Original Article Elevated Activity of Transcription Factor Nuclear Factor of Activated T-Cells 5 (NFAT5) and Diabetic Nephropathy Bingmei Yang,1 Andrea D. Hodgkinson,1 Peter J. Oates,2 Hyug Moo Kwon,3 Beverley A. Millward,1 and Andrew G. Demaine1
The expression of aldose reductase is tightly regulated by the transcription factor tonicity response element binding protein (TonEBP/NFAT5) binding to three osmotic response elements (OREs; OREA, OREB, and OREC) in the gene. The aim was to investigate the contribution of NFAT5 to the pathogenesis of diabetic nephropathy. Peripheral blood mononuclear cells (PBMCs) were isolated from the following subjects: 44 Caucasoid patients with type 1 diabetes, of whom 26 had nephropathy and 18 had no nephropathy after a diabetes duration of 20 years, and 13 normal healthy control subjects. In addition, human mesangial cells (HMCs) were isolated from the normal lobe of 10 kidneys following radical nephrectomy for renal cell carcinoma. Nuclear and cytoplasmic proteins were extracted from PBMCs and HMCs and cultured in either normal or high-glucose (31 mmol/l D-glucose) conditions for 5 days. NFAT5 binding activity was quantitated using electrophoretic mobility shift assays for each of the OREs. Western blotting was used to measure aldose reductase and sorbitol dehydrogenase protein levels. There were significant fold increases in DNA binding activities of NFAT5 to OREB (2.06 ⴞ 0.03 vs. 1.33 ⴞ 0.18, P ⴝ 0.033) and OREC (1.94 ⴞ 0.21 vs. 1.39 ⴞ 0.11, P ⴝ 0.024) in PBMCs from patients with diabetic nephropathy compared with diabetic control subjects cultured under high glucose. Aldose reductase and sorbitol dehydrogenase protein levels in the patients with diabetic nephropathy were significantly increased in PBMCs cultured in high-glucose conditions. In HMCs cultured under high glucose, there were significant increases in NFAT5 binding activities to OREA, OREB, and OREC by 1.38 ⴞ 0.22-, 1.84 ⴞ 0.44-, and 2.38 ⴞ 1.15-fold, respectively. Similar results were found in HMCs exposed to high glucose (aldose reductase 1.30 ⴞ 0.06-fold and sorbitol dehydrogenease 1.54 ⴞ 0.24-fold increases). Finally, the silencing of the NFAT5 gene in vitro reduced the expression of the aldose reductase gene. In conclusion, these results show that aldose reductase is
From the 1Molecular Medicine Research Group, Institute of Biological and Clinical Science, Peninsula Medical School, Universities of Exeter and Plymouth, Plymouth, U.K.; the 2Department of Cardiovascular and Metabolic Diseases, Pfizer Global Research and Development, Groton, Connecticut; and the 3Department of Medicine, University of Maryland, Baltimore, Maryland. Address correspondence and reprint requests to Professor Andrew G. Demaine, Molecular Medicine Research Group, The John Bull Building, Research Way, Peninsula Medical School, Universities of Exeter and Plymouth, Plymouth PL6 8BU, U.K. E-mail:
[email protected]. Received for publication 27 September 2005 and accepted in revised form 23 January 2006. HMC, human mesangial cell; NFAT5, nuclear factor of activated T-cells 5; ORE, osmotic response element; PBMC, peripheral blood mononuclear cell; SDH, sorbitol dehydrogenase; TonEBP, tonicity response element binding protein. DOI: 10.2337/db05-1260 © 2006 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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substantial number of patients with type 1 and type 2 diabetes will develop one or more microvascular complications during the course of the disease. The precise mechanisms that initiate and promote diabetic microvascular complications have still to be elucidated. It is well established that hyperglycemia is an important risk factor and that it stimulates increased metabolic flux through several biochemical pathways, notably the polyol pathway. Increased polyol pathway activity has been linked to abnormalities such as increased osmotic and oxidative stress factors that have been cited as promoters of diabetic microvascular disease (1–3). Aldose reductase (HUGO gene nomenclature: AKR1B1) is the first and rate-limiting enzyme of the polyol pathway, and its expression is tightly regulated by intracellular osmolality at the transcriptional level (4). This is mediated through the osmotic response elements (OREs) located in the 5⬘ flanking sequences of the AKR1B1 gene (5). There are three OREs (OREA, OREB, and OREC) that act as specific binding sites for the transcription factor, the tonicity response element binding protein (TonEBP), or nuclear factor of activated T-cells 5 (NFAT5) (5). NFAT5 activates the transcription of the AKR1B1, as well as the Na⫹/Cl⫺/betaine transporter (BGT1) and the Na⫹/myo-inositol cotransporter (SMIT) genes (6 – 8). NFAT5 is widely expressed in both the cytoplasm and nucleus of mammalian tissues including the kidney (9,10). Under hypertonic conditions, there is increased abundance of NFAT5 in the nucleus (9). Studies have shown that AKR1B1 mRNA and protein levels are increased in patients with diabetic microvascular complications (11,12). This effect is enhanced when peripheral blood mononuclear cells (PBMCs) from those patients with diabetic microvascular complications are cultured in high glucose compared with those without complications or normal control subjects (13,14). Patients with diabetic microvascular complications also have raised enzymatic activity of AKR1B1 compared with those without microvascular complications (15–17). Several groups have shown that AKR1B1 mRNA levels can be induced under hyperglycemic and hypertonic conditions (6,14,18,19). The aim was to determine whether ORE-dependent AKR1B1 gene expression is regulated by NFAT5 under high-glucose conditions and whether this expression is DIABETES, VOL. 55, MAY 2006
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TABLE 1 Clinical characteristics of patients with type 1 diabetes and normal control subjects
n Age (years) Age at onset of diabetes (years) Duration of diabetes (years) Fasting glucose (mmol/l) HbA1c (%) Male:female
Diabetic nephropathic subjects
Diabetic control subjects
Normal control subjects
26 40.2 ⫾ 2.5 (21–69) 13.6 ⫾ 1.4 (6–27) 28.0 ⫾ 2.3 (8–47) 11.5 ⫾ 1.4 8.8 ⫾ 0.3 8:18
18 44.2 ⫾ 3.6 (26–65) 16.1 ⫾ 2.8 (1–38) 29.1 ⫾ 2.4 (20–49) 9.7 ⫾ 1.1 8.4 ⫾ 0.2 5:13
13 36.4 ⫾ 3.5 (26–55) — — — — 6:7
Data are means ⫾ SE (ranges).
different between those patients with or without diabetic microvascular complications. RESEARCH DESIGN AND METHODS The following Caucasoid individuals were included in this study: 44 of patients with type 1 diabetes and 13 normal healthy control subjects (NC group). In addition, fresh tissue was obtained from the normal lobe of 10 nephrectomy samples taken from nondiabetic patients with renal cellular carcinoma. The tissue was used for isolating mesangial cells. All patients with type 1 diabetes, as defined by the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus (20), had attended the diabetes clinic at Derriford Hospital, Plymouth, U.K. The study was approved by the local research ethical committee, and informed consent was obtained from all subjects. Normal healthy control subjects were ethnically matched Caucasoid volunteers without family history of type 1 or type 2 diabetes. The criteria for diabetic microvascular complications has been previously published (21). Diabetic control subjects. Diabetic control subjects (DC group; n ⫽ 18) have been diagnosed with type 1 diabetes for at least 20 years but remain free of retinopathy (fewer than five dots or blots per fundus) and proteinuria (urine Albustix negative on the consecutive occasions over 12 months). Overt neuropathy was defined if there was any clinical evidence of peripheral or autonomic neuropathy. Diabetic nephropathic subjects. Diabetic nephropathic subjects (DN group; n ⫽ 26) have had type 1 diabetes for at least 8 years with persistent proteinuria (urine Albustix positive on at least three consecutive occasions over 12 months or three consecutive total urinary protein excretion rates ⬎0.5 g/24 h) in the absence of hematuria or infection on midstream urine samples. Diabetic nephropathy was always associated with retinopathy. Retinopathy was defined as more than five dots blots per eye; hard or soft exudates, new vessels, or fluorescein angiographic evidence of maculopathy or previous laser treatment for preproliferative or proliferative retinopathy; and maculopathy or vitreous hemorrage. Fundoscopy was performed by both a diabetologist and an ophthalmologist. Patients with renal cellular carcinoma. Ten patients with renal cell carcinomas were recruited from the Department of Urology, Derriford Hospital, Plymouth, U.K. Cell isolation and cultures. Peripheral venous blood samples (20 ml) were collected into 5% EDTA vacutainers (Becton Dickinson, Oxford, U.K.). The PBMCs were separated by using lymphoprep (Life Technologies, Paisley, U.K.) and grown in RPMI-1640 supplemented with 10% calf serum and 2 mmol/l L-glutamine, 100 units/ml penicillin G sodium, and 100 mg/ml streptomycin sulfate with phytohemagluttinin-P at 5 g/ml of concentration. The normal cortex was excised at the time of nephrectomy from the kidney of patients with renal cellular carcinoma. Primary cultures of human mesangial cells (HMCs) were established from collagenase-treated glomeruli as described in detail by Mene´ (22). Briefly, the cortex of the kidney was minced and passed through a series of graded sieves (120 and 75 m). The suspension was then transferred to a new tube by gentle aspiration through a 1-ml pipette tip followed by passing through a 21-gauge needle. Resulting glomeruli were digested with 750 units/ml solution of Worthington type I collagenase in RPMI-1640 medium with gentle stirring at 37°C in a water bath for ⬃5 min. The digested glomeruli were resuspended in a 200-ml flask with the growth medium consisting of RPMI-1640 supplemented with 17% FCS, 50 units/ml penicillin, 50 g/ml streptomycin, 0.25 g/ml amphotericin B, 300 g/ml glutamine, and 5 units/ml insulin. After 2–3 days ’ culture, the medium was changed once. HMCs appeared as outgrowth of the isolated glomeruli and obtained confluence after 7–10 days. Subculture was performed after a brief exposure to trypsin-EDTA (0.05 and 0.02%, respectively, in Hanks’ balanced salt solution without Ca2⫹ and Mg2⫹). The cells were characterized by DIABETES, VOL. 55, MAY 2006
morphology and immunohistochemical stains with ␣-smooth muscle actin and CD45 antibodies (Sigma, Dorset, U.K.). In morphology, they were stellate, spindle-shaped, “hills and valleys” when confluent. In immunohistochemical stains, they were positive for ␣-smooth muscle actin and negative for CD45 antibodies (22). At the second or third passages, uniformed populations were grown in the growth medium and used as detailed below. All PBMCs and HMCs were grown in a 37°C incubator with a controlled, humidified atmosphere of 95% air/5% CO2 and divided into two groups from each individual in 200-ml flasks. In group 1 (normal glucose conditions), cells were cultured in the appropriated medium without extra glucose and NaCl for 5 days. In group 2 (high-glucose conditions), extra D-glucose was added into the above-mentioned medium at a final concentration of 31 mmol/l and incubated at the same condition for 5 days. A portion of the PBMCs and HMCs were cultured under hypertonic conditions as positive controls: the extra NaCl 50 mmol/l was added into the normal culture medium after the 4th day’s incubation. Then the cells were continuously incubated at 37°C in 5% CO2 for another 20 h. At the end of the incubation time, all cells were harvested and nuclear and cytoplasmic proteins were extracted from PBMCs and mesangial cells, respectively, as below. Extraction of nuclear and cytoplasmic proteins. Cells were collected and resuspended in 100 l buffer A (10 mol/l HEPES, pH 7.9, 1.5 mmol/l MgCl2, 0.5 mmol/l dithiothreitol, 0.2% NP-40, 100 mmol/l AEBSF, 18.4 mg/ml sodium orthovanadate, 42 mg/ml sodium flouride, and 2.2 mg/ml aprotonin) and held on ice for 15 min. The resulting cell lysate was then centrifuged at 16,000g for 10 min. The supernatant containing cytoplasmic proteins was transferred into a fresh tube for Western blotting, and the nuclear pellets were resuspended in 50 l buffer C (20 mmol/l HEPES, pH 7.9, 25% glycerol, 0.42 mmol/l NaCl, 1.5 mmol/l MgCl2, 0.5 mmol/l dithiothreitol, 0.2 mmol/l EDTA, 100 mmol/l AEBSF, 18.4 mg/ml sodium orthovanadate, 42 mg/ml sodium fluoride, and 2.2 mg/ml aprotonin) and incubated on ice for 10 min. After centrifugation at 13,000 rpm for 10 min, the supernatant containing the nuclear protein was extracted and stored in a fresh tube at ⫺70°C until use. The concentrations of both nuclear and cytoplasmic proteins were determined using a Coomassie Plus Protein Assay kit (Pierce, Chester, U.K.). Electrophoretic mobility shift assay. Oligonucleotide sequences were OREA: 5⬘-TTACATGGAAAAATATCTGGG-3⬘, OREB: 5⬘-CTGTATAAATTTTTCCAGGAGGG-3⬘, and OREC: 5⬘-CACCAAATGGAAAATCAC CGGCATGG-3⬘, which are consensus sequences to OREA, OREB, and OREC of AKR1B1 gene and were labeled with [␣-32P]deoxy-ATP by T4 polynucleotude kinase (Amersham Pharmacia Biotech, Buckinghamshire, U.K.). The labeled ORE probes along with a gel binding buffer was incubated with 25 g nuclear proteins at room temperature for 20 min. The binding mixtures were resolved by electrophoresis on a 4% polyacrylamide gel at 100 V for 3– 4 h. The gel was exposed to X-Omax photographic paper. The specificity of the DNA binding protein for the putative binding sites was established by specific competitors and an antibody that is against human NFAT5 (University of Maryland, Baltimore, MD). Western blotting. Briefly, a total of 50 g cytoplasmic proteins were loaded onto a 12% SDS-PAGE, electrophoresed for 8 h at 150 V, and transferred to nitrocellular membrane (Amersham Pharmacia Biotech) overnight. Next, the membrane was blocked with 5% nonfat milk and 0.05% Tween 20-PBS for 1 h at room temperature. Immunoblotting was performed with primary rabbit antibodies (Cambridge Research Biochemicals, Cleveland, U.K.) against human AKR1B1 and sorbitol dehydrogenase (SDH) in 1:500 dilution and a secondary antibody against rabbit IgG of horseradish peroxidase conjugated in 1:5,000 dilution (Sigma). A chemiluminescence kit (Pierce) and Kodak X-Omat film (Amersham Pharmacia Biotech) were used to detect the amount of proteins. Meanwhile, in order to have an equal amount of proteins in all wells, -actin levels were measured by using a mouse antibody against human 1451
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-actin in 1:2,000 dilution (Sigma) and a secondary antibody against mouse IgG of horseradish peroxidase conjugated in 1:10,000 dilution (Sigma). Both DNA binding activities of NFAT5 and protein levels of AKR1B1 and SDH were analyzed and quantified using a phosphoimager (Biorad, Hertfordshire, U.K.) with multianalyst software. All results were expressed as means of fold increases due to high-glucose treatment calculated by dividing the amount of density in high-glucose–treated cells by the amount of density in untreated cells. SiRNA assay. To investigate effects of NFAT5 on the transcription of the AKR1B1 gene under high-glucose conditions, we silenced the expression of NFAT5 by RNA interference. siRNA duplex sequences were designed and validated according to previous study (23). 569R is targeted to the start codon at nucleotide position 569 of the NFAT5 cDNA. Inverted sequence (inv569R) of 569R that did not have an interference effect on NFAT5 transcription was used as a control (569R 5⬘-AUGGGCGGUGCUUGCAGCUCCUU; UUUAC CCGCCACGAACGUCGAGG-5⬘; inv569R 5⬘-CCUCGACGUUCGUGGCGGGU AUU; UUGGAGCUGCAAGCACCGCCCAU-5⬘). Transfection of siRNA was carried out using siFECT siRNA transfection reagent (Promega, Southampton, U.K.), and transfection of plasmids was performed by using Tfx-20 reagent (Promega). The day before transfection, HEK 293 cells (which express NFAT5 identified by RT-PCR, data not shown) were seeded at 50 –70% confluence in 96-well plates. Each transfection was performed in duplicate. SiRNA-569R or inv569R at 400 nmol/l or No-siRNA as controls was transfected into the HEK 293 cells. For analysis of AKR1B1 transcription, pGL3 reporter plasmids at 98 ng, which contain the promoter region with three OREs and different haplotypes of the AKR1B1 gene (24) and firefly luciferase gene downstream, were cotransfected with pRL-TK control plasmids at 2 ng, which contain the Renilla luciferase gene into the cells at 24 h after siRNA transfection. The next day, cells were divided into two groups: 1) cells maintained in original media and 2) cells switched to high-glucose conditions (31 mmol/l of D-glucose) for another 48 h or hypertonic conditions (extra 50 mmol/l NaCl) for another 20 h as positive controls. At the end of culture, cells were lysed using 20 l of lysis buffer (Promega). AKR1B1 transcription activity was measured using a dual-luciferase reporter assay system (Promega) in a MLX luminometer (Dynex Technologies). For each transfection, the relative activity was defined as luciferase activity normalized by the activity of the internal control (Renilla luciferase) as previously described (22). Statistical analysis. All data were expressed as the means ⫾ SE. Student’s t test was used to compare differences between two groups, and ANOVA was used to compare differences between more than two groups. A P value ⬍0.05 was considered to be significant.
RESULTS
FIG. 1. A: DNA binding activities of NFAT5 to OREA, OREB, and OREC in PBMCs, which were isolated from a patient who was a diabetic control subject (DC) and was cultured under normal conditions. Reactions were incubated with 25 g nuclear proteins (lane 1 and lanes 5–10) or without nuclear proteins (lanes 2– 4) and with 50 times competitors (corresponding to unlabeled probes OREA, lane 5; unlabeled OREB, lane 6; and unlabeled OREC, lane 7) or without competitors (lanes 8 –10), respectively. For competition assays, reactions were incubated with unlabeled probes at room temperature for 10 min, and then the 32P-ATP–labeled OREA, OREB, and OREC were added to each reaction followed by another 20-min incubation at room temperature. B: Supergel shift analysis of NFAT5. Lanes 1, 3, and 5 are reactive bands of NFAT5 to OREA, OREB, and OREC, respectively. Lanes 2, 4, and 6 are the reactive bands of NFAT5 to OREA, OREB, and OREC with poly-rabbit antibody against human NFAT5, respectively. 1452
Clinical characteristics of patients with type 1 diabetes and normal control subjects are shown in Table 1. There were no differences in age, sex, age at onset of diabetes, duration of diabetes, fasting glucose, or HbA1c among any of the patient groups. NFAT5 binding protein activity. The DNA binding activities to OREA, OREB, and OREC in PBMCs are shown in Fig. 1A. OREA displayed the highest NFAT5 binding affinity compared with OREB and OREC. The same pattern of binding activity was present in the HMCs (data not shown). By using 50 times corresponding unlabeled probes, binding complexes were partly abolished in OREA and completely abolished in OREB and OBEC (Fig. 1A). The supershift analysis of NFAT5 is shown in Fig. 1B. Following incubation with antibody against human NFAT5, the binding complexes tended to shift upwards as indicated in Fig. 1B. The DNA binding activities of NFAT5 to OREA, OREB, and OREC under normal, high-glucose, and hypertonic conditions in a representative patient with diabetic nephropathy are shown in Fig. 1C. High-glucose conditions C: Representative images of DNA binding to OREA, OREB, and OREC in patients with diabetes and nephropathy (DN), diabetic control subjects (DC), and normal healthy control subjects (NC). The PBMCs were incubated under normal (NG), high-glucose (HG; at 31 mmol/l D-glucose), or hypertonic (HT; extra 50 mmol/l NaCl was added into culture media) conditions, respectively. Probes OREA, OREB, and OREC were expressed as A, B, and C, respectively. DIABETES, VOL. 55, MAY 2006
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TABLE 2 DNA binding activities of NFAT5 to OREs in PBMCs and HMCs under high-glucose conditions
Probes
Diabetic nephropathic subjects
Diabetic control subjects
Normal control subjects
HMCs
n OREA OREB OREC
26 1.50 ⫾ 0.14 2.06 ⫾ 0.27* 1.94 ⫾ 0.21‡
18 1.18 ⫾ 0.09 1.33 ⫾ 0.18 1.39 ⫾ 0.11
13 1.40 ⫾ 0.13 1.11 ⫾ 0.11 0.95 ⫾ 0.05
10 1.38 ⫹ 0.22 1.84 ⫹ 044† 2.38 ⫹ 1.15§
Data are means of fold increases ⫾ SE under high-glucose conditions (31 mmol/l D-glucose) to normal conditions (the density value was defined as 1) in PBMCs and HMCs. *P ⫽ 0.033 and 0.023 vs. diabetic and normal control subjects, respectively. †P ⫽ 0.035 vs. normal conditions. ‡P ⫽ 0.024 and 0.002 vs. diabetic and normal control subjects, respectively. §P ⫽ 0.003 vs. normal conditions.
significantly increased the DNA binding activities of NFAT5 to the OREA, OREB, and OREC in all patients (data not shown). The fold increases of the binding activity of NFAT5 to OREA, OREB, and OREC in PBMCs exposed to high-glucose conditions in the different patient subgroups are shown in Table 2. There were significant increases in OREB binding in PBMCs exposed to highglucose conditions from diabetic nephropathy versus the DC and NC groups (2.06 ⫾ 0.27 vs. 1.33 ⫾ 0.18 and 1.11 ⫾ 0.11; P ⫽ 0.033 and P ⫽ 0.023, respectively). Similar results were obtained with OREC (1.94 ⫾ 0.21 vs. 1.39 ⫾ 0.11 and 0.95 ⫾ 0.05; P ⫽ 0.024 and P ⫽ 0.002, respectively). No significant difference in the binding activities to OREs was found between the DC and NC groups. NFAT5 binding in the HMCs exposed to high-glucose conditions was significantly increased for OREB (1.83 ⫾ 0.44-fold, P ⫽ 0.035) and OREC (2.38 ⫾ 1.15-fold, P ⫽ 0.003), respectively (Table 2). Exposure of the cells to hypertonic conditions was used as a positive control, and binding activities were significantly increased for OREA, OREB, and OREC in both PBMCs and HMCs (data not shown). Protein levels of AKR1B1 and SDH. The AKR1B1 and SDH levels were assessed by using Western blots (Fig. 2). In AKR1B1 and SDH protein levels in high glucose relative to normal glucose, high glucose significantly increased AKR1B1 and SDH protein levels in PBMCs from patients with diabetic nephropathy by 1.34- and 1.33-fold (could also say by 34 and 33%), respectively, consistent with previous findings of elevated AKR1B1 mRNA by high glucose in diabetic nephropathy (14). In contrast, the DC group did not show any elevation of AKR1B1 or SDH by high glucose (Table 3), a finding consistent with previous
AKR1B1 mRNA data (14). AKR1B1 and SDH levels in the HMCs were significantly increased under high-glucose conditions by 1.30 ⫾ 0.06 and 1.54 ⫾ 0.24, respectively, P ⬍ 0.0005) (Table 3). Silencing NFAT5 reduces AKR1B1 gene transcription. We confirmed that the recombinants with the Z-2/C106 AKR1B1 haplotype had the highest transcriptional activity among all haplotypes and that the Z/C-106 AKR1B1 haplotype had the second highest transcriptional activity in transfected HEK 293 cells (data not shown). High glucose significantly increased AKR1B1 transcription by 1.4- and 1.5-fold in the HEK293 cells transfected with Z-2/C-106 and Z/C-106 AKR1B1 haplotypes, respectively (Table 4). Transcriptional activities of AKR1B1 in these two AKR1B1/-106 recombinants were reduced by 44 and 36%, respectively, under high-glucose conditions in the presence of the 569R SiRNA (Table 4). There were no significant changes in transcriptional activities in transfected cells with the control inv569 SiRNA between normal and high-glucose conditions for both haplotypes. DISCUSSION
Chronic hyperglycemia is a major initiator of microvascular complications in diabetes. Accelerated flux through the polyol pathway may be a key to the development of diabetic nephropathy and other complications (3). There is strong evidence that those patients with diabetes and its complications have increased expression and activity of AKR1B1. What is a possible signaling pathway for this increase under hyperglycemic conditions? As the expression of AKR1B1 gene is ORE dependent, the expression of
FIG. 2. Western blotting results with antibodies against AKR1B1, SDH, and -actin, respectively. PBMCs from patients with diabetic nephropathy (DN) and diabetic control subjects (DC) and HMCs were cultured in normal (NG), high-glucose (HG; at 31 mmol/l D-glucose), or hypertonic (HT; extra 50 mmol/l NaCl was added into culture media) conditions, respectively. Standard AKR1B1 protein was indicated as S. DIABETES, VOL. 55, MAY 2006
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TABLE 3 Fold changes in the protein levels of aldose reductase (AKR1B1) and SDH in PBMCs and HMCs under high-glucose conditions
Diabetic nephropathic subjects Diabetic control subjects HMCs
AKR1B1
SDH
1.34 ⫾ 0.17 (n ⫽ 17)* 1.03 ⫾ 0.05 (n ⫽ 12) 1.30 ⫹ 0.06 (n ⫽ 10)
1.33 ⫾ 0.13 (n ⫽ 15)* 1.14 ⫾ 0.15 (n ⫽ 9) 1.54 ⫹ 0.24 (n ⫽ 10)§
Data are means of fold increases ⫾ SE under high-glucose conditions (31 mmol/l D-glucose) to normal conditions (the density value was defined as 1) in PBMCs and HMCs. *P ⫽ 0.032 and P ⫽ 0.029 vs. normal conditions in diabetic nephropathic subjects. †P ⫽ 0.0001 vs. normal conditions in HMCs.
the AKR1B1 gene may be regulated by NFAT5 under high-glucose conditions (25,26). Tonocity-responsive regulation of NFAT5 is not limited to the kidney, as it has been shown that T-cells express high levels of NFAT5 after activation of the T-cell receptor (19). The current study showed that the binding activity of NFAT5 to all three OREs was significantly increased under high-glucose conditions in both PBMCs (DN group) and HMCs. OREA displayed the highest binding activity, while OREB and OREC displayed lower binding activity for NFAT5. These OREs are functional, although the maximum response is found when all three fragments are present (5). It is possible that each individual ORE is stress-response specific. For instance, OREC may have increased activity in the osmotic response compared with OREA and OREB (7). We found that only OREB and OREC had significantly increased binding in the PBMCs of patients with diabetic nephropathy exposed to high glucose (Table 2). OREB and OREC may contribute to stress conditions in contrast to OREA (27). NFAT5 has at least four spliced isoforms (28) that may have different affinities to these OREs. The NFAT5 isoforms are differentially expressed in PBMCs of patients with diabetic nephropathy exposed to hypertonic conditions compared with those without diabetic nephropathy (29), implicating a role for these proteins in the pathogenesis of diabetic nephropathy. In mice, these isoforms have different gene target and are expressed differentially during development of mouse brain (30,31). Although splicing does not occur in the DNA-binding domain and may not affect binding directly, its action might be by modifying the tertiary structure of the protein. We had previously showed that mRNA levels of AKR1B1 and SDH were significantly increased in PBMCs from patients with type 1 diabetes and diabetic nephropathy compared with the DC group under high-glucose conditions (14). This suggests that the response to uptake and disposal of D-glucose is different between patients with type 1 diabetes and with or without diabetic nephropathy. The exact mechanisms have still to be elucidated and explored, but genetic factors such as 5⬘ ALR2 or GLUT1
gene polymorphisms probably make important contributions (14,32). Damage to the mesangial cells plays a central role in the pathogenesis of diabetic nephropathy (33). Here, we demonstrate that high glucose significantly increased NFAT5binding activities and protein levels of AKR1B1 and SDH in HMCs. This suggests that HMCs exposed to high glucose have increased expression of the enzymatic components of the polyol pathway and supports a previous report showing increased expression of AKR1B1 in glomeruli in diabetic patients (17). Our previous study (24) found a highly significant difference in the luciferase activities between the recombinants that contain different AKR1B1 gene haplotypes that had been transfected into Hep G2 cells exposed to high glucose. The present study confirmed that the Z-2/C-106 and Z/C-106 haplotypes have the highest transcription activities, and silencing of NFAT5 expression reduced the transcription activity of AKR1B1 even under high-glucose conditions. This supports the notion that NFAT5 contributes to the regulation of AKR1B1 expression under highglucose conditions. The exact pathways for NFAT5 regulation are largely unknown, although multiple kinase pathways such as p38 have previously been implicated (34 –37). It has been observed that high glucose increases GLUT1 expression and basal glucose uptake in cultured rat mesangial cells (38). Mesangial cells isolated from diabetic subjects had enhanced GLUT1 expression and glucose uptake (39). Therefore, increased glucose uptake may contribute to upregulation of NFAT5 and flux through the polyol pathway, especially in insulin-independent tissues such as the retina, nerves, and kidney. In diabetic conditions, despite the abnormally high level of sorbitol, these cells continue to express AKR1B1. In conclusion, high glucose increased binding activity of NFAT5 to the OREs in both PBMCs and HMCs. These increases were significantly higher in patients with diabetic nephropathy compared with diabetic control subjects and were accompanied with an increase of protein levels of AKR1B1 and SDH. These results support that
TABLE 4 Silencing of NFAT5 reduces transcription activity of AKR1B1 in transfected HEK 293 cells Z-2/C-106 No-siRNA 569R Inv569R
Z/C-106
Normal glucose
High glucose
Normal glucose
High glucose
87.4 ⫾ 14.9 (6) 81.4 ⫾ 1.9 (6) 105.5 ⫾ 10.2 (5)
119.4 ⫾ 39.0 (6)* 78.4 ⫾ 10.3 (6)‡ 122.4 ⫾ 8.1 (5)
35.1 ⫾ 8.3 (7) 33.1 ⫾ 8.8 (4) 44.2 ⫾ 7.9 (5)
51.4 ⫾ 8.9 (7)† 38.8 ⫾ 9.2 (4)§ 59.4 ⫾ 9.1 (5)
Data are means ⫾ SE (n experiments performed). Results are the relative activity of transcription and were defined as luciferase activity normalized by the activity of the internal control. Duplicated wells were performed for each transfection. Transfected cells were culture under normal and high-glucose conditions (31 mmol/l D-glucose). *P ⫽ 0.028 vs. normal glucose; †P ⫽ 0.018 vs. normal glucose; ‡P ⫽ 0.04 vs. high glucose with No-siRNA and inv569R; §P ⫽ 0.04 vs. high glucose with No-siRNA and Inv569R. 1454
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increased polyol pathway is involved in diabetes complications, and this pathway might be partly upregulated through NFAT5 under high-glucose conditions. ACKNOWLEDGMENTS
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