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Molecular Endocrinology, doi:10.1210/me.2005-0330
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Molecular Endocrinology 20 (10): 2548-2558
Copyright © 2006 by The Endocrine Society

Role of Nitric Oxide in High Glucose-Induced Mitogenic Response in Renal Fibroblasts

Lea-Yea Chuang, Jinn-Yuh Guh, Ke-Ann Wang, Yann-Jia Huang and Jau-Shyang Huang

Departments of Biochemistry (L.-Y.C.) and Internal Medicine (J.-Y.G.), Kaohsiung Medical University, Kaohsiung 807, Taiwan, Republic of China; and Department of Biological Science and Technology (K.-A.W., Y.-J.H., J.-S.H.), Chung Hwa College of Medical Technology, Tainan 717, Taiwan, Republic of China

Address all correspondence and requests for reprints to: Jau-Shyang Huang, Department of Biological Science and Technology, Chung Hwa College of Medical Technology, Tainan 717, Taiwan, Republic of China. E-mail: jaushyang12{at}hotmail.com.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nitric oxide (NO) has been suggested to be associated with tubulointerstitial fibrosis in diabetic nephropathy. Abnormal glucose handling in the tubulointerstitium may play an important role in the development of diabetic nephropathy. This study was designed to investigate the effect of NO generation and action in renal fibroblasts exposed to high glucose (HG). We found that HG (500 mg/dl) significantly decreased nitrite production compared with normal glucose (100 mg/dl) when the incubation period was for 12, 18, or 24 h. HG inhibited cGMP-dependent protein kinase (PKG) activation at 4, 8, and 12 h. Both NO donors and PKG activator treatment induced high levels of NO, inducible nitric oxide synthase, and PKG in HG-incubated cells. Interestingly, HG-induced Janus kinase 2-signal transducers and activators of transcription 1 (STAT1) activation but not STAT3 or STAT5 activation at 30 min were blocked by NO donors and PKG activator. Moreover, HG-enhanced Raf-1 and p42/p44 MAPK phosphorylation were markedly suppressed by NO donors or PKG activator. The ability of NO-PKG to inhibit HG-induced cell cycle progression was verified by the observation that NO donors and PKG activator inhibited cdk4 activation and increased p21Waf1/Cip1 and p16INK4a (but not p27Kip1) expression in HG-treated renal fibroblasts. Collectively, these data suggest that HG significantly blunted NO signaling, and activation of the NO-PKG pathway may modulate HG-enhanced mitogenic response via specific pathways.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TUBULOINTERSTITIAL FIBROSIS IS an important component of diabetic nephropathy, which is characterized by increased expression of interstitial extracellular matrix components and aberrant cell growth in renal tubulointerstitium (1). Renal tubular epithelial cells and interstitial fibroblasts are active participants in tubulointerstitial fibrosis (2). They can contribute to the increased expression of the basement membrane component collagen and fibronectin in tubulointerstitial fibrosis observed during diabetic nephropathy (1, 2, 3).

Hyperglycemia is responsible for the development and progression of diabetic nephropathy through metabolic derangements, including increased oxidative stress, renal polyol formation, activation of protein kinase C-MAPKs, and accumulation of advanced glycation end products (4, 5). Altered growth of renal cells is one of the early abnormalities detected after the onset of diabetes. Cell culture studies whereby renal cells are exposed to high glucose concentrations have provided a considerable amount of insight into mechanisms of growth (6).

The gaseous molecule nitric oxide (NO) modulates a large variety of physiological functions including vascular tone, intestinal motility, platelet aggregation, proliferation, apoptosis, and neurotransmission (7, 8). NO initiates diverse cellular signaling cascades which comprise nitrosylation of proteins, ADP ribosylation, or stimulation of soluble guanylyl cyclases which catalyze intracellular cGMP synthesis (9). cGMP activates cGMP-dependent protein kinases (PKGs) which mediate localized and global signaling. Activation of the NO-PKG pathway induces relaxation of smooth muscle by lowering the cytosolic calcium level and/or by calcium desensitization of the contractile elements (10). Furthermore, vascular endothelial growth factor (VEGF)-stimulated proliferation of cultured endothelial cells triggered by endothelial NO synthase activation was also shown to require intracellular signaling through PKG (11).

The Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway plays a central role in transducing the cytokine signal and regulates various biological processes such as cell proliferation, differentiation, migration, apoptosis, transformation, and immune response in mammalian cells (12, 13, 14). The effects of NO in cell growth are exerted through multiple mechanisms, which include interaction with cell signaling systems like JAK/STAT or Raf-1/MAPK-dependent signal transduction pathways (15, 16). They may also lead to modification of transcription factors activity and in this way modulate the expression of multiple other mediators of cellular mitogenesis.

Accumulating evidence suggests that the NO-PKG pathway modulates gene expression. This involves activation or inhibition of different transcription factors such as activator protein 1, c-Myc, nuclear factor-{kappa}B, and Sp1 (17, 18). However, the relationship between NO-PKG and JAK/STAT or Raf-1/MAPK pathways remains poorly understood. Therefore, in this study, we investigated the effect of high glucose (HG) on NO signaling and the antiproliferative mechanisms of NO-PKG in the HG-induced mitogenic response in renal fibroblasts. To better understand the mechanisms underlying these actions, we further assessed the role of NO-PKG activation in HG-induced JAK/STAT and Raf-1/MAPK pathways.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effects of HG on NO Production and PKG Activation
The goal of this study was to elucidate the mechanisms by which NO and its downstream signals mediate the HG-induced mitogenic response in renal fibroblasts (NRK-49F) cells. To investigate whether HG could affect NO production in NRK-49F cells, we first treated these cells with HG (500 mg/dl) and normal glucose (NG) (100 mg/dl) and analyzed endogenously NO production by the Griess method (11). We found that HG significantly decreased nitrite production compared with NG when the incubation period was for 12, 18, or 24 h (Fig. 1AGo). Raising the ambient D-glucose concentration causes a dose-dependent decrease in nitrite production (Fig. 1BGo). The inhibitory effect was not mediated by an increase in osmolarity, because raising the osmolarity by the addition of D-mannitol did not significantly decrease nitrite production. It is well established that the downstream target of the NO pathway is PKG, and that many of the biological effects of NO are mediated through the important kinase PKG. We examined whether NO blockade prevented PKG expression and found that HG significantly decreased PKG protein synthesis at 4, 8, and 12 h (Fig. 2AGo). We also found that the HG reduced the protein activity of PKG-I when compared with NG (Fig. 2BGo). These results indicate that the biological effects of HG in renal fibroblasts may be caused partially by inhibition of the NO-dependent PKG signaling.


Figure 1
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  		Fig. 1. Time Course and Dose-Dependent Effects of HG on NO Production in NRK-49F Cells

A, Serum-deprived NRK-49F cells (1.5 x 103 cells per well) were treated with serum-free (SF) medium, NG (100 mg/dl D-glucose), or HG (500 mg/dl D-glucose) for 0, 6, 12, 18, and 24 h. HG significantly decreased nitrite production at 12, 18, and 24 h. B, Raising the ambient D-glucose concentration causes a dose-dependent decrease in nitrite production at 12 h. The addition of an equimolar amount of D-mannitol does not significantly inhibit nitrite production. NO production was determined by monitoring nitrite production by Griess assay as described in Materials and Methods. Results were expressed as the mean ± SEM (n = 6). *, P < 0.01 vs. NG.

 

Figure 2
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  		Fig. 2. Modulation of PKG Activation by HG in NRK-49F Cells

Serum-deprived NRK-49F cells were treated with serum-free (SF) medium, NG (100 mg/dl D-glucose), or HG (500 mg/dl D-glucose) for 0, 2, 4, 8, and 12 h, and then assayed for PKG protein synthesis (A) and PKG activity (B) as described in Materials and Methods. Results were expressed as the mean ± SEM (n = 4). *, P < 0.01 vs. NG.

 
Effects of NO Donors and PKG Activator on HG-Inhibited NO-PKG Pathway
Several studies have reported that cytokine-induced increases in iNOS expression cause the subsequent production of pathophysiologically high levels of NO and PKG. Furthermore, NO donor drugs and cyclic nucleotide analogs increase PKG protein and activity in some cells. To investigate whether exogenously induced NO/PKG could affect the HG-inhibited NO-PKG pathway in NRK-49F cells, we treated these cells with PKG inhibitor, NO donors, and PKG activator and examined nitrite production, iNOS production, and PKG activation in the presence of HG. Figure 3Go illustrates that the NO donors S-nitroso-N-acetylpenicillamine (SNAP) (5 µM) and sodium nitroprusside (SNP) (5 µM) and the PKG activator 8-pCPT-cGMPs (2 µM) suppressed HG-inhibited nitrite production, iNOS synthesis, and PKG activation. A significant decrease in nitrite production was observed in 100 µM NG-monomethyl-L-arginine (L-NMMA)-treated cells when compared with control (NG) cells (Fig. 3AGo). Interestingly, the nitric oxide synthase (NOS) inhibitor L-NMMA may enhance the inhibitory effect of HG on nitrite production. On the other hand, the PKG inhibitor KT5823 (1 µM) did not alter HG-inhibited nitrite production and PKG activation. We also examined whether the NRK-49F cells express other NOS isoenzymes and found that the amount of endothelial NOS (eNOS) or neuronal NOS (nNOS) in the cells is very little and HG did not alter the expression of eNOS or nNOS when compared with NG (data not shown). Together, these results suggest that sustained NO generation was regulated by persistent activation of iNOS and PKG. NO inhibits HG-induced effects maybe involve the PKG-dependent mechanism in renal fibroblasts.


Figure 3
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  		Fig. 3. Effects of NOS Inhibitor, PKG Inhibitor, NO Donors, and PKG Activator on HG-Blocked NO Production, iNOS Synthesis, and PKG Activation

Serum-deprived NRK-49F cells were treated with L-NMMA (100 µM), KT5823 (1 µM), SNAP (5 µM), SNP (5 µM), and 8-pCPT-cGMPs (2 µM) for 12 h in the presence of NG (100 mg/dl D-glucose) or HG (500 mg/dl D-glucose), and then assayed for nitrite production by Griess assay (A). Cells treated with the same conditions for 4 h were assayed for iNOS and PKG synthesis by Western blotting (B) and assayed for PKG activity as described in Materials and Methods (C). These are representative experiments, each performed at least four times. *, P < 0.01 vs. HG.

 
Effects of NO Donors and PKG Activator on HG-Mediated JAK/STAT and Raf-1/MAPK Activation
Activating JAK/STAT and Raf-1/MAPK pathways has been implicated in diverse cellular processes, including cell growth, proliferation, differentiation, and survival. To determine whether the NO-PKG pathway played a role in HG-mediated signaling cascades, NO donors and PKG activator were used to pretreat NRK-49F cells in JAK/STAT and Raf-1/MAPK activities. We found that HG markedly induced tyrosine phosphorylation of JAK2 and STAT1 (but not STAT3 or STAT5) at 30 min (Figs. 4Go and 5Go). Interestingly, SNAP, SNP, and 8-pCPT-cGMPs significantly reduced phospho-JAK2 and phospho-STAT1 without affecting JAK2 and STAT1 protein levels in HG-treated cells. Surprisingly, activating the NO/PKG pathway did not affect phosphorylation of STAT3 or STAT5. On the other hand, we also tested whether NO-PKG regulates Raf-1/MAPK activity in NRK-49F cells treated with HG. We measured Raf-1 and p42/p44 MAPK activities after exposure of cultured NRK-49F cells to NG and HG. We found that HG markedly increased Raf-1 and p42/p44 MAPK phosphorylation at 60 min in comparison with NG (Fig. 6Go). Raf-1 and p42/p44 MAPK phosphorylation were suppressed by NO donors or PKG activator. These observations demonstrate that both the JAK2/STAT1 and the Raf-1/MAPK may play a role as important signal mediators in the HG-induced biological responses, and activating the NO/PKG pathway inhibited HG-enhanced phosphorylation of JAK2/STAT1 and Raf-1/MAPK.


Figure 4
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  		Fig. 4. Effects of PKG Inhibitor, NO Donors, and PKG Activator on HG-Induced Phosphorylation of JAK2, STAT1, STAT3, and STAT5

Total cell lysates from NRK-49F cells treated with KT5823 (1 µM), SNAP (5 µM), SNP (5 µM), and 8-pCPT-cGMPs (2 µM) in the presence of HG (500 mg/dl D-glucose) for 30 min. Proteins were separated by a polyacrylamide gel and immunoblotted with antiphospho-JAK2 (P-JAK2), antiphospho-STAT1 (P-STAT1), antiphospho-STAT3 (P-STAT3), and antiphospho-STAT5 (P-STAT5) antibodies (upper panels) or antibodies corresponding to the above antibodies (lower panels). These are representative experiments independently performed three times. IB, Immunoblot; IP, immunoprecipitation.

 

Figure 5
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  		Fig. 5. Laser Densitometry of the Gels Shown in Fig. 4Go and Two Additional Phosphorylation Experiments

Results are shown for JAK2 (A), STAT1 (B), STAT3 (C) and STAT5 (D), respectively. It is evident that NO donors and PKG activator significantly decreased HG-induced phosphorylation of JAK2 and STAT1 at 30 min. Results are expressed as arbitrary units plotted against time (mean ± SEM; n = 3). *, P < 0.01 vs. HG.

 

Figure 6
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  		Fig. 6. Effects of PKG Inhibitor, NO Donors, and PKG Activator on HG-Induced Phosphorylation of Raf-1 and p42/p44 MAPK

Total cell lysates from NRK-49F cells treated with KT5823 (1 µM), SNAP (5 µM), SNP (5 µM), and 8-pCPT-cGMPs (2 µM) in the presence of HG (500 mg/dl D-glucose) for 60 min. Proteins were separated by a polyacrylamide gel and immunoblotted with antiphospho-Raf-1 (Ser 338) and antiphospho-p42/p44 MAPK antibodies (upper panel) or antibodies corresponding to the above antibodies (lower panel). These are representative experiments independently performed three times. *, P < 0.01 vs. HG. IB, Immunoblot; IP, immunoprecipitation.

 
NO Donors and PKG Activator Block the Effects of HG on Cell Cycle Progression and Cell Proliferation
Because the induction of early growth response genes involves the JAK/STAT and the Raf-1/MAPK cascades, it was possible that NO/PKG activation involved in the growth effects of HG. To gain further insight into the mechanism exerted by NO/PKG, we next wished to determine whether activation of the NO/PKG pathway is responsible for inhibition of HG-mediated cell cycle progression in NRK-49F cells. HG-incubated cells were treated for the indicated times with KT5823, SNAP, SNP, and 8-pCPT-cGMPs, and then examined by flow-cytometric analysis. We found that a significant proportion of the cells entered S phase after the start of HG treatment, whereas control (NG) cells largely remained arrested in the G1 phase (Table 1Go). Clearly, SNAP, SNP, and 8-pCPT-cGMPs caused potent G1 arrest in HG-treated NRK-49F cells. These results demonstrate that HG can induce S phase entry, and activation of the NO-PKG pathway may also have the ability to induce cell cycle arrest from HG-treated cells. To investigate the possible role of NO-PKG in cell cycle progression, we examined the effects of SNAP, SNP, and 8-pCPT-cGMPs on HG-regulated expression of cell cycle regulatory molecules and cdk4 kinase activation. Western blot analysis revealed that SNAP, SNP, and 8-pCPT-cGMPs cannot prevent synthesis of p27Kip1, cyclin D1, and cdk4 (Fig. 7AGo). However, cdk4 kinase analysis showed that both NO donors and PKG activator markedly reduced HG-induced cdk4 kinase activation (Fig. 7BGo). Furthermore, NO donors and PKG activator significantly reversed HG-inhibited expression of p21Waf1/Cip1 (Fig. 7AGo). These results indicated that suppression of cdk4 activation and induction of p16INK4a and p21Waf1/Cip1 (but not p27Kip1) are the underlying mechanisms by NO donors and PKG activator to promote inhibition of cell cycle progression. To additionally test the HG-induced mitogenic effect inhibited by NO donors and PKG activator, we did the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and cell number analysis. Consistent with cell cycle studies, induction of NO/PKG expression resulted in a decline in HG-increased viable cell number after 5 d (Table 2Go). The MTT assay also showed that NO donors and PKG activator suppressed cell proliferation whereas PKG inhibitor had no effect. Taken together, these results indicate that activation of the NO/PKG pathway negatively regulates HG-stimulated cell growth by inducing G1 arrest.


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  		Table 1. Effects of PKG Inhibitor, NO Donors, and PKG Activator on HG-Induced Cell Cycle Progression in NRK-49F Cells

 

Figure 7
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  		Fig. 7. Effects of PKG Inhibitor, NO Donors, and PKG Activator on HG-Regulated Expression of Cell Cycle-Regulatory Molecules and cdk4 Kinase Activation

A, Total cell lysates from NRK-49F cells treated with KT5823 (1 µM), SNAP (5 µM), SNP (5 µM), and 8-pCPT-cGMPs (2 µM) in the presence of HG (500 mg/dl D-glucose) for 6 h were subjected to Western blot analysis for p27Kip1, p21Waf1/Cip1, p16INK4a, cyclin D1, and cdk4. B, Serum-deprived NRK-49F cells were treated with the same above test agents for 6 h, and then assayed for cdk4 kinase activation as described in Materials and Methods. The concentration of NG was 100 mg/dl. These are representative experiments, each performed at least three times. *, P < 0.01 vs. HG.

 

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  		Table 2. Effects of PKG inhibitor, NO Donors, and PKG Activator on HG-Induced Cellular Mitogenesis in NRK-49F Cells

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The current study determines the role of the NO/PKG pathway on HG-induced JAK/STAT and Raf-1/MAPK activation and mitogenic response in renal fibroblasts. NO/PKG is classically associated with antiproliferation (19, 20); therefore, increased activity of this pathway would be expected to occur with down-regulation of cell growth. In this study, we showed that HG markedly inhibited the NO/PKG activation. Induction of NO production and PKG activation in HG-treated cells under NO donors and PKG activator conditions resulted in significant decrease in the JAK2/STAT1 and the Raf-1/MAPK activities. Indeed our data support the hypothesis that areas of cross-talk exist between these pathways. Furthermore, we also found that NO donors and PKG activator significantly suppressed cdk4 activation and enhanced p21Waf1/Cip1 and p16INK4a expression in HG-regulated cell cycle progression.

The role of NO in renal disease is complicated. HG exerts it biological effects in association with NO, yet it is known that NO bioavailability is reduced in diabetes. VEGF stimulated NO production in a dose-dependent manner in bovine aortic endothelial cells (21), and this was inhibited by either HG or L-NAME treatment. Endothelial NOS phosphorylation by VEGF was also inhibited by HG. It is interesting that both HG and L-NAME enhanced the proliferative response of endothelial cells, which was prevented by an NO donor. Moreover, exposure of bovine aortic endothelial cells to HG for 24 h impaired insulin-mediated tyrosine phosphorylation of IRS-1, serine phosphorylation of Akt, activation of NOS, and production of NO (22). On the other hand, HG and osmotic control media increased DNA synthesis and cell cycle progression in cardiac fibroblasts (23). In glomerular mesangial cells, HG significantly induced cell growth, collagen IV production, and JAK2 activation and phosphorylation of STAT1 and STAT3 (24).

NO production by iNOS differs from the production by eNOS or nNOS (25, 26). iNOS produces very large, toxic amounts of NO in a sustained manner, whereas constitutive NOS isoforms produce NO within seconds, and its activities are direct and short acting. In our previous study, we found that iNOS (but not eNOS or nNOS) proteins were affected by advanced glycation end product and HG treatments in NRK-49F cells. The iNOS pathway may be the major source of NO produced by growth factors/cytokines stimulation in these cells. However, it is possible that NO can be generated from the iNOS-independent mechanism between reactive oxygen species and L-arginine (27). Large amounts of NO may target numerous proteins and enzymes critical for cell growth, survival, and apoptosis (28). These include signaling molecules involved in cytokine/growth factor signaling like JAK or STAT proteins, NF{kappa}B/I{kappa}B pathway as well as MAPK, and some G proteins and transcription factors (29). Nitration of cysteines in these proteins may lead to their activation or inactivation (30). Indeed there are multiple intracellular mechanisms through which NO may act as a tubulointerstitial fibrosis mediator (31).

Phosphorylation of JAKs leads to their activation, and activated JAKs phosphorylate and hence activate STATs (32). Specific subtypes of JAK and STAT molecules mediate different signals, resulting in specificity of responses (33). Thus, we examined the effect of HG on JAKs to determine the cause of STATs’ phosphorylation. In a previous study, we have found that JAK2 (but not JAK1, JAK3, or TYK2) was rapidly and markedly phosphorylated by HG in NRK-49F cells. Interestingly, this study showed that HG significantly induced phosphorylation of STAT1 (but not STAT3 or STAT5). In addition, Raf-1 and MAPK have been implicated in the signaling of HG action in NRK-49F cells. We found that NO donors and PKG activator diminished both HG-enhanced phosphorylation of JAK2/STAT1 and Raf-1/MAPK. However, further studies are needed to clarify how NO/PKG acts to inhibit HG-enhanced cellular hyperplasia/hypertrophy in renal tubulointerstitium. These studies will provide important information regarding NO-regulated tubulointerstitial fibrosis.

HG has been shown to trigger a series of events, including activation of JAK2/STAT1, protein kinase C, cdk4, and Raf-1/MAPK, elevation of reactive oxygen species, plus increased expression of cyclin D. The mechanism by which HG promotes JAK2 activation is speculative but may relate to an interaction of JAK2 with reactive oxygen species induced by HG. Therefore, given that JAK2/STAT1 activation promotes cyclin D gene expression and Raf-1/MAPK signaling, our findings in the current study suggest that the HG-induced activation of both JAK2 and STAT1 is linked to the HG-induced cyclin D/cdk4 activation and cell cycle progression.

We observed that NO induction, in addition to inhibiting cdk4 activation, strongly inhibited the HG-decreased expression of p21Waf1/Cip1 and p16INK4a. The protein levels of cyclin D1 and p27Kip1 after HG treatment were not altered by either NO donors or PKG activator. Inhibition of p21Waf1/Cip1 and p16INK4a expression by HG consistently correlated with enhanced cdk4 activation. Numerous studies have implicated that NO is capable of activating p21Waf1/Cip1 and p16INK4a via the cGMP/PKG pathway (34). It is possible that renal fibroblasts express the NO-sensitive isoform of soluble guanylyl cyclase, which responds to NO to generate cGMP and activate PKG and subsequently induce p21Waf1/Cip1 and p16INK4a expression. Whether p21Waf1/Cip1 and p16INK4a are direct targets for modification by NO or its derivatives are not known. These intriguing possibilities remain to be examined.

NO is involved in the regulation of glomerular filtration rate, sodium excretion, extracellular matrix accumulation, and the maintenance of renal structural integrity in the kidney (35). Diabetes-associated changes in the NO system have been investigated in isolated renal tissues; however, direct in vivo measurements of NO production in different compartments of the diabetic kidney and their changes in response to hyperglycemia remain controversial and poorly elucidated. In addition, one major general controversy exists between the in vitro findings, which generally suggest decreased bioavailability of NO in the diabetic kidney, and in vivo observations, which tend to suggest enhanced renal NO production and/or activity in diabetes, at least in the early stages (36, 37). It is believed that the diabetic milieu is complex and that in vitro approaches may miss some important mechanisms that comodulate NO activity in a particular system. Nevertheless, our studies and those of others in cultured renal cells have provided evidence that HG levels exert deleterious effects on NO bioavailability (21, 38, 39). Possible mechanisms whereby HG decreases NO bioavailibility include NO capture by glucose, L-arginine depletion, superoxide anion generation, and reduced tetrahydrobiopterin stability and availability (35, 37, 40).

In conclusion, our findings suggest that HG rapidly suppressed the NO/PKG signaling, resulting in renal fibroblasts proliferation. Our studies indicate that HG-stimulated JAK2/STAT1 or Raf-1/MAPK signaling probably mediates several important biological responses, including the NO/PKG signaling and cell cycle progression. NO and PKG are now emerging as important factors in signaling pathways and renal tubulointerstitial fibrosis. Our data provide significant new information regarding the molecular mechanisms underlying NO/PKG-inhibited HG-induced renal fibroblasts proliferation, and such knowledge will assist in the better understanding of the pathogenesis of diabetic nephropathy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents
Fetal bovine serum (FBS), DMEM, antibiotics, D-glucose, D-mannitol, molecular weight standards, trypsin-EDTA, trypan blue stain, and all medium additives were obtained from Life Technologies (Gaithersburg, MD). Anti-JAK2, -STAT1, -STAT3, -STAT5, -iNOS, -Raf-1, -phospho-Raf-1 (Ser 338), -p42/p44 MAPK, -cyclin D1, -cdk4, -p27Kip1, -p16INK4a, and -p21Waf1/Cip1 antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antiphospho-p42/p44 MAPK antibody was purchased from New England Biolabs (Beverly, MA). Anti-phospho-JAK2, -phospho-STAT1, -phospho-STAT3, and -phospho-STAT5 antibodies were obtained from Upstate Biotechnology Inc. (Charlottesville, VA). Protein A/G-coupled agarose beads, SNAP, SNP, 8-pCPT-cGMPs, KT5823, NG-monomethyl-L-arginine, and antiprotein kinase G-I antibody were purchased from Calbiochem (La Jolla, CA). Horseradish peroxidase-conjugated goat antirabbit or antimouse secondary antibody, [{gamma}-32P]ATP, and the enhanced chemiluminescence kit were obtained from Amersham Corp. (Arlington Heights, IL). N,N'-methylenebisacrylamide, acrylamide, sodium dodecyl sulfate, ammonium persulfate, Temed, and Tween 20 were purchased from Bio-Rad Laboratories (Hercules, CA). cGMP enzymatic immunoassay set was purchased from Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire, UK). Anti-ß-actin antibody, dimethylsulfoxide, MTT colorimetric assay kit, and all other chemicals were obtained from Sigma (St. Louis, MO).

Culture Conditions
NRK-49F cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were grown in culture flasks (Nunclon, Denmark) and maintained in DMEM (100 mg/dl D-glucose) supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin, and 5% FBS in a humidified 5% CO2 incubator at 37 C. In some experiments, cells were exposed to serum-free (0.1% FBS) DMEM supplemented with the specific PKG inhibitor, KT5823, for 16 h before timed exposure to FBS and HG. KT5823 was dissolved in dimethylsulfoxide. Cell viability was assessed by the trypan blue exclusion test and was routinely more than 92%. For cell number analysis, cells (1.0 x 105 cells per well) were cultured in six-well culture plates (Nunclon) and grown in the added test agents. Cells were harvested and counted with a hemocytometer. Cells between passages 10 and 35 were used in all experiments. Each experimental data point represents the mean of duplicate wells from three independent experiments.

NO Analysis
NO synthesis was determined by measuring the accumulation of nitrite (NO2–), a stable metabolite of NO, in culture supernatants using the Griess reaction. Briefly, 1.2 x 103 cells were plated in each well of a 96-well plate in DMEM with 5% FBS, and medium was deproteinized before assay. After being passed through 25-kDa ultrafilters, 20 µl of the medium was diluted with 120 µl assay buffer and mixed with 5 µl cofactor and 5 µl nitrate reductase (NO colorimetric assay kit, Calbiochem). After the medium had been kept at room temperature for 2 h to convert nitrate to nitrite, total nitrite was measured at 540 nm absorbance by reaction with Griess reagent (sulfanilamide and naphthalene-ethylene diamine dihydrochloride). Amounts of nitrite in the medium were estimated by a standard curve obtained from enzymatic conversion of NaNO3 to nitrite. The detection limit of the method is 0.2 µM, and the assay was reliable and reproducible with interassay and intraassay variation coefficients of 3.5% and 4.2%, respectively.

Western Blot Analysis
For protein analysis, 1.2 x 107 serum-deprived cells were treated with agents or HG as described above. Total cell lysates were harvested, resolved by 10% SDS-PAGE, and then transferred to Protran membranes (0.45 µm; Schleicher & Schuell, Keene, NH). The membranes were blocked in blocking solution and subsequently probed with primary antibody (1 µg/ml). The membrane was incubated in x4000 diluted horseradish peroxidase-conjugated goat antirabbit or antimouse secondary antibody. The protein bands were detected using the enhanced chemiluminescence (ECL) system, and the percentage of phosphorylated form of protein was determined using a scanning densitometer.

For JAK/STAT and Raf-1/MAPK activation assays, proteins were resolved by SDS-PAGE and transferred to Protran membranes. The membranes were probed with antiphospho-JAK2 (0.75 µg/ml), antiphospho-STAT1 (1 µg/ml), antiphospho-STAT3 (1 µg/ml), antiphospho-STAT5 (1 µg/ml), antiphospho-Raf-1 (0.75 µg/ml), antiphospho-p42/p44 MAPK (0.75 µg/ml), anti-JAK2 (1:1000), anti-STAT1 (1:1000), anti-STAT3 (1:1000), anti-STAT5 (1:1000), anti-Raf-1 (1:1000), and anti-p42/p44 MAPK (1:1000) antibodies. The antiphospho-STAT5 polyclonal antibody detects the tyrosine-phosphorylated forms of both STAT5a and STAT5b. Immunoreactive proteins were detected with the ECL system as described above.

Expression of PKG and Assay of PKG Activity
Cells were treated with agents or HG as described above. After the monolayer was rinsed with PBS, cells were harvested and homogenized with cold buffer consisting of 20 mM sodium phosphate, pH 6.8, 2 mM EDTA, 15 mM 2-mercaptoethanol, 150 mM NaCl, 2 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin A, and 10 µg/ml leupeptin. The suspension was centrifuged for 10 min at 14,000 rpm to obtain cell extract. Aliquots of extract were analyzed for PKG activity and also for Western blotting with an affinity-purified polyclonal rabbit anti-PKG-I antibody (1:500). The anti-PKG-I polyclonal antibody detects the activation forms of both PKG-I{alpha} and PKG-Iß. PKG activity was assayed as described by Pilz and Demple (17, 18) with a peptide substrate (RKISASEFDRPL) selective for PKG. The difference in the phosphorylation of substrate in the presence and absence of cGMP was taken as PKG activity.

Cdk4 Kinase Assay
For immunoprecipitation of cdk4 (41), cells were washed twice with ice-cold PBS, harvested in cdk4 lysis buffer [50 mM HEPES (pH 7.5), 10% glycerol, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM dithiothreitol, and 0.1% Tween 20, supplemented with the phosphatase and protease inhibitors 5 mM NaF, 0.1 mM sodium orthovanadate, 5 µg/ml leupeptin, 10 µg/ml aprotinin, 50 µg/ml phenylmethylsulfonyl fluoride, and 5 µg/ml pepstatin A], and lysed by repeated passages through a 25-gauge needle. Cellular debris was removed from soluble extracts by centrifugation at 16,000 x g for 10 min at 4 C. After normalization of protein content, lysates were precleared by incubation with protein A/G-agarose beads and preimmune rabbit serum for 30 min at 4 C. Endogenous cdk4-containing complexes were immunoprecipitated for 3 h at 4 C, using a rabbit polyclonal antihuman cdk4 antibody. Immunoprecipitates were washed twice with cdk4 lysis buffer and four times with glutathione S-transferase-RB (GST-RB) kinase buffer [50 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM dithiothreitol, 2.5 mM EGTA, 10 mM ß-glycerophosphate, 0.1 mM orthovanadate, 1 mM NaF] and then resuspended in 50 µl of GST-RB kinase buffer.

The kinase activity associated with anti-cdk4 immunocomplexes was assayed in 50 µl of GST-RB kinase buffer containing 2 µg of GST-RB substrate, and in each case supplemented with 2 mM EGTA and 2 µCi of [{gamma}-32P]ATP. Reactions were carried out for 30 min at room temperature, after which cold ATP (final concentration, 30 µM) was added to each reaction mixture to reduce background signal. Reactions were stopped by addition of Laemmli sample buffer, and the reaction products were electrophoresed in 12% SDS-PAGE, whereupon the gels were dried, visualized by autoradiography, and quantitated with a scanning densitometer.

MTT Assay
MTT assays were performed to evaluate the proliferation of NRK-49F cells. Cells (5 x 103 cells/dl) were plated and incubated for 24 h in wells of a 96-well plate. Various concentrations of each drug were then added to the wells. After 24 h incubation, 10 µl of sterile MTT dye was added, and the cells were incubated for 6 h at 37 C. Then 100 µl of acidic isopropanol (0.04 M HCl in isopropanol) was added and thoroughly mixed. Spectrometric absorbance at 595 nm (for formazan dye) was measured with the absorbance at 655 nm for reference.

Flow-Cytometric Analysis of the Cell Cycle
For flow-cytometric experiments to determine DNA content and the cell cycle profile, cells were plated at a density of 1 x 106 to 1.5 x 106 cells per T-25 flask. At various time points, cells were harvested and fixed with ice-cold 100% ethanol with vortexing at low speed, cells were then placed at –20 C for overnight. After fixation, cells were centrifuged and washed once with PBS containing 1% BSA. For staining with DNA dye, cells were resuspended in 0.5 to 1 ml of propidium iodide solution containing RNase and incubated at 37 C for 30 min, followed by overnight incubation at 4 C. Cell cycle profiles were obtained with a FACScan flow cytometer (Becton Dickinson, San Jose, CA.) and data were analyzed with ModFit software (Verity Software House, Inc., Topsham, ME) for cell cycle analysis.

Statistics
The results were expressed as the mean ± SEM. Unpaired Student’s t tests were used for the comparison between two groups. One-way ANOVA followed by unpaired t test was used for the comparison among more than three groups. A P value less than 0.05 was considered statistically significant.


   FOOTNOTES
 
This work was supported by Research Grant NSC-91–2314-B273-004 (to J.-S. H.) from the National Science Council, Republic of China.

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 8, 2006

Abbreviations: eNOS, Endothelial NOS; FBS, fetal bovine serum; GST, glutathione-S-transferase; HG, high glucose; iNOS, inducible NOS; JAK, Janus kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium; NG, normal glucose; NMMA, NG-monomethyl-L-arginine (L-NMMA)-nNOS, neuronal NOS; NO, nitric oxide; NOS, nitric oxide synthase; 8-pCPT-cGMPs, 8-para-chlorophenylthio-cGMPs; PKG, cGMP-dependent protein kinase; SNP, sodium nitroprusside; SNAP, S-nitroso-N-acetylpenicillamine; STAT, signal transducers and activators of transcription; VEGF, vascular endothelial growth factor.

Received for publication August 15, 2005. Accepted for publication May 31, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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