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Am J Physiol Renal Physiol 292: F804-F811, 2007. First published October 10, 2006; doi:10.1152/ajprenal.00258.2006
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Regulation of renal glucose transporters during severe inflammation

Christoph Schmidt,1 Klaus Höcherl,2 and Michael Bucher1

Departments of 1Anesthesiology and 2Pharmacology, Regensburg University, Regensburg, Germany

Submitted 10 July 2006 ; accepted in final form 5 October 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Severe sepsis is accompanied by acute renal failure (ARF) with renal tubular dysfunction and glucosuria. In this study, we aimed to determine the regulation of renal tubular glucose transporters during severe experimental inflammation. Male C57BL/6J mice were injected with LPS or proinflammatory cytokines, and renal perfusion, glomerular filtration rate (GFR), fractional glucose excretion, and expression of tubular glucose transporters were determined. We found a decreased plasma glucose concentration with impaired renal tissue perfusion and GFR and increased fractional glucose excretion associated with decreased expression of SGLT2, SGLT3, and GLUT2 after LPS injection. Similar alterations were observed after application of TNF-{alpha}, IL-1beta, IL-6, or IFN-{gamma}. To clarify the role of proinflammatory cytokines, we performed LPS injections in knockout mice with deficiencies for TNF-{alpha}, IL-1 receptor type 1, IFN-{gamma}, or IL-6 as well as LPS injections in glucocorticoid-treated wild-type mice. LPS-induced alterations of glucose transporters also were present in single-cytokine knockout mice. In contrast, glucocorticoid treatment clearly attenuated LPS-induced changes in renal glucose transporter expression and improved GFR and fractional glucose excretion. LPS-induced decrease of renal perfusion was not improved by glucocorticoids, indicating a minor role of ischemia in the development of septic renal dysfunction. Our results demonstrate modifications of tubular glucose transporters during severe inflammation that are probably mediated by proinflammatory cytokines and account for the development of ARF with increased fractional glucose excretion. In addition, our findings provide an explanation why single anti-cytokine strategies fail in the therapy of septic patients and contribute to an understanding of the beneficial effects of glucocorticoids on septic renal dysfunction.

sepsis; acute renal failure; tubular function; lipopolysaccharide; cytokines; glucocorticoids

ACUTE RENAL FAILURE (ARF) is defined as the abrupt decline in glomerular filtration rate (GFR) and tubular function affecting 5–7% of hospital patients (32, 46). Sepsis and septic shock are important risk factors for ARF and remain the most important trigger for ARF in intensive care units (7, 28, 41). Incidence of ARF is 20% in patients with severe sepsis and even 50% in patients with septic shock (36, 41), and the mortality rate of sepsis-related ARF is significantly high at 75% (29). Understanding the pathogenesis of sepsis-related ARF is of critical importance. Several in vivo and in vitro studies have suggested that the reduction of GFR in sepsis is secondary to altered glomerular hemodynamics (12, 30). In contrast, the pathophysiology of sepsis-associated renal tubular dysfunction with failure in urine concentration and increased fractional excretion of glucose with glucosuria has been poorly explained.

Adequate renal tubular function with the ability of potent urinary concentration and ~99.9% glucose reabsorption depends on the functional expression of several tubular transporters such as SGLT1, SGLT2, SGLT3, GLUT1, GLUT2, and Na+-K+-ATPase (27). Glucose is freely filtered by the glomerulus, and reabsorption occurs predominantly on the brush-border membrane of the convoluted segment of the proximal tubule by specific transporter proteins. More distal segments reabsorb almost all of the remainder, resulting in a fractional glucose excretion of <0.1% (44, 45). The sodium-dependent cotransporters (SGLTs), consisting of three subtypes, couple the uphill reabsorption of glucose from the renal tubule lumen with the downhill transport of sodium (19, 20). In the early part of the proximal tubule (S1 segment), a high-capacity/low-affinity transporter called SGLT2 mediates apical glucose uptake with a Na+-glucose stoichiometry of 1:1. In the later part of the proximal tubule (S3 segment), a high-affinity/low-capacity cotransporter called SGLT1 is responsible for apical glucose uptake. Because this transporter has a Na+-glucose stoichiometry of 2:1, it can generate a far larger glucose gradient across the apical membrane (45). In addition to SGLT1 and SGLT2, another closely related low-affinity sodium-glucose transporter, SGLT3, originally named SAAT1, has been identified in the proximal tubule, coupling two Na+ to one glucose molecule (15, 44, 45). Once inside the cell, glucose exits across the basolateral membrane via the insulin-dependent GLUT transporters, which are Na+ independent and move glucose by facilitated diffusion. Like the apical SGLTs, the basolateral GLUTs differ between early and late proximal tubule segments, with GLUT2 in the early and GLUT1 in the late proximal tubule (4). The electrochemical potential gradient for powering Na+-glucose symporter is restored when transported sodium is returned to the blood stream via the basolateral Na+-K+-ATPase along the whole nephron (44, 45).

Reports of altered glucose transporter expression in extrarenal tissue during endotoxemia directed our interest onto the regulation of renal tubular glucose transporters during experimental sepsis (51, 52). We hypothesized that endotoxemia alters renal expression of glucose transporters. On the basis of our previous findings that proinflammatory cytokines downregulate several vasoconstrictive receptors in renal tissue, we hypothesized that cytokines affect the expression of tubular glucose transporters (9–11). To test our hypotheses, we performed experiments with 1) lipopolysaccharide (LPS)-injected mice as a model for severe experimental gram-negative sepsis; 2) mice injected with the cytokines TNF-{alpha}, IL-1beta, IFN-{gamma}, or IL-6; 3) LPS-injected knockout mice with a deficiency for TNF-{alpha}, IL-1 receptor type 1, IFN-{gamma}, or IL-6; and finally, 4) LPS-injected mice without or with glucocorticoid pretreatment, which reduces LPS-induced cytokine production (6, 22, 23). We determined hemodynamic and renal parameters, especially GFR, fractional glucose excretion, tubular glucose reabsorption, renal tissue perfusion, and the expression of tubular glucose transporters.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal preparation. All animal experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mice were purchased from The Jackson Laboratory. C57BL/6J mice received NaCl (control) or LPS (Escherichia coli, serotype 0111:B4, 10 mg/kg; Sigma) intraperitoneally and were killed 6, 12, and 24 h (n = 6 per group) following LPS injection and determination of hemodynamic and renal parameters. Knockout mice for TNF-{alpha} (B6;129S6-Tnftm1Gk1) (8, 35), IL-1 receptor type 1 (B6.129S7-IL1r1tm1Imx) (40), IFN-{gamma} (B6.129S7-Ifngtm1Ts) (3), or IL-6 (B6.129S7-IL-6tm1Kopf) (25) and their wild-type strains, B6129SF2/J and C57BL/6J, received NaCl or LPS and were killed 12 h after injection (n = 6 per group), the point of time with the strongest effect. C57BL/6J mice were additionally treated with NaCl or TNF-{alpha}, IL-1beta, IFN-{gamma}, or IL-6 (1 µg/g; PeproTech) and killed 12 h after injection (n = 6 per group). In addition, C57BL/6J mice (n = 6 per group) received dexamethasone (10 mg/kg ip) alone or as a supplement 2 h before LPS injection. The doses of LPS, cytokines, and dexamethasone were chosen from data taken from the literature (11, 33, 48). Plasma was collected, and kidneys were extirpated and snap-frozen at –80°C until further processing.

Measurement of hemodynamic and renal parameters. Animals were anesthetized with Sevoflurane using a Trajan 808 (Dräger) 6, 12, or 24 h after injection. The right femoral artery was cannulated for continuous monitoring of mean arterial blood pressure (MAP) and heart rate (Siemens SC 9000). The left femoral vein was cannulated for maintenance infusion with 0.9% NaCl, and the bladder for collecting urine. FITC-inulin (0.5%; Sigma) was added to the infusion for determination of GFR. Plasma and urine FITC-inulin concentration were measured after an equilibration period of 30 min every 30 min for 2 h and averaged for calculation of inulin clearance. FITC-inulin in plasma and urine samples was measured using a spectrofluorometer (Shimadzu). Needle laser Doppler probes (type NS, diameter 0.58 mm; Transonic) were adjusted to renal cortex and medulla for measuring renal tissue perfusion. Both probes were connected to laser-Doppler flowmeters (Transonic BLF21), and renal tissue perfusion was measured 6, 12, and 24 h after sepsis induction with or without glucocorticoid treatment. GFR, fractional glucose excretion, and tubular glucose reabsorption were calculated using appropriate formulas.

mRNA extraction and real-time PCR analysis of tubular glucose transporters. Total kidney RNA was extracted and reverse transcribed, and real-time PCR was carried out using the LightCycler system (Roche Diagnostics) as described previously in brief (11). Each primer set (Table 1) was checked using a BLAST search to ascertain that the sequences were unique for each mouse transporter. beta-Actin was used as reference gene.


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  		Table 1. Primer set for glucose transporter analysis by real-time PCR

 
Protein preparation. Kidneys were homogenized in 10 volumes of ice-cold homogenization buffer in the presence of protease inhibitors, followed by centrifugation at 500 g for 15 min at 4°C. The resultant supernatant was centrifuged at 20,000 g for 30 min at 4°C and used for determination of tissue cytokine concentration. The resultant pellet was reconstituted in blotting buffer. After recentrifugation, the resultant pellet (membrane fraction) was reconstituted in blotting buffer and used for the determination of glucose transporter protein by Western blotting. Protein concentration was measured using the method of Lowry.

Concentrations of cytokines, insulin, and glucose. Tissue concentrations of TNF-{alpha}, IL-1beta, IFN-{gamma}, and IL-6 were determined using ELISA kits (R&D Systems) and set into proportion to total protein. Plasma concentration of insulin was assayed using ELISA (DRG Instruments); plasma and urinary glucose concentrations were assayed using the QuantiChrom glucose assay kit (BioAssay Systems).

Western blot analysis. Proteins (40 µg) were electrophoretically separated on a 10% polyacrylamide gel and transferred to nitrocellulose membrane, which was blocked overnight in 5% nonfat dry milk diluted in Tris-buffered saline with 0.2% Tween 20 and then incubated for 1 h at room temperature with rabbit polyclonal antibodies against SGLT1 (diluted 1:2,000; Chemicon), GLUT1 (diluted 1:1,000; Chemicon), GLUT2 (diluted 1:1,500; Chemicon), and Na+-K+-ATPase {alpha}1 (diluted 1:1,000; Upstate). After washing, the membrane was incubated for 90 min at room temperature with horseradish peroxidase-conjugated secondary antibodies (diluted 1:2,000; Santa Cruz Biotechnology) and subjected to a chemiluminescence detection system. The anti-SGLT1 antibody recognized a band of 75 kDa; anti-GLUT1 and anti-GLUT2 antibody recognized a band of 50 and 60 kDa. The anti-Na+-K+-ATPase antibody detected a band of 110 kDa. Quantitative assessment of bands was performed densitometrically.

Statistical analysis. Data were analyzed using ANOVA with multiple comparisons followed by two-tailed t-tests with Bonferroni's adjustment. P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Hemodynamic parameters. Hemodynamic parameters are presented in Table 2. Animals became lethargic and showed piloerection starting 2 h after LPS injection. MAP was 78.1 ± 1.1 mmHg in the control group and decreased at 6, 12, and 24 h after LPS injection to 81, 66, and 63% of control values, respectively. Heart rate of septic animals was significantly higher, with values between 520 and 619 beats/min at 6 and 24 h after LPS injection compared with the control group (474 beats/min). Plasma insulin values showed no differences between control and septic groups. However, animals presented significant lower blood glucose levels at 12 h (68.2 ± 1.6 mg/dl) and 24 h (63.2 ± 1.9 mg/dl) after LPS treatment than control level (113.1 ± 1.8 mg/dl). IL-1beta-injection also led to tachycardia and arterial hypotension with lowered blood glucose values at 12 h after injection.


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  		Table 2. Effect of LPS alone or with dexamethasone and effect of IL-1beta on hemodynamic and renal parameters

 
Injection of dexamethasone alone did not influence hemodynamic and blood glucose levels in wild-type mice at 6, 12, or 24 h after injection compared with control levels (data not shown). Supplementary treatment of LPS-injected animals with dexamethasone attenuated LPS-induced arterial hypotension compared with LPS treatment alone after 12 and 24 h. Blood glucose remained on control levels at 6, 12, and 24 h after treatment with LPS plus dexamethasone and differed significantly from those of LPS injection alone.

Renal parameters. Renal parameters are also presented in Table 2. GFR was lower in LPS-injected mice, and urine output decreased to 79, 55, and 60% of control animals at 6, 12, and 24 h after injection, respectively. Fractional glucose excretion increased ~15-fold in animals treated with LPS for 12 or 24 h compared with control animals. Tubular glucose reabsorption in the LPS-injected groups was considerably lower than in the control group, decreasing to 51, 14, and 11% of control levels at 6, 12, and 24 h after sepsis induction, respectively, with consequently increased urinary glucose excretion. Tissue perfusion in the renal medulla and especially in the renal cortex was decreased at 6, 12, and 24 h after sepsis induction (Fig. 1). Twelve hours after injection of IL-1beta, renal function was restricted compared with LPS-injected animals.


Figure 1
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  		Fig. 1. Effect of LPS (10 mg/kg), dexamethasone (10 mg/kg), and the combination of both on renal cortical (A) and renal medullary tissue perfusion (B) at 6, 12, and 24 h after intraperitoneal injection. Dexa, dexamethasone. Values are relative to data at control level and are given as percentages of vehicle control, expressed as means ± SE of 6 animals per group. *P < 0.05 vs. vehicle control.

 
Injection of dexamethasone alone showed no relevant changes in functional renal parameters at 6, 12, and 24 h after injection compared with controls (data not shown). Supplementary treatment of LPS-injected animals with dexamethasone attenuated renal injury as indicated by an increase in GFR and tubular glucose reabsorption and a decrease in fractional glucose excretion compared with LPS injection alone. Indeed, renal tissue perfusion in both the medulla and cortex increased smoothly, but not significantly, after supplementary glucocorticoid administration compared with LPS treatment alone (Fig. 1).

Effect of LPS on renal glucose transporter expression. Treatment with LPS for 6, 12, or 24 h decreased SGLT2, SGLT3, GLUT2, and Na+-K+-ATPase expression in the kidney. SGLT2 mRNA was downregulated to 67, 23, and 33% of control levels at 6, 12, and 24 h after LPS injection, respectively. GLUT2 mRNA levels showed a marked fall to 40, 13, and 39% of control levels at 6, 12, and 24 h after LPS administration, paralleled by a significant decrease of Na+-K+-ATPase mRNA to 40, 29, and 42% of control levels at 6, 12 and 24 h after LPS injection, respectively. SGLT3 mRNA values declined to 61% of control at 12 h after sepsis induction (Fig. 2). SGLT1 and GLUT1 mRNA concomitantly increased to 190 and 150% at 6 h, to 166 and 197% at 12 h, and to 140 and 193% at 24 h after LPS treatment.


Figure 2
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  		Fig. 2. Effect of LPS (10 mg/kg), dexamethasone (10 mg/kg), and the combination of both on the tubular transporters SGLT1 (A), SGLT2 (B), SGLT3 (C), GLUT1 (D), GLUT2 (E), and Na+-K+-ATPase {alpha}1 mRNA (F) in the kidney at 6, 12 and 24 h after intraperitoneal injection. Values are relative to signals obtained for beta-actin mRNA and are given as percentages of vehicle control, expressed as means ± SE of 6 animals per group. *P < 0.05 vs. vehicle control.

 
Protein expression of SGLTs, GLUTs, and Na+-K+-ATPase was determined by Western blot analysis (Fig. 3A). The lack of available antibodies limited the analysis of SGLT2 and SGLT3 protein expression in our study and previous investigations (37, 45). GLUT2 protein fell to 60, 31, and 21% of control levels at 6, 12, and 24 h after LPS injection, respectively. Western blot showed a decline of Na+-K+-ATPase protein to 54% of control level at 12 h and 59% of control at 24 h after LPS injection. The protein of SGLT1 increased to 155, 163, and 149% of control levels at 6, 12, and 24 h after LPS administration, respectively, and GLUT1 protein increased to 110, 148, and 174% of control levels at 6, 12, and 24 h after sepsis induction (Fig. 3, B–E).


Figure 3
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  		Fig. 3. Effect of LPS alone (10 mg/kg; A) as well as dexamethasone (10 mg/kg) alone or in combination with LPS (B–E) on SGLT1, GLUT1, GLUT2, and Na+-K+-ATPase {alpha}1 protein, respectively, in the kidney at 6, 12, and 24 h after intraperitoneal injection. Co, control. Representative immunoblots of SGLT1, GLUT1, GLUT2, and Na+-K+-ATPase {alpha}1 protein are shown. Values are relative to signals obtained for beta-actin protein and are given as percentages of vehicle control, expressed as means ± SE of 6 animals per group. *P < 0.05 vs. vehicle control. #P < 0.05 vs. LPS treatment.

 
Effect of cytokines on renal glucose transporter expression. For further characterization of the mechanisms along which tubular glucose transporters could be regulated during endotoxemia, renal gene expression in various cytokine-injected mice was examined. mRNAs of SGLT2, SGLT3, GLUT2, and Na+-K+-ATPase were strongly depressed after injection of TNF-{alpha}, IL-1beta, or IFN-{gamma} (apart from SGLT3). IL-6 only downregulated SGLT2 and GLUT2 mRNA. Inversely, SGLT1 and GLUT1 mRNA increased markedly after TNF-{alpha}, IL-1beta, or IFN-{gamma} injection but not after IL-6 treatment (Fig. 4).


Figure 4
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  		Fig. 4. Effect of LPS (10 mg/kg) and proinflammatory cytokines on SGLT1 (A), SGLT2 (B), SGLT3 (C), GLUT1 (D), GLUT2 (E), and Na+-K+-ATPase {alpha}1 mRNA (F) in the kidney at 12 h after intraperitoneal injection. Values are relative to signals obtained for beta-actin mRNA and are given as percentages of vehicle control, expressed as means ± SE of 6 animals per group. *P < 0.05 vs. vehicle control.

 
Effect of LPS on glucose transporter expression in cytokine knockout mice. To investigate whether LPS-induced alteration of renal glucose transporter expression could be inhibited by omission of single cytokine effects, we measured mRNA levels of glucose transporters in LPS-treated single cytokine knockout mice with a deficiency for TNF-{alpha}, IL-1 receptor type 1, IFN-{gamma}, or IL-6 and compared the effect to the impact on their wild-type control strain. NaCl-injected cytokine knockout and wild-type mice revealed comparable glucose transporter mRNA levels (data not shown). At 12 h after LPS injection, mRNA levels of SGLT2, SGLT3, GLUT2, and Na+-K+-ATPase were depressed (~21, 68, 14, and 47%, respectively, of wild-type and cytokine knockout control levels). mRNA levels of SGLT1 and GLUT1 (both ~166% of wild-type and cytokine knockout control levels) in all knockout species ascended to assimilable values as in LPS-treated wild-type mice. Hence, the regulative effect of LPS on all measured transporters could not be diminished in any knockout strain (Fig. 5).


Figure 5
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  		Fig. 5. Effect of LPS (10 mg/kg) on SGLT1 (A), SGLT2 (B), SGLT3 (C), GLUT1 (D), GLUT2 (E), and Na+-K+-ATPase {alpha}1 mRNA (F) in cytokine knockout mice at 12 h after intraperitoneal injection. IL-1r1, IL-1 receptor type 1. Values are relative to signals obtained for beta-actin mRNA and are given as percentages of vehicle control, expressed as means ± SE of 6 animals per group. *P < 0.05 vs. vehicle control.

 
Effect of dexamethasone on glucose transporter expression in LPS-treated mice. Since the absence of a single cytokine effect had no effect on LPS-induced alteration of glucose transporters, we performed experiments with dexamethasone-treated mice as a model of restricted release of multiple cytokines following LPS injection. As shown in Table 3, treatment of animals with dexamethasone markedly attenuated tissue cytokine concentration at 6, 12, and 24 h after LPS injection.


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  		Table 3. Effect of LPS alone or with dexamethasone on tissue cytokine concentrations

 
Administration of dexamethasone alone showed no differences in the expression of renal glucose transporters compared with control levels (Fig. 2, A–F). In animals treated for 6 h with LPS plus dexamethasone, gene expression of SGLT2, GLUT2, and Na+-K+-ATPase substantially increased related to treatment with LPS alone. At 12 and 24 h after injection, supplementary glucocorticoid treatment abolished the downregulatory effect of LPS concerning SGLT2, SGLT3, GLUT2, and Na+-K+-ATPase gene expression (Fig. 2, B, C, E, and F). On the other hand, administration of dexamethasone caused a significant attenuation of the LPS-induced upregulation of SGLT1 mRNA (Fig. 2A) but did not affect LPS-modified GLUT1 gene expression (Fig. 2D). In summary, supplementary injection of dexamethasone attenuated LPS-induced modification of renal glucose transporters and tended to result in abolishing the effect of LPS on glucose transporters by 24 h after LPS injection apart from GLUT1. Also, on a protein level, the positive effect of additional dexamethasone treatment on GLUT2, SGLT1, and Na+-K+-ATPase membrane protein was markedly distinguishable compared with that of LPS injection alone (Fig. 3, B, D, and E). Quantitative assessment of GLUT1 band densities showed no significant differences between LPS alone and supplementary glucocorticoid treatment (Fig. 3C).


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we aimed to characterize the regulation of renal tubular glucose transporters during severe experimental inflammation. A bolus of 10 mg/kg LPS in our in vivo model caused a time-dependent, pronounced arterial hypotension associated with tachycardia and an ARF with reduced GFR and tubular function, indicating the validity of our model of severe experimental endotoxemia. Noteworthy, glucosuria and lowered plasma glucose levels occurred without hyperinsulinemia in septic animals, indicating an insulin-independent renal glucose deprivation. This finding is in agreement with other studies reporting hypoglycemia in combination with nonaltered insulin levels in endotoxemic animals and postoperative septic patients (4, 14, 51, 52) as well as hypoglycemia paralleled by glucosuria in postischemic ARF (37). Fractional excretion of glucose and tubular glucose reabsorption were indicators of tubular function in this study. Decreased tubular glucose reabsorption with resulting glucosuria indicated tubular damage during experimental inflammation, which is in accordance with findings of previous investigations regarding tubular sodium reabsorption (16, 39).

In the present study, depressed renal tubular function was accompanied by a marked downregulation of SGLT2, SGLT3, GLUT2, and Na+-K+-ATPase, suggesting a possible causal link between impairment of renal glucose reabsorption and expression of these transporters. This hypothesis is strengthened by studies describing the coexistence of renal tubular dysfunction and downregulation of several tubular glucose transporters after renal ischemia reperfusion injury (31, 37). Remarkably, abatement of the high-capacity sodium-glucose transporter SGLT2, reabsorbing 98% of all filtrated glucose, and the Na+-K+-ATPase, creating the essential Na+ electrochemical gradient for glucose reabsorption, are mainly responsible for decreased tubular glucose reabsorption and glucosuria. The low-affinity SGLT3 transporter having characteristics of both SGLT1 and SGLT2 also decreased significantly after LPS treatment, indicating a contributive role of SGLT3 in renal glucose reabsorption underlying the currently accepted SGLT1/SGLT2 model (45). Expression of SGLT1 transporter, which is basically located in the S3 segment of the proximal tubule, rose significantly after sepsis induction, possibly trying to compensate for the decline of proximally situated glucose transporters as well as the developing glucosuria. However, a possible Na+ electrochemical gradient deficiency due to decreased Na+-K+-ATPase expression might reduce glucose influx through SGLT1 so that upregulation of SGLT1 with its low capacity for glucose reabsorption presumably could not countervail downregulation of SGLT2 and SGLT3, resulting in glucosuria and tubular dysfunction. A low intracellular glucose concentration on the basolateral membrane is the main cellular stimulus for GLUT1 (49), which should be increased in consequence of the intracellular glucose deficiency in our model. The raised GLUT1 values in our study are in accordance with previous studies reporting increased GLUT1 paralleled by decreased GLUT2 expression in endotoxemic animals as well as after glucose deprivation, and it is well known that GLUT1 is inversely regulated to GLUT2 in terms of intracellular glucose concentration (4, 13, 18, 49, 51, 52). In summary, our data indicate that the functional unit of the high-affinity/low-capacity SGLT1 transporter and GLUT1, normally responsible for reabsorbing the remaining 2% of the tubular glucose to a near-zero tubular glucose concentration (17), is not able to compensate the decline of SGLT2 and GLUT2 during endotoxemia.

We were further interested in the mechanisms along which septic terms lead to modified regulation of tubular glucose transporters. The hallmark of sepsis is the potent induction of proinflammatory cytokines such as TNF-{alpha}, IL-1beta, IFN-{gamma}, or IL-6 (34). Therefore, we were interested in the effect of these mediators on functional renal and hemodynamic parameters and on the expression of renal glucose transporters. Injection of IL-1beta caused a significant depression of hemodynamic and renal function similar to that caused by LPS. In addition, we found that TNF-{alpha}, IL-1beta, and IFN-{gamma} markedly downregulated expression of SGLT2, SGLT3, GLUT2, and Na+-K+-ATPase, whereas SGLT1 and GLUT1 expression significantly increased to levels comparable to those after LPS administration. IL-6 also inhibited expression of SGLT2 and GLUT2 but did not bias other glucose transporters. To our knowledge, there is no evidence in the published literature that proinflammatory cytokines decrease SGLT or GLUT activity in vitro. However, Kreydiyyeh et al. (26) demonstrated that IL-1beta reduces the activity and protein expression of Na+-K+-ATPase in LLC-PK1 cells, indicating an active signal transduction pathway for proinflammatory cytokines in renal proximal tubular cells (26).

We conclude from our results that cytokines possibly mediate the LPS-induced regulation of tubular glucose transporters. To further test this hypothesis, we performed additional experiments using knockout mice with a deficiency for TNF-{alpha}, IL-1 receptor type 1, IFN-{gamma}, or IL-6. However, it turned out that the absence of single-cytokine effects does not prevent LPS-induced downregulation of SGLT2, SGLT3, GLUT2, and Na+-K+-ATPase and upregulation of SGLT1 and GLUT1. Apparently, the LPS-mediated regulation of glucose transporters cannot be attributed to single cytokines, possibly because of overlapping actions of different proinflammatory cytokines.

To further clarify the role of cytokines in the regulation of tubular glucose transporters, we performed experiments using glucocorticoid-treated mice as a model of reduced LPS-induced upregulation of several proinflammatory cytokines. We found that glucocorticoid treatment attenuated LPS-induced alteration of renal glucose transporters and nearly abolished the endotoxemic alterations on glucose transporters in addition to GLUT1 gene expression. The main molecular mechanism responsible for this reversal may be the inhibition of the distribution of proinflammatory cytokines by glucocorticoids. This is in agreement with results from other studies reporting an almost reversing effect of glucocorticoids on inhibition of SGLT and Na+-K+-ATPase in chronically inflamed ileum (38, 43). In addition, glucocorticoid treatment alleviated the effect of LPS on GFR and fractional glucose excretion in our study and previous studies (47). These results emphasize the possible impact of proinflammatory cytokines in the pathogenesis of sepsis-induced ARF with tubular dysfunction and alterations of major renal glucose transporters. In healthy kidneys, glucocorticoids did not increase glucose uptake or renal glucose transporter expression in either our study or previous investigations (43, 50).

Because reduced tubular glucose transport with modifications of tubular glucose transporters has been demonstrated after renal ischemia reperfusion injury (21, 31, 37, 42), one could hypothesize that all the effects observed in the present study are due to ischemic kidney injury. However, in contrast to the results of the present study and those of another study in extrarenal tissue during endotoxemia (51), downregulation of SGLT1 with unmodified SGLT2 levels has been reported after ischemic ARF (37). The different regulation of SGLT1 and SGLT2 in ischemia- and LPS-induced ARF indicates that there may be different pathogenetic pathways. In addition, the results of the present study suggest that renal ischemia plays a minor role in mediating modifications of tubular glucose transporter expression given that glucocorticoid treatment improved LPS-induced alterations of tubular glucose transporters without affecting renal perfusion.

In conclusion, our data demonstrate that expression of tubular glucose transporters SGLT1–3, GLUT1, GLUT2, and Na+-K+-ATPase is regulated during experimental sepsis and suggest that this modification is mediated by proinflammatory cytokines. The regulation of glucose transporters likely contributes to the development of glucosuria and ARF during sepsis. Because of overlapping actions of different proinflammatory cytokines, the regulation of glucose transporters cannot be attributed to single cytokines, providing an explanation why single anti-cytokine strategies do not improve the outcome of septic patients. In addition, our findings contribute to an explanation and understanding of the beneficial effects of glucocorticoids in septic patients (1, 2, 5, 24).


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was financially supported by grants from the German Research Foundation (SFB 699, Projects B4 and B5).


   ACKNOWLEDGMENTS
 
The expert technical assistance provided by Maria Hirblinger is gratefully acknowledged.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Bucher, Dept. of Anesthesiology, Regensburg Univ., 93042 Regensburg, Germany (e-mail: michael.bucher{at}klinik.uni-regensburg.de)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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