Glucose stimulates O2 consumption, NOS, and Na/H exchange in diabetic rat proximal tubules

Andrew Baines and Patrick Ho

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada M5G 1L5


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 · min-1 · mg-1) and oligomycin-sensitive O2 consumption (QO2; 16.0 ± 1.7 vs. 11.3 ± 0.7 nmol · min-1 · mg-1). Ethylisopropyl amiloride-inhibitable QO2 (6.5 ± 0.6 vs. 2.4 ± 0.3 nmol · min-1 · mg-1) accounted for increased oligomycin-sensitive QO2 in diabetic tubules. NG-monomethyl-L-arginine methyl ester (L-NAME) inhibited most of the increase in 86Rb uptake and QO2 in diabetic tubules. L-NAME had little effect on nondiabetic tubules. Inhibition of QO2 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 QO2 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (QO2) in isolated tubule fragments.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 QO2, 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 QO2. NG-monomethyl-L-arginine methyl ester (L-NAME) and NG-monomethyl-D-arginine methyl ester (10 mM) were added 30 min before measurement of QO2. 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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).

                              
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Table 1.   Nondiabetic compared with diabetic rats

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).


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Fig. 1.   Western blot analysis stained for endothelial nitric oxide synthase (NOS) and neuronal NOS in homogenates of proximal tubules from 4 nondiabetic and 4 diabetic rats.

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.


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Fig. 2.   NOS activity in proximal tubule homogenates. A: calcium-sensitive citrulline production from [14C]arginine. Proximal tubule fragments from diabetic and nondiabetic rats were incubated at 37°C for 30 min with either high glucose (25 mM) or low glucose (5 mM glucose + 20 mM mannitol) before homogenization. B: calcium-insensitive citrulline production. Ca-sensitive citrulline production was significantly increased in the diabetic tubules incubated with high glucose. Values are means ± SE. P = 0.02, ANOVA.


                              
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Table 2.   Citrulline produced by proximal 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.

                              
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Table 3.   Nitric oxide concentration in incubation medium of diabetic and 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.

                              
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Table 4.   86Rb uptake by proximal tubule fragments

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 QO2 with nondiabetic tubules (24.7 ± 1.1 vs. 24.9 ± 0.9 nmol · min-1 · mg-1), but high glucose modestly increased QO2 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 QO2 (16.0 ± 1.7 vs. 11.3 ± 0.7 nmol · min-1 · mg-1, P = 0.017) and EIPA-sensitive QO2 (6.5 ± 0.6 vs. 2.4 ± 0.3 nmol · min-1 · mg-1, P < 0.001) (Fig. 3). Increased oligomycin-sensitive QO2 can be entirely attributed to the increase in EIPA-inhibited QO2 (Figs. 3 and 4). The phloridzin-sensitive component of QO2 was not increased in diabetic tubules (2.2 ± 0.6 vs. 1.8 ± 0.7 nmol · min-1 · mg-1) (Fig. 3).


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Fig. 3.   Effect of inhibitors on oxygen consumption (QO2) in diabetic and nondiabetic proximal tubules incubated with 25 mM glucose. A: inhibition by 20 µM oligomycin. B: inhibition by 100 µM phloridzin or 10 µM ethylisopropyl amiloride (EIPA). Values are means ± SE. *P < 0.05, ***P < 0.001, unpaired Student's t-test.



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Fig. 4.   Components of QO2 in nondiabetic and diabetic tubules incubated with high glucose (25 mM). Inhibitors were 20 µM oligomycin, 100 µM phloridzin, and 10 µM EIPA.

L-NAME-inhibited QO2 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 QO2 appreciably (+0.7 and -0.9 ± 0.6 nmol · min-1 · mg-1 in nondiabetic and diabetic tubules, respectively).


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Fig. 5.   Inhibition of proximal tubule QO2 by 10 µM EIPA, 10 mM NG-monomethyl-L-arginine methyl ester (L-NAME), or EIPA and L-NAME together. Tubules were incubated with high glucose. Repeated measures were made with 3 nondiabetic and 6 diabetic tubule preparations. Repeated-measures ANOVA showed significant inhibitory effects of L-NAME and EIPA and an interaction between L-NAME and EIPA. Values are means ± SE. Inhibition was significantly greater in tubules from diabetic rats (P values for a comparison of diabetic with nondiabetic tubules are shown above error bars, Student's unpaired t-test).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 QO2 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 QO2 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 QO2 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 QO2. Diabetes increased 86Rb uptake (Table 4) and QO2 (Fig. 3). Both the oligomycin-inhibited and oligomycin-insensitive components of QO2 increased (Fig. 4) (6). Oligomycin-inhibited QO2 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 QO2. [Actually, the ratio underestimates the efficiency of QO2 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 QO2 in tubule fragments were not due to increased Na-glucose cotransport. Inhibiting Na-glucose cotransport with phloridzin reduced QO2 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 QO2.

EIPA-inhibited QO2, probably reflecting ATP consumption secondary to Na entry through Na/H exchange, accounted for 19% of the oligomycin-inhibited QO2 in nondiabetic tubules and 40% in diabetic tubules (Fig. 4). When EIPA-inhibited QO2 is subtracted from the oligomycin-sensitive QO2, 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 QO2 (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 QO2 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 QO2 for purposes other than ATP production (oligomycin-insensitive QO2, 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 QO2 by 32% in rat proximal tubule cells, with 45% of the increase being insensitive to ouabain. The increase of oligomycin-insensitive QO2 in diabetic tubules may be related to increased expression of uncoupling protein 2 and increased levels of free fatty acid, which uncouple QO2 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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