| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
University of Montréal, Maisonneuve-Rosemont Hospital Research Center (S.-L.Z., X.C., J.G.F., J.S.D.C.), Montréal, Québec, Canada H1T 2M4; and Harvard Medical School, Pediatric Nephrology Unit, Massachusetts General Hospital (S.-S.T., J.R.I.), Boston, Massachusetts 02114-3117
Address all correspondence and requests for reprints to: Dr. John S. D. Chan, University of Montréal, Centre Hospitalier de l’Université de Montréal, Hôtel-Dieu Hospital, Pavillon Masson, 3850 St. Urbain Street, Montréal, Québec, Canada H2W 1T8. E-mail: john.chan{at}umontreal.ca
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
35
mM) also stimulated the phosphorylation of p38 MAPK, but
did not stimulate ANG secretion or gene expression. GF 109203X (an
inhibitor of protein kinase C) blocked the stimulatory effect of high
levels of glucose and PMA on ANG gene expression, whereas it did not
block the effect of high levels of glucose, sorbitol, or
2-deoxy-D-glucose on p38 MAPK phosphorylation in IRPTCs.
These studies demonstrate that the stimulatory effect of a high level
of glucose (25 mM) on ANG gene expression in IRPTCS may be
mediated at least in part via activation of p38 MAPK signal
transduction pathway and is protein kinase C independent. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Previous studies have shown that high levels of glucose and/or
angiotensin II (Ang II) may directly or indirectly be responsible for
the renal proximal tubular hypertrophy observed in diabetes, as
demonstrated by incubation of murine proximal tubular cells in a high
glucose medium and/or Ang II (i.e.
10-8
M) (4, 5, 6, 7, 8, 9, 10). Such observations
suggest that high glucose levels and Ang II may play an important role
in the pathogenesis of diabetic nephropathy. Indeed, this hypothesis is
supported by the observation that administration of
angiotensin-converting enzyme (ACE) inhibitors or Ang II receptor
antagonists reduces proteinuria and slows the progression of
nephropathy in diabetic patients (11, 12, 13, 14, 15).
The presence of a local intrarenal RAS has been generally accepted. The messenger RNA (mRNA) components of the RAS, including angiotensinogen (ANG), renin, ACE, and Ang II receptors (AT-1 and AT-2 receptors), are expressed in murine, mouse, and rat immortalized proximal tubular cell lines (16, 17, 18, 19, 20). We previously reported that the ANG protein is secreted from rat immortalized renal proximal tubular cells (IRPTCs) (21), supporting the idea that intrarenal Ang II is derived from the ANG that is synthesized within the renal proximal tubular cells in vivo.
We recently reported that the expression of ANG in IRPTCs is stimulated by high levels of glucose (i.e. 25 mM) (22). Inhibitors of aldose reductase (i.e. Tolrestat) and protein kinase C (PKC; i.e. staurosporine or H-7) block the stimulatory effect of glucose (22), suggesting that the stimulatory effect of high glucose levels on the expression of the ANG gene in IRPTCs is mediated at least in part via the de novo synthesis of diacylglycerol, an activator of the PKC signal transduction pathway. More recently, we demonstrated that insulin inhibits the stimulatory effect of glucose on ANG gene expression via the activation of p44/42 mitogen-activated protein kinase (MAPK) in IRPTCs (23), suggesting that MAPK signal transduction pathway may be involved in regulating ANG gene expression in IRPTCs.
Recent studies have shown that high levels of glucose stimulate the phosphorylation of p38 MAPK in mesangial cells (24), but the physiological role(s) of phosphorylated p38 MAPK in mesangial cells is not clear. In the present studies we investigated whether the stimulatory effect of high glucose levels on the expression of the ANG gene in IRPTCs might be mediated at least in part via the activation of p38 mitogen-activated protein kinase (p38 MAPK). Our studies showed that a high level of glucose (25 mM) stimulated the phosphorylation of p38 MAPK and ANG gene expression in IRPTCs. SB 203580 (an inhibitor of p38 MAPK) blocked the stimulatory effect of glucose. The addition of GF 109203X (an inhibitor of PKC), however, did not block the phosphorylation of p38 MAPK, but inhibited high glucose stimulation of ANG gene expression in IRPTCs.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
[
-32P]ATP (3000 Ci/mol) and
Na125I were purchased from DuPont/NEN Life Science Products (Boston, MA). Restriction and modifying enzymes
were purchased from Life Technologies, Inc., Roche Molecular Biochemicals (Dorval, Canada), or Pharmacia Biotech (Baie d’Urfé, Canada).
The Phospho Plus p38 MAPK antibody kit was purchased from New England Biolabs, Inc. (Mississauga, Canada). This kit was used for the rapid analysis of p38 MAPK (Thr180/Tyr182) phosphorylation status that functions in the stress-activated protein kinase cascade.
RIA for rat angiotensinogen
The RIA for rat ANG (RIA-rANG) was developed in our laboratory
(by J.S.D.C.), and the procedure has been previously described in
detail (21). Purified rat plasma ANG (i.e.
>90% pure, as analyzed by SDS-PAGE) and iodinated rANG were used as
the hormone standard and tracer, respectively. This RIA is specific for
intact rat (62–65 kDa) rANG and has no cross-reactivity with pituitary
hormone preparations or other rat plasma proteins (21).
The lower limit of detection for the RIA is approximately 2 ng rANG.
The intra- and interassay coefficients of variation were 9% (n =
10) and 14% (n = 10), respectively.
Cell culture
IRPTCs at passages 12–18 were used in the present study. The
characteristics of IRPTCs have been previously described
(25). These cells express the mRNA and protein of ANG,
renin, angiotensin-converting enzyme, and angiotensin II receptors
(25).
IRPTCs were grown in 100 x 20-mm plastic petri dishes (Life Technologies, Inc.) in normal glucose (i.e. 5 mM glucose DMEM, pH 7.45) supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin until study (see below). The cells were grown in a humidified atmosphere in 95% air/5% CO2 at 37 C. For subculturing, cells were trypsinized (0.5% trypsin and EDTA) and plated at 2.5 x 104 cells/cm2 in 100 x 20-mm petri dishes.
Effect of glucose, sorbitol, 2-deoxy-D-glucose, and PMA
on the secretion of IR-rANG in IRPTCs
IRPTCs were plated at a density of 1–2 x
105 cells/well in six-well plates and incubated
overnight in normal glucose (i.e. 5
mM) and DMEM containing 10% FBS. Cell growth was
arrested by incubating the cells in serum-free medium with 5
mM glucose DMEM for 24 h.
D(+)Glucose (10–35 mM),
D-mannitol (10–35 mM),
D-(+)sorbitol (10–35 mM),
2-deoxy-D-glucose (10–35
mM), or PMA
(10-7
M) was added to a normal (5
mM) glucose culture medium containing 1%
depleted FBS, and the cells were incubated for an additional 24 h.
At the end of the incubation period, media were collected and stored at
-20 C until assayed for IR-rANG. The depleted FBS (i.e.
depleted of endogenous steroid and thyroid hormones) was prepared by
incubation with 1% activated charcoal and 1% AG 1 x 8 ion
exchange resin (Bio-Rad Laboratories, Inc., Richmond, CA)
for 16–24 h at room temperature as described by Samuels et
al. (26).
To study whether the stimulatory effect of high glucose levels (25 mM) or PMA (10-7 M) is mediated via the PKC or p38 MAPK signal transduction pathway, IRPTCs were incubated in a fresh 5-mM glucose medium plus PMA (10-7 M) or 25 mM glucose medium plus PMA (10-7 M) in the presence or absence of GF 109203X (10-6 M) or SB 203580 (10-6 M) for 24 h. At the end of the incubation period, media were collected and stored at -20 C until assayed for IR-rANG.
Phosphorylation of p38 MAPK in IRPTCs
The effect of glucose, sorbitol, 2-deoxy-D-glucose,
D-mannitol, or PMA on the activation of p38 MAPK signal
transduction pathways in IRPTCs was evaluated by the phosphorylation of
p38 MAPK by employing the Phospho Plus p38 MAPK antibody kit. Briefly,
cells were plated at 5 x 104 cells/well in
six-well plates in 5 mM glucose/DMEM containing 10% FBS
and were synchronized in a 5-mM glucose medium for 24
h. Subsequently, the cells were incubated in a medium containing 5
mM glucose, 5 mM glucose plus various
concentrations (10–35 mM) of sorbitol or
2-deoxy-D-glucose, or D-mannitol, 25
mM glucose, 25 mM glucose, and PMA
(10-7 M) in
the absence or presence of SB 203580
(10-6 M) or GF
109203X (10-6
M) for various time periods. Cells were lysed in 100 µl
lysis buffer [62.5 mM Tris-HCl, pH 6.8, containing 2% SDS
(wt/vol), 10% glycerol, 50 mM dithiothreitol, and 0.1%
bromophenol blue (wt/vol)] and transferred into Eppendorf
tubes. The cell lysates were sonicated for 2 sec, heated at 95 C for 5
min, and then centrifuged at 12,000 x g for 2 min at 4
C. Small aliquots (20–50 µl) of the supernatants were subjected to
SDS-PAGE (10%) and then transferred onto a polyvinylidene difluoride
membrane (Hybond-P, Amersham Pharmacia Biotech, Oakville,
Canada). The membrane was then blotted for the phosphorylated p38 MAPK
by employing the Phospho Plus p38 MAP kinase antibody kit.
Expression of ANG mRNA in IRPTCs
To study the effects of glucose, PMA, and sorbitol on the
expression of ANG mRNA in IRPTCs, the cells were incubated in 5
mM glucose plus 20 mM D-mannitol,
25 mM glucose medium, 25 mM glucose medium plus
PMA (10-7 M),
or 5 mM glucose medium plus sorbitol (35 mM) in
the absence or presence of SB 203580
(10-6 M) or GF
109203X (10-6
M) for 24 h. At the end of the incubation period,
cells were collected, and total RNA was isolated using TRIzol reagent
(Life Technologies, Inc.) according to the protocol of the
supplier. The total RNA was used in a RT-PCR to quantify the amount of
ANG mRNA expressed in IRPTCs as described previously (23).
The forward primer, 5'-CCT CGC TCT CTG GAC TTA TC-3', and the reverse
primer, 5'-CAG ACA CTG AGG TGC TGT TG-3', corresponding to the
nucleotide sequence of N+676 to N+695 and N+882 To N+901 of the rat
complementary DNA (27), were used for PCR. Furthermore,
primers specific for rat ß-actin (28) (forward and
reverse primers, 5'-ATG CCA TCC TGC GTC TGG ACC TGG C-3' and 5'-AGC ATT
TGC GGT GCA CGA TGG AGG G-3', corresponding to nucleotide N+155 to
N+139 of exon 3 and nucleotide N+115 to N+139 of exon 5 of rat
ß-actin) were used in another PCR reaction as internal controls. The
RT-PCR reaction mixtures were then separated on a 1.5% agarose gel and
transferred onto a Hybond XL nylon membrane (Amersham Pharmacia Biotech). Subsequently, 32P-labeled
oligonucleotides 5'-GAG GGG GTC AGC ACG GAC AGC ACC-3' and 5'-TCC TGT
GGC ATC CAT GAA ACT ACA TTC-3' corresponding to the nucleotide N+775 to
N+798 of the rat ANG complementary DNA (27) and
nucleotides N+9 to N+35 of exon 4 of rat ß-actin (28),
respectively, were used to hybridize the products on the membrane.
Finally, the membrane was washed and exposed to autoradiography. The
relative densities of the PCR bands were determined with a computerized
laser densitometer.
Statistical analysis
Two or three separate experiments per protocol were performed,
and each treatment group was assayed in triplicate. The data were
analyzed by Student’s t test or ANOVA. P 
0.05 was considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
;
P 0.01) after a 24-h incubation period. The
secretion of IR-rANG was decreased at 40 mM
glucose. The addition of sorbitol (10–35 mM;
Fig. B) or 2-deoxy-D-glucose (10–35
mM; Fig. 1C), however, did not increase the
secretion of IR-rANG by IRPTCs into the culture medium. The addition of
SB 203580 (a specific inhibitor of p38 MAPK) to the culture medium
abolished the high glucose (25 mM)-stimulated
secretion of IR-rANG in IRPTCs in a dose-dependent manner (Fig. 2A), with a maximal effect observed at
10-7–10-6
M. This dose of SB 203580
(10-6
M) was then routinely used in all subsequent
experiments. Furthermore, SB 202474 (a negative control for SB 203580)
at concentrations ranging from
10-13–10-6
M had no detectable effect on the
glucose-stimulated secretion of IR-rANG in IRPTCs (Fig. 2B). These
studies demonstrate that the high glucose levels that stimulated the
secretion of IR-rANG in IRPTCs are mediated at least in part via the
activation of p38 MAPK.
|
|
shows that the addition of PMA
(10-7 M)
stimulated the secretion of IR-rANG (Fig. 3A) and further enhanced the
stimulatory effect of 25 mM glucose (Fig. 3B) on the
secretion of IR-rANG in IRPTCs. This stimulatory effect of PMA or a
combination of PMA and high glucose was blocked by the presence of SB
203580, with a maximal effective dose at
10-7 or
10-6 M. These
results further support the idea that the stimulatory effect of high
glucose levels and/or PMA on the secretion of IR-rANG in IRPTCs may be
mediated at least in part via the p38 MAPK signal transduction
pathway.
|
shows that glucose
activated the phosphorylation of p38 MAPK in IRPTCs in a time-dependent
(Fig. 4A) and dose-dependent (Fig. 4B) manner. It is evident that 25
mM glucose after a 60-min incubation period was the optimal
condition for the activation of the phosphorylation of p38 MAPK (Fig. 4A). There was no significant difference in glucose-induced
phosphorylation of p38 MAPK in IRPTCs after a 24-h incubation period.
It appears that glucose at a concentration of 40 mM did not
stimulate the phosphorylation of p38 MAPK to the same extent as 25
mM in IRPTCs (Fig. 4B). The stimulatory effect of high
glucose (25 mM) was blocked in the presence of SB 203580
(10-6 M), but
not in the presence of GF 109203X
(10-6 M; Fig. 5). Interestingly, incubation of IRPTCs
in 5 mM glucose plus high levels of
2-deoxy-D-glucose, sorbitol, or D-mannitol
also stimulated the phosphorylation of p38 MAPK at levels comparable to
those evoked by 5 mM D-glucose (Fig. 6). This stimulatory effect of 5
mM glucose plus 35 mM
2-deoxy-D-glucose (Fig. 7) or
35 mM sorbitol (Fig. 8) was
also blocked by SB 203580
(10-6 M), but
was not blocked by GF 109203X
(10-6 M).
Similarly, SB 20358 (10-6
M) blocked the stimulatory effect of 35 mM
D-mannitol on the phosphorylation of p38 MAPK in IRPTCs,
but GF 109203X (10-6
M) had no effect (not shown). These studies demonstrate
that the stimulatory effect of 25 mM glucose on the
phosphorylation of p38 MAPK in IRPTCs is mediated at least in part via
high osmolarity and is PKC independent.
|
|
|
|
|
) or 25
mM glucose medium (Fig. 10). The addition of SB 203580
(10-6 M)
completely blocked the stimulatory effect of PMA on the phosphorylation
of p38 MAPK in IRPTCs incubated in 5 mM glucose medium
(Fig. 9) or in a 25-mM glucose medium (Fig. 10). The
addition of GF 109203X
(10-6 M)
blocked the stimulatory effect of PMA in 5 mM glucose (Fig. 9), but only partially blocked the stimulatory effect of PMA and 25
mM glucose on the phosphorylation of p38 MAPK (Fig. 10).
These results demonstrate that the effect of PMA on the phosphorylation
of p38 MAPK is at least in part PKC dependent.
|
|
shows that a high
glucose concentration (25 mM) stimulated the accumulation
of ANG mRNA to levels that were 2.5-fold higher than those found in
control cells cultured in a medium containing 5 mM glucose
plus 20 mM D-mannitol (P
0.01). The addition of SB 203580
(10-6
M) or GF 109203X
(10-6
M) abolished the stimulatory effect of 25
mM glucose. Figure 12 shows that PMA
(10-7
M) further enhanced the stimulatory effect of 25
mM glucose (P 0.05). The
addition of SB 203580
(10-6
M) or GF 109203X
(10-6
M) abolished the effect of PMA. These data
demonstrate that the stimulatory effect of high levels of glucose
and/or PMA on the levels of cellular ANG mRNA is mediated at least in
part via the activation of both PKC and p38 MAPK signal transduction
pathways. Sorbitol at a concentration of 35 mM
did not stimulate the accumulation of ANG mRNA in IRPTCs (not
shown).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The secretion of IR-rANG from IRPTCs was increased 1.5-fold in the presence of a high glucose (25 mM) medium compared with a normal glucose (5 mM) medium when IRPTCs were cultured for a 24-h period. This level of stimulation is consistent with our previous studies, which showed that a high level of glucose (25 mM) stimulates expression of the ANG gene 1.5-fold in opossum kidney (OK) cells (29) and in IRPTCs (22, 23). In contrast, 2-deoxy-D-glucose and sorbitol did not stimulate the secretion of IR-rANG from IRPTCs at different concentrations (10–35 mM). These studies demonstrate that high glucose specifically stimulates the secretion of IR-rANG from IRPTCs. At present, we do not know the exact reasons why glucose at 40 mM showed a relatively smaller stimulation of IR-rANG secretion compared with 25 mM glucose. One reason may be that 40 mM glucose down-regulates or desensitizes signal transduction pathways, i.e. PKC. This possibility is supported by our previous finding that overnight preincubation of IRPTCs with 10-6 M PMA abolished the response to high levels of glucose (22). Nevertheless, additional studies are definitely required to explain this observation.
p38 MAPK is a member of the MAPK superfamily [i.e. extracellular signal-regulated kinase (ERKs or the p42/p44 MAPKs), stress-activated protein kinases(s) (SAPK/JNK), and p38 MAPK] (30). These three kinases exhibit distinct, but related, signal transduction pathways by which kinases are activated by extracellular signals and then translocated into the nucleus to stimulate specific gene expression (31). For example, studies have shown that p42/p44 MAPK is activated by a variety of growth and neurotropic factors and is linked to growth and differentiation signals with gene transcription in the nucleus (32). The SAPK/JNK and p38 MAPK pathways have been implicated in the cellular response to environmental stress such as high extracellular osmolarity, UV irradiation, heat shock, and inflammatory cytokines (33, 34, 35, 36, 37). SB 203580 has been identified as a selective and potent inhibitor of p38 MAPK (38, 39). As this compound does not inhibit SAPK/JNK, it can therefore be of use in the identification of those functional activities that are mediated by SAPK/JNK or p38 MAPK.
Our present study demonstrates that SB 203580 blocked the stimulatory effect of 25 mM glucose, PMA (10-7 M), or a combination of 25 mM glucose and PMA (10-7 M) on the secretion of IR-rANG in IRPTCs, whereas SB20474 had no effect. These studies suggest that the stimulatory effect of high glucose (25 mM) and/or PMA on the secretion of IR-rANG may be mediated at least in part via the p38 MAPK signal transduction pathway.
To confirm further that the stimulatory effect of high glucose levels and PMA are mediated at least in part via the p38 MAPK signal transduction pathway, we performed Western blot analysis of phosphorylated p38 MAPK in IRPTCs. Our studies showed that 25 mM glucose stimulated the phosphorylation of p38 MAPK in IRPTCs in a time-dependent and dose-dependent manner, and that SB 203580 completely prevented the phosphorylation of p38 MAPK. These results are in agreement with those of other investigators who have reported that high glucose stimulates the phosphorylation of p38 MAPK in rat mesangial cells and vascular smooth muscle cells (24, 39).
Surprisingly, our results revealed that the addition of GF 109203X did not block the stimulatory effect of high glucose (25 mM) on the phosphorylation of p38 MAPK in IRPTCs. These findings indicate that high osmolarity alone is sufficient to stimulate the phosphorylation of p38 MAPK in IRPTCs that is independent of PKC activation. Indeed, this idea is further supported by the findings that high levels of 2-deoxy-D-glucose (10–35 mM), sorbitol (10–35 mM), or D-mannitol (10–35 mM) stimulated the phosphorylation of p38 MAPK. The stimulatory effect of high 2-deoxy-D-glucose or sorbitol was inhibited in the presence of SB 203580, but not in the presence of GF 109205X. Similar results were observed with high concentrations of D-mannitol (our unpublished results). Interestingly, PMA also stimulated the phosphorylation of p38 MAPK in IRPTCs cultured in DMEM containing 5 mM glucose or 25 mM glucose. The addition of SB 203580 completely inhibited the stimulatory effect of PMA on the phosphorylation of p38 MAPK, whereas GF 109203X inhibited the stimulatory effect of PMA in 5 mM glucose medium, but only partially blocked the stimulatory effect of PMA on the phosphorylation of p38 MAPK in 25 mM glucose. These results demonstrate that the stimulatory effect of high levels of glucose on the phosphorylation of p38 MAPK is PKC independent, whereas the effect of PMA is mediated at least in part via the PKC-dependent pathway. Our results are in agreement with the studies of Igarashi et al. (39), who also reported that glucose at elevated levels could activate p38 MAPK by hyperosmolarity via a PKC-independent pathway.
Finally, the effects of high glucose (25 mM) and p38 MAPK
on ANG appear to occur at the level of mRNA. Exposure of IRPTCs to 25
mM glucose stimulated the expression of ANG mRNA by
2.5-fold (P 0.01) compared with levels in control
cells (i.e. cells cultured in 5 mM
glucose plus 20 mM
D-mannitol). The addition of PMA
(10-7
M) further enhanced the stimulatory effect of 25
mM glucose. The addition of SB 2103580
(10-6
M) or GF 109203X
(10-6
M) completely blocked the stimulatory effect of
25 mM glucose and 25 mM
glucose plus PMA on cellular ANG mRNA. These studies provide evidence
that the effect of high levels of glucose on the expression of the ANG
gene in IRPTCs may be mediated at least in part via both the PKC and
p38 MAPK signal transduction pathways. At present, it is not clear
whether high levels of glucose and/or p38 MAPK increase the levels of
transcription or might affect the stability of the ANG mRNA in IRPTCs.
Studies are underway in our laboratory to investigate these
possibilities.
In the present studies we have not quantified the levels of Ang II in
the medium. Our recent studies, however, have shown that the levels of
immunoreactive Ang II in IRPTCs incubated in a 25-mM
glucose medium were at least 2-fold higher (P 0.05)
than levels in IRPTCs incubated in a 5-mM glucose
medium (40). Furthermore, high levels of glucose
(i.e. 25 mM) induced the hypertrophy
in IRPTCs, and this stimulatory effect was blocked in the presence of
ACE inhibitors and an Ang II receptor blocker (40). Taken
together, our studies suggest that the molecular mechanism(s) of action
of high glucose on the hypertrophy of renal proximal tubular cells in
early diabetes may be mediated at least in part via local renal ANG
gene expression and the activation of RAS.
At present, we do not know the exact molecular mechanism(s) for the stimulatory effect of high glucose (that is, the downstream pathway after the activation of PKC and p38 MAPK) on the expression of rat ANG gene in IRPTCs. One possibility might be that high glucose induces the phosphorylation of nuclear activating transcription factor-2 (ATF-2) via the p38 MAPK signal transduction pathway. Indeed, previous studies have shown that p38 MAPK phosphorylates the transcription factor ATF-2 (41, 42). The phosphorylated ATF-2 then forms the heterodimer complex with the phosphorylated cAMP response element (CRE)-binding protein (CREB) [note: the phosphorylation of CREB is induced by high levels of glucose via the PKC signal transduction pathway, as demonstrated by Kreisberg et al. (43)]. The phosphorylated ATF-2/CREB heterodimer then binds to the CRE in the 5'-flanking region of the rat ANG gene (44, 45) and subsequently enhances gene expression. This possibility is supported by our recent studies that showed that the 43-kDa CREB enhances ANG gene expression in OK cells (46), and high levels of glucose stimulated the phosphorylation of 43-kDa CREB (47). Studies are ongoing in our laboratory to explore the downstream molecular mechanism(s) of the stimulatory effect of high glucose on expression of the ANG gene in IRPTCs.
In summary, our present studies showed that high glucose levels directly stimulate expression of the ANG gene in IRPTCs. The addition of SB 203580 blocked the stimulatory effect of high glucose, implicating involvement of the p38 MAPK signal transduction pathway in the up-regulated expression of the renal ANG gene under hyperglycemic conditions. These results suggest that the blockade of p38 MAPK may represent a novel therapeutic approach to prevent or attenuate glucose-induced ANG gene expression and, consequently, the development of diabetic nephropathy. However, it remains to be shown whether long-term blockade of p38 MAPK may indeed be beneficial in the treatment of diabetic nephropathy. This interesting possibility certainly warrants further investigation.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received April 24, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  V. A Stevens, S. Saad, P. Poronnik, C. A Fenton-Lee, T. S Polhill, and C. A Pollock The role of SGK-1 in angiotensin II-mediated sodium reabsorption in human proximal tubular cells Nephrol. Dial. Transplant., June 1, 2008; 23(6): 1834 - 1843. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-C. Wei, S.-L. Zhang, Y.-W. Chen, D.-F. Guo, J. R. Ingelfinger, K. Bomsztyk, and J. S. D. Chan Heterogeneous Nuclear Ribonucleoprotein K Modulates Angiotensinogen Gene Expression in Kidney Cells J. Biol. Chem., September 1, 2006; 281(35): 25344 - 25355. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-C. Wei, D.-F. Guo, S.-L. Zhang, J. R. Ingelfinger, and J. S.D. Chan Heterogenous Nuclear Ribonucleoprotein F Modulates Angiotensinogen Gene Expression in Rat Kidney Proximal Tubular Cells J. Am. Soc. Nephrol., March 1, 2005; 16(3): 616 - 628. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T.-J. Hsieh, P. Fustier, C.-C. Wei, S.-L. Zhang, J. G Filep, S.-S. Tang, J. R Ingelfinger, I G. Fantus, P. Hamet, and J. S D Chan Reactive oxygen species blockade and action of insulin on expression of angiotensinogen gene in proximal tubular cells J. Endocrinol., December 1, 2004; 183(3): 535 - 550. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Fujita, S. Omori, K. Ishikura, M. Hida, and M. Awazu ERK and p38 mediate high-glucose-induced hypertrophy and TGF-{beta} expression in renal tubular cells Am J Physiol Renal Physiol, January 1, 2004; 286(1): F120 - F126. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T.-J. Hsieh, P. Fustier, S.-L. Zhang, J. G. Filep, S.-S. Tang, J. R. Ingelfinger, I. G. Fantus, P. Hamet, and J. S. D. Chan High Glucose Stimulates Angiotensinogen Gene Expression and Cell Hypertrophy via Activation of the Hexosamine Biosynthesis Pathway in Rat Kidney Proximal Tubular Cells Endocrinology, October 1, 2003; 144(10): 4338 - 4349. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Sengul, C. Zwizinski, and V. Batuman Role of MAPK pathways in light chain-induced cytokine production in human proximal tubule cells Am J Physiol Renal Physiol, June 1, 2003; 284(6): F1245 - F1254. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Singh, A. K. Singh, N. Alavi, and D. J. Leehey Mechanism of Increased Angiotensin II Levels in Glomerular Mesangial Cells Cultured in High Glucose J. Am. Soc. Nephrol., April 1, 2003; 14(4): 873 - 880. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-L. Zhang, X. Chen, C.-C. Wei, J. G. Filep, S.-S. Tang, J. R. Ingelfinger, and J. S. D. Chan Insulin Inhibits Dexamethasone Effect on Angiotensinogen Gene Expression and Induction of Hypertrophy in Rat Kidney Proximal Tubular Cells in High Glucose Endocrinology, December 1, 2002; 143(12): 4627 - 4635. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T.-J. Hsieh, S.-L. Zhang, J. G. Filep, S.-S. Tang, J. R. Ingelfinger, and J. S. D. Chan High Glucose Stimulates Angiotensinogen Gene Expression via Reactive Oxygen Species Generation in Rat Kidney Proximal Tubular Cells Endocrinology, August 1, 2002; 143(8): 2975 - 2985. [Abstract] [Full Text] |
