Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada M5G 1L5
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Endothelial
nitric oxide synthase (NOS) and neuronal NOS protein increased in
proximal tubules of acidotic diabetic rats 3-5 wk after
streptozotocin injection. NOS activity (citrulline production) was
similar in nondiabetic and diabetic tubules incubated with low glucose
(5 mM glucose + 20 mM mannitol); but after 30 min with high
glucose (25 mM), Ca-sensitive citrulline production had increased 23%
in diabetic tubules. Glucose concentration did not influence citrulline
production in nondiabetic tubules. High glucose increased
carboxy-2-phenyl-4,4,5,5,-tetramethylimidazoline 1-oxyl-3-oxide
(cpt10)-scavenged NO sevenfold in a suspension of diabetic tubules but
did not alter NO in nondiabetic tubules. Diabetes increased
ouabain-sensitive 86Rb uptake (141 ± 9 vs. 122 ± 6 nmol · min1 · mg
1) and
oligomycin-sensitive O2 consumption
(
O2; 16.0 ± 1.7 vs. 11.3 ± 0.7 nmol · min
1 · mg
1).
Ethylisopropyl amiloride-inhibitable
O2
(6.5 ± 0.6 vs. 2.4 ± 0.3 nmol · min
1 · mg
1)
accounted for increased oligomycin-sensitive
O2 in diabetic tubules.
NG-monomethyl-L-arginine methyl
ester (L-NAME) inhibited most of the increase in
86Rb uptake and
O2 in
diabetic tubules. L-NAME had little effect on
nondiabetic tubules. Inhibition of
O2 by
ethylisopropyl amiloride and L-NAME was only 5-8%
additive. Uncontrolled diabetes for 3-5 wk increases NOS protein
in proximal tubules and makes NOS activity sensitive to glucose
concentration. Under these conditions, NO stimulates Na-K-ATPase and
O2 in proximal tubules.
oxygen consumption; nitric oxide synthase; sodium-hydrogen exchange; kidney; streptozotocin; oligomycin; ouabain; rubidium uptake; ketoacidosis; NG-monomethyl-L-arginine methyl ester; ethylisopropyl amiloride
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CONTROVERSY SURROUNDS THE effect of diabetes on renal nitric oxide (NO) production. Is expression of NO synthase (NOS) increased (22, 34, 37) or decreased (19, 20, 31)? Is NO production increased, decreased, or unchanged? Does NO stimulate or inhibit Na transport by proximal tubules? Some of this ambiguity can be attributed to changes in NO production as diabetes progresses. In the first 1-2 wk of streptozotocin-induced diabetes, NOS mRNA and protein expression are unchanged or decreased (19, 20, 31). After 2 wk, NOS expression increases in the renal cortex and outer medulla (32) endothelium, mesangium, and macula densa (22, 34, 37). Endothelial NOS (eNOS) protein also increases in proximal convoluted and straight segments, and neuronal NOS (nNOS) protein increases in the straight proximal segment (32).
NO production depends not only on NOS protein expression but also on cofactors [calcium, reduced NADP (NADPH), tetrahydrobiopterin, and FAD] and on posttranscriptional modification by kinases (10, 17, 19). Changes in one or more cofactors or phosphorylation by kinases may account for increased NO production without increased NOS protein in rat renal cortex 14 days after streptozotocin injection (19).
There is also controversy about the effect of NO on proximal tubule
function. Basal NO production stimulates Na/H exchange in proximal
tubules of nondiabetic rats (4, 14, 38, 39), whereas
higher NO concentrations from exogenous sources inhibit Na/H
exchange and Na reabsorption [reviewed by Liang and Knox (25)]. Hypoxia enhances inhibition by NO
(21). These observations suggest that NO might either
enhance or diminish adaptation of Na/H exchange as diabetic
ketoacidosis progresses (3, 15). In the experiments
described below, we examined the connections between NO and Na/H
exchange in proximal tubules of mildly acidotic diabetic rats 3-5
wk after streptozotocin injection. We measured NOS protein expression,
NO production, and the effects of inhibiting NO production on
86Rb uptake and oxygen consumption
(O2) in isolated tubule fragments.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Male Wistar rats (250 g; Charles River) were injected with streptozotocin (55 mg/kg body wt ip). They were considered to be diabetic if their tail blood glucose, collected in the morning, exceeded 18 mM. Three to five weeks after the streptozotocin injection, the rats were anesthetized with pentobarbital sodium (100 mg/kg body wt ip), venous blood was collected, and kidneys were removed. Plasma was analyzed for glucose, Na, K, Cl, HCO3, and creatinine with a COBAS Integra. NO2 + NO3 in urine was measured with a modified Griess reaction (30). Some rats were housed in metabolic cages, and urine was collected daily for 3-5 days before removal of their kidneys.
Tubules were prepared from outer cortical slices as previously described (5). The kidneys were cleared of blood by infusing 60 ml of isotonic saline through the aorta. Slices were removed from the outer cortex with a Stadie-Riggs microtome, placed in ice-cold saline, and minced finely with a razor blade. The minceate was incubated at 37°C for 30 min in 6 ml of Krebs-Henseleit buffer containing 7.2 mg of collagenase (Sigma) and 30 mg of bovine serum albumin. The reaction was stopped with ice-cold buffer solution. The tissue was passed through a tea strainer, washed four times with buffer, and suspended in 30 ml of 45% Percoll in Krebs-Henseleit buffer. Tubules were separated from glomeruli by centrifugation in a 60Ti rotor at 20,000 rpm for 20 min. Microscopic examination showed that the bottom layer, containing 80-90% proximal tubule fragments with virtually no glomeruli, was washed four times with buffer and passed once through a 100-µm sieve. Tubules were kept on ice in a modified Krebs-Henseleit solution, which contained (in mM) 136 Na, 5 K, 111 Cl, 25 HCO3, 0.5 Mg, 1 Ca, 5 or 25 glucose, 2 lactate, 0.2 pyruvate, 2 glutamine, 1 arginine, 1 alanine, and 1 heptanoic acid, as well as 10 g/l bovine serum albumin. The pH was 7.4 at 37°C when equilibrated with 95% air-5% CO2.
Before measurement of O2, tubules were
incubated at 37°C with 95% air-5% CO2 for 30 min in the
modified Krebs-Henseleit solution with either high glucose (25 mM) or
low glucose (5 mM glucose+20 mM mannitol). Inhibitors,
ethylisopropyl amiloride (EIPA; 10 µM), phloridzin (100 µM), and
oligomycin (20 µM), were added to the incubation solution 2-5
min before measurement of
O2.
NG-monomethyl-L-arginine methyl
ester (L-NAME) and
NG-monomethyl-D-arginine methyl
ester (10 mM) were added 30 min before measurement of
O2. Sigma (St. Louis, MO)
provided chemicals and inhibitors unless otherwise noted. Measurements
were started by injecting tubules (0.8-1.6 mg of protein) into the
0.6-ml analytic chamber (Diamond General Development, Ann Arbor, MI)
containing buffer solution with or without inhibitors preequilibrated
with 95% air-5% CO2 at 37°C.
PO2 was recorded polarographically with a YSI
model 5300 biological oxygen monitor. Comparisons with and without
inhibitors were done in duplicate when responses with high and low
glucose were obtained with the same tubule preparation. When one
glucose concentration was used, the result for each tubule preparation
was the average of triplicate or quadruplicate measurements. Tubules
were washed from the chamber with saline for protein measurement.
Ouabain-sensitive 86Rb uptake by proximal tubule fragments was used to measure Na-K-ATPase activity (5). 86Rb (PerkinElmer, Boston, MA) was added to produce an activity of ~1 µCi/ml with ~2 mg of tubule protein. Uptake was measured with and without 2.5 mM ouabain. Uptake was terminated after 1 min by layering the tubule suspension onto 0.5 ml of a 2:1 mixture of dibutyl-dioctylphthalate in a 1.5-ml centrifuge tube and centrifuging for 10 s in an Eppendorf 5414 centrifuge. The medium above the oil layer was removed, and the tubule was rinsed five times with distilled water without disturbing of the oil. The pellet of tubules was dissolved in 1 ml of 0.1 N NaOH, and 200 µl of the solution were added to 10 ml of liquid scintillation cocktail (Ready Safe, Beckman Coulter, Fullerton, CA) for counting in a liquid scintillation counter. In preliminary experiments, [3H]inulin was added with 86Rb. Less than 1% of the 3H passed through the oil with the tubules; therefore, in subsequent experiments, we did not include [3H]inulin and did not correct for trapped extracellular fluid.
NOS activity was measured in three ways: citrulline production in tubule homogenates, citrulline production in intact tubule fragments, and NO concentration in the incubation medium. Before NOS activity was measured, the tubules were incubated in either high- or low-glucose solution at 37°C for 30 min. Tubules were briefly washed in ice-cold saline before they were homogenized according to the method of Chin et al. (11). NOS activity in the homogenate was measured in 100 µl of a solution containing (in mM) 50 KH2PO4, 1 MgCl2, 1 CaCl2, 1 NADPH, and 0.022 L-arginine, as well as 3 µmol/l of U-[14C]arginine HCl (Amersham). Duplicate samples were incubated for 1 h at 37°C with no inhibitors, with 4 mmol/EGTA, and with 4 mM EGTA+5 mM L-NAME. The reaction was stopped by adding 1.5 ml of a 1:1 suspension of Dowex 50W (Na form) in water. The solution was centrifuged, 5 ml water were added, and the solution was recentrifuged. Two hundred microliters of the supernatant were used for radioactive counting. In pilot experiments, we found that adding 50 mM valine, 100 mM biopterin, 100 mM FAD, and 1 µg/ml calmodulin did not alter the results; therefore, these cofactors were omitted from the assay mixture. NOS activity in intact tubule fragments was measured by incubating tubules with [3H]arginine (59 Ci/mmol; Amersham) in Krebs-Henseleit buffer gassed with 95% air-5%CO2 at 37°C for 1 h. The reaction was stopped with 1 ml of ice-cold 4 mM EDTA, and tubules were homogenized for 30 s with a Polytron. After centrifugation, citrulline and arginine were separated with Dowex as described above.
NO produced by proximal tubule fragments was measured with a Clark-type electrode (Diamond General) in 2.5 ml of HEPES-buffered Krebs-Henseleit containing 1,000 U/ml superoxide dismutase, at 36.5°C in a shaking water bath (42, 43). NO for daily calibration was generated from NaNO2 (0.05 mM) in KI, H2SO4, and K2SO4. The electrode reacted linearly to the NO standard and to increasing concentrations of S-nitroso-N-acetylpenicillamine (SNAP). NO concentration in the bathing medium was calculated from the decrease produced by adding 50 µg carboxy-2-phenyl-4,4,5,5,-tetramethylimidazoline 1-oxyl-3-oxide (cpt10; Research Biochemicals International, Natick, MA). NO consumption by tubules was assessed by adding SNAP (1 µM) to the tubule suspension. The area under the curve of SNAP concentration (concentration × time), measured by cutting out and weighing the chart paper, was similar for diabetic and nondiabetic tubules.
For Western blotting, tubule fragments were incubated at 37°C with high or low glucose for 30 min before homogenization in boiling 1% SDS, 10 mM Tris, pH 7.4, and 1 mM Na orthovanadate. Protein samples (200 µg) were run on 7.5% Tris · HCl Ready Gels (Bio-Rad) in a Bio-Rad minigel apparatus at 200 constant V for ~30 min. Proteins were transferred to Immobilon-P membranes (Millipore, Bedford, MA) at 100 V for 3 h at 4°C. The membranes were blocked with 5% nonfat dry milk, 10 mM Tris, pH 7.5, 100 mM NaCl, and 0.1% Tween 20 for 1 h at room temperature. They were then incubated overnight with 1:2,500 dilution of mouse monoclonal antibodies against nNOS, inducible NOS (iNOS), and eNOS (Transduction Laboratories) and visualized with 1:1,000 horseradish peroxidase-labeled goat anti-mouse with enhanced chemiluminescence immunodetection (Amersham).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Three to five weeks after streptozotocin injection, diabetic rats
appeared healthy, although they had not gained weight as rapidly as the
untreated group (Table 1). Plasma
unmeasured anion gap (Na-Cl-HCO3), creatinine clearance,
and NO2 + NO3 excretion (NOx) were higher
and plasma bicarbonate was lower (26.7 ± 0.8 vs. 29.3 ± 0.6 mM, P = 0.03) in the diabetic rats. Part of the increase in creatinine clearance and NOx excretion can be attributed to
increased renal mass. NOx excretion correlated with creatinine clearance in both groups of rats (nondiabetic,
r2 = 0.28; diabetic,
r2= 0.33).
|
Western blot analysis showed 3.5-fold more eNOS (P < 0.001) and 1.5-fold more nNOS protein (P = 0.017) in
diabetic tubules but no iNOS in tubules from nondiabetic or diabetic
rats (Fig. 1). The tubules used
to prepare Fig. 1 were incubated with high glucose for 30 min
before being homogenized. In other experiments, we incubated duplicate
aliquots from diabetic tubule preparations with high or low glucose.
Glucose concentration did not influence NOS protein expression
(n = 4 pairs of duplicates, data not shown).
|
After 30 min with low glucose, NOS activity was similar in proximal
tubule homogenates from diabetic and nondiabetic rats. Roughly
two-thirds of the citrulline production was calcium sensitive (Fig.
2A). Thirty minutes with high
glucose increased calcium-sensitive citrulline production by 23% in
diabetic tubules, but there was no change in nondiabetic tubules. High
glucose did not alter calcium-insensitive citrulline production (Fig.
2B). High glucose also stimulated NO production when intact
diabetic tubule fragments were incubated without NADPH or other
cofactors (Table 2). High glucose did not
alter citrulline production by nondiabetic tubule fragments.
|
|
We used NO released from SNAP to examine the effects of different
incubation media and tubules on NO consumption. The pattern of NO
release from SNAP was identical in low glucose (5 mM glucose + 20 mM mannitol) and high glucose (25 mM). Diabetic and nondiabetic tubules
decreased the amount of NO detected from SNAP to the same extent. With
high glucose, the areas under the curves for NO released from SNAP were
51 ± 10 (n = 8) and 55 ± 10 (n = 8) arbitrary units for nondiabetic and diabetic
tubules, respectively. Ambient NO concentration in the tubule
suspension was estimated from the decrease in the signal produced by
the NO scavenger cpt10 (Table 3). High
glucose increased cpt10-scavenged NO with diabetic tubules (P < 0.001) but numerically decreased NO concentration
with nondiabetic tubules.
|
Ouabain inhibited 75 ± 4% of 86Rb uptake in
nondiabetic tubules and 79 ± 3% in diabetic tubules
(P = 0.09). Diabetes increased ouabain-sensitive
86Rb uptake by 16% (Table
4). Incubation with L-NAME
(10 mM) for 20 min reduced 86Rb uptake in diabetic
tubules by 16% but had little effect on control tubules. Consequently,
the difference in 86Rb uptake between diabetic and
nondiabetic tubules was largely obliterated by L-NAME.
|
Tubules from diabetic rats consumed 36% more oxygen than tubules
from nondiabetic rats when both were incubated in high glucose (P < 0.001). Glucose concentration had no effect on
O2 with nondiabetic tubules (24.7 ± 1.1 vs. 24.9 ± 0.9 nmol · min
1 · mg
1), but
high glucose modestly increased
O2 by
diabetic tubules (32.8 ± 1.0 vs. 30.2 ± 1.1 nmol · min
1 · mg
1). The
interaction between glucose and diabetes was significant (2-way
repeated measures ANOVA, P = 0.02). Diabetes
substantially increased oligomycin-sensitive
O2 (16.0 ± 1.7 vs. 11.3 ± 0.7 nmol · min
1 · mg
1,
P = 0.017) and EIPA-sensitive
O2 (6.5 ± 0.6 vs. 2.4 ± 0.3 nmol · min
1 · mg
1,
P < 0.001) (Fig. 3).
Increased oligomycin-sensitive
O2 can be
entirely attributed to the increase in EIPA-inhibited
O2 (Figs. 3 and
4). The phloridzin-sensitive component of
O2 was not increased in diabetic tubules
(2.2 ± 0.6 vs. 1.8 ± 0.7 nmol · min
1 · mg
1) (Fig.
3).
|
|
L-NAME-inhibited O2
was more than doubled in diabetic tubules compared with
nondiabetic tubules [4.5 ± 0.7 (n = 10) vs. 2.0 ± 0.5 nmol · min
1 · mg
1
(n = 9), P = 0.01]. In a smaller
number of experiments, inhibition by EIPA and L-NAME was
compared by using paired analyses with the same tubule preparations
(Fig. 5). According to repeated-measures ANOVA, there was an interaction between EIPA and
L-NAME; however, the combined effect was only 5-8% of
the expected increase if the inhibitors were additive when used
together (Fig. 5).
NG-monomethyl-D-arginine methyl
ester did not alter
O2 appreciably (+0.7
and
0.9 ± 0.6 nmol · min
1 · mg
1 in
nondiabetic and diabetic tubules, respectively).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
NOS RNA and protein are unchanged or decrease for the first 1-2 wk after streptozotocin injection (19, 20, 31). Thereafter, NOS expression increases in the renal cortex and outer medulla (22, 32, 34, 37), and immunohistochemical staining of eNOS and nNOS increases in proximal tubules (32). NOS activity, measured as citrulline production, rises after 2 wk, even before there is a detectable change in NOS protein (19). Three to five weeks of uncontrolled diabetes not only increases NOS proteins in proximal tubules (Fig. 1) but also increases NOS activity and makes it sensitive to glucose concentration (Fig. 2, Tables 1-3). NOS activity in tubules from nondiabetic rats is not sensitive to glucose concentration.
High glucose also increases the steady-state concentration of NO that
can be scavenged by cpt10 (Table 3). This observation is consistent
with the effect of glucose on citrulline production. When measuring NO,
we added superoxide dismutase (SOD) to remove reactive oxygen species
(19, 35). SOD was not added to the incubation mixture for
measurement of O2 and 86Rb
uptake; therefore, the NO measurements may not reflect NO
concentrations during those measurements. In diabetic tubules,
increased reactive oxygen species production is partially
counterbalanced by increased SOD expression (19). The net
effect on NO concentration in vivo is unknown. Binding of NO to glucose
(8) did not produce differences between low- and
high-glucose measurements, because the low-glucose solution contained
20 mM mannitol, which also binds NO. In summary, both cpt10-scavenged
NO and citrulline production show increased NO production by diabetic
tubules but only in the presence of high glucose concentration.
Adaptation to prolonged hyperglycemia alters signal transduction (12) and metabolism in ways that could affect NOS activity. For example, brief exposure to high glucose increases protein kinase C (PKC) activity in LLC-PK1 and opossum kidney (OK/E) cells after they have been grown with high glucose for several days. High glucose does not increase PKC activity in cells that have been grown with low glucose (12). PKC, activated by high glucose, could stimulate NO production by phosphorylating the NOS enzyme or a cofactor (16, 17, 19). Metabolic adaptation to constant hyperglycemia could also sensitize NOS activity to glucose concentration by altering the level of essential metabolic cofactors. In our experiments, the assay mixture did not include calmodulin, FAD, and reduced tetrahydrobiopterin; therefore, NOS activity was determined by the availability of these cofactors in the tubule fragments or homogenate. Cellular levels of reduced tetrahydrobiopterin may be the most vulnerable to modulation by prolonged hyperglycemia. NO production requires the formation of a complex between reduced tetrahydrobiopterin and NOS (40). Reduction of tetrahydrobiopterin depends on NADPH concentration, which may be altered by increased glucose flux through aldose reductase and pentose phosphate pathways in diabetic tubules. Preincubation of diabetic tubules with high glucose could facilitate the production of the active NOS-reduced tetrahydrobiopterin complex.
The remainder of this discussion deals with the effects of NO
production on proximal tubule function. Endogenous NO production has
been linked to stimulation of Na reabsorption through Na/H exchange in
proximal tubules (4, 14, 38, 39). However, a number of
other studies indicate that NO reduces reabsorption by inhibiting Na/H
exchange, Na-K-ATPase, or mitochondrial oxidative phosphorylation
(1, 25, 26, 29). Inhibition is inversely proportional to
PO2 (21). When
PO2 falls below 40 mmHg, physiologically relevant NO concentrations [~100 nM (24)] begin to
inhibit O2 by proximal tubules, and
inhibition increases as PO2 approaches zero. In
our experiments, the PO2 in tubule suspensions
equilibrated with 95% air was ~140 mmHg. The highest concentration
of cpt10-scavenged NO in diabetic hyperglycemic tubule suspensions was
similar to concentrations found in proximal tubule fluid of nondiabetic
rats in vivo (24). NO in such a low concentration is
unlikely to inhibit
O2 and Na transport
under the conditions of high PO2 that pertained
to our experiments. However, a similar low-NO concentration might be
inhibitory under hypoxic conditions, such as those found in slices of
kidney cortex (1, 7).
To explore the consequences of inhibiting NO production on proximal
tubule function, we measured Na-K-ATPase (86Rb uptake) and
O2. Diabetes increased 86Rb
uptake (Table 4) and
O2 (Fig. 3). Both
the oligomycin-inhibited and oligomycin-insensitive components of
O2 increased (Fig. 4) (6).
Oligomycin-inhibited
O2 reflects
O2 used for oxidative phosphorylation to produce ATP, most
of which is consumed by Na-K-ATPase. The efficiency of O2
use for K transport by Na-K-ATPase can be estimated from the ratio of
86Rb uptake to oligomycin-sensitive
O2. [Actually, the ratio underestimates the efficiency of
O2 to provide ATP for
Na-K-ATPase, because oligomycin inhibits the production of ATP used for
all ATPases, including, for example, H+-ATPase
(9).] Theory predicts that if each O2
generates 6 ATP molecules and each ATP transports 2 K (or
86Rb) equivalents, then the ratio will be 12. For
nondiabetic tubules with high glucose, the ratio was 122:11.3 or 10.8, and for diabetic tubules it was 141:16 or 8.8. Diabetes is associated
with increased serum-free fatty acids and increased fatty acid
metabolism by proximal tubules (18). Fatty acid oxidation
produces only four ATP molecules for each O2 consumed;
therefore, a lower ratio is to be expected in the diabetic tubules.
In vivo hyperglycemia increases proximal tubule reabsorption by
delivering more glucose in glomerular filtrate for reabsorption along
the proximal tubule (36). However, the effects of diabetes on O2 in tubule fragments were not due
to increased Na-glucose cotransport. Inhibiting Na-glucose cotransport
with phloridzin reduced
O2 to a similar
extent in diabetic and nondiabetic tubules incubated with high glucose
(Fig. 3). Five millimolar glucose exceeds the 1.6 mM
Km for glucose transport with Na in proximal tubule S1 and S2 segments (33). Therefore, raising glucose
from 5 to 25 mM had no effect on phloridzin-inhibited
O2.
EIPA-inhibited O2, probably reflecting
ATP consumption secondary to Na entry through Na/H exchange, accounted
for 19% of the oligomycin-inhibited
O2
in nondiabetic tubules and 40% in diabetic tubules (Fig. 4). When
EIPA-inhibited
O2 is subtracted from the
oligomycin-sensitive
O2, the remainder
is almost identical in diabetic and nondiabetic tubules (9.5 and 9.3 nmol · min
1 · mg
1,
respectively). Thus it appears that Na/H exchange (28) was responsible for increased ATP consumption by diabetic tubule fragments. Diabetes might increase the inhibitory effect of EIPA on Na-K-ATPase, but this is less likely than an effect on Na/H exchange, which we know
is increased with untreated streptozotocin diabetes (15).
Inhibiting NO production decreased Na-K-ATPase activity
[86Rb uptake (Table 4) and
O2 (Fig. 5)] in diabetic tubules but
had little effect in nondiabetic tubules. The small response in
nondiabetic tubules is consistent with some stimulation of Na transport
through Na/H exchange as observed in nondiabetic tubules by others
(13). If NO acts by stimulating Na/H exchange (39,
4, 25), then inhibition of NO production should have no
additional effect on
O2 when Na/H
exchange is blocked by EIPA. This supposition is supported by the
finding that L-NAME inhibition and EIPA inhibition are only
5-8% additive (Fig. 5). Increased Na/H exchange is a well-known
consequence of metabolic acidosis (3, 41). The recent
observation that nNOS knockout mice develop metabolic acidosis (39) suggests that NO production could play a role in
long-term adaptation to diabetic metabolic acidosis. Acidosis increases NOS expression in brain cells (27), but the effect of
chronic acidosis on renal NOS expression has not been reported to our knowledge. Increased osmolality associated with hyperglycemia might
also contribute to increased expression of the Na/H exchanger (2). Further experiments are required to establish the
relative roles of acidosis and hyperglycemia in long-term regulation of NOS expression, NO production, and their relationship to Na/H exchange.
Diabetes also increased O2 for purposes
other than ATP production (oligomycin-insensitive
O2, 15.9 ± 0.5 vs. 12.2 ± 0.6 nmol · min
1 · mg
1,
P < 0.001, unpaired Student's
t-test). Similar results were obtained by Korner et
al. (23), who found that streptozotocin diabetes increased
O2 by 32% in rat proximal tubule cells,
with 45% of the increase being insensitive to ouabain. The increase of
oligomycin-insensitive
O2 in diabetic
tubules may be related to increased expression of uncoupling protein 2 and increased levels of free fatty acid, which uncouple
O2 from ATP production (6).
In summary, streptozotocin-induced diabetes increases eNOS and nNOS protein expression, and high glucose concentrations stimulate NO production in diabetic proximal tubules. Increased NO production under hyperglycemic conditions stimulates Na/H exchange. The role of NO in the adaptation of Na/H exchange to the diabetic state deserves further investigation.
![]() |
ACKNOWLEDGEMENTS |
---|
This research was supported by the Kidney Foundation of Canada.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: A. Baines, Dept. of Laboratory Medicine and Pathobiology, Univ. of Toronto, Rm. 408, 100 College St., Toronto, ON, Canada M5G 1L5 (E-mail: andrew.baines{at}utoronto.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
January 29, 2002;10.1152/ajprenal.00330.2001
Received 30 October 2001; accepted in final form 24 January 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Adler, S,
Huang H,
Loke KE,
Xu X,
Tada H,
Laumas A,
and
Hintze TH.
Endothelial nitric oxide synthase plays an essential role in regulation of renal oxygen consumption by NO.
Am J Physiol Renal Physiol
280:
F838-F843,
2001
2.
Ambuhl, P,
Amemiya M,
Preisig PA,
Moe OW,
and
Alpern RJ.
Chronic hyperosmolality increases NHE3 activity in OKP cells.
J Clin Invest
101:
170-177,
1998
3.
Ambuhl, PM,
Amemiya M,
Danczkay M,
Lotscher M,
Kaissling B,
Moe OW,
Preisig PA,
and
Alpern RJ.
Chronic metabolic acidosis increases NHE3 protein abundance in rat kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
271:
F917-F925,
1996
4.
Amorena, C,
and
Castro AF.
Control of proximal tubule acidification by the endothelium of the peritubular capillaries.
Am J Physiol Regul Integr Comp Physiol
272:
R691-R694,
1997
5.
Baines, AD,
Drangova R,
and
Ho P.
Role of diacylglycerol in adrenergic-stimulated 86Rb uptake by proximal tubules.
Am J Physiol Renal Fluid Electrolyte Physiol
258:
F1133-F1138,
1990
6.
Baines, AD,
Fayoda T,
Lieu CC,
and
Ho P.
Streptozotocin-diabetes increases, Na-proton exchange, uncoupled oxygen consumption and UCP2 in rat proximal tubules.
J Am Soc Nephrol
11:
635A,
2000.
7.
Balaban, RS,
Soltoff SP,
Storey JM,
and
Mandel LJ.
Improved renal cortical tubule suspension: spectrophotometric study of O2 delivery.
Am J Physiol Renal Fluid Electrolyte Physiol
238:
F50-F59,
1980
8.
Brodsky, SV,
Morrishow AM,
Dharia N,
Gross SS,
and
Goligorsky MS.
Glucose scavenging of nitric oxide.
Am J Physiol Renal Physiol
280:
F480-F486,
2001
9.
Chambrey, R,
Paillard M,
and
Podevin RA.
Enzymatic and functional evidence for adaptation of the vacuolar H(+)-ATPase in proximal tubule apical membranes from rats with chronic metabolic acidosis.
J Biol Chem
269:
3243-3250,
1994
10.
Chen, ZP,
Mitchelhill KI,
Michell BJ,
Stapleton D,
Rodriguez-Crespo I,
Witters LA,
Power DA,
Ortiz De Montellano PR,
and
Kemp BE.
AMP-activated protein kinase phosphorylation of endothelial NO synthase.
FEBS Lett
443:
285-289,
1999[ISI][Medline].
11.
Chin, SY,
Pandey KN,
Shi SJ,
Kobori H,
Moreno C,
and
Navar LG.
Increased activity and expression of Ca2+-dependent NOS in renal cortex of ANG II-infused hypertensive rats.
Am J Physiol Renal Physiol
277:
F797-F804,
1999
12.
Cole, JA,
Walker REW,
Yordy MR,
and
Walker RE.
Hyperglycemia-induced changes in Na+/myo-inositol transport, Na+-K+-ATPase, and protein kinase C activity in proximal tubule cells.
Diabetes
44:
446-452,
1995[Abstract].
13.
De Nicola, L,
Blantz RC,
and
Gabbai FB.
Nitric oxide and angiotensin. II. Glomerular and tubular interaction in the rata.
J Clin Invest
89:
1248-1256,
1992[ISI][Medline].
14.
Diaz-Sylvester, P,
Mac Laughlin M,
and
Amorena C.
Peritubular fluid viscosity modulates H+ flux in proximal tubules through NO release.
Am J Physiol Renal Physiol
280:
F239-F243,
2001
15.
El-Seifi, S,
Freiberg JM,
Kinsella J,
Cheng L,
and
Sacktor B.
Na+-H+ exchange and Na+-dependent transport systems in streptozotocin diabetic rat kidneys.
Am J Physiol Regul Integr Comp Physiol
252:
R40-R47,
1987
16.
Ferrer, M,
Marin J,
and
Balfagon G.
Diabetes alters neuronal nitric oxide release from rat mesenteric arteries. Role of protein kinase C.
Life Sci
66:
337-345,
2000[ISI][Medline].
17.
Fulton, D,
Gratton JP,
McCabe TJ,
Fontana J,
Fujio Y,
Walsh K,
Franke TF,
Papapetropoulos A,
and
Sessa WC.
Regulation of endothelium-derived nitric oxide production by the protein kinase Akt.
Nature
399:
597-601,
1999[ISI][Medline].
18.
Guder, WG,
Schmolke M,
and
Wirthensohn G.
Carbohydrate and lipid metabolism of the renal tubule in diabetes mellitus.
Eur J Clin Chem Clin Biochem
30:
669-674,
1992[ISI][Medline].
19.
Ishii, N,
Patel KP,
Lane PH,
Taylor T,
Bian K,
Murad F,
Pollock JS,
and
Carmines PK.
Nitric oxide synthesis and oxidative stress in the renal cortex of rats with diabetes mellitus.
J Am Soc Nephrol
12:
1630-1639,
2001
20.
Keynan, S,
Hirshberg B,
Levin-Iaina N,
Wexler ID,
Dahan R,
Reinhartz E,
Ovadia H,
Wollman Y,
Chernihovskey T,
Iaina A,
and
Raz I.
Renal nitric oxide production during the early phase of experimental diabetes mellitus.
Kidney Int
58:
740-747,
2000[ISI][Medline].
21.
Koivosto, A,
Pittner J,
Froelich M,
and
Persson AEG
Oxygen-dependent inhibition of respiration in isolated renal tubules by nitric oxide.
Kidney Int
55:
2368-2375,
1999[ISI][Medline].
22.
Komers, R,
Lindsley JN,
Oyama TT,
Allison KM,
and
Anderson S.
Role of neuronal nitric oxide synthase (NOS1) in the pathogenesis of renal hemodynamic changes in diabetes.
Am J Physiol Renal Physiol
279:
F573-F583,
2000
23.
Korner, A,
Eklof AC,
Celsi G,
and
Aperia A.
Increased renal metabolism in diabetes. Mechanism and functional implications.
Diabetes
43:
629-633,
1994[Abstract].
24.
Levine, DZ,
Iacovitti M,
Burns KD,
and
Zhang X.
Real-time profiling of kidney tubular fluid nitric oxide concentrations in vivo.
Am J Physiol Renal Physiol
281:
F189-F194,
2001
25.
Liang, M,
and
Knox FG.
Production and functional roles of nitric oxide in the proximal tubule.
Am J Physiol Regul Integr Comp Physiol
278:
R1117-R1124,
2000
26.
Linas, SL,
and
Repine JE.
Endothelial cells regulate proximal tubule epithelial cell sodium transport.
Kidney Int
55:
1251-1258,
1999[ISI][Medline].
27.
Najarian, T,
Marrache AM,
Dumont I,
Hardy P,
Beauchamp MH,
Hou X,
Peri K,
Gobeil F,
Varma DR,
and
Chemtob S.
Prolonged hypercapnia-evoked cerebral hyperemia via K+ channel- and prostaglandin E-2-dependent endothelial nitric oxide synthase induction.
Circ Res
87:
1149-1156,
2000
28.
Nascimento-Gomes, G,
Zaladek GF,
and
Mello-Aires M.
Alterations of the renal handling of H+ in diabetic rats.
Kidney Blood Press Res
20:
251-257,
1997[ISI][Medline].
29.
Roczniak, A,
and
Burns KD.
Nitric oxide stimulates guanylate cyclase and regulates sodium transport in rabbit proximal tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F106-F115,
1996
30.
Schmidt, HHHW,
and
Kelm M.
Determination of nitrite and nitrate by the Griess reaction.
In: Methods in Nitric Oxide Research, edited by Feelisch M,
and Stamler JS.. New York: Wiley, 1996, p. 491-497.
31.
Schwartz, D,
Schwartz IF,
and
Blantz RC.
An analysis of renal nitric oxide contribution to hyperfiltration in diabetic rats.
J Lab Clin Med
137:
107-114,
2001[ISI][Medline].
32.
Shin, SJ,
Lai FJ,
Wen JD,
Hsiao PJ,
Hsieh MC,
Tzeng TF,
Chen HC,
Guh JY,
and
Tsai JH.
Neuronal and endothelial nitric oxide synthase expression in outer medulla of streptozotocin-induced diabetic rat kidney.
Diabetologia
43:
649-659,
2000[ISI][Medline].
33.
Silverman, M,
and
Turner RJ.
Glucose transport in the renal proximal tubule.
In: Handbook of Physiology. Renal Physiology. Bethesda, MD: Am Physiol Soc, 1992, sect. 8, vol. II, chapt. 43, p. 2017-2038.
34.
Sugimoto, H,
Shikata K,
Matsuda M,
Kushiro M,
Hayashi Y,
Hiragushi K,
Wada J,
and
Makino H.
Increased expression of endothelial cell nitric oxide synthase (ecNOS) in afferent and glomerular endothelial cells is involved in glomerular hyperfiltration of diabetic nephropathy.
Diabetologia
41:
1426-1434,
1998[ISI][Medline].
35.
Thuraisingham, RC,
Nott CA,
Dodd SM,
and
Yaqoob MM.
Increased nitrotyrosine staining in kidneys from patients with diabetic nephropathy.
Kidney Int
57:
1968-1972,
2000[ISI][Medline].
36.
Vallon, V,
Richter K,
Blantz RC,
Thomson S,
and
Osswald H.
Glomerular hyperfiltration in experimental diabetes mellitus: potential role of tubular reabsorption.
J Am Soc Nephrol
10:
2569-2576,
1999
37.
Veelken, R,
Hilgers KF,
Hartner A,
Haas A,
Bohmer KP,
and
Sterzel RB.
Nitric oxide synthase isoforms and glomerular hyperfiltration in early diabetic nephropathy.
J Am Soc Nephrol
11:
71-79,
2000
38.
Wang, T.
Nitric oxide regulates HCO
39.
Wang, T,
Inglis FM,
and
Kalb RG.
Defective fluid and HCO
40.
Witteveen, CFB,
Giovanelli J,
and
Kaufman S.
Reactivity of tetrahydrobiopterin bound to nitric-oxide synthase.
J Biol Chem
274:
29755-29762,
1999
41.
Wu, MS,
Biemesderfer D,
Giebisch G,
and
Aronson PS.
Role of NHE3 in mediating renal brush border Na+-H+ exchangeadaptation to metabolic acidosis.
J Biol Chem
271:
32749-32752,
1996
42.
Yamauchi, M,
Omote K,
and
Ninomiya T.
Direct evidence for the role of nitric oxide on the glutamate-induced neuronal death in cultured cortical neurons.
Brain Res
780:
253-259,
1998[ISI][Medline].
43.
Yaqoob, M,
Edelstein CL,
Wieder ED,
Alkhunaizi AM,
Gengaro PE,
Nemenoff RA,
and
Schrier RW.
Nitric oxide kinetics during hypoxia in proximal tubules: effects of acidosis and glycine.
Kidney Int
49:
1314-1319,
1996[ISI][Medline].