Effects of advanced glycation end products on renal fibrosis and oxidative stress in cultured NRK-49F cells

Diabetic nephropathy (DN), a major microvascular complication of diabetes mellitus (DM), has been a leading cause of end stage renal disease (ESRD).¹ There is growing evidence suggesting that advanced glycation end products (AGEs), formed by nonenzymatic reaction of reducing sugar with the amino group of proteins, lipids and nucleic acids, play a critical role in the development of DN.^2,3 However, the mechanisms underlying the role of AGEs in DN are not fully understood.

Transforming growth factorβ (TGF-β) is an important fibrogenic growth factor, and connective tissue growth factor (CTGF) is a downstream factor mediating fibrotic activity of TGF-β. TGF-β and CTGF can induce renal extracelluar matrix (ECM, such as fibronectin (Fn) and collagen) accumulation and renal fibrosis.^3-5 The expression of TGF-β, CTGF and components of ECM were upregulated in response to AGEs has been reported.^2-6 Reactive oxygen species (ROS) may play a critical role in AGEs induced growth factor expression.⁶ In 2005, Brownlee⁷ showed that hyperglycaemia induced overproduction of superoxide by the mitochondrial electron transport chain was the unifying mechanism in the pathogenesis of diabetic complications.

Ginkgo biloba extract (EGb) has numerous pharmacological actions. In particular, flavonol glycosides, an essential component of EGb, act as a free radical scavenger and an inhibitor of nearly all ROS.⁸ Recently, several in vivo studies have indicated that EGb has a protective effect on interstitial fibrosis.^9-11 It has been reported that EGb attenuates liver fibrosis induced by biliary obstruction or CCl₄ in rats through its antioxidant action.^10,11

The present study investigated whether AGEs could upregulate expression of TGF-β1, CTGF, Fn and formation of ROS in cultured normal rat kidney interstitial fibroblast (NRK-49F) cells and whether AGEs-induced TGF-β1 CTGF and Fn expression was inhibited by antioxidant.

METHODS

Preparation of AGEs-bovine serum albumin (BSA)
AGEs-BSA was prepared according to Vlassara et al.¹² Briefly, BSA was incubated under sterile conditions with 0.5 mol/L D-glucose in phosphate buffered saline (PBS) (pH 7.2) at 37˚C for 8 weeks in the presence of protease inhibitors (phenylmethylsulfonylfluoride 1.5 mmol/L and EDTA 0.5 mol/L) and antibiotics (penicillin 100 U/L and streptomaycin 100 mg/L). BSA was incubated under the same condition except for the presence of D-glucose as control. At the end of incubation, unincorporated sugar was removed by dialysis against PBS. Fluorescent intensity of AGEs-BSA was measured with fluorospectrometer (Hitachi, Japan) at excitation of 370 nm and emission of 440 nm ((141.10±0.61) U/mg protein for AGEs-BSA, (13.53±0.01) U/mg protein for control BSA). The concentration of AGEs-BSA and BSA were measured by Coomassie Brilliant Blue G assay.

Cell culture and intervention
NRK-49F cells, gifted from Professor CHEN Nan (Medical School of Shanghai Jiaotong University, Shanghai, China) were grown in DMEM (Gibco, USA) containing 5.5 mmol/L glucose and 10% foetal bovine serum under standard cell cultured conditions (humidified atmosphere, 5% CO₂, 37˚C), passaged as usual. AGEs and antioxidants treatment were carried out in a serum-free medium.

Cells were treated with serum-free medium for 24 hours as control and treated with different doses of AGEs-BSA (50, 100, 200 µg/ml) for 24 hours, or 100 µg/ml AGEs-BSA for different times (0, 12, 24, 48 hours). Cells were treated with high glucose (25 mmol/L) or high glucose adding 100 µg/ml AGEs-BSA for 24 hours to determine synergistic effects between AGEs and glucose.

To investigate the role of antioxidant, cells were pretreated with control medium or medium containing N-acetylcysteine (NAC, Wako Pure Chemical Industries Ltd., Japan, 10 mmol/L) or EGb (50 mg/L or 100 mg/L, Schwabe, Germany) for 24 hours and then treated with 100 µg/ml AGEs-BSA for another 24 hours.

Cell growth evaluation
The Alamarblue dye assay was used to analyze the cell growth. The colour of AlamarBlue stock is blue but changes to red when oxidized by cellular metabolites. Cells were plated at a density of 1×10⁵/ml per well in 96-well plates. When grown to 85% confluence, the cells were incubated with 25, 50, 100, 200 or 400 µg/ml AGEs-BSA for 24 hours. The medium containing 10% Alamarblue was added to the cells. After 4-hour incubation, the cell growth was evaluated as the absorbance using a plate reader (LabSystems Multiskan Ascent 354, Finland). An excitation wavelength of 540 nm was used, and the emission was read at 620 nm. Alamarblue reduction rates were then calculated.

Measurement of intracellular ROS generation
The intracellular generation of ROS was detected using the fluorescent probe CM-H₂DCFDA. After various treatments, cells were loaded with 20 µmol/L CM-H₂DCFDA and incubated for 30 minutes at 37˚C, and analysed by flow cytometry (excitation/emission 488/525 nm) using Cell Quest software. Ten thousand events were analyzed for each sample and in every experiment a control tube with cells alone was analyzed.

Measurement of intracellular glutathione level
Intracellular level of glutathione (GSH) was estimated using fluorescent reagent orthophthaladehyde.¹³ After various treatments, 20% phosphoric acid was added to cells. Then precipitated proteins were centrifuged at 4˚C at 12 000 g for 10 minutes and the supernatants were treated with PBS (0.1 mol/L)-EDTA (5 mmol/L) buffer (pH 8.0); and the fluorescence was measured after the addition of orthophthaladehyde (1 mg/ml, Sigma, USA) using spectrofluorometer (excitation/emission 350/420 nm). The results were expressed in milligram per milligram protein.

Semiquantitative RT-PCR
Expression of TGF-β1 and CTGF mRNA were performed by a semiquantitative RT-PCR technique. Total RNA was extracted using UNIQ-10 Column Total RNA Isolation Kit (Shengong, China) according to the manufacturer's instructions. Ultraviolet spectrophotometry was used to estimate the purity and concentration of total RNA. RT-PCR was performed on PTC-100 programmable thermal controller (MJ Research Inc, USA) using RNA PCR Kit (AMV V3.0, TaKaRa, Japan). Total RNA (0.3 µg) was reverse transcribed into single stranded DNA with AMV RNase Reverse Transcriptase, and oligo (dT)₁₅ primer at 50˚C for 30 minutes, and 99˚C for 5 minutes. PCR amplification was performed with 0.5 U Taq DNA polymerase in a final volume of 50 µg. The primer sequences were as follows: TGF-β1, forward primer 5'-GGA CTA CTA CGC CAA AGA AG-3', reverse primer 5'-TCA AAA GAC AGC CAC TCA GG-3' (PCR product 293 bp); CTGF, forward primer 5'-CTA AGA CCT GTG GGA TGG GC-3', reverse primer 5'-CTC AAA GAT GTC ATT GTC CCC-3' (PCR product 383 bp); Fn, forward primer 5'-TCA GCT GTA CCA TTG CAA ATC-3', reverse primer 5'-CAA TGA TCA GGA CAC CAG GA-3' (PCR product 315 bp); GAPDH, forward primer 5'-ACC ACA TCC ATG CCA TCA C-3', reverse primer 5'-TCC ACC ACC CTG TTG CTG TA-3' (PCR product 452 bp). PCR program was 35 cycles of 94˚C for 30 seconds, 55˚C for 30 seconds, and 72˚C for 1 minutes. PCR products were separated by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. Data were analyzed with GDS8000 gel analyser and Gelworks 1D software.

Statistical analysis
All values were presented as mean ± standard deviation (SD). Statistical analysis was carried out using SPSS 12.0. Multiple comparisons between experimental groups were performed with one way analysis of variance (ANOVA). A level of P<0.05 was considered statistically significant.

RESULTS

Effects of AGEs on cell growth
Compared with the control, the growth of NRK-49F cells was inhibited by various concentrations of AGEs (25, 50, 100, 200 or 400 µg/ml). The Alamarblue reduction rate markedly decreased by incubation with 400 µg/ml AGEs-BSA for 24 hours (P<0.05, Fig. 1).

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Fig.1. Effects of AGEs on the growth of NRK-49F cells. ^*P<0.05 compared with the control group.

Intracellular ROS generation induced by AGEs and effects of antioxidants
Compared with control, incubation of cells with AGEs or high glucose stimulated intracellular ROS generation. Furthermore, AGEs increased ROS generation in dose- and time-dependent manners. Compared with high glucose, treatment with AGEs (100 or 200 µg/ml) or AGEs (100 µg/ml) + high glucose significantly increased ROS generation in NRK-49F cells (P<0.05). Twenty- four-hour preincubation with NAC (10 mmol/L) decreased ROS generation induced by AGEs (100 µg/ml, P<0.05). Furthermore, pretreatments with EGb (50 or 100 mg/L) decreased ROS generation induced by AGEs (100 µg/ml) in a dose-dependant manner in NRK-49F cells (Figs. 2 and 3).

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Fig. 2. Intracellular ROS generation in NRK-49F cells measured by flow cytometry. Cells were treated with serum-free medium (a) or AGEs-BSA 50 μg/ml (c), 100 μg/ml (d) or 200 μg/ml (e) for 24 hours. Alternatively, cells were treated with high glucose (b) or medium containing high glucose + 100 μg/ml AGEs-BSA (f) for 24 hours. To investigate the effects of antioxidant, cells were pretreated with medium containing 10 mmol/L NAC (g), 50 mg/L EGb (h) and 100 mg/L EGb (i) for 24 hours, and then treated with AGEs-BSA (100 μg/ml ) for another 24 hours. ^*P<0.05, ^**P<0.01 vs the control group (a), ^#P<0.05 vs high glucose group (b), ⁺P<0.05, ⁺⁺P<0.01 vs 100 μg/ml AGEs-BSA group (d).

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Fig. 3. Effects of AGEs-BSA 100 μg/ml on the intracellular ROS generation in a time-dependent manner in NRK-49F cells. Cells were treated with 100 μg/ml AGEs-BSA for different times (0, 12, 24 or 48 hours). Then intracellular ROS generation was measured by flow cytometry. *P<0.05, **P<0.01 vs 0 hour group.

Effects of AGEs and antioxidants on intracellular GSH level
Compared with control, AGEs (100 or 200 µg/ml), high glucose, or both high glucose and 100 µg/ml AGEs significantly decreased intracellular GSH level (P<0.05). In addition, AGEs decreased GSH level in a time-dependent manner. Twenty-four-hour preincubation with NAC (10 mmol/L) significantly increased GSH level (P<0.01). Furthermore, compared with untreated group, pretreatment with 50 mg/L EGb increased GSH level, but not significantly; while pretreatment with 100 mg/L EGb significantly increased GSH level (P<0.05, Fig. 4).

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Fig. 4. Effects of AGEs and antioxidants on intracellular GSH level. A: Cells were treated with serum-free medium (a) or AGEs-BSA 50 μg/ml (c), 100 μg/ml (d) or 200 μg/ml (e) for 24 hours. Alternatively, cells were treated with high glucose (b) or medium containing high glucose + 100 μg/ml AGEs-BSA (f) for 24 hours. Cells were pretreated with medium containing 10 mmol/L NAC (g), 50 mg/L EGb (h) and 100 mg/L EGb (i) for 24 hours, and then treated with AGEs-BSA (100 μg/ml) for another 24 hours. B: Cells were treated with 100 μg/ml AGEs-BSA for different times (0, 12, 24 or 48 hours). ^*P<0.05 vs control, ^#P<0.05, ⁺P<0.05, ⁺⁺P<0.01 vs AGEs-BSA 100 μg/ml group, ^#P<0.05, ^##P<0.01 vs 0 hour group.

Effects of AGEs, high glucose and antioxidants on TGF-β1, CTGF and Fn mRNA expression
Compared with control medium, AGEs induced the expression of TGF-β1, CTGF and Fn mRNA in dose- and time-dependent manners. Compared with high glucose, AGEs (100 or 200 µg/ml) significantly increased TGF-β1, CTGF and Fn mRNA expression. In addition, compared with high glucose or AGEs (100 µg/ml) alone, expression of TGF-β1, CTGF and Fn mRNA induced by AGEs + high glucose (25 mmol/L) significantly increased (P<0.05). Twenty-four-hour preincubation with NAC (10 mmol/L) decreased the upregulation of TGF-β1, CTGF and Fn mRNA induced by AGEs (100 µg/ml, P<0.05). Furthermore, preincubation of cells with EGb (50 or 100 mg/L) decreased expression of TGF-β1, CTGF, Fn mRNA induced by AGEs in a dose-dependant manner (Figs. 5 and 6).

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Fig. 5. Effects of AGEs-BSA, high glucose and antioxidants on TGF-β1, CTGF and Fn mRNA expression in NRK-49F cells. A: Cells were treated with serum-free medium as control, and treated with different doses of AGEs-BSA (50, 100 or 200 μg/ml) for 24 hours. Alternatively, cells were treated with high glucose (25 mmol/L) or medium containing both high glucose and 100 μg/ml AGEs-BSA for 24 hours. B: Cells were incubated with control medium or medium containing NAC (10 mmol/L) or EGb (50 or 100 mg/L) for 24 hours, and then treated with AGEs-BSA (100 μg/ml) for another 24 hours. HG: high glucose. ^*P<0.05, ^**P<0.01 vs control; ^#P<0.05, ^##P<0.01 vs HG; ^ΔP<0.05 vs AGEs 100 μg/ml group, ⁺P<0.05, ⁺⁺P<0.01 vs AGEs group.

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Fig. 6. Effects of AGEs-BSA (100 μg/ml) on TGF-β1, CTGF and Fn mRNA expression in a time-dependent manner in NRK-49F cells. Cells were treated with 100 μg/ml AGEs-BSA for different times (0, 12, 24 or 48 hours). Then total RNA was extracted and the level of TGF-β1, CTGF and Fn mRNA expression was assessed by semiquantitative RT-PCR. ^*P<0.05, ^**P<0.01 vs 0 hour group.

DISCUSSION

DN is characterized by hypertrophy of glomerulus and tubules, basement membrane thickening of glomerulus and tubules, mesangial extracelluar matrix accumulation and tubulointerstitial fibrosis. The tubulointerstitium accounts for more than 90% of the kidney volume, so tubulointerstitial atrophy is responsible for renal enlargement in DN.¹⁴ Hypertrophy of tubular epithelial cells and tubular basement membrane thickening have already occurred in early stage of DN and the extent of tubulointerstitial injury is associated closely with prognosis in DN. In DN, AGEs mainly deposit in nodular glomerulosclerosis area, glomerular mesangium and basement membrane, also in tubulointerstitium and vessel wall.¹⁵ Several studies have indicated the effects of AGEs on glomerulus, but the effects of AGEs on tubulointerstitium are still not fully understood. We studied the major cell types of tubulointerstitium and fibroblasts to investigate the effects of AGEs on tubulointerstitial injury.

AGEs play a critical role in development of DN as in vitro and in vivo studies have already indicated that AGEs can stimulate growth factors, such as TGF-β, synthesis and lead to imbalance between syntheses and degradation of ECM inducing renal fibrosis,^2,3,5,6 and these effects can be prevented by AGEs formation inhibitor aminoguanidine.⁵ Yamamoto et al¹⁶ used a double transgenic mouse, which overexpressed human receptor for AGE (RAGE) and developed insulin dependent diabetes, glomerular hypertrophy, increased albuminuria, mesangial expansion, advanced glomerulosclerosis and increased serum creatinine compared with diabetic mouse without the RAGE transgene. In present study, AGEs significantly upregulated TGF-β1, CTGF and Fn mRNA expression in dose- and time-dependent manners in renal fibroblasts. TGF-β is a major aetiological factor in the pathogenesis of renal fibrosis in DN. TGF-β stimulates the synthesis of ECM such as collagen and laminin, and blocks ECM degradation through inhibition of matrix metalloproteinase.¹⁷ TGF-β1 also possesses antiproliferative function.¹⁸ This study also indicated that AGEs inhibited NRK-49F cell growth. We deduce that AGEs upregulate TGF-β1 expression, alter the cell cycle, and inhibit cell proliferation, resulting in cell hypertrophy and overproduction of ECM thus playing a critical role in progressive tubulointerstitial fibrosis. CTGF, a cysteine rich polypeptide, is one of the recently characterized growth factors and acts as a downstream factor mediating fibrotic activity of TGF-β. CTGF has become a new detected antifibrotic target of DN. Zhou et al⁵ reported that AGE induced CTGF expression through a TGF-β1 independent pathway and played a pivotal role in DN. Fn is a key component of ECM, which accumulates in glomerular mesangium and tubulointerstitium and leads to renal fibrosis. Our findings suggest that AGEs can upregulate the expression of fibrogenic growth factor TGF-β1, CTGF and ECM component Fn in renal interstitial fibroblast. It may be one of the mechanisms underlying the role of AGEs in tubulointerstitial injury in DN.

In DN, activation of neutrophils and monocytes induces “respiratory burst” and the generation of ROS: the overproduction of ROS may cause imbalance between oxidation and antioxidation in body.¹⁹ Oxidative stress is recognized as a key component in the development of diabetic complications⁷ and increases in proportion to the AGEs accumulation. AGEs can generate ROS directly through catalytic sites in their molecular structure²⁰ and stimulation of membrane bound NAD(P)H oxidase via the RAGE receptor²¹ and induction of mitochondrial dysfunction.²² Our findings show that AGEs increase intracellular generation of ROS and decrease intracellular GSH level in time-dependent manner. Preincubation with NAC, an antioxidant that can boost GSH level in cells, significantly increase GSH level and decrease generation of ROS. These suggest an oxidant role for AGEs. GSH, a key antioxidant in cell, is an important constituent of intracellular protective mechanisms against oxidative stress. On the other hand, reduced GSH is known to be a major low relative molecular mass scavenger of free radicals in the cytoplasm. It has been found that AGEs can augment the formation of ROS through depletion of glutathione peroxidase.²³ We also find that preincubation with NAC attenuates AGEs induced upregulation of TGF-β1, CTGF and Fn mRNA. These suggest that ROS may be an important mediator of AGEs induced renal fibrosis. Fukami et al⁶ indicated that AGE/RAGE mediated ROS generation activates TGF-β-Smad signal pathway and subsequently induces cell hypertrophy and fibronectin synthesis by autocrine production of angiotensin II in rat mesangial cells. In addition, Yamagishi et al²⁴ demonstrated that AGE stimulated TGF-β mRNA expression through overgeneration of intracellular ROS in proximal tubular epithelial cells.

The present study also indicates that compared with high glucose and AGEs separately, the presence of AGEs and high glucose significantly increases the expression of TGF-β1, CTGF, Fn mRNA and intracellular generation of ROS, suggesting that there is a synergistic relationship between AGEs and high glucose. The AGEs-induced interstitial fibrosis injury might be accelerated in hyperglycaemia. Thus, we infer that both AGEs and high glucose upregulate expression of TGF-β1, CTGF and Fn through the same signal transduction pathways and those processes of signal transduction are amplified in presence of both. The overproduction of superoxide by mitochondria activates the major pathways of hyperglycaemic damage,⁷ so perhaps AGEs and high glucose effect the expression of fibrogenic factors and ECM through the acceleration of oxidative stress. These possibilities need to be followed up in future studies.

EGb, effective components of which are ginkgo flavonol glycosides and bilobalide, is an extract of dried leaves of Ginkgo biloba. EGb is an effective therapy for cardiovascular diseases, ischaemic cerebral impairment and asthma. It has been used in treating cardiovascular diseases and nephropathy in recent years. Wu²⁵ reported that oral administration of EGb reduced the level of blood urea and creatinine, suggesting that EGb might improve the renal function of glomerulonephritis patients. Lu et al²⁶ have shown that compared with control group, urine protein excretion rate significantly decreased and endogenous creatinine clearance rate, blood lipids and haemorrheology indices improved after injection of EGb in incipient DN patients. The mechanisms underlying the effects of EGb in DN correlate with its range of pharmacological actions: acting as a free radical scavenger and an inhibitor of lipid peroxidation; protecting mitochondrial respiration and oxidative phosphorylations; regulating the release of endothelial factors and the catecholaminergic system; exerting a specific and potent platelet activating factor antagonist activity; and regulating dyslipidaemia and anti- inflammation.^8,27 Here, we provide the first evidence that EGb can reduce AGEs induced TGF-β, CTGF and Fn mRNA overexpression, in a dose-dependent manner through inhibiting ROS and increasing GSH level in renal fibroblasts. These suggest that attenuation of AGEs induced profibrotic growth factor overexpression in renal fibroblasts, through scavenging of free radicals and inhibition of oxidative stress, may be one of the mechanisms underlying EGb in prevention and treatment of DN. Several studies have confirmed the effect of EGb in fibrosis. A study in an animal model of progressive glomerular sclerosis by Tang et al²⁸ states that compared with sclerotic group, sclerotic index of glomerulus and proliferating cell nuclear antigen in tubulointerstitium significantly reduce, and the content of hydroxyproline in renal tissue is lower in EGb treatment group, suggesting that EGb may ameliorate glomerulosis and tubulointerstitial injury. Sener et al¹⁰ reported that EGb could elevate the decreased hepatic glutathione level and attenuate the increased tissue malondialdehyde level, myeloperoxidase activity and collagen content in an animal model of liver fibrosis induced by bile obstruction. In CCl₄ induced liver fibrosis in rats, the histopathological score of fibrosis and the levels of plasma hyaluronic acid and laminin significantly improved and the activities of superoxide dismutase and glutathione peroxidase notably elevated by treated with EGb.¹¹

In conclusion, the present study demonstrates that AGEs can upregulate expression of TGF-β1, CTGF and Fn mRNA and simultaneously stimulate generation of intracellular ROS in NRK-49F cells. Antioxidant NAC decreases overexpression of TGF-β1, CTGF and Fn expression induced by AGEs, suggesting that ROS might be a mediator of AGEs induced renal injury. There is an interaction between AGEs and high glucose in upregulation of TGF-β1, CTGF and Fn mRNA expression. The present study also suggests that EGb attenuates AGEs-induced TGF-β1, CTGF and Fn mRNA overexpression through inhibiting the oxidative stress. Antioxidant therapy, especially ginkgo biloba extract treatment, shows promise in preventing oxidative damage and renal fibrosis in DN.

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