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*Yonsei University, College of Science, Department of Biochemistry, Hyonam Kidney Laboratory, Soon Chun Hyang University, Yonsei University, College of Science, Department of Chemistry, Yonsei University, College of Medicine, Department of Endocrinology, ¶Yonsei University, College of Medicine, Department of pathology, Seoul, Korea.
Correspondence to Dr. Hyun Chul Lee and Dr. Young Dong Cho, Department of Internal Medicine, and Department of Biochemistry, Yonsei University, 134 Shinchon-Dong, Seodaemoon-Ku, P.O. Box 120-749, Seoul, Korea. Phone: 822-361-5425; Fax: 822-393-6884;
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1 (TGF-1) is the final mediator of ECM accumulation, reactive oxygen species (ROS) and protein kinase C (PKC) are the upstream signaling molecules that mediate hyperglycemia-induced ECM expansion. Magnesium lithospermate B (LAB) is an active component isolated from Salvia miltiorrhizae with known renoprotective properties due to its antioxidative effects. Thus, the present study examined the effects of LAB on renal injury in streptozotocin-induced diabetic rats (STZR) and on the activation of mesangial cells cultured under high glucose conditions. Ten micrtograms of LAB/kg per day was started 8 wk after streptozotocin injection and continued for a period of 8 wk. It significantly suppressed renal malondialdehyde (MDA), microalbuminuria, glomerular hypertrophy, mesangial expansion, and the upregulation of renal TGF-1, fibronectin, and collagen in STZR without significantly affecting plasma glucose. Both 30 mM of glucose and 100 uM of H2O2 significantly increased TGF-1 and fibronectin protein secretion by mesangial cells. LAB at 10 µg/ml inhibited high glucose- and H2O2-induced TGF-1 and fibronectin secretion. LAB also inhibited glucose-induced intracellular ROS generation and PKC activation in mesangial cells, but it did not directly inhibit PKC activity at dosages that inhibited ROS generation. The in vitro data of this study show that LAB inhibits ROS generation leading to PKC activation and TGF-1 and fibronectin upregulation in mesangial cells cultured under high glucose conditions. Moreover, delayed treatment with LAB was found to significantly suppress the progression of renal injury in STZR. LAB may become a new therapeutic agent for the treatment of diabetic nephropathy. E-mail: endohclee@yumc.yonsei.ac.kr |
Introduction |
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High glucose (30 mM) is the main determinant of diabetic nephropathy in both type 1 (2) and type 2 (3) diabetes. Though transforming factor-1 (TGF-1) is the final mediator of extracellular matrix (ECM) expansion in diabetic nephropathy (4–6), increased reactive oxygen species (ROS) generation (7,8) and protein kinase C (PKC) activation (9,10) in association with increased glucose metabolism are considered to be the main upstream signaling molecules of glucose-induced vascular injury, which includes diabetic nephropathy.
ROS generation (8,11,12) and PKC activation (13–15) are elevated in mesangial cells cultured under high glucose conditions. The kidneys of experimental diabetic animals also exhibit increased lipid peroxidation (16), which is a marker of increased ROS generation, and PKC activation (16). In addition, exogenously administered ROS (8,17) and PKC activator (8) lead to TGF-1 and ECM upregulation in mesangial cells. Moreover, antioxidants (18–26) and PKC inhibition (27,28) have been reported to prevent or attenuate both glucose-induced mesangial cell activation and renal injury in diabetes.
Salvia miltiorrhizae radix is a Chinese herbal medicine that has been traditionally used for the treatment of diabetic complications. Lithospermate B (LAB) was recently isolated from S. miltiorrhizae and found to have beneficial effects on the renal function of nephrectomized rats (29), possibly by reducing ROS generation (30). It is tetramer of caffeic acid (Figure 1). Thus, the present study was undertaken to examine the effects of delayed LAB treatment on the renal function of streptozotocin-induced diabetic rats (STZR) and to evaluate the role of the antioxidative effects of LAB on protection against diabetic renal injury using mesangial cells cultured under high glucose conditions.
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Isolation and Purification of Lithospermate B (LAB)
Magnesium LAB was isolated from S. miltiorrhizae as described previously (31).
Induction of Experimental Diabetes Mellitus
Male, 10-wk-old, Sprague-Dawley rats were obtained from the animal facility of Yonsei University College of Medicine, Seoul, Korea. The animals were housed 2 to 3 per cage in an animal room controlled at 23 ± 2°C and 55 ± 5% room humidity, under a 12 h-light 12 h-dark cycle. All rats were maintained on standard rat chow (Samyang rat chow, Seoul, Korea) and tap water ad libitum.
Diabetes was induced by injecting 60 mg/kg of streptozotocin (STZ) intravenously (dissolved in pH 4.5 citrate buffer immediately before injection) into the tail vein, as described previously (21). Control rats (CR: 10 rats) received the same volume of citrate buffer (2.5 ml/kg). Induction of the diabetic state was confirmed by determining plasma glucose with commercial enzymatic test strips (SureStep, LIFESCAN INC.) two days after the streptozotocin injection. A total of 16 rats with plasma glucose concentrations > 300 mg/dl were defined as diabetic rats. Separate groups of streptozotocin-induced diabetic rats (STZR) and CR were treated with LAB starting 8 wk after the streptozotocin or citrate buffer treatment. LAB was orally administered daily at a dose of 10 mg/kg for an additional 8 wk. The effects of different concentrations of LAB (1 to 20 mg/kg/d) on albuminuria in diabetic STZR were compared during our preliminary study. LAB at dose 10 mg/kg per d exhibited better effect than 1 mg/kg per d but similar effect as 20 mg/kg per d, 10 mg/kg per d was used in further studies. All STZR were survived without insulin treatment in the present study. We did not administer insulin to avoid non-metabolic effects of insulin.
Cell cultures
Murine mesangial cells obtained from the American Type Cell Collection (ATCC: MES-13, cloned from mice transgenic for the early region of the SV-40 virus, passage 25) were grown in DMEM containing 5% fetal bovine serum (FBS), 1% streptomycin-penicillin mixture, 0.5 µg/ml fungizone, 44 mM NaHCO3, and 14 mM HEPES in an atmosphere of 5% CO2 and 95% air at 37°C in a humidified incubator. Results from MES-13 were confirmed by primary rat mesangial cell isolated and cultured as described previously (32). Subcultures were prepared from confluent cultures by trypsinization in Hanks’ balanced salt solution containing 0.5 mM EDTA and 0.25% trypsin. These mesangial cells were then grown in DMEM containing 5% FBS to near confluence (80%), and further incubated in serum-free medium for 24 h to arrest and synchronize cell growth. The medium was then changed to serum-free DMEM containing different concentrations of glucose or H2O2 in the presence or absence of LAB. Treatment with LAB up to 40 µg/ml did not show any discernable cytotoxicity, as estimated by the MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay (control: 100 ± 7.6%; 40 µg/ml LAB treatment: 92.3 ± 9.1%) and LDH release (control: 9.9 ± 2.4%; 40 µg/ml LAB treatment: 12.5 ± 4.9%). Cells were stimulated for 1 h to determine ROS generation or for 48 h for PKC activity and TGF-1 and fibronectin protein secretion. We previously demonstrated that the secretion of TGF-1 and fibronectin protein by mesangial cells is significantly increased 48 h after the addition of 30 mM glucose (32). After incubation, the media were collected, aliquoted, and frozen at -70°C until required for TGF-1 and fibronectin protein determinations. Cellular protein concentrations in cell lysates were measured by the Bio-Rad assay using the Bradford method (33).
Measurement of Urinary Albumin
Urine samples collected over a 24-h period for three consecutive days were centrifuged at 3000 x g for 10 min, and urinary albumin in the supernatants was determined by ELISA using a Nephrat microalbumin assay kit (Exocell Inc.). All samples were assayed in triplicate and the mean value from a given rat was calculated.
Measurement of Malondialdehyde (MDA) in the Renal Cortex
A modification of the thiobarbituric acid method of Ohkawa et al. (34) was used to measure the level of lipid peroxidation, as described previously (35). The renal cortex was excised and homogenized with extraction solution (iNtron, Seoul, Korea). Aliquots of homogenates (80 µl) were mixed with 80 µl of 8% SDS and a reaction mixture consisting of 120 µl of 0.8% 2-thiobarbituric acid and 120 µl of 20% acetic acid. This solution was placed in a water bath and kept at 95°C for 60 min. After stopping the reaction by cooling with tap water, the mixture was centrifuged at 15,000 x g for 5 min to precipitate interfering particulate materials. The amount of MDA formed was determined by spectrofluorometry (SPF-500C, SLM Instruments) at an emission wavelength of 553 nm at an excitation wavelength of 515 nm and calculated using a tetraethoxypropane standard curve.
Glomerular Histology and Morphometry
For each rat, quantitative morphometry analyses of the different glomerular domains were performed as described previously (36). A 3-µm-thick section was obtained from kidney transversely cut through the hilus in each rat. On this section, 30 different superficial glomeruli were randomly sampled for morphometric analysis as follows: The microscopic slide was scanned clockwise along the superficial cortex, and one glomerulus out of three was analyzed. For every investigated glomerulus, the following measurements were obtained with an automated image analysis system: (1) the total glomerular profile area limited; (2) the glomerular tuft area; (3) the mesangial matrix area (36).
Immunohistochemical Staining for TGF-1 and Fibronectin in Renal Cortex
TGF-1 and fibronectin were immunolocalized as described previously (37). Fifty-micrometer-thick Vibratome sections taken through the entire kidney were mounted in Epon between polyethylene vinyl sheets. Sections from the cortex were excised and glued on blocks of Epon, and 3-µm sections were cut for light microscopy. Vibratome sections were washed with 50 mM NH4Cl in PBS three times for 15 min; before incubation with the primary antibody, the sections were incubated for 3 h with PBS that contained 1% bovine serum albumin, 0.05% saponin, and 0.2% gelatin (solution A). The tissue sections then were incubated overnight at 4°C with polyclonal antibody against TGF-1 and fibronectin obtained from Santa Cruz (Delaware, CA). Antibodies were diluted 1:1000 for TGF-1 and fibronectin in PBS containing 1% bovine serum albumin (solution B). After several washes in solution A, the sections were incubated for 2 h in peroxidase-conjugated goat anti-rabbit and anti-mouse IgG Fab fragment (Jackson ImmunoResearch Laboratories) diluted 1:100 in solution B. The sections were then rinsed in solution A and subsequently in 0.05 M Trisbuffer (pH 7.6). To detect horseradish peroxidase, the sections were incubated in 0.1% 3,3'-diaminobenzidine in 0.05 M Tris buffer for 5 min. H2O2 was then added to a final concentration of 0.01%, and the incubation continued for 10 min. After washing with 0.05 M Tris buffer three times, the sections were dehydrated in a graded series of ethanol, embedded in Epon, and photographed under an Olympus photomicroscope (Tokyo, Japan) that was equipped with differential interference contrast optics.
Analysis of the percentage of area occupied by TGF-1, and fibronectin positive staining was performed using computer-assisted image analysis software (Meta Morph, version 4.6, Universal Imaging Corporation). For each kidney, more than 30 glomerular profiles, cut in equatorial section planes and successively appearing in the visual field of the microscope, were examined.
Staining for Collagen Protein in Renal Cortex: Masson Trichrome Stain
Paraffin-embedded sections were stained with Masson Trichrome for collagen. Extent of collagen accumulation was analyzed using Meta Morph software as described in above.
Assay for TGF-1 Protein: ELISA
The TGF-1 in the renal cortex homogenate and in culture media supernatant were converted to the active form by HCl treatment (final concentration, 0.2 M) for 30 min at room temperature followed by neutralization with equimolar NaOH. The amount of TGF-1 was determined by quantitative sandwich enzyme immunoassay using the TGF-1 Emax ImmunoAssay System (Promega) according to the manufacturer’s description. This TGF-1 Emax ImmunoAssay System has a detection limit of 32 pg/ml for TGF-1.
Assay for Fibronectin Protein: Western Blot Analysis
Immunoblot analysis was used to determine fibronectin in the renal cortex homogenate and in the culture media supernatant as described previously (32). In brief, aliquots of the renal cortex homogenate and conditioned media were mixed with sample buffer containing SDS and -mercaptoethanol and heated at 95°C for 15 min. Respective samples were then applied to a 5% polyacrylamide gel and subjected to electrophoresis. A prestained SDS-PAGE standard (broad range, Bio-Rad) was used as a molecular weight marker. The proteins were transferred onto a nitrocellulose membrane using a transblot chamber with Tris buffer, and the membranes incubated with rabbit anti-human fibronectin (HRP-conjugated, DAKO, Glostrup, Denmark) diluted 1:10000 in PBS containing 1% bovine serum albumin for 2 h at room temperature and subjected to ECL western blotting (Amersham Life Science, Little Chalfont, UK). Positive immunoreactive bands were quantified densitometrically and compared with the controls.
Assay for Intracellular ROS
Intracellular ROS production was measured using the method of Bass et al. (38) modified for confocal microscopy as described previously (12). In brief, coverglasses of confluent cells obtained 1 h after high glucose stimulation were washed with Dulbecco’s PBS and incubated in the dark for 5 min in Krebs-Ringer solution containing 5 mM of 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA, Molecular Probes Inc). CM-H2DCFDA is a nonpolar compound that readily diffuses into cells, where it is hydrolyzed to the nonfluorescent polar derivative 2'7'-dichlorofluorescin (DCF). Culture dishes were transferred to a Leica DM IRB/E inverted microscope, equipped with a x20 Fluotar objective and a Leica TCS NT confocal attachment, and ROS generation was detected (excitation, 488 nm; emission, 515 to 540 nm). The effect of DCFH photoxidation was minimized by collecting the fluorescence image with a single rapid scan (line average, 4; total scan time, 5.2 s), and identical parameters, such as contrast and brightness, were used for all determinations. Cells were imaged by differential interface contrast microscopy.
Assay for PKC activity
Membrane and cytosolic fractions were obtained by the method described by Kikkawa et al. (13). PKC activities were measured using a Pep Tag Non-Radioactive Assay kit (Promega). Fluorescence peptide (D-L-S-R-T-L-S-V-A-A-K), which is highly specific for PKC, was used as a substrate. The phosphorylation of this specific substrate by PKC alters the peptide’s net charge from +1 to -1. The phosphorylated substrate so obtained was separated in agarose gel at pH 8.0, and negatively charged bands from the gel were removed and heated at 95°C until the gel slice melted. Absorbance of the solubilized slice was measured using a spectrophotometer at 570 nm.
To investigate whether LAB acts as a direct inhibitor of PKC, LAB (5 to 200 µg/ml) was added directly to reaction mixtures containing rat brain PKC supplied by manufacturer or membrane fractions of mouse mesangial cell cultured under high glucose for 48 h.
Statistical Analysis
All results are expressed as means ± SD. ANOVA was used to assess the intergroup differences. If the F statistics proved to be significant, the mean values obtained from each group were compared using Fisher least significant difference method. A P value of less than 0.05 was used as the criterion for statistically significant difference.
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Results |
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The administration of LAB, started 8 wk after STZ treatment for 8 wk significantly ameliorated absolute kidney weight, kidney weight expressed as a function of body weight, glomerular hypertrophy (Table 1), and albuminuria (Figure 3) in STZR, although kidney weight expressed as a function of body weight and urinary albumin excretion in LAB+STZR remained statistically higher than in CR.
Immunostaining for TGF-1 (Figure 4A) and fibronectin (Figure 4B) and Masson Trichrome staining for collagen (Figure 4C) demonstrated that increases in TGF-1, fibronectin, and collagen deposition in both glomeruli and tubulointerstitium of the renal cortex in diabetic STZR. Semiquantitative analyses for each parameter in the glomeruli and in the tubules from different experimental group are summarized in Table 2. Delayed treatment with LAB effectively inhibited all diabetes-associated TGF-1, fibronectin, and collagen upregulation in the renal cortex. Upregulation of TGF-1 and fibronectin in the cortex were confirmed by ELISA and Western blot analysis, respectively (Figure 5, A and B).
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As summarized in Figure 6, MDA was sevenfold higher in the renal cortex of STZR than in CR at 16 wk after STZ treatment. MDA in LAB+STZR was significantly lower than in STZR but statistically higher than in CR and LAB+CR.
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1 and Fibronectin Protein Secretion by Mesangial Cells Cultured under High Glucose or H2O21 showed that the exposure of mesangial cells to 30 mM glucose (Figure 7A) or 100 µM H2O2 (Figure 7B) significantly induced TGF-1 protein secretion after 48 h. LAB inhibited high glucose-induced TGF-1 secretion by mesangial cells in a dose-dependent manner (Figure 7A). LAB at above 20 µg/ml decreased TGF-1 protein secretion by mesangial cells cultured in control 5.6 mM glucose (data not shown), 10 µg/ml of LAB was used to determine the effects on H2O2-induced TGF-1 secretion (Figure 7B).
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1 secretion. Exposure of mesangial cells to 30 mM glucose (Figure 8A) or 100 µM H2O2 (Figure 8B) significantly increased fibronectin secretion after 48 h. LAB at 10 µg/ml inhibited both high glucose- and H2O2-induced, but not basal, fibronectin secretions by mesangial cells.
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Effects of LAB Intracellular ROS in Mesangial Cells Cultured under High Glucose or H2O2
The incubation of mesangial cells with 30 mM of glucose for 1 h (Figure 9A) or H2O2 for 15 min (Figure 9B) increased intracellular ROS. Quantitative analyses of ROS showed that the exposure of mesangial cells to high glucose increased intracellular ROS 11-fold compared with control and that this was effectively inhibited by the addition of LAB 10 µg/ml; 100 µM of H2O2 increased intracellular ROS tenfold compared with control, and this was also effectively inhibited by the addition of LAB at 10 µg/ml.
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As summarized in Figures 11A and 11B, exogenously administered LAB into purified rat brain PKC or membrane fraction of mouse mesangial cell did not inhibit PKC activity at concentrations up to 40 µg/ml. Concentrations of LAB higher than 100 µg/ml directly inhibited PKC activity in a dose-dependent manner.
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Discussion |
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1, fibronectin, and collagen expression in STZR. In view of the fact that in the clinical situation treatment is often initiated after the onset of diabetic nephropathy, the present finding suggests that LAB may prove to be a new therapeutic agent for the treatment of diabetic nephropathy.
Renal TGF-1, fibronectin, and collagen upregulations in STZR were totally blocked by delayed LAB treatment. The levels of urinary albumin excretion and MDA in the renal cortices of LAB+STZR were also significantly lower than that of the STZR but statistically higher than those of CR in the present study. These differences may have resulted from the different time courses among TGF-1, fibronectin, and collagen upregulation in the renal cortex, albuminuria, or MDA accumulation in STZR and/or the sensitivity of each measurement used in present study. The concentration of MDA and the expression of TGF-1, fibronectin, and collagen in renal cortex at 8 wk after STZ injection but before treatment with LAB could not be measured. UAE in diabetic rats in the present study are similar to those in a previous study (39).
We, therefore, examined the antioxidative property of LAB by using of mesangial cells cultured under high glucose, an accepted in vitro model for diabetic nephropathy. LAB inhibited high glucose-induced intracellular ROS, and LAB, at a dose-inhibiting ROS generation, inhibited both high glucose- and H2O2-induced TGF-1 and fibronectin protein secretion by mesangial cells; therefore, the protective effects of LAB on experimental diabetic nephropathy may be largely due to its antioxidative effects.
The inhibition of H2O2-induced intracellular DCF-sensitive fluorescence by LAB (Figure 9B) is in general agreement with previous reports, which demonstrated that S. miltiorrhizae normalizes catalase activity and restores superoxide dismutase levels (30). Previous studies have demonstrated that high glucose increases intracellular ROS as a result of glucose uptake and metabolism in mesangial cells (12) and found that different antioxidants ameliorate high glucose-induced TGF-1 and ECM synthesis in mesangial cells (18,20). In agreement with these studies, we found that LAB, at concentrations that inhibited high glucose-induced intracellular ROS generation, effectively blocked TGF-1 and fibronectin upregulation in mesangial cells cultured under high glucose conditions.
Although LAB treatment inhibited high glucose-induced PKC activation in mesangial cells, LAB at concentrations up to 40 µg/ml, did not directly inhibit PKC activity. This suggests that the inhibition of high glucose-induced PKC activation by LAB may occur indirectly through the inhibition of intracellular ROS generation. PKC activation under hyperglycemic conditions is largely related to an increase in the de novo synthesis of diacylglycerol (DAG), the major endogenous activator of PKC, and results from an increased glucose metabolism (10). However, PKC can be sensitively regulated by redox modification, which is independent of DAG. The N-terminal regulatory domain of PKC contains zinc-binding, cystein-rich motifs that are susceptible to oxidative modification, and which are readily oxidized by peroxide. When oxidized, the autoinhibitory function of the regulatory domain is compromised and cellular PKC activity is consequently stimulated. In addition, H2O2 activates PKC-
through the activation of tyrosine kinase (40). In this context, a recent study by Nishikawa et al. (7) demonstrated that ROS play an important role in PKC activation in endothelial cells cultured under high glucose conditions. PKC activations in mesangial cells cultured under high glucose conditions (20) and in STZ-induced diabetic rats (41) were effectively inhibited by antioxidants.
Although the chemical structure of LAB is known (Figure 1), it is not clear at present whether mesangial cells take up LAB. Further studies are needed to determine the exact mechanisms involved in inhibition of intracellular ROS by LAB.
Potential toxic effects of LAB should also be investigated in detail, because the mean body weight of LAB+STZR was slightly lower, although it is not statistically different, than that of STZR (Figure 2). Data from the present study provided that LAB did not affect survival, SGOT, and SGPT in diabetic STZR as well as CR (data not shown).
In conclusion, LAB inhibited the ROS generation, and this led to PKC activation and TGF-1 and fibronectin upregulation in mesangial cells cultured under high glucose conditions. Moreover, delayed treatment with LAB significantly suppressed the progression of renal injury in diabetic rats. LAB, an active component of S. miltiorrhizae radix, may prove to be a new therapeutic agent for the treatment of diabetic nephropathy.
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Acknowledgments |
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in rat glomerular mesangial cells cultured under high glucose conditions. Diabetologia 37: 838–841, 1994[Medline]
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antibody in db/db diabetic mice. Proc Nat Acad Sci 97: 8015–8020, 2000 isoforms and their receptors in acute and chronic experimental diabetes in rats. Endocrinology 141: 1196–1208, 2000 and C-
by oxidative stress in early diabetic rat kidney. Am J Kid Dis 38 [Suppl 1]: S204–S207, 2001[Medline]This article has been cited by other articles:
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