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In human kidneys, the mechanisms underlying angiotensinogen (AGT) augmentation by interleukin 6 (IL-6) are poorly understood and the only information available is in HK-2, immortalized human renal proximal tubular epithelial cells. Therefore, the present study was performed to elucidate the effects of IL-6 on AGT expression in primary cultured human renal proximal tubular epithelial cells (RPTEC) after characterization of HK-2 and RPTEC. RPTEC showed low basal AGT mRNA (11±1%) and protein (7.0±0.9%) expression, high IL-6 receptor (IL-6R) expression (282±17%), and low basal NF-κB (43±7%) and STAT3 (43±7%) activities compared to those in HK-2. In RPTEC, AGT mRNA and protein expressions were enhanced by IL-6 (172±31% and 378±39%, respectively). This AGT augmentation was attenuated by an IL-6R antibody. STAT3 phosphorylation (366±55% at 30 min) and translocation were enhanced by IL-6. The AGT augmentation was attenuated by a STAT3 inhibitor. These data indicate that IL-6 increases AGT expression via STAT3 pathway in RPTEC.
Angiotensinogen (AGT) is regarded as an important factor for the systemic renin-angiotensin system (RAS) activity (Corvol and Jeunemaitre, 1997). Angiotensin II (Ang II) increases AGT expression via nuclear factor-κB (NF-κB) activation in hepatocytes (Brasier et al., 2000; Jamaluddin et al., 2000; Li and Brasier, 1996). Ang II induces secretion of cytokines including tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β)and interleukin 6 (IL-6) (Recinos et al., 2007). IL-6 augments AGT expression via signal transducer and activator of transcription 3 (STAT3) activation in hepatocytes (Jain et al., 2007; Ohtani et al., 1992; Ray et al., 2005). These data suggest that Ang II upregulates circulating AGT via IL-6 augmentation, which may worsen vascular inflammation (Brasier et al., 2002).
There is growing recognition that the intrarenal RAS is an independent mechanism of the systemic RAS and is involved in the regulation of individual organs in a tissue-specific manner (Dzau and Re, 1994; Paul et al., 2006; Re, 2004; Navar et al., 2002). An in vitro study showed that Ang II induces AGT expression in rat renal proximal tubular cells which is the main source of intrarenal AGT (Ingelfinger et al., 1999; Ingelfinger et al., 1990; Terada et al., 1993). Several in vivo studies also demonstrated that the expression of renal AGT mRNA and protein are enhanced in Ang II-infused rats and human renin/human AGT double transgenic mice (Schunkert et al., 1992; Kobori et al., 2001; Kobori et al., 2007b). These data provide a firm foundation for the hypothesis that the Ang II-induced AGT augmentation in renal proximal tubular cells contributes to further increases in intrarenal Ang II levels (Kobori et al., 2007a).
Intrarenal TNF-α and IL-6 levels are elevated in the kidneys of Ang II-infused hypertensive rats (Ruiz-Ortega et al., 2002). Moreover, Ang II stimulates IL-6 secretion from cultured mesangial cells (Moriyama et al., 1995). Enalapril, an angiotensin converting enzyme (ACE) inhibitor, abrogates enhanced expression of TNF-α, IL-1β and IL-6 in the renal cortex of diabetic rats (Navarro et al., 2006). In IL-6 knockout mice, the magnitudes of Ang II-induced hypertension and albumin excretion are attenuated (Lee et al., 2006). These findings suggest that the augmented intrarenal Ang II as well as circulating Ang II induces intrarenal cytokines which leads to the development of renal injury probably accompanied by the activation of Ang II-AGT augmentation mechanism. However, little is known about direct interaction between AGT expression and IL-6 in the kidney.
We recently reported that Ang II and IL-6 synergistically induce human AGT expression through the activation of NF-κB and STAT3 in HK-2 cells, immortalized human renal proximal tubular cells. In contrast, while augmentation of AGT by IL-6 alone (Jain et al., 2007; Ohtani et al., 1992; Ray et al., 2005) has been reported in hepatocytes, stimulation with IL-6 alone did not increase AGT expression in HK2 cells (Satou et al., 2008). However, HK-2 cells have high basal activity of NF-κB which may limit the ability of these cells to respond further to stimulatory agents (Satou et al., 2008; de Haij et al., 2003). In further studies, we observed that basal activities of NF-κB and STAT3 are much lower in primary cultured human renal proximal tubular epithelial cells (RPTEC). Thus, further studies were performed to compare the basal expression levels of AGT and IL-6 receptor (IL-6R) and basal activities of NF-κB and STAT3 in HK-2 cells and RPTEC. Then, we performed more detailed studies on action of IL-6 to augment AGT in RPTEC.
HK-2 cells were obtained from ATCC. The cells were cultured as previously described (Satou et al., 2008). RPTEC were obtained from Cambrex. The cells were grown in Renal Epithelial Cell Growth Medium (Cambrex) supplemented with 0.5% heat-inactivated fetal calf serum as recommended by the manufacturer. RPTEC were used within passage 7. Cells were plated at a density of 2×105 cells/well in 6-well plates. Prior to stimulation, the cells were exposed to serum-free medium for 24 hr in both cell lines. Thereafter, RPTEC were treated with 10 ng/ml human TNF-α (Pierce), 10 ng/ml human IL-1β (Peprotech), 0.625–20.0 ng/ml human IL-6 (Peprotech) and 10−7 M Ang II for up to 24 hr in a medium containing 0.05% serum. In addition, cells were treated with 10−7 M Ang II and 10 ng/ml for 24 hr to test synergistic effects of Ang II and IL-6 on AGT expression. To investigate the influence of STAT3 on human AGT expression, cells were treated with 0.2 μM JSI-124 (Calbiochem). The treatment with JSI-124 was started before 3 hr of stimulations with IL-6 because the inhibitory effect of JSI-124 is slower than STAT3 activation by IL-6 (van Kester et al., 2008). To determine the contribution of IL-6R to the change in AGT expression and STAT3 activation by IL-6, RPTEC was pretreated with 0.1–10 μg/ml anti-IL-6R antibody (R & D Systems) for 1 hr; thereafter, cells were treated with 10 ng/ml IL-6.
Quantitative real-time RT-PCR (qRT-PCR) was performed to evaluate human AGT mRNA expression using the TaqMan PCR system. For total RNA isolation, treated cells were washed with 3 ml of PBS and harvested at each experimental time point. Total RNA was isolated from the cells using the BIO-ROBOT EZ 1 (Qiagen). Subsequently, qRT-PCR was performed as previously described (Kobori et al., 2005). In brief, 20 ng total RNA from each sample was applied to the Mx3000P System (Stratagene) equipped with the Brilliant Single-Step QRT-PCR Master Mix II Kit (Stratagene). All samples were analyzed in triplicate, and the data were normalized based on the expression level of the human GAPDH mRNA. The sequences are as follows: human AGT mRNA: forward primer, 5′-GAG AGA GCC CAC AGA GTC TA-3′; reverse primer, 5′-GCT TTG ATC ATA CAC AGC AA-3′; and probe, 5′/6-FAM/CCA ACA GCT TAA CAA GCC TGA GGT/BHQ1/3′ and human GAPDH mRNA; forward primer, 5′-ATC ATC CCT GCC TCT ACT GG-3′; reverse primer, 5′-CTG CTT CAC CAC CTT CTT GA-3′; and probe, 5′/HEX/ACC TGA CCT GCC GTC TAG AAA AAC CTG/BHQ2/3′.
To quantify human AGT protein in the cell lysate and the culture medium, human AGT ELISA was performed. HK-2 and RPTEC was cultured in 6-well plates with 1.5 ml of medium/well. The media were collected into tubes, and the supernatants were collected after centrifugation of the media for 5 min at 5,000 rpm. Cell lysates of RPTEC were prepared as previously described (Satou et al., 2008). The ELISA for AGT was performed as previously described (Katsurada et al., 2007). Total protein concentrations in the media were also quantified using Micro BCA Protein Assay Kit (Pierce). For comparison of basal AGT expressions between HK-2 cells and RPTEC, data from human AGT ELISA were normalized based on total volume of medium. To examine the effect of IL-6 on AGT expression in RPTEC, data were normalized based on total protein concentration of the media.
To detect the protein expression levels of IL-6R, western blots were performed as previously described (Satou et al., 2008). After treatment with serum-free medium for 24 hr, HK-2 cells and RPTEC were washed 3 times with 3 ml PBS. The cells were harvested with 80 μL lysis buffer containing 1% Triton X-100, 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Nonidet P-40, 1 mmol/L Na3VO4, and 0.25% Protease Inhibitor Cocktail (Sigma). The lysates were sonicated 3 times for 10 sec each and centrifuged at 13,000 rpm at 4°C for 30 min. Total protein concentration of the supernatant was quantified using Micro BCA Protein Assay Kit (Pierce). Then, 20 μg of total protein was applied to a pre-cast NuPAGE 4–12% gel (Invitrogen). The separated proteins were transferred to a nitrocellulose membrane (Bio-Rad). A goat anti-IL-6R antibody (1:150, R & D Systems) and an IRDye labeled anti-goat IgG antibody (1:15000, Li-Cor) were used for the detection of human IL-6R. Detection was performed using the Odyssey System (Li-Cor). Data were normalized based on human β-actin protein expression levels. To determine NF-κB activity, a mouse anti-phospho-p65 (Ser 536) antibody (1:1000, Cell Signaling) and an IRDye labeled anti-mouse IgG antibody (1:15000, Li-Cor) were used for the detection of phosphorylated p65. Expression level of total p65 protein was evaluated using a rabbit anti-p65 antibody (1:1000, Cell Signaling) and an IRDye labeled anti-rabbit IgG antibody (Li-Cor). The level of phosphorylated p65 was normalized based on total p65.
Phosphorylation of STAT3 was detected using western blot analysis in order to elucidate participations of the transcriptional factor in the effects mediated by IL-6 on AGT expression in RPTEC. Cells were treated with 10 ng/ml IL-6 for up to 60 min and for 24 hr. A mouse anti-phospho-STAT3 (Tyr 705) antibody (1:250, BD Transduction Laboratories) and an IRDye labeled anti-mouse IgG antibody (1:15000, Li-Cor) were used for the detection of phosphorylated STAT3. A rabbit anti-STAT3 antibody (1:250, Thermo) and an IRDye labeled anti-rabbit IgG antibody (Li-Cor) were used to detect total STAT3. The level of phosphorylated STAT3 was normalized based on total STAT3.
RPTEC were cultured in 4-well chamber (Lab-Tek). After treatment with 10 ng/ml IL-6 for 30 min, cells were rinsed with PBS, then, fixed for 20 min by 4% formaldehyde. After incubated with 0.2% Triton X-100 for 4 min, the chambers were blocked by Image-iT FX signal enhancer (Invitrogen). The cells were incubated with a rabbit anti-STAT3 antibody (1:100, Thermo) for 30 min. After washing with PBS, the cells were incubated with an Alexa Fluor 488 goat anti-rabbit antibody (Invitrogen). ProLong Gold anti-fade reagent with DAPI (Invitrogen) was used to stain nuclei and as a mount reagent. Localization of STAT3 in RPTEC was observed and photographed under a fluorescence microscope (Olympus BX51, Olympus Optical Co. Ltd.).
Data are expressed as mean ± SD. The data were analyzed using student t test or one-way ANOVA followed by Bonferroni/Dunn multiple comparison post hoc test. P<0.05 was considered statistically significant.
Basal AGT mRNA expression in RPTEC was markedly lower than in HK-2 cells (0.11 ± 0.01, ratio to HK-2, Fig. 1A). This lower expression level of AGT in RPTEC than in HK-2 cells was also observed at the protein level (HK-2: 12.04 ± 0.35 ng/ml, RPTEC: 0.84 ± 0.11 ng/ml, Fig. 1A). IL-6R protein expression in RPTEC was higher than in HK-2 cells (282 ± 17%, compared with HK-2, Fig. 1B).
NF-κB activation involves phosphorylation of p65, a subunit of NF-κB (Sakurai et al., 1999) Basal phosphorylation level of p65 in RPTEC was lower than in HK-2 cells (43 ±7%, compared with HK-2) although total p65 expression levels were similar in both cell lines (Fig. 2A). Moreover, basal phosphorylation level of STAT3 in RPTEC was lower than in HK-2 cells (43 ± 7, compared with HK-2) although total STAT3 expression levels were similar in both cell lines (Fig. 2B).
Treatment with 10 ng/ml TNF-α suppressed AGT mRNA expression at 24 hr (Fig. 3A, 0.14 ± 0.03, ratio to control). Furthermore, the same concentration of IL-1β decreased AGT mRNA expression (Fig. 3A, 0.14 ± 0.03, ratio to control). Only IL-6 (10 ng/ml) augmented AGT mRNA expression (1.72 ± 0.31, ratio to control) in cytokines tested in this study (Fig. 3A). Treatment with 10−7 M Ang II did not change AGT mRNA expression. Additionally, Ang II did not further augment IL-6-increased AGT expression (Fig. 3B).
Treatment with 10 ng/ml IL-6 increased AGT mRNA expression in a time-dependent manner (Fig. 4A). A significant increase compared to the control was observed after 8 hr. This increment in AGT mRNA expression induced by IL-6 was dose-dependent at 24 hr (Fig. 4B).
In control cells, accumulated AGT protein in the medium was not detectable at 12 hr. In contrast, AGT protein level following treatment with 10 ng/ml IL-6 was detectable at the same time point (Fig. 5, 141.9 ± 25.6 ng AGT/mg total protein). AGT protein levels were detectable in 24 hr accumulation in both groups. AGT protein levels were significantly increased by treatment with IL-6 in medium at the time (Fig. 5, 61.4 ± 1.0 and 232.4 ± 23.9 ng AGT/mg total protein, control vs. IL-6 treatment). However, intracellular AGT protein was below the detection level both in control and IL-6 treatment groups at any time point tested.
Treatment with 10 ng/ml IL-6 alone increased AGT mRNA expression. This augmentation of AGT was attenuated by 2 and 10 μg/ml IL-6R antibody at 24 hr (Fig. 6). Treatment with 10 μg/ml IL-6R antibody alone did not have any effect on AGT mRNA.
STAT3 phosphorylation (Tyr705) by 10 ng/ml IL-6 was increased compared to control at 5, 10, 15 and 30 min (Fig 7A, 2.21 ± 0.61, 3.15 ± 0.87, 3.34 ± 1.54 and 3.36 ± 0.55, respectively, ratio to 0 min). The difference of phosphorylation level between control and IL-6 treatment was not observed at 24 hr (data not shown).
To determine the contribution of IL-6R to the IL-6-induced STAT3 phosphorylation, cells were pretreated with anti-IL-6R antibody for 1hr before treatment with IL-6 for 30 min. The STAT3 phosphorylation by IL-6 was attenuated by pretreatment with 10 μg/ml IL-6 antibody (Fig. 7B).
Using immunofluorescence staining, we observed translocation of STAT3 by IL-6. In control condition, STAT3 was localized primarily in cytoplasm (Fig. 7C left panels). Translocation of STAT3 into nuclei occurred with IL-6 treatment (10 ng/ml) was confirmed by merged images (Fig. 7C, center and lower panels). In the absence of primary antibody against STAT3, there was no fluorescence except for nuclei by 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining (Fig. 7C, right panels).
JSI-124, an inhibitor of STAT3 activation (Blaskovich et al., 2003), inhibited STAT3 phosphorylation by treatment with 10 ng/ml IL-6 for 30 min (Fig. 8A). Treatment with JSI-124 alone did not affect STAT3 phosphorylation compared with control. The inhibitor attenuated increment in AGT mRNA expression by treatment with 10 ng/ml IL-6 (Fig. 8B). Treatment with JSI-124 alone did not show effect on basal AGT expression.
The present data demonstrates the effects of IL-6 on human AGT expression in primary cultured human renal proximal tubular epithelial cells. Several in vivo studies in rodents have demonstrated Ang II-induced augmentation of renal AGT expression (Schunkert et al., 1992; Kobori et al., 2001; Kobori et al., 2007b). Moreover, Ang II increases renal IL-6 levels (Ruiz-Ortega et al., 2002). It has also been demonstrated that NF-κB and STAT3 activation contribute to the AGT augmentation by Ang II in hepatocytes (Brasier et al., 2000; Jamaluddin et al., 2000; Li and Brasier, 1996). In the present study, we compared basal expression levels of AGT and IL-6R, and basal activities of NF-κB and STAT3 between HK-2 cells and RPTEC to characterize these cell lines. AGT mRNA and protein expression levels in HK-2 cells were higher than those in RPTEC (Fig. 1A). Furthermore, basal NF-κB and STAT3 activities in HK-2 cells were higher than those in RPTEC (Fig. 2A and B). The high basal NF-κB activity in HK-2 cells has been shown previously (Navarro et al., 2006; Satou et al., 2008). These results indicate that high basal NF-κB and STAT3 activities contribute to high basal AGT expression in HK-2 cells. The basal NF-κB and STAT3 activities in HK-2 cells might be caused by immortalization or culture conditions of this cell line.
Basal IL-6R expression in RPTEC was higher than in HK-2 cells (Fig. 1B); in contrast, basal STAT3 activity in RPTEC was lower than in HK-2 cells (Fig. 2B). Based on these characteristics of RPTEC, we examined the contribution of IL-6 to AGT regulation and its mechanism in this cell line. In comparing the effects of cytokines on AGT expression in RPTEC, we observed TNF-α suppressed AGT mRNA expression (Fig. 3). Cardiac restricted overexpression of TNF-α reduced cardiac AGT expression (Flesch et al., 2003). In human adipocytes, TNF-α suppressed AGT expression (Wang et al., 2005). The present results are consistent with these reports. In contrast, it has been reported that TNF-α contributes to an augmentation of AGT in several tissues (Nyui et al., 1997). Therefore, the effects of TNF-α on AGT expression may be cell- and tissue-specific. Endogenous AGT expression was not affected by IL-1β in primary cultured rat hepatocytes (Ohtani et al., 1992). Recently, it was reported that IL-1β abrogated Ang II-induced connective tissue growth factor production in mesangial cells (Sanchez-Lopez et al., 2008). In contrast to the effects of IL-6, we observed that IL-1β decreased AGT mRNA expression (Fig. 3). These data suggest a possible antagonistic activity of IL-1β on RAS activity.
Of the cytokines examined in this study, only IL-6 increased AGT mRNA expression (Fig. 3). The effects of IL-6 on AGT expression were in time- and dose-dependent (Fig. 4). Moreover, AGT protein expression and accumulation in medium was elevated by IL-6 (Fig. 5). These results suggest that intrarenal IL-6 increases AGT expression in human renal proximal tubular cells. Because renal proximal tubular cells are the main source of AGT in the kidney (Ingelfinger et al., 1990; Terada et al., 1993), IL-6-AGT axis in the cells may be able to play an important role in intrarenal Ang II generation. Increases in blood pressure and albumin excretion elicited by Ang II infusions in IL-6 knockout mice were weaker than those in wild type mice although it was shown that the increase in blood pressure was not due to renal injury caused by Ang II infusion (Lee et al., 2006). Therefore, the intrarenal IL-6-AGT axis suggested by the present results may help explain the development of hypertension and/or renal injury.
Our previous study showed that co-stimulation with Ang II and IL-6 increased AGT expression (Satou et al., 2008). Moreover, stimulation with IL-6 alone did not have any effect on AGT expression in HK-2 (Satou et al., 2008). The present study demonstrated that Ang II did not cause further augmentation of IL-6-induced AGT upregulation. Although more detailed experiments will be required to elucidate effect of Ang II on IL-6-induced AGT upregulation, the different effects of Ang II and IL-6 on AGT expression in these two human renal proximal tubular epithelial cell lines, HK-2 and RPTEC, may be due to differences in basal NF-κB and STAT3 activities and in basal AGT expression level. In particular, the difference in basal IL-6R expression is strongly associated with the differences in response to IL-6 because anti-IL-6R antibody completely neutralized the AGT augmentation by IL-6 in RPTEC (Fig. 6). On the other hand, expression levels of β-actin, NF-κB and STAT3 were the same in both cell lines indicating that basal characteristics of the two cell lines are similar as human renal proximal tubular cells. These findings suggest that the mechanism underlying the contribution of IL-6 to the augmentation may be dependent on the activities of signal transducers and the receptor expression levels in renal proximal tubular cells. However, further study will be necessary to elucidate mechanisms underlying AGT augmentation by IL-6 in human kidneys under different clinical status.
After binding of IL-6 to membrane IL-6R or soluble IL-6R, the complex and GP130 protein synergistically stimulate Janus kinase 2. Thereafter, the activated kinase activates STAT3 (Hodge et al., 2005). In human hepatocytes, IL-6 augments AGT expression via STAT3 activation (Jain et al., 2007; Ohtani et al., 1992; Ray et al., 2005). In the present study, STAT3 was phosphorylated by IL-6 in RPTEC (Fig. 7A). The anti-IL-6R antibody attenuated the STAT3 phosphorylation by IL-6 (Fig. 7B) indicating that IL-6R participates in the STAT3 activation by IL-6 in RPTEC. Furthermore, STAT3 was translocated into nuclei by the treatment (Fig. 7C). An inhibitor of STAT3 activation attenuated STAT3 phosphorylation (Fig. 8A) and increment in AGT mRNA expression (Fig. 8B) by treatment with 10 ng/ml IL-6. These data indicate that the AGT augmentation by IL-6 is mediated by STAT3 activation in RPTEC as well as in hepatocytes.
In summary, we have demonstrated that IL-6 stimulates human AGT expression through STAT3 activation in human renal proximal tubular epithelial cells, suggesting that IL-6 plays an important role in augmentation of AGT by Ang II in human renal proximal tubular epithelial cells which may contribute to the development of hypertension and renal injury. Therefore, elucidation of the mechanisms underlying this augmentation may lead to new strategies to treat hypertension, and cardiovascular and renal diseases.
This study was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (R01DK072408), National Center for Research Resources (P20RR017659), and National Heart, Lung, and Blood Institute (R01HL026371). The authors acknowledge the valuable comments and excellent technical assistance from Toshie Saito, MD and Akemi Katsurada, MS (Tulane University).
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