyzed. The data are shown as ratios (%) relative to the normal group and are expressed as mean ± SD of three independent experiments.

 
Expression of IP-10 and CXCR3 Was Heightened in PAN and ANA Nephropathy, Whereas that of Nephrin and Podocin Was Lowered
The amount of 24-h proteinuria in PAN nephropathy was as follows: Day 1, 1.73 ± 0.7; day 9, 255.4 ± 43.2; and day 28, 20.1 ± 13.9. That in ANA nephropathy was as follows: Day 1, 48.8 ± 29.4; day 5, 188.9 ± 122.1; and day 14, 6.0 ± 3.5. mRNA expression of IP-10 was already heightened at 1 h in both PAN and ANA nephropathy. Although the heightened expression transiently declined on day 1, a clear increase in IP-10 was observed on day 9 of PAN and on day 5 of ANA nephropathy, when the amount of proteinuria peaked. In the ANA model, the mRNA expression of CXCR3 gradually increased from hour 1 to day 5, and the heightened expression continued to day 14. A CXCR3 expression pattern similar to that of the ANA model was seen in the PAN model, except for the lowered expression pattern on day 28.

By contrast, the mRNA expression of nephrin significantly decreased on day 1 and gradually recovered in both models. Lowered expression of podocin was also detected on day 1 in both models. The kinetics of the mRNA expression of these molecules is shown in Figure 2.


Figure 2
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  		Figure 2. The kinetics of the mRNA expression of IP-10, CXCR3, and SD components in puromycin aminonucleoside (PAN) and anti–nephrin antibody (ANA) nephropathy. The mRNA expressions of IP-10, CXCR3, nephrin, and podocin were semiquantified by RT-PCR. The ratios of their densitometric signals to that of the internal control (GAPDH) were analyzed. The data are shown as ratios (%) relative to the RVG1-injected control group and are expressed as mean ± SD of three independent experiments. The mRNA expression of IP-10 and CXCR3 was heightened on days 9 and 5 after the induction of PAN and ANA nephropathy, respectively, when massive proteinuria was detected. By contrast, the mRNA expression of nephrin and podocin was significantly lower already on day 1 in both models and recovered or was heightened on days 28 and 14 of PAN and ANA nephropathy, respectively, at which time the proteinuria was normalized.

 
Figure 3 shows the kinetics of IF staining of IP-10, CXCR3, nephrin, and podocin in PAN and ANA nephropathy. The immunostaining intensity of IP-10 and CXCR3 was clearly heightened on day 5 in ANA and on day 9 in PAN nephropathy, when massive proteinuria was observed. The heightened staining was observed in the form of a quasi-linear pattern along the glomerular capillary wall. By contrast, the immunostaining intensity of nephrin and podocin was dramatically decreased at the peak of proteinuria in both models.


Figure 3
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  		Figure 3. The kinetics of immunofluorescence (IF) staining of IP-10, CXCR3, nephrin, and podocin in PAN and ANA nephropathy. The immunostaining intensity of IP-10 and CXCR3 was clearly higher on day 9 of PAN and on day 5 in ANA nephropathy, when massive proteinuria was observed. The intensified staining was observed in the form of a linear pattern along the glomerular capillary wall. By contrast, the immunostaining intensity of nephrin and podocin was dramatically lower at the peak of proteinuria in both models. Magnification, x400.

 
Blocking of IP-10 Exacerbates PAN and ANA Nephropathy
The anti–IP-10 mAb treatment resulted in significantly exacerbated proteinuria on days 7 and 9 in PAN and on days 3 and 5 in ANA nephropathy (Figure 4A). Anti–IP-10 mAb treatment enhanced the decrease in the mRNA expression of podocin and podoplanin in both models (Figure 4B). Anti–IP-10 mAb also lowered the mRNA expression of IP-10 in PAN nephropathy. Western blot quantification showed that anti–IP-10 treatment decreased the protein levels of podocin and nephrin in both PAN and ANA nephropathy (Figure 4C).


Figure 4
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  		Figure 4. The effect of anti–IP-10 mAb treatment on PAN and ANA nephropathy. Effect of anti–IP-10 mAb (5 mg/100 g body wt) treatment on the kinetics of proteinuria (A), the mRNA expression of podocyte-associated proteins (B), and the protein level of nephrin and podocin (C) were analyzed. Daily injection with anti–IP-10 mAb exacerbated the proteinuria on days 7 and 9 and on days 3 and 5 after the induction of PAN and ANA nephropathy, respectively (A). mRNA (B) and Western blot (C) samples were prepared from the rats of days 9 and 5 after the induction of PAN and ANA nephropathy, respectively, when massive proteinuria was observed. The anti–IP-10 mAb treatment enhanced the decrease in the mRNA and protein levels of podocyte-associated molecules in both models (B and C). Equal amounts (250 µg) of solubilized glomerular lysate from the anti–IP-10 treatment group and the control group were loaded onto each lane. For ensuring equal loading, the translated membrane of each group was stained with Coomassie Brilliant Blue. Each Western blot was performed three times. Data are expressed as mean ± SD (n = 5; *P<0.05, **P < 0.01 versus the control group).

 
IF Staining of p27Kip1 and Cyclins E and A Altered in PAN and ANA Nephropathy
The expression of p27Kip1 was clearly detected in normal glomeruli, and it gradually decreased from day 1 to day 9 after induction of PAN. The gradually decreasing staining of p27Kip1 was also observed in ANA nephropathy. The expression of cyclins E and A was weakly detected in normal glomeruli, and it gradually increased from day 1. The intensified staining of cyclins A and E became more remarkable on days 5 and 9 after the induction of PAN and ANA nephropathy, respectively, when massive proteinuria was observed (Figure 5).


Figure 5
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  		Figure 5. The kinetics of IF staining of p27Kip1 and cyclins E and A in PAN and ANA nephropathy. The expression of p27Kip1 was clearly detected in normal glomerular section and gradually decreased from day 1 to day 9 and day 5 after induction of PAN and ANA nephropathy, respectively. Heightened expression of cyclins E and A was already detected on day 1 after disease induction in both models. The intensified staining of cyclins E and A became more remarkable on days 9 and 5 after the induction of PAN and ANA nephropathy, respectively, when massive proteinuria was observed. Magnification, x400.

 
rIP-10 Treatment Altered Expression of Cell-Cycle Regulatory Proteins in Nonpermissive Conditioned Podocytes and Inhibited Proliferation of Permissive Conditioned Podocytes
Heightened expression of p27Kip1 and lowered expression of cyclins A and E were detected in the cultured podocytes treated with rIP-10 for 24 h. Anti–IP-10 mAb pretreatment inhibited the effect of rIP-10. Anti–IP-10 mAb treatment without subsequent rIP-10 incubation resulted in a decrease in the p27Kip1 level, and the treatment clearly increased the protein level of cyclin E, cyclin A, and pRb. No specific changes in the expression of p57Kip2 and Rb were detected after these treatments (Figure 6A). The treatment with rIP-10 for 48 h inhibited the proliferation of permissive conditioned podocyte, whereas the treatment with anti–IP-10 for 24 and 48 h enhanced the proliferation (Figure 6B).


Figure 6
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  		Figure 6. The effect of the treatments of rIP-10 and anti–IP-10 mAb on the expression of the cell-cycle regulatory proteins and on the proliferation. (A) Heightened expression of p27Kip1 and lowered expression of cyclins E and A were detected in the nonpermissive conditioned cultured podocytes treated with rIP-10 for 24 h (second lane from the left). Anti–IP-10 mAb pretreatment inhibited this effect of rIP-10 (third lane). Anti–IP-10 mAb treatment without subsequent rIP-10 incubation lowered the p27Kip1 level while clearly heightening the protein level of cyclin E, cyclin A, and retinoblastoma protein (pRb; fourth lane). No specific changes in the expression of p57Kip2 and Rb were detected as a result of the intervention of IP-10. The ratios of their densitometric signals to that of the internal control (beta-actin) were analyzed. The data are shown as ratios (%) relative to the normal group and are expressed as mean ± SD of three independent experiments. (B) Proliferation assay was carried out with permissive conditioned cultured podocyte. The treatment with anti–IP-10 for 24 and 48h enhanced the proliferation of the cells, whereas the treatment of rIP-10 for 48 h reduced the number of the cells.

 
siRNA of Cyclin A and p27Kip1 Affected Expression of IP-10 and SD Components
The treatment with siRNA for cyclin A and p27Kip1 clearly silenced the expression of each target molecule in both mRNA and protein levels in nonpermissive conditioned podocyte. The treatment with siRNA for cyclin A enhanced the expressions in both mRNA (Figure 7A) and protein (Figure 7B) levels of IP-10 and SD components, whereas the siRNA treatment for p27Kip1 lowered them. The treatment with siRNA for cyclin A for 48 h inhibited the proliferation of permissive conditioned podocyte, whereas the treatment with siRNA of p27Kip1 for 48 h enhanced it (Figure 7C).


Figure 7
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  		Figure 7. The effect of the treatment of small interfering RNA (siRNA) of cyclin A and p27Kip on the expression of IP-10 and SD components and on the proliferation. siRNA of cyclin A and p27Kip1 clearly lowered the expression of each target molecule in both mRNA and protein levels in nonpermissive conditioned podocyte. siRNA treatment of cyclin A heightened the expression of IP-10 and SD components in both mRNA (A) and protein (B) levels, but the siRNA p27Kip1 lowered the expression of these molecules. The treatment with siRNA for cyclin A for 48 h inhibited the proliferation of permissive conditioned podocyte, whereas the treatment with siRNA of p27Kip1 for 48 h enhanced it (C).

 
Daily Anti–IP-10 mAb Injections into Normal Rats Lowered Expression of p27Kip1 and Enhanced Expression of Cyclins E and A
Daily anti–IP-10 mAb treatment for 5 d lowered the expression of p27Kip1, whereas the anti–IP-10 mAb treatment enhanced the expression of cyclins E and A in the glomeruli (Figure 8).


Figure 8
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  		Figure 8. The effect of anti–IP-10 mAb treatment on the IF staining of p27Kip1 and cyclins E and A. The expression of p27Kip1 decreased in rats that received daily injections of anti–IP-10 mAb for 5 d, whereas the expression of cyclins E and A in the glomerular podocyte clearly heightened in rats treated with anti–IP-10 mAb. No altered expression of them was observed in rats treated with control IgG1 (RVG1). Magnification, x200 in upper lane; x400 in lower lane.

 
Blocking of IP-10 Heightened Expression of Cyclin A in PAN and ANA Nephropathy
Daily injection with anti–IP-10 mAb promoted the increase of cyclin A expression on days 5 and 9 after the induction of ANA and PAN nephropathy, respectively, when massive proteinuria was observed (Figure 9).


Figure 9
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  		Figure 9. The effect of anti–IP-10 mAb treatment on the IF staining of cyclin A in PAN and ANA nephropathy. Heightened expression of cyclin A was observed on day 9 and day 5 after the induction of PAN and ANA nephropathy, respectively, when massive proteinuria was observed (irrelevant IgG1, RVG1-treated group). Daily injection with anti–IP-10 mAb enhanced the increase of cyclin A expression of both PAN and ANA nephropathy. The left top figure (normal) indicates the finding of normal rat. Magnification, x400.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Podocytes, which are highly specialized, terminally differentiated cells, are characterized by the SD, a unique cell–cell junction structure. The SD plays a critical role in maintaining the barrier function of the glomerular capillary wall, preventing the leak of plasma proteins into urine. It is now accepted that SD dysfunction is involved in the development of proteinuria in a variety of diseases (11,30). IP-10, a CXC chemokine, is reported to have multiple functions, such as the regulation of the adhesion molecules and the inhibition of cell proliferation and angiogenesis. In the previous report, we showed that IP-10 contributes to the regulation of the expression of SD components. We also showed that anti–IP-10 function-blocking antibody treatment exacerbates mesangial alteration by disturbing the podocyte function (9). In this study, we first investigated whether the regulation of the IP-10 function can be a therapeutic target in podocyte injuries that manifest massive proteinuria.

We started this study to investigate whether rIP-10 treatment affects the expression of the differentiated podocyte molecules of the cultured podocytes, because in the previous report, we did not offer direct evidence for the capacity of IP-10 to enhance the expression of these molecules. As shown in Figure 1, IP-10 enhanced the mRNA expression of the SD components nephrin and podocin and also the protein expression of podocin, and this effect was inhibited by anti–IP-10 mAb. These in vitro findings and the in vivo findings in the previous report clearly show that IP-10 is involved in maintaining the differentiated podocyte phenotype in the physiologic state. Next, we analyzed the expression of IP-10 and its receptor CXCR3 in the podocyte in pathologic states. In this study, we adopted two rat models of podocyte injury, PAN nephropathy and ANA nephropathy. PAN nephropathy is widely used as a model of human minimal change-type nephropathy (31). ANA nephropathy is a model of proteinuria that is caused directly by nephrin dysfunction. In concordance with the previous report, the expression of nephrin and podocin clearly decreased in both models (16,26), when massive proteinuria was detected (Figures 2 and 3). By contrast, the expression of IP-10 and CXCR3 was markedly heightened at those time points (Figures 2 and 3). It is conceivable that the heightened expression of IP-10/CXCR3 is a protective response by which podocytes maintain their function, because the findings obtained in this and the previous studies suggest that IP-10 plays a role in maintaining the expression of SD components. To clarify this mechanism, we then analyzed the effect of anti–IP-10 function-blocking antibody administration on the severity of these podocyte injuries. We observed that anti–IP-10 function-blocking antibody treatment exacerbated proteinuria in both models by promoting the decrease in the expression of SD components (Figure 4), which suggests that IP-10 contributes to the expression of SD components not only in the physiologic state but also in pathologic states. Although the expression of IP-10 in human glomeruli is not precisely outlined yet in other reports, we have found that IP-10 is expressed in the form of an epithelial pattern along the glomerular capillary wall in humans as well as in rats (data not shown). All of these findings suggest that the heightening of the IP-10 function could be a therapeutic target in nephrotic syndrome.

The next important question that should be asked is how the podocyte maintains its differentiated phenotype. It was reported recently that the differentiated podocyte phenotype is maintained by the cell-cycle balance (19). In this study, we analyzed the immunohistochemical staining of the cell-cycle regulatory proteins of the podocyte in the normal rat and in experimental models of podocyte injury with massive proteinuria, PAN and ANA nephropathy. Although p27Kip1 staining of the normal rat glomeruli was detected mainly in the mesangial area, the staining along the capillary wall was also observed (Figure 5). The decreased expression of p27Kip1 in glomeruli was observed when massive proteinuria was detected. No Rb or pRb staining was detected in the glomeruli. By contrast, cyclins A and E were observed in the form of podocyte patterns along the glomerular capillary wall. Heightened expression of cyclins A and E was already detected on day 1 after disease induction in both models, when abnormal proteinuria had not occurred yet. In both models, the intensified staining of cyclins A and E became more remarkable when massive proteinuria was detected. In both models, the staining of cyclins A and E returned to normal when proteinuria was normalized (Figure 5). These findings clearly show that the cell-cycle balance of the podocyte is altered in these proteinuric states caused by SD dysfunction. The causal relationship between the cell-cycle balance and the expression of SD components is uncertain. It is generally observed that the expression of differentiated functional molecules is lower in cells whose negative regulation of cell-cycle balance is altered. Several investigations have suggested that cell-cycle regulatory proteins may regulate the expression of differentiated functional molecules (22,23). Conversely, some reports have shown that the expression of the intercellular junctional complex plays a role in regulating the cell cycle (32,33). It is conceivable that the cell-cycle balance and the expression of molecules that appear in the differentiated phenotype of the cells must reciprocally regulate each other. However, it should be noted that the expression of cyclin A, the positive regulator of the cell cycle, was clearly heightened already on day 1 after PAN injection (Figure 5), when the lowered expression of SD components was not yet remarkable (Figure 3). This suggests that the altered cell-cycle balance gives rise to a lowered expression of SD components in PAN nephropathy. Heightened expression of cyclin A was detected in the early phase (day 1) in ANA nephropathy as well. Although the proteinuria in ANA nephropathy is considered to result directly from the SD dysfunction caused by antibody binding, this finding suggests that the altered cell-cycle balance of the podocyte contributes to the development of proteinuria in ANA nephropathy as well.

Then, we investigated whether IP-10 regulates the cell-cycle balance. The effects of the rIP-10 treatment and the anti–IP-10 function-blocking antibody treatment on the expression of the cell-cycle regulatory proteins in the cultured podocytes were analyzed. Western blot analysis showed that rIP-10 treatment heightened the expression of p27Kip1 and p57Kip2 in the cultured podocytes, whereas it lowered the expression of cyclin E, cyclin A, and pRb. The p27Kip1 that is enhanced by IP-10 may downregulate the expression of cyclin E, cyclin A, and pRb (Figure 6A). We also showed that rIP-10 treatment inhibited the proliferation of permissive conditioned podocyte (Figure 6B). It is not clear whether this effect of IP-10 is through CXCR3 receptor, because the expression of CXCR3 was rarely detected in permissive conditioned podocyte. Whatever their precise mechanism is, these findings clearly indicate that IP-10 contributes to the regulation of the expression of cell-cycle regulatory proteins and also to the inhibition of the proliferation of podocyte. These effects of rIP-10 were neutralized or reversed by co-incubation with anti–IP-10 function-blocking antibody. Anti–IP-10 mAb treatment without rIP-10 pretreatment clearly heightened the expression of cyclins E and A and lowered the expression of p27Kip1 and p57Kip2, which means that anti–IP-10 mAb blocked endogenous IP-10 in the cultured podocytes. Recently, siRNA has become a specific and useful technique to turn off the expression of target genes (34). To reduce the expression of cyclin A and p27Kip1, we used siRNA targeting the two molecules. The expression of IP-10 and SD components was heightened by the treatment of siRNA of cyclin A. The treatment with siRNA p27Kip1 lowered the expression of IP-10 and SD components (Figure 7). The finding clearly showed the link between cell-cycle protein and IP-10 and SD components. The effects of IP-10 on the regulation of the cell cycle were confirmed by in vivo studies on rats treated with anti–IP-10 mAb. The rats that received injections of anti–IP-10 mAb showed a clearer expression of cyclins E and A than the rats that received injections of irrelevant antibodies (Figure 8). These data clearly show that, in vivo, IP-10 is involved in the regulation of the cell-cycle pathways. Luster et al. (35) stated that IP-10 inhibits endothelial cell proliferation as well as platelet factor 4 (PF4). Romagnani et al. (36) reported that CXCR3 expression is limited to the S/G2-M phase of the endothelial cell. They also reported that PF4, which shares the binding for CXCR3 with IP-10, upregulates the p21 level. However, no reports providing direct evidence that IP-10 is associated with the expression of cell-cycle regulatory proteins have been published. Our study is the first report to demonstrate that IP-10 plays a role in the regulation of the cell-cycle balance. More important, we have demonstrated here that anti–IP-10 antibody injection enhances the expression of cyclin A in the podocytes of proteinuric rats as well as normal rats. On the basis of these findings, we think that anti–IP-10 mAb treatment exacerbates podocyte injuries by disturbing the cell-cycle balance. We propose IP-10 as a possible therapeutic target candidate not only in podocyte injury but also in several diseases in which the negative regulation of the cell-cycle balance is broken down, although additional studies with other kinds of cell lines and tissues are needed to confirm this.

Finally, the question of whether IP-10 functions in the podocyte by binding its receptor, CXCR3, should be discussed. We have demonstrated here that the expression of CXCR3 increases in the injured podocyte in parallel with that of IP-10 (Figure 3), which suggests that IP-10 functions in the podocyte through CXCR3. It is reported that chemokines other than IP-10, which share the binding to CXCR3, can inhibit the proliferation of human microvascular endothelial cells as well and that the effect is inhibited by anti-CXCR3 antibody (36). Recently, Lasagni et al. (37) reported that CXCR3 has an alternative splicing variant (CXCR3-B) and that CXCR3-B mediates the inhibitory activity of IP-10 on the growth of human endothelial cells. Further characterization of CXCR3 may allow the development of new effective therapeutic strategies for podocyte injuries and other diseases that are caused by altered cell-cycle balance.

In conclusion, our study has demonstrated for the first time that IP-10 plays a pivotal role in maintaining the SD function by regulating the cell-cycle balance of the podocyte. IP-10/CXCR3 could be an attractive therapeutic target for nephrotic syndrome and a variety of diseases in which the negative cell-cycle balance has been disturbed.


   Acknowledgments
 
This work was supported by Grant-Aids for Scientific Research (B) (13557084 to H. Kawachi), Grant-Aids for Scientific Research (B) (14370317 to H. Kawachi), and Grant-Aids for Scientific Research (B) (15390268 to F. Shimizu) from Ministry of Education, Science, Culture and Sports of Japan.

We are grateful to Dr. Peter Mundel for willingness to provide valuable material for this study. We express our gratitude to Dr. Yumiko Fujioka, Dr. Naoko Miyauchi, Dr. Tamaki Karasawa, and Dr. Yutaka Harita for helpful discussions. We also thank Mutsumi Kayaba and Chiharu Nagasawa for tremendous technical assistance.


   Footnotes
 
Published online ahead of print. Publication date available at www.jasn.org.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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