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【摘要】 Cultured podocytes easily lose expression of nephrin. In this report, we developed optimum media for recovery and maintenance of nephrin gene expression in murine podocytes. Using reporter podocytes, we found that activity of the nephrin gene promoter was enhanced by DMEM/F12 or -MEM compared with RPMI-1640. In any of these basal media, addition of 1,25-dihydroxyvitamin D 3, all- trans -retinoic acid or dexamethasone significantly increased activity of the nephrin promoter. The effects of the supplemental components were synergistic, and the maximum activation was achieved by DMEM/F12 supplemented with three agents. This culture medium was designated as v itamin D 3, r etinoic a cid and d examethasone-supplemented D MEM/F12 (VRADD). In reporter podocytes that express nephrin, VRADD induced activation of the nephrin gene promoter up to 60-fold. Even in podocytes that have lost nephrin expression during multiple passages, expression of nephrin mRNA was dramatically recovered by VRADD. However, VRADD caused damage of podocytes in prolonged cultures, which was avoided in the absence of dexamethasone (designated as VRAD). VRAD maintained expression of nephrin for extended periods, which was associated with the differentiated phenotype of podocytes. Using the VRAD-primed podocytes, we revealed that expression of nephrin mRNA as well as nephrin promoter activity was suppressed by a putative dedifferentiation factor of podocytes, hepatocyte growth factor.
【关键词】 retinoic acid vitamin D dexamethasone hepatocyte growth factor
THE SLIT DIAPHRAGM LOCATED at the interface of interdigitated foot processes in podocytes plays a crucial role as the glomerular filtration barrier ( 27, 39 ). Previous investigation identified nephrin, a podocyte-specific protein, as a key component involved in the structure and function of the slit diaphragm ( 35 ). The level of nephrin decreases in various proteinuric glomerular diseases including minimal change disease, focal segmental glomerulosclerosis, and diabetic nephropathy ( 6, 13 ). It is important to identify mechanisms involved in the downregulation of nephrin in disease states. For this purpose, in vitro investigation using cultured podocytes should provide a powerful tool. Several podocyte cell lines, as well as primary cultures of podocytes, have been established and utilized to date ( 22, 29, 32 ). However, dedifferentiation of podocytes often occurs in vitro, including rapid loss of specialized foot processes ( 40, 48 ) and attenuation of differentiation marker expression ( 20, 21 ). In particular, among several differentiation markers of podocytes, expression of nephrin is lost very easily. In primary cultures, podocytes express nephrin only at early passages and in low abundance ( 34, 47 ). During establishment of conditionally immortalized podocytes, Schiwek et al. ( 32 ) reported that expression of nephrin mRNA was detectable only in 2 of 30 podocyte clones that were positive for both WT-1 and podocalyxin. It means that 93% of established podocyte clones had selectively lost expression of nephrin mRNA. Of note, podocytes with very low levels of nephrin gene expression have been used in several previous reports ( 4, 10, 28 ). It is the current, crucial problem for investigating biological function of nephrin as well as regulation of the nephrin gene in cultured podocytes. To overcome this problem, we aimed in the present study at developing culture media optimized for recovery and maintenance of nephrin gene expression in podocytes. For this purpose, we used conditionally immortalized reporter podocytes that produce secreted alkaline phosphatase (SEAP) under the control of the nephrin gene promoter ( 46 ). Using this reporter system, we examined effects of different basal culture media supplemented with different substances that may allow for recovery and maintenance of nephrin gene expression.
We recently reported that, when added to RPMI-1640, 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ], all- trans -retinoic acid (ATRA) and dexamethasone significantly upregulated activity of the nephrin gene promoter as well as expression of nephrin mRNA during short-term treatments ( 46 ). In the present investigation, these substances were added singly or in combination to different basal media including RPMI-1640, DMEM/F12, and -MEM, and their effects on nephrin gene expression and cellular differentiation were tested using short-term and long-term cultivation protocols. In this report, we first demonstrate that v itamin D 3, r etinoic a cid and d examethasone-supplemented DMEM/F12 (VRADD) is the most potent, optimum medium for recovery of nephrin gene expression in podocytes. We next show that DMEM/F12 supplemented with vitamin D 3 and retinoic acid (VRAD) is suitable for the maintenance of nephrin gene expression and cellular differentiation in prolonged cultures. Finally, using VRAD-primed differentiated podocytes that express nephrin abundantly, we provide evidence that hepatocyte growth factor (HGF), a recently reported putative dedifferentiation factor of podocytes ( 25 ), depresses expression of the nephrin gene as well as activity of the nephrin gene promoter in murine podocytes.
MATERIALS AND METHODS
Cells and stable transfectants. Conditionally immortalized murine podocyte cell lines ( 22, 32 ) were kindly provided by Drs. Karlhans Endlich (University of Heidelberg, Heidelberg, Germany) and Peter Mundel (Albert Einstein College of Medicine). The cells established by Schiwek et al. ( 32 ) were generally used for experiments ( passages 13-25 ). For maintenance and/or propagation, the podocytes were cultured at 37°C in type I collagen-coated flasks using RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% FBS and 10 U/ml mouse IFN- (PeproTech, Rocky Hill, NJ). Reporter podocytes REPON5.4 were established by stable transfection of murine podocytes with pN5.4-SEAP, which introduces the SEAP gene under the control of the 5.4-kb murine nephrin gene promoter ( 19, 46 ).
Pharmacological treatments. Podocytes were seeded onto collagen-coated plates and preincubated at 37°C for 48 h in RPMI-1640 supplemented with 10% FBS in the absence of IFN-. The cells were then cultured for 24 h-1 mo in 10% FBS-containing basal media (RPMI-1640, DMEM/F12, GIBCO BRL, Gaithersburg, MD; or -MEM, Invitrogen) supplemented with 1,25(OH) 2 D 3 (10-100 nM, Chugai Pharmaceutical, Tokyo, Japan), ATRA (1 µM, Genzyme, Cambridge, MA), and/or dexamethasone (0.1 µM, Sigma-Aldrich Japan, Tokyo, Japan). Effects of sodium pyruvate (0.1-1 mM; Sigma-Aldrich Japan), D -pantothenic acid (0.1-1 µM, Sigma-Aldrich Japan), folic acid (0.1-1 µM, Sigma-Aldrich Japan), vitamin B 12 (1-10 µg/ml, Wako Pure Chemical Industries, Osaka, Japan), or human recombinant HGF (10-100 ng/ml, PeproTech EC, London, UK) were also tested. After incubation, the cells and culture media were subjected to RT-PCR and chemiluminescent assay for SEAP, respectively.
RT-PCR. Total RNA was extracted using TRIzol Reagent (Invitrogen), and 1 µg of RNA was subjected to reverse transcription using Omniscript Reverse Transcriptase (Qiagen, Tokyo, Japan). Reaction mixtures without reverse transcriptase were used as negative controls. The following primers were purchased from Operon (Tokyo, Japan) and used for analyses.
Nephrin: forward, 5'-CCCCAACATCGACTTCACTT-3'; reverse, 5'-GGCAGGACATCCATGTAGAG-3'.
Podocalyxin: forward, 5'-GAAAGGAGCCCTCTGGATGA-3'; reverse, 5'-GGGCTCAGGCACAAGTAGGT-3'.
P-cadherin: forward, 5'-TGTCAACGAAGCCCCTGTGT-3'; reverse, 5'-CGGTGGAGTTGGGTGATGTC-3'.
NEPH1: forward, 5'-CGTCTAATGACCTGCCAATC-3'; reverse, 5'-CCTCATTCATACTGCGACAG-3'.
GAPDH: forward, 5'-ACCACAGTCCATGCCATCAC-3'; reverse, 5'-TCCACCACCCTGTTGCTGTA-3'.
PCR was performed using TaKaRa Ex Taq Hot Start Version (Takara, Kyoto, Japan) and the following conditions: 94°C for 15 s, 57°C for 15 s and 72°C for 30 s with 25-45 cycles, and 72°C for 10 min for final extension.
SEAP assay. Activity of SEAP in the culture medium was evaluated by a chemiluminescent method using a Great EscAPe SEAP detection kit (BD Bioscience, Palo Alto, CA), as described before ( 11, 17 ).
Formazan assay. The number of viable cells was assessed by a formazan assay using Cell Counting Kit-8 (Dojindo Laboratory, Kumamoto, Japan). In brief, after the collection of culture media for the SEAP assay, cells in 96-well plates were incubated at 37°C for 2 h in medium containing 10% Cell Counting Kit-8 assay solution. Absorbance (450 nm) of formazan generated from WST-8 was measured by Spectra Max 340 (Nihon Molecular Devices, Tokyo, Japan).
Western blot analysis. Western blot analysis was performed as described before ( 46 ) using goat polyclonal anti-human nephrin antibody (N-20: sc-19000, Santa Cruz Biotechnology, Santa Cruz, CA). Nephrin protein was visualized using the enhanced chemiluminescence system (Amersham Biosciences, Buckinghamshire, UK). As a loading control, the level of -actin was evaluated using an anti- -actin antibody (Sigma-Aldrich Japan).
Statistical analysis. Assays were performed in quadruplicate. Data are expressed as means ± SE. Statistical analysis was performed using a nonparametric Mann-Whitney U -test to compare data in different groups. A P value <0.05 was considered to indicate a statistically significant difference.
RESULTS
Effects of basal culture media on the activity of the nephrin gene promoter. RPMI-1640 has been used as the standard culture medium for podocytes ( 22, 29, 32 ). We first compared effects of basal culture media on the activity of the nephrin gene promoter. Reporter podocytes REPON5.4 that express SEAP under the control of the nephrin gene promoter ( 46 ) were incubated for 24-48 h in RPMI-1640, DMEM/F12, or -MEM supplemented with 1% FBS, and the activity of SEAP was evaluated. When reporter podocytes were incubated in DMEM/F12 or -MEM, activity of the nephrin promoter was significantly upregulated by 2.3- to 3.2-fold during the initial 24 h and 3.2- to 3.7-fold during the next 24 h compared with the cells incubated in RPMI-1640 ( Fig. 1 A ). To exclude possible influences of cell death and proliferation, the number of viable cells was evaluated by formazan assay, and the levels of SEAP were normalized. Compared with RPMI-1640, -MEM modestly but significantly increased the number of podocytes ( Fig. 1 B ). After normalization of the SEAP values, activity of the nephrin gene promoter was still higher in DMEM/F12 (3.1-fold) and -MEM (2.7-fold) than in RPMI-1640 ( Fig. 1 C ).
Fig. 1. Effects of basal culture media on the activity of the nephrin gene promoter. A : reporter podocytes were incubated for 24-48 h in RPMI-1640 (RPMI), DMEM/F12, or -MEM supplemented with 1% FBS, and activity of secreted alkaline phosphatase (SEAP) in culture media during the initial and next 24 h (0-24 h and 24-48 h, respectively) was evaluated. B : cells were subsequently subjected to formazan assay to evaluate the number of viable cells. C : SEAP activity (24-48 h) was normalized by the number of viable cells. Assays were performed in quadruplicate, and data are expressed as means ± SE. RLU, relative light units. *Statistically significant differences ( P < 0.05) vs. RPMI.
To identify components responsible for the activation of the nephrin gene promoter, effects of constituents enriched in DMEM/F12 and -MEM were examined. Those include sodium pyruvate, D -pantothenic acid, folic acid, and vitamin B 12. Reporter podocytes were cultured for 24 h in RPMI-1640 supplemented with these constituents individually, and the culture media were subjected to SEAP assay. The results, however, showed that none of these components induced activation of the nephrin gene promoter (data not shown).
Effects of 1,25(OH) 2 D 3, ATRA, and dexamethasone in combination with different basal media. 1,25(OH) 2 D 3, ATRA, and dexamethasone have been known as potentially antiproteinuric substances that may upregulate expression of nephrin ( 18, 24, 41, 46 ). To develop an optimum culture condition for expression of the nephrin gene, reporter podocytes were cultured in three types of basal media supplemented with these agents. As shown in Fig. 2, in all three basal media, 1,25(OH) 2 D 3 significantly induced activity of SEAP. The maximum effect was observed at 10-100 nM (data not shown). 1,25(OH) 2 D 3 slightly decreased the number of viable cells only in RPMI-1640. Normalization of the SEAP values by the number of viable cells revealed that the effect of 1,25(OH) 2 D 3 was most prominent in DMEM/F12 compared with other basal media ( Fig. 2 ). Figure 3 shows effects of different culture media supplemented with ATRA or dexamethasone. Like 1,25(OH) 2 D 3, significant elevation of SEAP activity was also observed with ATRA or dexamethasone in all basal media. The maximum effects were observed with 1 µM ATRA and 0.1 µM dexamethasone (data not shown). Dexamethasone, but not ATRA, significantly decreased the number of viable cells in DMEM/F12 and -MEM. Normalization of the SEAP values by the number of viable cells revealed that the effects of ATRA and dexamethasone were most prominent in DMEM/F12 compared with other basal media ( Fig. 3 ).
Fig. 2. Effects of 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] in combination with different basal media on the activity of the nephrin promoter. Reporter podocytes were incubated for 48 h in RPMI, DMEM/F12, or -MEM supplemented (+) or not supplemented (-) with 100 nM 1,25(OH) 2 D 3, and culture media and cells were subjected to SEAP assay and formazan assay. SEAP activity was normalized by the number of viable cells, and the results were shown. Assays were performed in quadruplicate, and data are expressed as means ± SE. *Statistically significant differences ( P < 0.05).
Fig. 3. Effects of all- trans -retinoic acid (ATRA) and dexamethasone in combination with different basal media on the activity of the nephrin promoter. Reporter podocytes were incubated for 48 h in RPMI, DMEM/F12, or -MEM supplemented with 1 µM ATRA or 0.1 µM dexamethasone (Dex), and culture media and cells were subjected to SEAP assay and formazan assay, respectively. Activity of SEAP was normalized by the number of viable cells, and the results are shown. Assays were performed in quadruplicate, and data are expressed as means ± SE. *Statistically significant differences vs. untreated controls ( P < 0.05).
Combinational effects of 1,25(OH) 2 D 3, ATRA, and dexamethasone on nephrin gene expression. We examined combinational effects of 1,25(OH) 2 D 3, ATRA, and dexamethasone on the promoter activity of the nephrin gene in podocytes cultured in DMEM/F12. As shown in Fig. 4 A, the maximum induction of SEAP was achieved by combination of the three agents. The induction was 20-fold greater than the value of DMEM/F12 alone and 60-fold over the value of RPMI-1640 alone. Although the normalized values of SEAP in the 1,25(OH) 2 D 3 +ATRA+Dex group was not significantly different from that in the 1,25(OH) 2 D 3 +Dex group, the absolute values without normalization in the former was significantly higher than that in the latter (data not shown). Consistently, RT-PCR showed dramatic upregulation of nephrin mRNA by VRADD ( Fig. 4 B ), the DMEM/F12-based medium supplemented with three agents.
Fig. 4. Combinational effects of 1,25(OH) 2 D 3, ATRA, and dexamethasone on podocytes cultured in DMEM/F12. A : reporter podocytes were incubated for 24 h in DMEM/F12 supplemented with 10 nM 1,25(OH) 2 D 3, 1 µM ATRA, and/or 0.1 µM dexamethasone (Dex), and culture media and cells were subjected to SEAP assay and formazan assay. Activity of SEAP was normalized by the number of viable cells, and the results are shown. Assays were performed in quadruplicate, and data are expressed as means ± SE. *Statistically significant differences ( P < 0.05). B : reporter podocytes were cultured in RPMI alone or in DMEM/F12 supplemented with 10 nM 1,25(OH) 2 D 3, 1 µM ATRA, and 0.1 µM dexamethasone [ v itamin D 3, r etinoic a cid, and d examethasone-supplemented D MEM/F12 (VRADD)] for 24 h, and expression of nephrin mRNA was evaluated by RT-PCR. Molecular markers are indicated on the left, and expression of GAPDH is shown as a loading control. RT (-), reaction without reverse transcriptase.
Effects of VRADD on podocyte cell lines that do not express nephrin mRNA after multiple passages. Effects of VRADD were tested in podocytes established by Schiwek et al. ( 32 ) that have lost expression of nephrin mRNA after multiple passages. RT-PCR could not detect nephrin mRNA in this cell line cultured in RPMI-1640 at passage 25. However, when these cells were cultured in VRADD for 24 h, dramatic recovery of nephrin mRNA was observed ( Fig. 5 A ). We also tested another podocyte cell line established by Mundel et al. ( 22 ). Like the cell line described above, expression of nephrin mRNA was not detectable in this podocyte line after prolonged culture. When the medium was replaced with DMEM/F12, modest recovery of nephrin mRNA was observed. When the medium was changed to VRADD, expression of nephrin mRNA was dramatically recovered ( Fig. 5 B ). These results suggested that VRADD allows for recovery of nephrin gene expression in murine podocytes in which expression of nephrin has been lost after multiple passages.
Fig. 5. Effects of VRADD on other podocyte cell lines. Murine podocyte cell lines established by Schiwek et al. (32; A ) and by Mundel et al. (22; B ) were cultured in RPMI, DMEM/F12, or VRADD for 24 h, and expression of nephrin mRNA was evaluated by RT-PCR. Expression of GAPDH is shown as a loading control.
Effects of VRADD on other differentiation markers in podocytes. Nephrin is known to be a differentiation marker for podocytes. The recovery of nephrin by VRADD may be associated with differentiation of podocytes. To examine this possibility, we investigated expression of other differentiation markers in podocytes including podocalyxin, P-cadherin, and NEPH1 ( 23 ) by RT-PCR. Constitutive expression of P-cadherin, podocalyxin, and NEPH1 mRNAs was observed in podocytes even after multiple passages. Like nephrin, expression of P-cadherin and NEPH1 mRNAs was modestly upregulated by VRADD ( Fig. 6 ). Constitutive expression of podocalyxin mRNA was not obviously affected by VRADD. These results indicated that VRADD caused not only recovery of nephrin gene expression but also a modest shift toward differentiation in cultured podocytes.
Fig. 6. Effects of VRADD on the expression of differentiation markers for podocytes. Reporter podocytes were cultured in RPMI or VRADD for 24 h, and expression of podocalyxin, P-cadherin, and NEPH1 mRNAs was evaluated by RT-PCR. Expression of GAPDH is shown at the bottom as a loading control.
Effects of VRADD on podocytes in prolonged cultures. As described, we identified that VRADD is the most potent medium for the recovery of the nephrin gene expression in murine podocytes in short-term cultures. However, we found that, when reporter podocytes were cultured in VRADD for prolonged periods, the levels of SEAP declined. For example, incubation of the cells in VRADD for 48 h resulted in a dramatic increase in SEAP from 6,197 ± 428 to 393,471 ± 17,880 relative light units (RLU; means ± SE, P < 0.05), whereas incubation for 1 wk resulted in much less increase from 2,907 ± 188 to 38,750 ± 2,393 RLU ( Fig. 7 A ). Formazan assay revealed that the blunted response of SEAP was due to cellular damage ( Fig. 7 B ). Morphological examination revealed that incubation of podocytes in VRADD for 1 wk, but not for 24-48 h, caused cellular death characterized by accumulation of vacuoles in the cytoplasm ( Fig. 7 C ). Subsequent experiments revealed that a similar phenomenon was also observed in podocytes exposed to dexamethasone for 1 wk and that VRAD (VRADD lacking dexamethasone) did not cause cellular injury. These results suggested that, although VRADD was the most potent for the induction of the nephrin gene, it did not allow for maintenance of nephrin expression in prolonged cultures. VRAD may be more useful for the maintenance of nephrin gene expression and, possibly, cell differentiation for extended periods of cultures.
Fig. 7. Effects of VRADD on podocytes in prolonged cultures. A : reporter podocytes were incubated for 48 h or 1 wk in RPMI or VRADD in the presence of 10% FBS. After incubation, the cells were further cultured in individual media supplemented with 1% FBS for 24 h, and culture media were subjected to SEAP assay. Assays were performed in quadruplicate, and data are expressed as means ± SE. *Statistically significant differences ( P < 0.05). B : after the collection of culture media in A, the cells were subjected to formazan assay to evaluate the number of viable cells. C : morphological features of podocytes incubated in RPMI or VRADD for 48 h or 1 wk using phase-contrast microscopy.
Effects of VRAD on podocytes in prolonged cultures. We tested effects of VRAD on podocytes in prolonged cultures. First, reporter podocytes were incubated in RPMI-1640, VRAD, or VRADD for 1 wk, and activity of SEAP during the last 24 h was evaluated. As shown in Fig. 8 A, activity of SEAP in the cells incubated in VRAD was markedly increased, and it was significantly higher than the cells in VRADD (79,058 ± 5,082 RLU in VRAD vs. 38,750 ± 2,393 RLU in VRADD, P < 0.05). Morphological examination revealed that incubation of podocytes in VRAD for 1 wk did not cause cellular damage ( Fig. 8 B, top right ). Murine podocytes cultured in RPMI-1640 for prolonged periods showed fibroblast-like spindle shapes ( Fig. 8 B, bottom left ), even in the absence of IFN-. However, the cells maintained in VRAD showed the digital cell shape with fine, small processes typical of podocytes during early passages ( Fig. 8 B, bottom right ).
Fig. 8 Effects of VRADD without dexamethasone (VRAD) on podocytes. A : reporter podocytes were incubated for 1 wk in RPMI, VRADD, or VRAD in the presence of 10% FBS. After incubation, the cells were further cultured for 24 h in individual media supplemented with 1% FBS and subjected to SEAP assay. Assays were performed in quadruplicate, and data are expressed as means ± SE. *Statistically significant difference ( P < 0.05). B : morphological features of reporter podocytes after incubation in RPMI or VRAD for 1 wk using phase-contrast microscopy. C : reporter podocytes were cultured in RPMI or VRAD for 24 h, and expression of nephrin mRNA was evaluated by RT-PCR. D : after reporter podocytes were cultured for 1 mo in RPMI or VRAD, the cells were incubated for 8 h in individual fresh media supplemented with 1% FBS and subjected to SEAP assay. During the prolonged cultures in VRAD, cells were maintained with once or twice passages per week. Data are expressed as means ± SE. *Statistically significant difference ( P < 0.05). E : reporter podocytes were cultured in RPMI or VRAD for 48 h in the presence of 1% FBS, and protein levels of nephrin were evaluated by Western blot analysis. The level of -actin is shown at the bottom as a loading control. F : reporter podocytes were cultured in RPMI or VRAD for 24 h, and expression of podocalyxin, P-cadherin, and NEPH1 mRNAs was evaluated by RT-PCR. Expression of GAPDH is shown at the bottom as a loading control.
RT-PCR revealed that, like with VRADD, nephrin mRNA was substantially induced by VRAD (24 h incubation) in dedifferentiated podocytes lacking nephrin expression ( Fig. 8 C ). Furthermore, the expression of the nephrin gene was maintained by VRAD for longer periods. As shown in Fig. 8 D, after incubation of reporter cells in RPMI-1640 for 1 mo, the levels of SEAP were very low. In contrast, maintenance of the cells in VRAD for 1 mo still showed high levels of nephrin promoter activity ( Fig. 8 D ). These results suggested that VRAD is suitable not only for the recovery of the nephrin expression but also for the maintenance of nephrin expression in prolonged cultures.
To examine whether VRAD upregulates not only nephrin mRNA but also its protein level, reporter podocytes ( passage 15 ) were cultured in either RPMI-1640 or VRAD, and Western blot analysis was performed. As shown in Fig. 8 E, in the cells cultured in RPMI-1640, low levels of nephrin protein were detectable. The level of nephrin protein was increased in the podocytes cultured in VRAD. We also examined expression of other differentiation markers in podocytes cultured in VRAD by RT-PCR. Like VRADD, VRAD modestly increased the level of P-cadherin whereas it did not affect the level of podocalyxin ( Fig. 8 F ). Together with the facts that VRAD led to 1 ) dramatic recovery of nephrin gene expression ( Fig. 8 C ), 2 ) upregulation of nephrin protein ( Fig. 8 E ), and 3 ) morphological differentiation ( Fig. 8 B ), this result suggested that VRAD may also induce the phenotypic shift of cultured podocytes toward differentiation.
Suppression of nephrin gene expression by HGF. As described before, cultured podocytes easily lose expression of the nephrin gene, and it is the crucial problem especially when repressors of nephrin expression are sought using cultured podocytes. To overcome this problem, we used reporter podocytes maintained in VRAD. Using this culture condition, we tested effects of HGF on expression of nephrin mRNA as well as activity of the nephrin gene promoter. Recently, Rampino et al. ( 25 ) reported a possibility that HGF may be a pathogenic factor responsible for crescent formation in experimental glomerulonephritis. They showed that cultured podocytes express the c-Met HGF receptor and that recombinant HGF induced migration, proliferation, and epithelial-to-mesenchymal transdifferentiation in murine podocytes. We hypothesized that HGF may be a dedifferentiation factor that decreases expression of nephrin in podocytes. To examine this hypothesis, reporter podocytes maintained in VRAD were treated with serial concentrations of HGF for 24 h, and the levels of SEAP were evaluated. As shown in Fig. 9 A, untreated control cells cultured in VRAD exhibited high levels of SEAP, and it was downregulated by HGF in a dose-dependent manner. The number of viable cells was not affected by the treatment with HGF when examined by formazan assay ( Fig. 9 B ). Consistently, the reduction in SEAP activity was correlated with abrogation of nephrin mRNA when examined by RT-PCR ( Fig. 9 C ). These results clearly showed that HGF has the potential to depress expression of nephrin via transcriptional suppression. This experiment also evidenced usefulness of VRAD for identification of pathogenic repressors of nephrin gene expression using podocytes that easily lose expression of nephrin mRNA.
Fig. 9. Suppression of nephrin gene expression by hepatocyte growth factor (HGF). Reporter podocytes cultured in VRAD for 1 mo were treated with human recombinant HGF (0-100 ng/ml) for 24 h, and culture media and cells were subjected to SEAP assay ( A ), formazan assay ( B ), and RT-PCR ( C ), respectively. In A and B, assays were performed in quadruplicate, and data are expressed as means ± SE. *Statistically significant differences vs. untreated control ( P < 0.05).
DISCUSSION
In the present report, we developed the optimum medium, VRADD, for the recovery of a differentiated phenotype of podocytes. First, compared with RPMI-1640 (conventional basal culture medium used for podocytes), DMEM/F12 and -MEM had the potential for increasing nephrin gene expression. This effect was not due to substances enriched in these media, including sodium pyruvate, D -pantothenic acid, folic acid, and vitamin B 12. Other unknown factors (e.g., saccharides, inorganic salts, amino acids, other vitamins) may be responsible for the effects of DMEM/F12 and -MEM.
We recently reported that, in the conventional culture medium RPMI-1640, short-term exposures to 1,25(OH) 2 D 3, ATRA, and dexamethasone synergistically increased expression of nephrin mRNA as well as activity of the nephrin gene promoter in murine podocytes ( 46 ). Based on these findings, we tested effects of the three basal media supplemented with these substances individually or in combination on the promoter activity of the nephrin gene. Our findings were 1 ) in any of basal media tested, addition of 1,25(OH) 2 D 3, ATRA, or dexamethasone significantly increased activity of the nephrin promoter; and 2 ) the effects of supplemental components were most potent when they were added to DMEM/F12. 1,25(OH) 2 D 3, ATRA, and dexamethasone are ligands of nuclear receptors, and these ligands bind to the vitamin D receptor, retinoic acid receptor, and glucocorticoid receptor, respectively. The complexes formed by these ligands and nuclear receptors then translocate to the specific response elements in the nucleus and initiate transcription of target genes ( 5, 33, 45 ). The 5'-regulatory region of the nephrin gene contains putative retinoic acid-response elements, vitamin D-response elements, and a glucocorticoid-response element ( 46 ). Possibly, activation of the nephrin gene promoter and induction of nephrin gene expression by VRADD were caused through these regulatory elements.
VRADD induced activation of the nephrin gene promoter up to 60-fold in nephrin-expressing reporter cells. Even in podocytes that have lost nephrin expression during multiple passages, expression of nephrin mRNA was dramatically recovered by VRADD. Expression of other podocyte markers P-cadherin and NEPH1 was also upregulated by VRADD, suggesting that VRADD influenced podocytes toward differentiation. However, VRADD caused damage of podocytes in prolonged cultures. The similar effect was observed in dexamethasone-treated podocytes, but not the cells maintained in VRADD lacking dexamethasone. These results suggested that, although VRADD is most potent for the transient induction of nephrin gene expression, it is not competent for long-term maintenance of podocytes in the differentiated state. Cellular damages caused by dexamethasone have been reported in several cell types, including lymphoid cells and other immune cells, osteoblasts and osteocytes, germ cells, vascular pericytes, and pancreatic -cells ( 12, 26, 30, 43, 44 ), and a variety of molecular mechanisms have been postulated to date to explain its proapoptotic effects. Currently, however, mechanisms involved in the dexamethasone-induced injury of podocytes are unknown. Recent reports showed that, in keratinocytes and chondrocytes, dexamethasone suppressed the phosphatidylinositol 3-kinase (PI3K)-Akt pathway ( 7, 16 ), which is one element of the cytoprotective machinery in podocytes ( 3 ). Dexamethasone is also a well-known inhibitor of NF- B ( 2 ), another crucial element of cytoprotective machinery in various cells ( 36 ). Because cultured podocytes have basal activity of PI3K and NF- B ( 3, 31 ), the induction of podocyte damage by dexamethasone may be due, at least in part, to suppression of the basal activity of these cytoprotective molecules.
To overcome the limitation observed in VRADD, we tested the potential of VRAD (lacking dexamethasone). Although activation of the nephrin gene promoter was less than VRADD, VRAD significantly induced and maintained activity of the nephrin gene promoter as well as expression of nephrin mRNA for extended periods. This defined medium, therefore, enables one to investigate regulation of the nephrin gene expression in podocytes even after multiple passages. It is especially important when we use stably transfected podocytes that can be established after prolonged cultures and multiple passages. Genetically engineered podocytes maintained in VRAD would provide a useful tool in seeking endogenous, nephrotic factors that cause downregulation of nephrin expression, a phenomenon that is often observed in various human glomerular diseases associated with proteinuria ( 6, 8, 13, 38 ).
Rampino et al. ( 25 ) recently reported a possibility that HGF may be a pathogenic factor responsible for crescent formation in experimental glomerulonephritis. They found that cultured podocytes express the c-Met HGF receptor and that recombinant HGF induced migration, proliferation, and epithelial-to-mesenchymal transdifferentiation of podocytes. Using the VRAD-primed, stably transfected reporter podocytes, we revealed in this report that expression of nephrin mRNA as well as nephrin promoter activity were suppressed by HGF. Currently, molecular mechanisms involved in this phenomenon are unclear, but several possibilities may be postulated. Previous reports showed that the basal expression of nephrin gene in podocytes is maintained by WT1, the Wilms' tumor suppressor ( 9, 42 ). The transacting potential of WT1 is dependent on its phosphorylation states; i.e., nonphosphorylated WT1 is functional, whereas its phosphorylated form loses the transacting potential ( 49 ). The phosphorylation of WT1 is induced by activation of protein kinase A and protein kinase C ( 49 ), and HGF may have the ability to induce activation of these kinases ( 1, 15, 37 ). Taken together, these data raise a possibility that HGF may have downregulated transcription of the nephrin gene via activation of these kinases and subsequent suppression of WT1 function.
Recently, Kusumoto et al. ( 14 ) provided evidence that recurrent, in vivo administration with HGF induced proteinuria. They examined therapeutic effects of HGF on experimental liver cirrhosis caused by dimethylnitrosamine. The authors found that repeated administration of HGF ameliorated hepatic fibrosis but, unexpectedly, led to an increase in the level of urinary albumin without affecting serum creatinine levels. Excretion of urinary albumin returned to baseline after cessation of HGF ( 14 ). Our current data might provide a possible explanation through which HGF induced proteinuria.
In summary, our current results demonstrated that VRAD allows for the recovery and maintenance of nephrin expression in cultured podocytes. This defined medium should provide a useful tool to investigate biological function of nephrin as well as transcriptional regulation of the nephrin gene in cultured podocytes.
ACKNOWLEDGMENTS
We thank Drs. Karlhans Endlich (University of Heidelberg), Peter Mundel (Albert Einstein College of Medicine), Lawrence Holzman (University of Michigan), and Sei Kakinuma (Tokyo Medical and Dental University) for kindly providing experimental materials. We also thank Dr. Michio Nagata (University of Tsukuba) for support and helpful discussion.
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作者单位：Departments of 1 Molecular Signaling and 2 Internal Medicine, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi, Japan